Combination vaccine against coronavirus, influenza and / or RSV infections

Abstract

Specifically, in one embodiment, the present disclosure relates to a method for inducing an immune response in a subject against coronavirus spike proteins (S proteins), particularly the S protein of SARS-CoV-2, and influenza proteins, particularly the hemagglutinin (HA) proteins of influenza A and B viruses, comprising administering to the subject (i) a bivalent RNA vaccine encoding a peptide or protein comprising an epitope of the S protein of SARS-CoV-2, and (ii) a tetravalent RNA vaccine encoding a peptide or protein comprising an epitope of HA.

Description

[Technical Field]

[0001] Priority claims This application claims priority under 35 U.S.C. § 120 to each of the following applications, the disclosures of each of which are incorporated by reference in their entirety: U.S. Provisional Application No. 63 / 416,933, filed October 17, 2022; U.S. Provisional Application No. 63 / 431,615, filed December 9, 2022; U.S. Provisional Application No. 63 / 437,967, filed January 9, 2023; U.S. Provisional Application No. 63 / 465,516, filed May 10, 2023; and U.S. Provisional Application No. 63 / 469,473, filed May 29, 2023. [Background technology]

[0002] Viral infections represent a major threat to human health and well-being. For example, coronaviruses are a class of RNA viruses that cause respiratory infections that can range from mild to fatal. Mild illness in humans includes some cases of the common cold (which is also caused by other viruses, primarily rhinoviruses), while more deadly varieties can cause SARS, MERS, and COVID-19.

[0003] Influenza, commonly known as "the flu," is an infectious disease caused by influenza viruses, a family of negative-sense RNA viruses. Symptoms range from mild to severe and often include fever, runny nose, sore throat, muscle aches, headache, cough, and fatigue. In a typical year, 5-15% of the population contracts influenza, resulting in 3-5 million severe cases and 650,000 respiratory-related deaths worldwide each year. Summary of the Invention

[0004] The present disclosure relates to simultaneous delivery of multiple antigenic polypeptides to a subject (e.g., a human subject) and related techniques (e.g., methods) for preventing and / or treating multiple infectious agents. In some embodiments, such infectious agents may include, but are not limited to, infectious bacterial and viral agents.

[0005] In some embodiments, the present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) that achieve simultaneous delivery of such multiple antigenic polypeptides. In some embodiments, the present disclosure provides several combination compositions that will be particularly useful for effective vaccination. In some embodiments, such combination compositions comprise multiple RNAs that encode antigenic polypeptides of (e.g., that induce or promote immunity against) at least two different infectious diseases (e.g., in some embodiments, infectious respiratory diseases).

[0006] In some embodiments, an antigenic polypeptide described herein is a polypeptide comprising at least one antigenic epitope. In some embodiments, the antigenic polypeptide is a full-length antigen. In some embodiments, the antigenic polypeptide is an immunogenic fragment of a full-length antigen. In some embodiments, the antigenic polypeptide can comprise one or more modifications (e.g., in some embodiments, substitutions) compared to the full-length or immunogenic fragment, e.g., as found in the relevant infectious agent. In some embodiments, the antigenic epitope or antigenic polypeptide is cross-reactive with (e.g., induces and / or enhances an immune response against) a corresponding epitope or polypeptide in an infectious agent (e.g., in a virus, e.g., a respiratory virus). The present disclosure provides, among other things, the recognition that annual vaccination programs for some infectious diseases (e.g., influenza, respiratory syncytial virus disease, and / or coronavirus disease) may occur at similar times during the year, and that existing vaccine vectors and conventional technologies may limit the production time from selection of seasonal strains. For example, while influenza and coronavirus vaccines are currently available, the present disclosure identifies the source of problems with current vaccination programs and further provides improved vaccination techniques, including specific vaccine compositions and strategies. Because a lack of neutralizing antibody titers has been observed over time after the initial series of COVID-19 vaccines, booster doses are recommended to restore or maintain robust immunity and disease protection. Moreover, as new SARS-CoV-2 variants continue to evolve, vaccine strain changes are inevitable. Influenza vaccines are also updated periodically (typically annually). Existing influenza vaccines have limitations, including, for example, production time from seasonal strain selection, manufacturing complexity, and limited efficacy. The present disclosure provides, among other things, the recognition that combination RNA vaccines for some infectious diseases (e.g., infectious respiratory diseases) may be beneficial in addressing some of the limitations of existing individual vaccines for some infectious diseases.For example, in one aspect, the present disclosure provides the recognition that combination RNA vaccines may offer several advantages, including, but not limited to, potentially faster manufacturing, e.g., by eliminating a genetic reassortment step, and / or reduced chance of the vaccine being mismatched with seasonal circulating strains, and / or improved efficacy compared to currently licensed vaccines due to the induction of strong T cell responses (e.g., CD4+ and / or CD8+ T cell responses).

[0007] In one aspect, the present disclosure relates to technologies (e.g., compositions and methods) for vaccination against coronavirus and influenza virus infection or disease and for inducing effective coronavirus and influenza virus antigen-specific immune responses, such as antibody and / or T cell responses. In some embodiments, such technologies based on RNA technology are particularly useful for preventing or treating coronavirus and influenza virus infection and / or disease. By administering the RNA disclosed herein to a subject, the subject can be protected from coronavirus infection and / or influenza virus infection (e.g., the likelihood of infection and / or disease resulting from exposure is reduced).

[0008] In some embodiments, the present disclosure provides techniques (e.g., compositions and / or methods) for protecting against coronavirus and influenza virus infection by administering to a subject RNA encoding a coronavirus antigenic polypeptide and RNA encoding an influenza antigenic polypeptide. Specifically, in one embodiment, the present disclosure relates to a method comprising administering to a subject RNA encoding a coronavirus peptide or protein, particularly the S protein of SARS-CoV-2, containing an epitope of the S protein and RNA encoding a peptide or protein containing an epitope of the HA protein, i.e., vaccine RNA encoding a vaccine antigen, to induce an immune response against the coronavirus spike protein (S protein) and an immune response against the hemagglutinin (HA) protein of the coronavirus. Administering the RNA encoding the vaccine antigen to a subject can result in the vaccine antigen (after expression of the RNA by appropriate target cells) being provided in the subject for inducing an immune response against the vaccine antigen (and disease-associated antigens).

[0009] The present disclosure provides, among other things, insights for achieving effective delivery of multiple antigenic polypeptides (e.g., antigenic polypeptides from different infectious agents) to a patient and / or for providing a robust immune response against various infectious diseases. In some embodiments, the present disclosure provides insights regarding RNA vaccine technology, such as antigen combinations, sequences used to encode antigenic polypeptides, non-coding elements, nanoparticle formulations, pharmaceutical compositions, and dosing regimens that can provide effective delivery of multiple antigenic polypeptides (e.g., antigenic polypeptides from different infectious agents) to a subject and / or provide a robust immune response against various infectious diseases.

[0010] In some embodiments, the insights provided herein result in RNA compositions that can generate effective immune responses against multiple infectious agents. In some embodiments, the insights provided herein result in RNA compositions that can generate immune responses against at least two infectious agents that are comparable to (e.g., within 70%, 80%, 90%, 95%, or more, and up to 100%) or better (e.g., at least 30%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 1.1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, or more) the efficacy of the respective single (e.g., monovalent) RNA vaccine or reference vaccine, e.g., a non-RNA vaccine (e.g., an inactivated virus vaccine). In some embodiments, the insights provided herein result in RNA compositions that are capable of generating a superior (e.g., at least 30%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 1.1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, or more increased) immune response against at least one, and in some embodiments each, of at least two infectious agents, compared to the efficacy of the respective RNA vaccine alone (e.g., monovalent) and thus the efficacy of the respective RNA vaccine alone when administered at the same dose.In some embodiments, the insights provided herein can even be used to produce RNA compositions comprising RNAs encoding at least two antigenic polypeptides of at least two infectious agents, each at a dose lower than the dose used in their respective single (e.g., monovalent) RNA vaccines, that are capable of generating a superior (e.g., at least 30%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 1.1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, or more increased) immune response against at least one, and in embodiments each, of the at least two infectious agents, compared to those single (e.g., monovalent) RNA vaccines having higher doses.

[0011] In some embodiments, the present disclosure provides insight that using the same RNA backbone construct (e.g., one with the same combination of non-coding elements, e.g., the same 5' cap, proximal cap sequence, 5' UTR sequence, 3' UTR sequence, and polyA sequence, excluding the sequence encoding the antigenic payload, relative to the mRNA) and / or the same nanoparticle formulation (e.g., the same lipid formulation) encapsulating the RNA to deliver one or more antigenic polypeptides from at least two different infectious agents (e.g., respiratory infectious agents) in a single composition may offer one or more particular advantages. For example, in some embodiments, such an approach may enable such RNA to remain stable in a single composition after storage at a non-zero temperature or higher for at least 24 hours or more (e.g., in some embodiments, exposure to 30°C for a period of time followed by maintenance at 2-8°C for a period of time). In some embodiments, the present disclosure provides the insight that using the same RNA backbone construct (e.g., one with the same combination of non-coding elements, e.g., one with the same 5' cap, proximal cap sequence, 5' UTR sequence, 3' UTR sequence, and polyA sequence, except for the sequence encoding the antigenic payload, relative to the mRNA) and / or the same nanoparticle formulation (e.g., the same lipid formulation) in which the RNA is encapsulated to deliver one or more antigenic polypeptides from at least two different infectious agents (e.g., respiratory infectious agents) in a single composition can result in comparable pharmacokinetics and / or pharmacodynamics of such RNAs and / or reduce or minimize interfering immunogenicity of the encoded antigenic polypeptides compared to RNAs with different combinations of non-coding elements and / or different nanoparticle formulations.

[0012] In some embodiments, the present disclosure provides the insight that in a composition comprising two or more polynucleotides, each comprising a nucleotide sequence encoding an antigenic polypeptide associated with a different infectious agent, when the two or more polynucleotides comprise the same combination of non-coding elements (e.g., the same 5' cap, near-cap sequence, 5' UTR sequence, 3' UTR sequence, and polyA sequence, excluding the sequence encoding the antigenic payload, relative to the mRNA) and / or are formulated in the same nanoparticle formulation (e.g., the same lipid formulation), superior immune responses can be induced against each target infectious agent compared to RNAs with different combinations of non-coding elements and / or different nanoparticle formulations.

[0013] In some embodiments, the present disclosure provides insight that the multivalency of combination vaccines may have an adjuvant effect. While not wishing to be bound by a particular theory, in some embodiments, such an adjuvant effect may be due to the increased concentration of nanoparticles (e.g., lipid nanoparticles) encapsulating RNA, which may lead to a dose-sparing effect. Thus, in some embodiments, a composition comprising two or more polynucleotides, each comprising a nucleotide sequence encoding an antigen associated with a different infectious agent, and each formulated (together or separately) in a nanoparticle formulation (e.g., a lipid nanoparticle formulation), may induce a superior immune response than that induced by a corresponding monovalent composition (e.g., a composition comprising only one of the polynucleotides in the same nanoparticle formulation). In some embodiments, the immune response induced by such a composition may be stronger than that induced by the same amount of a monovalent composition. In some embodiments, a smaller amount of such a composition may be required to generate an immune response of the same strength as a monovalent product.

[0014] While not wishing to be bound by any particular theory, the present application provides the insight that, in some embodiments, separate encapsulation (e.g., within delivery vehicle(s), e.g., LNP(s)) of individual mRNAs encoding different antigenic polypeptides (e.g., antigenic polypeptides from different infectious agents, e.g., SARS-CoV-2 versus influenza, or antigenic polypeptides corresponding to different variants of the same infectious agent, e.g., different variants of the SARS-CoV-2 spike protein) may provide certain advantages, e.g., certain immunological benefits, compared to co-encapsulation (e.g., two or more individual mRNAs encoding distinct antigenic polypeptides) within the same delivery vehicle(s). For example, the present disclosure proposes that, in some embodiments, separate encapsulation may provide improved expression of one or more of the encoded antigenic polypeptides and / or an improved immune response to one or more of the encoded antigenic polypeptides. Without wishing to be bound by any particular theory, in some embodiments, separate encapsulation can facilitate separate uptake of individual mRNAs into cells (e.g., separate APCs) in a subject. Without wishing to be bound by any particular theory, the present application notes that co-encapsulation of two or more RNAs, each encoding a different antigenic polypeptide, can lead to co-uptake by cells (e.g., APCs), which can result in competition for translation of one or more of the encoded antigenic polypeptides, resulting in reduced expression and / or a reduced immune response thereto.In some embodiments, separate encapsulation of mRNAs encoding influenza antigens (e.g., separately encapsulating each mRNA encoding an influenza antigen; encapsulating mRNA(s) encoding influenza A antigens in a first population of nanoparticles (e.g., LNPs) and mRNA(s) encoding influenza B antigens in a second population of nanoparticles (e.g., LNPs); or encapsulating mRNA(s) encoding influenza A antigens in a first population of nanoparticles (e.g., LNPs) and each mRNA encoding influenza B antigens in a separate population of nanoparticles (e.g., LNPs)) may provide advantages over compositions comprising mRNAs encoding co-encapsulated influenza antigens. In some embodiments, a composition comprises: (i) two or more different RNAs, each encoding an antigenic polypeptide (e.g., an HA protein) of an influenza A virus (e.g., such that the two or more different RNAs together encode two or more different influenza A HA polypeptides); and (ii) two or more different RNAs, each encoding an antigenic polypeptide (e.g., an HA protein) of an influenza B virus (e.g., such that the two or more different RNAs together encode two or more different influenza B HA polypeptides). each RNA encoding an antigenic polypeptide of influenza A virus is encapsulated in a first population of nanoparticles and each RNA encoding an antigenic polypeptide of influenza B virus is encapsulated in a second population of nanoparticles; Each RNA is encapsulated in a separate nanoparticle; or each RNA encoding an influenza A antigenic polypeptide is encapsulated in a first population of nanoparticles, and each of the two RNAs encoding influenza B antigenic polypeptides is encapsulated in a separate population of nanoparticles; In this way, they are formulated into nanoparticles (e.g., LNPs).

[0015] In some embodiments, the present disclosure also provides insights for generating immune responses that broadly neutralize various influenza virus strains (e.g., result in high neutralization titers and / or seroconversion rates against influenza A and / or B viruses (e.g., clinically meaningful levels of neutralization titers and / or seroconversion rates (e.g., (i) neutralization titers that are similar to or better than those previously shown to prevent influenza symptoms, and / or (ii) neutralization titers and / or seroconversion rates that are similar to or better than those induced by an appropriate comparator (e.g., a commercially licensed influenza vaccine or an influenza RNA vaccine administered without a SARS-CoV-2 vaccine))). The present disclosure also provides exemplary doses of RNA that can produce a strong immune response against both types of influenza virus (e.g., clinically meaningful levels of neutralization titers and / or seroconversion rates (e.g., (i) neutralization titers similar to or better than those previously shown to prevent influenza symptoms, and / or (ii) neutralization titers and / or seroconversion rates similar to or better than those induced by an appropriate comparator (e.g., a commercially licensed influenza vaccine or an influenza RNA vaccine administered without a SARS-CoV-2 vaccine))).

[0016] Coronaviruses are positive-sense single-stranded RNA ((+)ssRNA) enveloped viruses that encode a total of four structural proteins: spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The spike protein (S protein) is responsible for receptor recognition, cell attachment, infection via the endosomal pathway, and genome release facilitated by fusion of the viral membrane with the endosomal membrane. Although the sequence differs among different family members, conserved regions and motifs exist in the S protein, allowing it to be divided into two subdomains, S1 and S2. While S2 is responsible for membrane fusion via its transmembrane domain, the S1 domain recognizes virus-specific receptors and binds to target host cells. The receptor-binding domain (RBD) has been identified in several coronavirus isolates, and the general structure of the S protein has been defined (Figure 1).

[0017] In December 2019, a pneumonia outbreak of unknown cause occurred in Wuhan, China, and a novel coronavirus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) was identified as the underlying cause. The genetic sequence of SARS-CoV-2 was made available to the WHO and the public (MN908947.3), and the virus was classified into the Betacoronavirus subfamily. Sequence analysis revealed a phylogenetic tree showing that severe acute respiratory syndrome (SARS) virus isolates are more closely related to the SARS virus than to another coronavirus that infects humans, namely the Middle East respiratory syndrome (MERS) virus.

[0018] SARS-CoV-2 infection and the resulting disease, COVID-19, have spread globally, with the number of affected countries continuing to grow. On March 11, 2020, the WHO deemed the COVID-19 outbreak a pandemic. As of December 1, 2020, there were more than 63 million confirmed cases of COVID-19 worldwide, with over 1.4 million deaths and 191 affected countries / territories. The ongoing pandemic continues to pose a major challenge to global public health and economic stability.

[0019] There is no pre-existing immunity to SARS-CoV-2, so all individuals are at risk of infection. After infection, some, if not all, individuals develop protective immunity in terms of neutralizing antibody responses and cell-mediated immunity. However, the extent and duration of this protection are currently unknown. According to the WHO, 80% of infected individuals recover without needing hospital care, but 15% develop more severe disease and 5% require intensive care. Increasing age and underlying health conditions are considered risk factors for developing severe disease.

[0020] COVID-19 symptoms typically include cough and fever, with ground-glass or patchy opacities on chest radiographs. However, many patients present without fever or radiographic changes, and infection may be asymptomatic, warranting infection control. For symptomatic patients, disease progression may lead to acute respiratory distress syndrome requiring mechanical ventilation and subsequent multiple organ failure and death. Common symptoms in hospitalized patients (in order of frequency from most to least common) include fever, dry cough, shortness of breath, fatigue, myalgia, nausea / vomiting or diarrhea, headache, weakness, and rhinorrhea. Anosmia (loss of smell) or loss of taste may be the only symptom present in approximately 3% of individuals with COVID-19.

[0021] Although people of all ages can develop the disease, notably, the case fatality rate (CFR) is higher in people over 60 years of age. Comorbidities are also associated with increased CFR, including cardiovascular disease, diabetes, hypertension, and chronic respiratory disease. Healthcare workers represent a large proportion of COVID-19 patients due to their occupational exposure to infected patients.

[0022] In most situations, molecular tests are used to detect SARS-CoV-2 and confirm infection. Reverse transcription-polymerase chain reaction (RT-PCR) tests targeting SARS-CoV-2 viral RNA are the gold standard in vitro method for diagnosing suspected cases of COVID-19. Samples to be tested are collected from the nasal cavity and / or throat via swabs.

[0023] Influenza is a major cause of morbidity and mortality worldwide, occurring in annual seasonal epidemics and, occasionally, pandemics (Cunha BA. Influenza: historical aspects of epidemics and pandemics. Infect Dis Clin North Am. 2004;18(1):141-55). Symptomatic influenza virus infection causes a febrile illness accompanied by respiratory and systemic symptoms (Monto AS, Gravenstein S, Elliott M, et al. Clinical signs and symptoms predicting influenza infection. Arch Intern Med. 2000;160(21):3243-7). However, influenza virus infection is also frequently asymptomatic (Cowling BJ, Chan KH, Fang VJ, et al. Comparative epidemiology of pandemic and seasonal influenza A in households. N Engl J Med. 2010;362(23):2175-84). The risk of influenza-related complications and hospitalization is higher in people aged 65 years and older, children, and those with certain underlying medical conditions. On average, more than 200,000 hospitalizations per year in the United States are associated with influenza, while estimated annual deaths worldwide range from approximately 300,000 to more than 600,000 (Iuliano AD, Roguski KM, Chang HH, et al. Estimates of global seasonal influenza-associated respiratory mortality: a modeling study. Lancet. 2018;391(10127):1285-300).

[0024] Signs and Symptoms Seasonal influenza is characterized by the sudden onset of fever, cough (usually dry), headache, muscle and joint pain, severe discomfort (feeling unwell), sore throat, and runny nose. The cough can be severe and may last for two weeks or more. Most people recover from fever and other symptoms within a week without needing medical attention, but influenza can cause severe illness or death, especially in high-risk individuals.

[0025] In industrialized countries, most influenza-related deaths occur in people over the age of 65. Epidemics can lead to high levels of work / school absenteeism and lost productivity. Clinics and hospitals can be overwhelmed during peak disease periods.

[0026] The impact of seasonal influenza epidemics in developed countries is not fully understood, but studies estimate that 99% of influenza-associated lower respiratory tract infection deaths in children under 5 years of age occur in developed countries.

[0027] Epidemiology People of all ages can contract influenza infection, although some populations are at higher risk than others. Those at higher risk of severe disease or complications if infected include pregnant women, children under 59 months of age, the elderly, individuals with chronic medical conditions (e.g., chronic cardiac, renal, metabolic, neurodevelopmental, liver, or hematological disorders), and individuals with immunosuppressive conditions (e.g., HIV / AIDS, chemotherapy or steroids, or malignancies).

[0028] Healthcare workers are also at high risk of contracting influenza virus infection due to their exposure to patients and may further spread the disease to particularly vulnerable individuals.

[0029] Seasonal influenza is easily transmitted and spreads rapidly in crowded places, such as schools and daycare centers. When an infected person coughs or sneezes, droplets containing the virus (infectious droplets) are dispersed into the air, which can travel up to one meter, infecting anyone in the immediate vicinity who inhales these droplets. The virus can also be spread by hands that are contaminated with the influenza virus. To prevent transmission, people should cover their mouth and nose with a tissue when coughing and should wash their hands regularly.

[0030] In temperate climates, seasonal epidemics occur primarily during the winter, but in tropical regions, influenza can occur throughout the year, causing more irregular outbreaks. The time between infection and illness, known as the incubation period, is approximately 2 days, but can range from 1 to 4 days.

[0031] diagnosis Most cases of human influenza are diagnosed clinically. To establish a definitive diagnosis, appropriate respiratory specimen collection and laboratory diagnostic testing are typically recommended. Appropriate collection, storage, and transportation of respiratory specimens are typically the first step for laboratory detection of influenza virus infection. Influenza infection is typically confirmed using samples from throat, nasal, and nasopharyngeal secretions, or respiratory aspirates or washes, using, for example, direct antigen detection, virus isolation, or detection of influenza-specific RNA by reverse transcriptase polymerase chain reaction (RT-PCR). Laboratory technique guidelines are known in the art and can be found, for example, on the World Health Organization (WHO) website. Rapid influenza diagnostic tests (RIDTs) are sometimes used in clinical settings, but they may be less sensitive than RT-PCR, and their reliability depends primarily on the conditions of their use. The present disclosure provides, among other things, insights into the immune responses elicited by compositions comprising (i) two or more antigenic polypeptides, each associated with a different infectious agent, or (ii) two or more polynucleotides, each comprising a sequence encoding an antigenic polypeptide associated with a different infectious agent. These insights enable the development of combination products and / or combination therapies that can induce potent immune responses against multiple infectious agents (e.g., combination products that induce immune responses similar to, and even superior to, those induced by monovalent products). In particular, the present disclosure provides insights into the immune responses elicited by compositions comprising (i) one or more antigenic polypeptides associated with a coronavirus and one or more antigenic polypeptides associated with an influenza virus, or (ii) one or more polynucleotides, each comprising a nucleotide sequence encoding an antigenic polypeptide associated with an influenza virus.

[0032] In some embodiments, the present disclosure provides a composition comprising: (i) an RNA comprising a first nucleotide sequence that comprises a modified uridine and encodes a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein the first nucleotide sequence is at least 85% identical to SEQ ID NO:9; (ii) an RNA comprising a second nucleotide sequence that comprises a modified uridine and encodes a second SARS-CoV-2 spike (S) polypeptide from a variant of a SARS-CoV-2 strain, wherein the second nucleotide sequence is at least 85% identical to SEQ ID NO: 70; (iii) RNA comprising a third nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the third nucleotide sequence is at least 85% identical to SEQ ID NO: 92; (iv) RNA comprising a fourth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein the fourth nucleotide sequence is at least 85% identical to SEQ ID NO: 97; (v) RNA comprising a fifth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the fifth nucleotide sequence is at least 85% identical to SEQ ID NO: 102; and (vi) an RNA comprising a sixth nucleotide sequence that comprises a modified uridine and encodes an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the sixth nucleotide sequence is at least 85% identical to SEQ ID NO: 107. The composition comprises:

[0033] In some embodiments, the present disclosure provides a composition comprising: (i) RNA comprising a nucleotide sequence that contains a modified uridine and encodes a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein the nucleotide sequence is at least 85% identical to SEQ ID NO: 20; (ii) RNA comprising a nucleotide sequence that includes a modified uridine and encodes a second SARS-CoV-2 spike (S) polypeptide from a variant of a SARS-CoV-2 strain, the nucleotide sequence being at least 85% identical to SEQ ID NO: 72; (iii) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the RNA comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 94; (iv) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from an influenza A H3N2 strain, said RNA comprising a nucleotide sequence that is at least 85% identical to SEQ ID NO: 99; (v) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from the influenza B Victoria strain, said RNA comprising a nucleotide sequence that is at least 85% identical to SEQ ID NO: 104; and (vi) RNA comprising a modified uridine and a nucleotide sequence encoding an influenza hemagglutinin antigen from influenza B Yamagata strain, said RNA comprising a nucleotide sequence at least 85% identical to SEQ ID NO: 109. The composition comprises:

[0034] In some embodiments, the influenza A H1N1 strain is influenza A / Wisconsin / 588 / 2019. In some embodiments, the influenza A H3N2 strain is influenza A / Cambodia / e0826360 / 2020. In some embodiments, the influenza B Victoria strain is influenza B / Washington / 02 / 2019. In some embodiments, the influenza B Yamagata strain is influenza B / Phuket / 3073 / 2013.

[0035] In some embodiments, disclosed herein is a composition comprising: (i) an RNA comprising a first nucleotide sequence that comprises a modified uridine and encodes a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein the first nucleotide sequence is at least 85% identical to SEQ ID NO:9; (ii) an RNA comprising a second nucleotide sequence that comprises a modified uridine and encodes a second SARS-CoV-2 spike (S) polypeptide from a variant of a SARS-CoV-2 strain, wherein the second nucleotide sequence is at least 85% identical to SEQ ID NO: 70; (iii) RNA comprising a third nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the third nucleotide sequence is at least 85% identical to SEQ ID NO: 92; (iv) RNA comprising a fourth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein the fourth nucleotide sequence is at least 85% identical to SEQ ID NO: 82; (v) RNA comprising a fifth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the fifth nucleotide sequence is at least 85% identical to SEQ ID NO: 87; and (vi) an RNA comprising a sixth nucleotide sequence that comprises a modified uridine and encodes an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the sixth nucleotide sequence is at least 85% identical to SEQ ID NO: 107. The composition comprises:

[0036] In some embodiments, the present disclosure provides a composition comprising: (i) an RNA comprising a first nucleotide sequence comprising a modified uridine and encoding a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein the first nucleotide sequence is at least 85% identical to SEQ ID NO: 20; (ii) an RNA comprising a second nucleotide sequence comprising a modified uridine and encoding a second SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein the first nucleotide sequence is at least 85% identical to SEQ ID NO: 72; (iii) RNA comprising a third nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the third nucleotide sequence is at least 85% identical to SEQ ID NO: 94; (iv) RNA comprising a fourth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein the fourth nucleotide sequence is at least 85% identical to SEQ ID NO: 84; (v) RNA comprising a fifth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the fifth nucleotide sequence is at least 85% identical to SEQ ID NO: 89; and (vi) RNA comprising a sixth nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the sixth nucleotide sequence is at least 85% identical to SEQ ID NO: 109. The composition comprises:

[0037] In some embodiments, the influenza A H1N1 strain is influenza A / Wisconsin / 588 / 2019. In some embodiments, the influenza A H3N2 strain is influenza A / Darwin / 6 / 2021. In some embodiments, the influenza B Victoria strain is influenza B / Austria / 1359417 / 2021. In some embodiments, the influenza B Yamagata strain is influenza B / Phuket / 3073 / 2013.

[0038] In some embodiments, the SARS-CoV-2 strain is the Wuhan strain. In some embodiments, the SARS-CoV-2 strain variant is an Omicron BA.4 / 5 variant. In some embodiments, the SARS-CoV-2 strain variant is an Omicron XBB.1.5 variant.

[0039] In some embodiments, each RNA in the composition contains the same non-coding elements, including the same 5' cap, cap proximal sequence, 5' UTR sequence, 3' UTR sequence, and poly A sequence.

[0040] In some embodiments, the mass ratio of RNA(i)-(ii) to RNA(iii)-(vi) is 1:5-5:1, 1:4-4:1, 1:3-3:1, or 1:2-2:1. In some embodiments, the mass ratio of RNA(iii)-(iv) to RNA(v)-(vi) is 1:1-1:5. In some embodiments, the mass ratio of RNA(i) to RNA(ii) is 1:1. In some embodiments, RNA(iii), (iv), (v), and (vi) are present in a mass ratio of 1:1:1:1 or 1:1:5:5.

[0041] In some embodiments, the total mass of RNA(i)-(vi) in the composition is about 30 μg to about 100 μg. In some embodiments, the total mass of RNA(i) and RNA(ii) is about 3 μg to about 60 μg (e.g., about 3 μg, about 10 μg, about 30 μg, or about 60 μg). In some embodiments, the total mass of RNA(iii)-(vi) is about 30 μg to about 60 μg (e.g., about 30 μg or about 60 μg).

[0042] In some embodiments, RNA(i) and RNA(ii) are each present in an amount of about 15 μg, and RNA(iii)-(vi) are each present in an amount of about 7.5 μg. In some embodiments, RNA(i) and (ii) are each present in an amount of about 30 μg, and RNA(iii)-(vi) are each present in an amount of about 7.5 μg. In some embodiments, RNA(i) and (ii) are each present in an amount of about 15 μg, and RNA(iii)-(vi) are each present in an amount of about 11.25 μg. In some embodiments, RNA(i) and (ii) are each present in an amount of about 15 μg, RNA(iii) and (iv) are each present in an amount of about 5 μg, and RNA(v) and (vi) are each present in an amount of about 25 μg. In some embodiments, RNA(i) and (ii) are each present in an amount of about 15 μg, RNA(iii) and (iv) are each present in an amount of about 2.5 μg, and RNA(v) and (vi) are each present in an amount of about 12.5 μg. In some embodiments, RNA(i) and (ii) are each present in an amount of about 30 μg, RNA(iii) and (iv) are each present in an amount of about 2.5 μg, and RNA(v) and (vi) are each present in an amount of about 12.5 μg. In some embodiments, RNA(i)-(vi) are each present in an amount of about 15 μg.

[0043] In some embodiments, the composition comprises: (i) an RNA comprising a first nucleotide sequence comprising a modified uridine and encoding a SARS-CoV-2 spike (S) polypeptide, wherein the first nucleotide sequence is at least 85% identical to SEQ ID NO: 129; (ii) an RNA comprising a second nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the second nucleotide sequence is at least 85% identical to SEQ ID NO: 92; (iii) RNA comprising a modified uridine and a third nucleotide sequence encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein the third nucleotide sequence is at least 85% identical to SEQ ID NO: 99; (iv) RNA comprising a fourth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the fourth nucleotide sequence is at least 85% identical to SEQ ID NO: 102; and (v) RNA comprising a fifth nucleotide sequence that comprises a modified uridine and encodes an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the fifth nucleotide sequence is at least 85% identical to SEQ ID NO: 107. Includes.

[0044] In some embodiments, the composition comprises: (i) RNA comprising a nucleotide sequence that contains a modified uridine and encodes a SARS-CoV-2 spike (S) polypeptide, wherein the nucleotide sequence is at least 85% identical to SEQ ID NO: 132; (ii) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the RNA comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 94; (iii) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein the RNA comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 99; (iv) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from the influenza B Victoria strain, said RNA comprising a nucleotide sequence that is at least 85% identical to SEQ ID NO: 104; and (v) RNA comprising a modified uridine and a nucleotide sequence encoding an influenza hemagglutinin antigen from influenza B Yamagata strain, said RNA comprising a nucleotide sequence at least 85% identical to SEQ ID NO: 109. Includes.

[0045] In some embodiments, the composition comprises RNA encoding a hemagglutinin antigen from an influenza A H1N1 strain, wherein the influenza A H1N1 strain is influenza A / Wisconsin / 588 / 2019.

[0046] In some embodiments, the composition comprises RNA encoding a hemagglutinin antigen from an influenza A H3N2 strain, wherein the influenza A H3N2 strain is influenza A / Cambodia / e0826360 / 2020.

[0047] In some embodiments, the composition comprises RNA encoding a hemagglutinin antigen from the influenza B Victoria strain, wherein the influenza B Victoria strain is influenza B / Washington / 02 / 2019.

[0048] In some embodiments, the composition comprises RNA encoding a hemagglutinin antigen from influenza B Yamagata strain, wherein the influenza B Yamagata strain is influenza B / Phuket / 3073 / 2013.

[0049] In some embodiments, the composition comprises: (i) an RNA comprising a first nucleotide sequence comprising a modified uridine and encoding a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein the first nucleotide sequence is at least 85% identical to SEQ ID NO: 130; (ii) an RNA comprising a second nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the second nucleotide sequence is at least 85% identical to SEQ ID NO: 92; (iii) RNA comprising a third nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein the third nucleotide sequence is at least 85% identical to SEQ ID NO: 82; (iv) RNA comprising a fourth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the fourth nucleotide sequence is at least 85% identical to SEQ ID NO: 87; and (v) RNA comprising a fifth nucleotide sequence that comprises a modified uridine and encodes an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the fifth nucleotide sequence is at least 85% identical to SEQ ID NO: 107. Includes:

[0050] In some embodiments, the composition comprises: (i) an RNA comprising a first nucleotide sequence comprising a modified uridine and encoding a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein the first nucleotide sequence is at least 85% identical to SEQ ID NO: 132; (iii) RNA comprising a second nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the second nucleotide sequence is at least 85% identical to SEQ ID NO: 94; (iv) RNA comprising a modified uridine and a third nucleotide sequence encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein the third nucleotide sequence is at least 85% identical to SEQ ID NO: 84; (v) RNA comprising a fourth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the fourth nucleotide sequence is at least 85% identical to SEQ ID NO: 89; and (vi) RNA comprising a fifth nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the fifth nucleotide sequence is at least 85% identical to SEQ ID NO: 109. Includes:

[0051] In some embodiments, the composition comprises RNA encoding a hemagglutinin antigen from an influenza A H3N2 strain, wherein the influenza A H3N2 strain is influenza A / Darwin / 6 / 2021.

[0052] In some embodiments, the composition comprises RNA encoding a hemagglutinin antigen from the influenza B Victoria strain, wherein the influenza B Victoria strain is influenza B / Austria / 1359417 / 2021.

[0053] In some embodiments, the composition comprises RNA encoding a hemagglutinin antigen from influenza B Yamagata strain, wherein the influenza B Yamagata strain is influenza B / Phuket / 3073 / 2013.

[0054] In some embodiments, the composition comprises RNA encoding the SARS-CoV-2 S protein from the Wuhan strain.

[0055] In some embodiments, the composition comprises RNA encoding a SARS-CoV-2 S protein from the Omicron BA.4 / 5 variant.

[0056] In some embodiments, the composition comprises RNA encoding a SARS-CoV-2 S protein from the XBB.1.5 variant.

[0057] In some embodiments, each of the RNAs in the composition comprises the same non-coding elements (eg, comprising the same 5' cap, proximal cap sequence, 5' UTR sequence, 3' UTR sequence, and polyA sequence).

[0058] In some embodiments, the composition comprises RNA (i) and RNAs (ii) to (v) in a mass ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:1.

[0059] In some embodiments, the composition comprises RNA (i) and RNAs (ii) to (v) in a mass ratio of 1.1 to 1.5.

[0060] In some embodiments, the composition comprises RNA (ii), (iii), (iv), and (v) in a mass ratio of 1:1:1:1, or 1:1:5:5.

[0061] In some embodiments, the composition comprises one or more RNAs encoding a SARS-CoV-2 S protein and one or more RNAs encoding an influenza HA protein, wherein the mass ratio of (i) the one or more RNAs encoding the SARS-CoV-2 S protein to (ii) the one or more RNAs encoding the influenza HA protein is 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:1 (e.g., 1:2 or 2:1).

[0062] In some embodiments, the composition comprises one or more RNAs encoding a SARS-CoV-2 S protein, wherein the one or more RNAs have a mass of about 30 μg. In some embodiments, the composition comprises one or more RNAs encoding a SARS-CoV-2 S protein, wherein the one or more RNAs have a mass of about 60 μg.

[0063] In some embodiments, the composition comprises one or more RNAs encoding influenza HA proteins, wherein the mass of the one or more RNAs encoding influenza HA proteins is 30 μg. In some embodiments, the composition comprises one or more RNAs encoding influenza HA proteins, wherein the mass of the one or more RNAs encoding influenza HA proteins is 60 μg.

[0064] In some embodiments, the composition comprises: (a) RNA (i) in an amount of about 30 μg, and RNAs (ii) to (v) in an amount of about 7.5 μg each; (b) RNA (i) in an amount of about 60 μg, and RNAs (ii)-(v) in an amount of about 7.5 μg each; (c) RNA (i) in an amount of about 30 μg, and RNAs (ii) to (v) in an amount of about 11.25 μg each; (d) RNA (i) in an amount of about 30 μg, RNAs (ii) and (iii) in an amount of about 5 μg each, and RNAs (iv) and (v) in an amount of about 25 μg each; (e) RNA (i) in an amount of about 30 μg, RNAs (ii) and (iii) in an amount of about 2.5 μg each, and RNAs (iv) and (v) in an amount of about 12.5 μg each; (f) RNA (i) in an amount of about 30 μg, RNAs (ii) and (iii) in an amount of about 2.5 μg each, and RNAs (iv) and (v) in an amount of about 12.5 μg each; or (g) RNA (i) in an amount of about 30 μg, and RNAs (ii) to (v) in an amount of about 15 μg each. Includes.

[0065] In some embodiments, the compositions described herein comprise: (i) a coronavirus RNA vaccine comprising one or more RNAs, each containing a nucleotide sequence encoding a SARS-CoV-2 antigen; and (ii) An influenza RNA vaccine comprising one or more RNAs, each RNA comprising one or more nucleotide sequences encoding an influenza antigen, wherein the influenza RNA vaccine encodes at least four influenza antigens, each influenza antigen being from a distinct influenza virus predicted to be circulating during a particular hemisphere's influenza season. Including, Each RNA in the composition contains the same non-coding elements, including the same 5' cap, cap proximal sequence, 5' UTR sequence, 3' UTR sequence, and poly A sequence.

[0066] In some embodiments, each of the one or more RNAs in the coronavirus RNA vaccine and each of the one or more RNAs in the influenza RNA vaccine comprises one or more modified uridines.

[0067] In some embodiments, the present disclosure provides a composition comprising: a coronavirus RNA vaccine that is at least bivalent, the coronavirus RNA vaccine comprising one or more RNAs that comprise nucleotide sequences encoding at least two SARS-CoV-2 antigens; and An influenza RNA vaccine that is at least tetravalent, the influenza RNA vaccine comprising one or more RNAs that include nucleotide sequences encoding at least four influenza antigens, each influenza antigen from a distinct influenza virus predicted to be circulating during a particular hemisphere flu season. Including, Each RNA in the coronavirus RNA vaccine and the influenza RNA vaccine contains the same non-coding elements, including the same 5' cap, cap adjacent sequence, 5' UTR sequence, 3' UTR sequence, and polyA sequence. The composition is provided.

[0068] In some embodiments, each RNA in the at least bivalent coronavirus RNA vaccine and each RNA in the at least tetravalent influenza RNA vaccine comprises a modified uridine in place of a uridine.

[0069] In some embodiments, the at least bivalent SARS-CoV-2 vaccine comprises or encodes at least two SARS-CoV-2 antigens that are or comprise a SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain and a SARS-CoV-2 S polypeptide from a variant of the SARS-CoV-2 strain.

[0070] In some embodiments, a quadrivalent influenza vaccine comprises or encodes at least four influenza antigens, each of which is or comprises a hemagglutinin antigen from a distinct influenza virus predicted to circulate during the flu season, hi some embodiments, each distinct influenza virus predicted to circulate during the flu season based on human serology data from the Northern or Southern Hemisphere.

[0071] In some embodiments, the coronavirus RNA vaccine encodes at least two SARS-CoV-2 antigens, each from a distinct SARS-CoV-2 strain or variant.

[0072] In some embodiments, the at least bivalent SARS-CoV-2 vaccine comprises RNA encoding at least two SARS-CoV-2 antigens, each of which is encoded by a separate RNA.

[0073] In some embodiments, the at least tetravalent influenza vaccine comprises RNA encoding at least four influenza antigens, each of which is encoded by a separate RNA.

[0074] In some embodiments, the RNA in the at least bivalent coronavirus vaccine and the RNA in the at least tetravalent influenza vaccine are present in a mass ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:1.

[0075] In some embodiments, the at least tetravalent influenza vaccine comprises RNA encoding at least four influenza antigens, including at least two hemagglutinin antigens from influenza A virus and at least two hemagglutinin antigens from influenza B virus, each influenza antigen encoded by a separate RNA. In some embodiments, the RNA encoding the hemagglutinin antigens from influenza A virus and the RNA encoding the hemagglutinin antigens from influenza B virus are present in a mass ratio of 1:1 to 1:5. In some embodiments, the at least tetravalent influenza vaccine comprises at least four RNAs present in a mass ratio of 1:1:1:1.

[0076] In some embodiments, the at least bivalent coronavirus vaccine comprises at least two RNAs, each encoding a different coronavirus antigen, and the two RNAs are present in a 1:1 mass ratio.

[0077] In some embodiments, the composition comprises a total amount of RNA of about 30 μg to about 100 μg (eg, about 30 μg, about 45 μg, about 60 μg, about 75 μg, or about 90 μg).

[0078] In some embodiments, the composition comprising at least a bivalent coronavirus vaccine and at least a tetravalent influenza vaccine comprises a total amount of RNA between 30 μg and 100 μg.

[0079] In some embodiments, the present disclosure provides a composition comprising: one or more first RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a first infectious agent; one or more second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent; Including, The second infectious agent is different from the first infectious agent; each of the first and second RNAs in the composition comprises the same non-coding elements, including the same 5' cap, cap proximal sequence, 5' UTR sequence, 3' UTR sequence, and polyA sequence; At least one of the same non-coding elements (i) a 5'-UTR sequence that is or includes a modified human alpha globin 5'-UTR; (ii) a 3'-UTR sequence that is or includes a first sequence from an amino terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; (iii) a polyA sequence comprising at least 100 A nucleotides, wherein the first RNA and the second RNA each do not comprise a stretch of at least 30 consecutive C nucleotides between the 3' UTR and the polyA sequence; (iv) a polyA sequence comprising a staggered sequence of A nucleotides, optionally comprising 30 adenine nucleotides (SEQ ID NO: 174) followed by 70 adenine nucleotides (SEQ ID NO: 175), wherein the 30 adenine nucleotides (SEQ ID NO: 174) and the 70 adenine nucleotides (SEQ ID NO: 175) are separated by a linker sequence; or (v) a 5' cap containing a Cap1 structure and a cap-proximal sequence including positions +1, +2, +3, +4, and +5 of the RNA; is or contains; (a) The Cap1 structure comprises m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2, where A1 is the +1 position of the RNA and G2 is the +2 position of the RNA; (b) the cap-proximal sequence comprises a sequence comprising A1 and G2 of the Cap1 structure and A3N4N5 at positions +3, +4, and +5 of the RNA, respectively, wherein N4 and N5 are each independently selected from A, G, C, and U; The composition is provided.

[0080] In some embodiments, the present disclosure provides a composition comprising: one or more first RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a first infectious agent; one or more second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent that is different from the first infectious agent; Includes; each of the first and second RNAs in the composition comprises the same non-coding elements, including the same 5' cap, cap proximal sequence, 5' UTR sequence, 3' UTR sequence, and polyA sequence; Each of the first and second RNAs is (i) the level of immune response induced by the RNA in the composition is at least 80% of the level of immune response induced by the same RNA when administered alone; and / or (ii) the level of immune response induced by the RNA in the composition is at least 80% of the level of immune response induced by the same RNA when administered separately from other RNAs to a different location in the subject's body; and / or (iii) the level of immune response induced by the RNA in the composition is at least 80% of the level of immune response induced by the respective reference composition; The composition is characterized by:

[0081] In some embodiments, each reference composition is an inactivated viral vaccine.

[0082] In some embodiments, the immune response induced by the one or more first RNA(s) and one or more second RNA(s) is each at least 100% of the level of the immune response induced by the same RNA(s) when the one or more first RNA(s) and one or more second RNA(s) are administered separately.

[0083] In some embodiments, the immune response induced by the one or more first RNA(s) and the one or more second RNA(s) is each greater than the immune response induced by the same RNA(s) administered individually.

[0084] In some embodiments, the one or more first RNA(s) and one or more second RNA(s) are each present at a lower dose compared to the dose of the same RNA(s) administered individually, and the immune response induced by the lower dose of the one or more first RNA(s) and one or more second RNA(s) each is substantially the same as or greater than the immune response induced by a larger dose of the same RNA(s) administered individually.

[0085] In some embodiments, disclosed herein is a composition comprising: one or more first RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a first infectious agent; one or more second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent that is different from the first infectious agent; Includes; each of the first and second RNAs in the composition comprises the same non-coding elements, including the same 5' cap, cap proximal sequence, 5' UTR sequence, 3' UTR sequence, and polyA sequence; each of the first and second RNAs is encapsulated separately or together in a nanoparticle; The composition comprises: (i) the RNA content of the composition is at least 95% of the initial RNA content after storage for 24 hours; (ii) RNA encapsulation remains at least 95% of the initial RNA encapsulation after 24 hours of storage; (iii) the nanoparticles encapsulating the first and second RNAs maintain substantially the same particle size after 24 hours of storage; (iv) the nanoparticles encapsulating the first and second RNAs maintain a polydispersity of 0.3 or less after 24 hours; and / or (v) the mass ratio of the first RNA to the second RNA remains substantially the same after 24 hours of storage. characterized by: This is the composition.

[0086] In some embodiments, compositions disclosed herein include nanoparticles comprising lipid nanoparticles, polyplexes (PLX), lipid-linked polyplexes (LPLX), liposomes, or polysaccharide nanoparticles. In some embodiments, compositions disclosed herein include nanoparticles comprising lipid nanoparticles. In some embodiments, the lipid nanoparticles each comprise a cationizable lipid, one or more neutral lipids, and a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid comprises a PEG-conjugated lipid. In some embodiments, the nanoparticles have an average diameter of about 50-150 nm.

[0087] In some embodiments, for each of (i)-(v), the first 12 hours of storage are at 30°C and the remaining 12 hours of storage are at 2-8°C.

[0088] In some embodiments, the one or more first RNAs comprise at least two first RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with different strains or variants of the first infectious agent.

[0089] In some embodiments, the one or more second RNAs comprise at least two second RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with different strains or variants of the second infectious agent.

[0090] In some embodiments, the one or more second RNAs comprise at least three second RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with different strains or variants of the second infectious agent.

[0091] In some embodiments, the one or more second RNAs comprise at least four second RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with different variants or strains of the second infectious agent.

[0092] In some embodiments, disclosed herein is a composition comprising: a plurality (e.g., at least two, at least three, at least four, or at least five or more) of first RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with the first infectious agent of different strains and / or variants thereof; one or more second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent that is different from the first infectious agent; Includes; each of the first and second RNAs is formulated, separately or together, in the same nanoparticle formulation; (i) the first RNA and the second RNA are present in a mass ratio of 1:2 to 2:1, and / or (ii) the first RNA and the second RNA are present in a total amount of about 10 μg to about 100 μg per dose; the one or more first RNAs each comprise a nucleotide sequence encoding one or more antigenic polypeptides associated with a first infectious agent; the one or more second RNAs each comprise a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent, the second infectious agent being different from the first infectious agent; each of the first and second RNAs in the composition comprises the same non-coding elements, including the same 5' cap, cap proximal sequence, 5' UTR sequence, 3' UTR sequence, and polyA sequence; At least one of the same non-coding elements (i) a 5'-UTR sequence that is or includes a modified human alpha globin 5'-UTR; (ii) a 3'-UTR sequence that is or includes a first sequence from an amino terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; (iii) a polyA sequence comprising at least 100 A nucleotides, wherein the first RNA and the second RNA each do not comprise a stretch of at least 30 consecutive C nucleotides between the 3' UTR and the polyA sequence; (iv) a polyA sequence comprising a staggered sequence of A nucleotides, optionally comprising 30 adenine nucleotides (SEQ ID NO: 174) followed by 70 adenine nucleotides (SEQ ID NO: 175), wherein the 30 adenine nucleotides (SEQ ID NO: 174) and the 70 adenine nucleotides (SEQ ID NO: 175) are separated by a linker sequence; or (v) a 5' cap containing a Cap1 structure and a cap-proximal sequence including positions +1, +2, +3, +4, and +5 of the RNA; is or contains; (a) The Cap1 structure comprises m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2, where A1 is the +1 position of the RNA and G2 is the +2 position of the RNA; (b) the cap-proximal sequence comprises a sequence comprising A1 and G2 of the Cap1 structure and A3N4N5 at positions +3, +4, and +5 of the RNA, respectively, wherein N4 and N5 are each independently selected from A, G, C, and U; This is the composition.

[0093] In some embodiments, each first RNA in the composition is co-formulated in the same nanoparticle formulation. In some embodiments, each second RNA in the composition is co-formulated in the same nanoparticle formulation. In some embodiments, the first RNA and the second RNA in the composition are formulated in separate nanoparticle populations. In some embodiments, each first RNA and each second RNA in the composition are co-formulated together in the same nanoparticle formulation.

[0094] In some embodiments, the first infectious agent is or comprises a coronavirus.

[0095] In some embodiments, the composition comprises one or more first RNAs, including (i) an RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a first coronavirus, and (ii) an RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second coronavirus.

[0096] In some embodiments, the composition comprises multiple second RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent of different strains and / or variants thereof.

[0097] In some embodiments, the composition comprises at least two second RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent of a different strain and / or variant thereof.

[0098] In some embodiments, the composition comprises at least three second RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent of a different strain and / or variant thereof.

[0099] In some embodiments, the composition comprises at least four second RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent of a different strain and / or variant thereof.

[0100] In some embodiments, the second infectious agent is or comprises a bacterial infectious agent, hi some embodiments, the bacterial infectious agent is Streptococcus pneumoniae.

[0101] In some embodiments, the second infectious agent is or comprises a viral infectious agent. In some embodiments, the viral infectious agent causes an infectious respiratory disease. In some embodiments, the viral infectious agent is or comprises an influenza virus, a pneumoviridae virus, or a Paramyxoviridae virus. In some embodiments, the Pneumoviridae virus is a respiratory syncytial virus (RSV). In some embodiments, the infectious respiratory disease is or comprises an influenza A, B, and / or C virus. In some embodiments, the infectious respiratory disease is or comprises an influenza A and / or B virus.

[0102] In some embodiments, the composition comprises (i) at least one RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with influenza A virus, and (ii) at least one RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with influenza B virus.

[0103] In some embodiments, the composition comprises at least two RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with different strains of influenza A virus, and at least two RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with different strains of influenza B virus.

[0104] In some embodiments, the antigenic polypeptide(s) associated with an influenza virus, a hemagglutinin (HA) polypeptide, a neuraminidase (NA) polypeptide, or a combination thereof or immunogenic fragments thereof.

[0105] In some embodiments, the strain(s) of influenza A and / or influenza B virus are predicted to be or are pandemic strains in the upcoming flu season, e.g., based on human serology data.

[0106] In some embodiments, the strain(s) of influenza A virus is / are selected from H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N7, and H10N8 viruses. In some embodiments, the strain(s) of influenza A virus is / are selected from H1N1, H3N2, H5N1, and H5N8 viruses.

[0107] In some embodiments, the composition comprises one or more second RNAs comprising an RNA comprising a nucleotide sequence encoding an antigenic polypeptide associated with an H1N1 virus. In some embodiments, the H1N1 virus is A / Wisconsin / 588 / 2019. In some embodiments, the antigenic polypeptide associated with A / Wisconsin / 588 / 2019 is an HA polypeptide and comprises an amino acid sequence at least 85% identical to SEQ ID NO: 90. In some embodiments, the antigenic polypeptide associated with A / Wisconsin / 588 / 2019 is an HA polypeptide and the RNA encoding the HA polypeptide comprises a nucleotide sequence at least 85% identical to SEQ ID NO: 92.

[0108] In some embodiments, the composition comprises one or more second RNAs comprising RNA comprising a nucleotide sequence encoding an antigenic polypeptide associated with an H3N2 virus. In some embodiments, the H3N2 virus is A / Cambodia / e0826360 / 2020. In some embodiments, the antigenic polypeptide associated with A / Cambodia / e0826360 / 2020 is an HA polypeptide and comprises an amino acid sequence at least 85% identical to SEQ ID NO: 95. In some embodiments, the antigenic polypeptide associated with A / Cambodia / e0826360 / 2020 is an HA polypeptide, and the RNA encoding the HA polypeptide comprises a nucleotide sequence at least 85% identical to SEQ ID NO: 92. In some embodiments, the H3N2 virus is A / Darwin / 6 / 2021. In some embodiments, the antigenic polypeptide associated with A / Darwin / 6 / 2021 is an HA polypeptide and comprises an amino acid sequence at least 85% identical to SEQ ID NO: 80. In some embodiments, the antigenic polypeptide associated with A / Darwin / 6 / 2021 is an HA polypeptide, and the RNA encoding the HA polypeptide comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:82.

[0109] In some embodiments, the composition comprises one or more second RNAs comprising RNA comprising a nucleotide sequence encoding an antigenic polypeptide associated with a B / Yamagata or B / Victoria lineage virus. In some embodiments, the B / Victoria lineage influenza virus is B / Washington / 02 / 2019. In some embodiments, the antigenic polypeptide associated with B / Washington / 02 / 2019 is an HA polypeptide and comprises an amino acid sequence at least 85% identical to SEQ ID NO: 100. In some embodiments, the antigenic polypeptide associated with B / Washington / 02 / 2019 is an HA polypeptide, and the RNA encoding the HA polypeptide comprises a nucleotide sequence at least 85% identical to SEQ ID NO: 102. In some embodiments, the B / Victoria lineage influenza virus is B / Austria / 1359417 / 2021. In some embodiments, the antigenic polypeptide associated with B / Austria / 1359417 / 2021 is an HA polypeptide and comprises a sequence at least 85% identical to SEQ ID NO: 85. In some embodiments, the antigenic polypeptide associated with B / Austria / 1359417 / 2021 is an HA polypeptide, and the RNA encoding the HA polypeptide comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:87.

[0110] In some embodiments, the B / Yamagata lineage influenza virus is B / Phuket / 3073 / 2013. In some embodiments, the antigenic polypeptide associated with B / Phuket / 3073 / 2013 is an HA polypeptide and comprises an amino acid sequence at least 85% identical to SEQ ID NO: 105. In some embodiments, the antigenic polypeptide associated with B / Phuket / 3073 / 2013 is an HA polypeptide, and the RNA encoding the HA polypeptide comprises a nucleotide sequence at least 85% identical to SEQ ID NO: 107.

[0111] In some embodiments, the first infectious agent is a coronavirus. In some embodiments, the coronavirus is an alphacoronavirus, betacoronavirus, gammacoronavirus, or deltacoronavirus. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the betacoronavirus is a sarbecovirus, merbecovirus, enbecovirus, nobecovirus, or hibecovirus. In some embodiments, the sarbecovirus is SARS-CoV-1 or SARS-CoV-2. In some embodiments, the sarbecovirus is SARS-CoV-2. In some embodiments, the merbecovirus is MERS-CoV.

[0112] In some embodiments, the compositions comprise one or more first RNAs that include RNA that includes a nucleotide sequence encoding one or more antigenic polypeptides associated with a SARS-CoV-2 variant that is prevalent in the relevant population or that has been identified as a variant of concern at the time of administration.

[0113] In some embodiments, the composition comprises one or more first RNAs, wherein the first RNA comprises an RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with an Omicron SARS-CoV-2 variant (e.g., a BA.1, BA.2, BA.4 / 5, or XBB.1.5 variant).

[0114] In some embodiments, the compositions disclosed herein comprise (i) RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a first SARS-CoV-2 strain, wherein the first SARS-CoV-2 strain is an ancestral SARS-CoV-2 strain (Wuhan strain), and (ii) RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second SARS-CoV-2 variant, wherein the second SARS-CoV-2 is a variant of the ancestral SARS-CoV-2 strain and is circulating in a relevant population or has been identified as a variant of concern at the time of administration.

[0115] In some embodiments, the composition comprises (i) RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a first SARS-CoV-2 variant, and (ii) RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second SARS-CoV-2 variant, wherein the first and second SARS-CoV-2 variants are each identified as prevalent in a relevant population or as variants of concern at the time of administration. In some embodiments, the second SARS-CoV-2 variant is an omicron variant of SARS-CoV-2. In some embodiments, the omicron variant of SARS-CoV-2 is or includes omicron BA.1, BA.2, or BA.4 / 5. In some embodiments, the antigenic polypeptide(s) associated with the coronavirus is a spike (S) polypeptide, or an immunogenic fragment or variant thereof. In some embodiments, the S polypeptide is a prefusion stabilized S polypeptide. In some embodiments, the prefusion stabilized S polypeptide comprises at least two proline substitutions. In some embodiments, the two proline substitutions comprise proline residues at positions corresponding to residues 986 and 987 of SEQ ID NO: 1. In some embodiments, the pre-fusion stabilized S polypeptide comprises at least six proline substitutions. In some embodiments, the pre-fusion stabilized S polypeptide comprises proline residues at positions corresponding to residues 817, 892, 899, and 942 of SEQ ID NO: 1. In some embodiments, the RNA encoding one or more antigenic polypeptides associated with an Omicron SARS-CoV-2 variant encodes an S protein associated with the XBB.1.5 strain and comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 129.

[0116] In some embodiments, the RNA encoding the one or more antigenic polypeptides associated with the SARS-CoV-2 ancestral strain encodes an S protein associated with the Wuhan strain and comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 1. In some embodiments, the RNA encoding SEQ ID NO: 1 comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 9.

[0117] In some embodiments, the RNA encoding one or more antigenic polypeptides associated with the second SARS-CoV-2 variant encodes an S protein that is associated with the BA.4 / 5 variant and comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 69. In some embodiments, the RNA encoding SEQ ID NO: 69 comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 70.

[0118] In some embodiments, the compositions disclosed herein comprise: (i) (a) RNA comprising a nucleotide sequence encoding a SARS-CoV-2 spike (S) polypeptide from an omicron variant of SARS-CoV-2 (e.g., a BA.1, BA.2, BA.4 / 5, or XBB.1.5 variant); or (b) RNA comprising a nucleotide sequence encoding a SARS-CoV-2 spike (S) polypeptide from an ancestral strain of SARS-CoV-2 (Wuhan strain) and RNA comprising a nucleotide sequence encoding a SARS-CoV-2 spike (S) polypeptide from an omicron variant of SARS-CoV-2 (e.g., a BA.1, BA.2, BA.4 / 5, or XBB.1.5 variant); and (ii) RNA comprising a nucleotide sequence encoding an HA polypeptide from an influenza A / H1N1 virus, RNA comprising a nucleotide sequence encoding an HA polypeptide from an influenza A / H3N2 virus, RNA comprising a nucleotide sequence encoding an HA polypeptide from an influenza B / Victoria lineage virus, and RNA comprising a nucleotide sequence encoding an HA polypeptide from an influenza B / Yamagata virus. Includes:

[0119] In some embodiments, the H1N1 virus is A / Wisconsin / 588 / 2019. In some embodiments, the HA polypeptide associated with A / Wisconsin / 588 / 2019 comprises a sequence at least 85% identical to SEQ ID NO: 90. In some embodiments, the HA polypeptide associated with A / Wisconsin / 588 / 2019 is encoded by RNA comprising a nucleotide sequence at least 85% identical to SEQ ID NO: 92.

[0120] In some embodiments, the H3N2 virus is A / Cambodia / e0826360 / 2020. In some embodiments, the HA polypeptide associated with A / Cambodia / e0826360 / 2020 comprises a sequence at least 85% identical to SEQ ID NO: 95. In some embodiments, the HA polypeptide associated with A / Cambodia / e0826360 / 2020 is encoded by RNA comprising a sequence at least 85% identical to SEQ ID NO: 97.

[0121] In some embodiments, the B / Victoria lineage influenza virus is B / Washington / 02 / 2019. In some embodiments, the HA polypeptide associated with B / Washington / 02 / 2019 comprises an amino acid sequence at least 85% identical to SEQ ID NO: 100. In some embodiments, the HA polypeptide associated with B / Washington / 02 / 2019 is encoded by RNA comprising a nucleotide sequence at least 85% identical to SEQ ID NO: 102.

[0122] In some embodiments, the B / Yamagata lineage influenza virus is B / Phuket / 3073 / 2013. In some embodiments, the HA polypeptide associated with B / Phuket / 3073 / 2013 comprises an amino acid sequence at least 85% identical to SEQ ID NO: 105. In some embodiments, the HA polypeptide associated with B / Phuket / 3073 / 2013 is encoded by RNA comprising a sequence at least 85% identical to SEQ ID NO: 107.

[0123] In some embodiments, the S polypeptide associated with the Wuhan strain comprises a sequence that is at least 85% identical to SEQ ID NO:7.

[0124] In some embodiments, the S polypeptide associated with the Wuhan strain comprises a sequence that is at least 85% identical to SEQ ID NO:9.

[0125] In some embodiments, the omicron variant is a BA.4 / 5 variant. In some embodiments, the S polypeptide associated with the BA.4 / 5 omicron variant comprises a sequence at least 85% identical to SEQ ID NO: 69. In some embodiments, the S polypeptide associated with the BA.4 / 5 omicron variant is encoded by an RNA comprising a sequence at least 85% identical to SEQ ID NO: 70.

[0126] In some embodiments, the Omicron variant is an XBB.1.5 variant. In some embodiments, the S polypeptide associated with the XBB.1.5 Omicron variant comprises a sequence at least 85% identical to SEQ ID NO: 69. In some embodiments, the S polypeptide associated with the XBB.1.5 variant is encoded by an RNA comprising a sequence at least 85% identical to SEQ ID NO: 130.

[0127] In some embodiments, each of the RNAs in the compositions disclosed herein comprises the same non-coding elements, wherein at least one of the non-coding elements is (i) a 5'-UTR sequence that is or includes a modified human alpha globin 5'-UTR; (ii) a 3'-UTR sequence that is or includes a first sequence from an amino terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; (iii) a polyA sequence comprising at least 100 A nucleotides, wherein the first RNA and the second RNA each do not comprise a stretch of at least 30 consecutive C nucleotides between the 3' UTR and the polyA sequence; (iv) a polyA sequence comprising a staggered sequence of A nucleotides, optionally comprising 30 adenine nucleotides (SEQ ID NO: 174) followed by 70 adenine nucleotides (SEQ ID NO: 175), wherein the 30 adenine nucleotides (SEQ ID NO: 174) and the 70 adenine nucleotides (SEQ ID NO: 175) are separated by a linker sequence; or (v) a 5' cap containing a Cap1 structure and a cap-proximal sequence including positions +1, +2, +3, +4, and +5 of the RNA; is or contains; (a) The Cap1 structure contains m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2, where A1 is the +1 position of the RNA and G2 is the +2 position of the RNA; (b) The cap-proximal sequence includes A1 and G2 of the Cap1 structure and a sequence including A3N4N5 at positions +3, +4, and +5 of the RNA, respectively, where N4 and N5 are each independently selected from A, G, C, and U.

[0128] In some embodiments, each RNA in the composition comprises the same non-coding elements, and at least one of the same non-coding elements is (i) a 5'-UTR sequence that is or includes a modified human alpha globin 5'-UTR; (ii) a 3'-UTR sequence that is or includes a first sequence from an amino terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; (iii) a polyA sequence comprising at least 100 A nucleotides, wherein the first RNA and the second RNA each do not comprise a stretch of at least 30 consecutive C nucleotides between the 3' UTR and the polyA sequence; (iv) a polyA sequence comprising a staggered sequence of A nucleotides, optionally comprising 30 adenine nucleotides (SEQ ID NO: 174) followed by 70 adenine nucleotides (SEQ ID NO: 175), wherein the 30 adenine nucleotides (SEQ ID NO: 174) and the 70 adenine nucleotides (SEQ ID NO: 175) are separated by a linker sequence; and (v) a 5' cap containing a Cap1 structure and a cap-proximal sequence including positions +1, +2, +3, +4, and +5 of the RNA; is or contains; (a) The Cap1 structure contains m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2, where A1 is the +1 position of the RNA and G2 is the +2 position of the RNA; (b) The cap-proximal sequence includes A1 and G2 of the Cap1 structure and a sequence including A3N4N5 at positions +3, +4, and +5 of the RNA, respectively, where N4 and N5 are each independently selected from A, G, C, and U.

[0129] In some embodiments, each RNA in the composition comprises, from 5' to 3', a 5' cap, a cap proximal sequence, a 5' UTR sequence, a 3' UTR sequence, and a polyA sequence.

[0130] In some embodiments, each RNA in the composition is m2 7,3’-O Gppp(m1 2’-O ) A 5'-cap that is or contains ApG.

[0131] In some embodiments, each RNA in the composition comprises a 5'UTR that comprises or consists of a human alpha globin 5'-UTR. In some embodiments, the human alpha globin 5'-UTR comprises SEQ ID NO: 12.

[0132] In some embodiments, each RNA in the composition comprises a 3' UTR that comprises or consists of a first sequence from an amino terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA, hi some embodiments, each RNA in the composition comprises a 3' UTR that comprises or consists of a sequence according to SEQ ID NO: 13.

[0133] In some embodiments, each RNA in the composition comprises a poly-A tail sequence that is a truncated poly-A tail sequence. In some embodiments, the truncated poly-A tail sequence comprises 30 adenine nucleotides (SEQ ID NO: 174) followed by 70 adenine nucleotides (SEQ ID NO: 175), where the 30 adenine nucleotides (SEQ ID NO: 174) and the 70 adenine nucleotides (SEQ ID NO: 175) are separated by a linker sequence. In some embodiments, the truncated poly-A tail sequence comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 14.

[0134] In some embodiments, each RNA in the composition comprises a modified uridine in place of every uridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine.

[0135] In some embodiments, the composition comprises one or more first RNAs and one or more second RNAs in a mass ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:1.

[0136] In some embodiments, each RNA in the composition is formulated in a nanoparticle.

[0137] In some embodiments, all of the first RNAs in the composition are co-formulated together in the same nanoparticle population, all of the second RNAs in the composition are co-formulated together in the same nanoparticle population, and the first and second RNAs are formulated in separate nanoparticle populations.

[0138] In some embodiments, all of the first RNAs and all of the second RNAs in the composition are co-formulated together in the same population of nanoparticles.

[0139] In some embodiments, the nanoparticles comprise lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles. In some embodiments, the nanoparticles comprise lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise a cationizable lipid, one or more neutral lipids, and a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid comprises a PEG-conjugated lipid. In some embodiments, the nanoparticles have an average diameter of about 50-150 nm.

[0140] In some embodiments, the compositions disclosed herein further comprise one or more third RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a third infectious agent that is distinct from the first infectious agent and the second infectious agent. In some embodiments, the compositions disclosed herein further comprise one or more antigenic polypeptides associated with a third infectious agent that is distinct from the first infectious agent and the second infectious agent. In some embodiments, the third infectious agent is a respiratory virus (e.g., a respiratory virus that is not SARS-CoV-2 or an influenza virus). In some embodiments, the third infectious agent is a respiratory syncytial virus (RSV).

[0141] In some embodiments, the composition comprises one or more RNAs, each encoding a RSV polypeptide. In some embodiments, the composition comprises one or more RSV polypeptides. In some embodiments, the composition comprises one or more RNAs, each encoding a RSV F protein, a variant thereof, or an immunogenic fragment of the RSV F protein or variant thereof. In some embodiments, the composition comprises one or more RSV F proteins, immunogenic variants thereof, or immunogenic fragments of the RSV F protein or variant thereof.

[0142] In some embodiments, the compositions described herein comprise: (i) one or more RNAs each encoding a polypeptide of a RSV subtype A virus (e.g., an F protein, a variant thereof, or an immunogenic fragment of an F protein or a variant thereof), and one or more RNAs each encoding a polypeptide of a RSV subtype B virus (e.g., an F protein, a variant thereof, or an immunogenic fragment of an F protein or a variant thereof); or (ii) one or more polypeptides of a RSV subtype A virus (e.g., an F protein, a variant thereof, or an immunogenic fragment of an F protein or a variant thereof), and one or more polypeptides of a RSV subtype B virus (e.g., an F protein, a variant thereof, or an immunogenic fragment of an F protein or a variant thereof). Includes.

[0143] In some embodiments, the RSVF protein, variant, or immunogenic fragment is stabilized in pre-fusion confirmation. In some embodiments, the composition comprises or represents RSVpreF (also known as Abrysvo™) and Arexvy™.

[0144] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a composition disclosed herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a cryoprotectant, optionally the cryoprotectant is or comprises sucrose. In some embodiments, the pharmaceutical composition comprises an aqueous buffer solution, optionally the aqueous buffer solution comprises one or more of Tris base, Tris-HCl, NaCl, KCl, NaHPO, and KHPO.

[0145] In some embodiments, the pharmaceutical composition is formulated to provide a dose of 100 μg or less of total RNA. In some embodiments, the pharmaceutical composition is formulated to provide a dose of 90 μg of total RNA. In some embodiments, the pharmaceutical composition is formulated to provide a dose of 60 μg of total RNA. In some embodiments, the pharmaceutical composition is formulated to provide a dose of 30 μg of one or more first RNAs and a dose of 60 μg of one or more second RNAs. In some embodiments, the pharmaceutical composition is formulated to provide a dose of 60 μg of one or more first RNAs and a dose of 30 μg of one or more second RNAs. In some embodiments, the pharmaceutical composition is formulated to provide a dose of 30 μg of one or more first RNAs and a dose of 30 μg of one or more second RNAs.

[0146] In some embodiments, the pharmaceutical composition comprises four second RNAs, each comprising a nucleotide sequence encoding an antigenic polypeptide associated with a different influenza virus, and the pharmaceutical composition is formulated to provide a 15 μg dose of each second RNA.

[0147] In some embodiments, the pharmaceutical composition comprises four second RNAs, each comprising a nucleotide sequence encoding an antigenic polypeptide associated with a different influenza virus, and the pharmaceutical composition is formulated to provide a dose of 7.5 μg of each second RNA.

[0148] In some embodiments, the pharmaceutical composition comprises two first RNAs, each comprising a nucleotide sequence encoding an antigenic polypeptide associated with a different coronavirus virus, and the pharmaceutical composition is formulated to provide a 15 μg dose of each first RNA.

[0149] In some embodiments, the pharmaceutical composition comprises two first RNAs, each comprising a nucleotide sequence encoding an antigenic polypeptide associated with a different coronavirus virus, and the pharmaceutical composition is formulated to provide a 30 μg dose of each first RNA.

[0150] In some embodiments, disclosed herein are methods, the methods comprising administering to a subject a composition or pharmaceutical composition disclosed herein.

[0151] In some embodiments, disclosed herein are methods comprising administering to a subject one or more doses of a pharmaceutical composition disclosed herein.

[0152] In some embodiments, the methods disclosed herein are methods for treating coronavirus and influenza diseases. In some embodiments, the methods disclosed herein are methods for treating coronavirus and RSV diseases. In some embodiments, the methods disclosed herein are methods for treating coronavirus, influenza, and RSV diseases.

[0153] In some embodiments, the methods disclosed herein are methods for preventing coronavirus and influenza diseases. In some embodiments, the methods disclosed herein are methods for preventing coronavirus and RSV diseases. In some embodiments, the methods disclosed herein are methods for preventing coronavirus, influenza, and RSV diseases.

[0154] In some embodiments, the methods disclosed herein are methods for inducing an immune response against one or more coronaviruses and one or more influenza viruses. In some embodiments, the methods disclosed herein are methods for inducing an immune response against one or more coronaviruses and one or more RSV. In some embodiments, the methods disclosed herein are methods for inducing an immune response against one or more coronaviruses, one or more influenza viruses, and one or more RSV.

[0155] In some embodiments, one or more doses of the composition or pharmaceutical composition are co-administered with a vaccine against a third infectious agent. In some embodiments, the third infectious agent is a virus that can cause respiratory disease. In some embodiments, the third infectious agent is RSV. In some embodiments, the vaccine against the third infectious agent is Arexvy™ or Abrysvo™.

[0156] In some embodiments, the vaccine against the third infectious agent is mixed with one or more doses of the composition or one or more doses of the pharmaceutical composition immediately prior to administration to the subject. In some embodiments, the vaccine against the third infectious agent is administered separately from the one or more doses of the composition or one or more doses of the pharmaceutical composition (e.g., the vaccine against the third infectious agent and the one or more doses of the composition or one or more doses of the pharmaceutical composition are administered to the subject at separate injection sites (e.g., in opposing arms).

[0157] In some embodiments, disclosed herein are compositions or pharmaceutical compositions for use in treating a coronavirus disease, an influenza disease, and / or a RSV disease (e.g., a coronavirus disease and an influenza disease; a coronavirus disease and an RSV disease; or a coronavirus disease, an influenza disease, and an RSV disease), comprising administering one or more doses of the composition or pharmaceutical composition to a subject.

[0158] In some embodiments, disclosed herein are compositions or pharmaceutical compositions for use in preventing coronavirus disease, RSV disease, and / or influenza disease (e.g., coronavirus disease and influenza disease; coronavirus disease and RSV disease; or coronavirus disease, influenza disease, and RSV disease), comprising administering one or more doses of the composition or pharmaceutical composition to a subject. In some embodiments, the use comprises administering two or more doses of the composition or pharmaceutical composition to a subject. In some embodiments, the two doses are administered at least about 21 days apart.

[0159] In some embodiments, the methods or uses disclosed herein comprise administering three or more doses of the composition or pharmaceutical composition to the subject.

[0160] In some embodiments, the method or use comprises administering to a subject who has previously been exposed to a coronavirus and / or influenza virus (e.g., by vaccination or by infection).

[0161] In some embodiments, the method or use induces an immune response in a subject against one or more coronaviruses, one or more RSV, and / or one or more influenza viruses (e.g., one or more coronaviruses and one or more influenza viruses; one or more coronaviruses and one or more RSV; or one or more coronaviruses, one or more influenza viruses, and one or more RSV). In some embodiments, the immune response comprises a B cell response. In some embodiments, the B cell response comprises the production of antibodies directed against one or more antigens. In some embodiments, the immune response comprises a T cell response. In some embodiments, the T cell response is or comprises a CD4+ T cell response. In some embodiments, the T cell response is or comprises a CD8+ T cell response.

[0162] In some embodiments, disclosed herein are methods or uses of the pharmaceutical compositions disclosed herein to treat coronavirus disease, RSV disease, and / or influenza disease (e.g., coronavirus disease and influenza disease; coronavirus disease and RSV disease; or coronavirus disease, influenza disease, and RSV disease).

[0163] In some embodiments, disclosed herein are compositions (e.g., compositions described herein) or pharmaceutical compositions (e.g., pharmaceutical compositions described herein) for use in preventing coronavirus disease, RSV disease, and / or influenza disease (e.g., coronavirus disease and influenza disease; coronavirus disease and RSV disease; or coronavirus disease, influenza disease, and RSV disease). In some embodiments, disclosed herein are compositions or pharmaceutical compositions for use in inducing an immune response in a subject against one or more coronaviruses, one or more RSV, and one or more influenza viruses (e.g., one or more coronaviruses and one or more influenza viruses; one or more coronaviruses and one or more RSV; or one or more coronaviruses, one or more influenza viruses, and one or more RSV).

[0164] In some embodiments, the composition comprises: one or more RNAs, each encoding a polypeptide of a first infectious agent; and One or more polypeptides of a second infectious agent Includes:

[0165] In some embodiments, the composition comprises one or more RNAs, each encoding a polypeptide of a coronavirus (e.g., SARS-CoV-2 virus). In some embodiments, the composition comprises one or more RNAs, each encoding the SARS-CoV-2 S protein, a variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or a variant thereof. In some embodiments, the composition comprises one or more RNAs, each encoding the SARS-CoV-2 S protein of the Wuhan strain or a SARS-CoV-2 variant (e.g., an Omicron variant (e.g., an Omicron BA.1, BA.2, BA.4 / 5, or XBB.1.5 variant (e.g., an RNA described herein))), a variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or a variant thereof). In some embodiments, the composition comprises one or more polypeptides of an influenza virus. In some embodiments, the composition comprises one or more polypeptides of one or more influenza viruses (e.g., one or more polypeptides of two or more influenza virus strains (e.g., one or more polypeptides of four or more influenza virus strains that are circulating or predicted to be circulating in the jurisdiction of interest)). In some embodiments, the composition comprises a commercially available influenza virus (e.g., a recombinant commercially available influenza virus or an inactivated virus vaccine described herein). In some embodiments, the commercially available influenza virus is Flublok or Fluzone. In some embodiments, the composition comprises one or more polypeptides of RSV. In some embodiments, the composition comprises one or more polypeptides associated with a first RSV subtype and one or more polypeptides of a second RSV subtype. In some embodiments, the composition comprises one or more RSV F proteins, variants thereof, or immunogenic fragments of the RSV F protein or variants thereof. In some embodiments, the composition comprises a RSV F protein or variant thereof comprising one or more mutations that stabilize pre-fusion confirmation of the F protein. In some embodiments, the composition comprises Arexvy™ or ABRYSVO™.In some embodiments, the composition comprises one or more polypeptides of a third infectious agent.

[0166] In some embodiments, the composition comprises: one or more RNAs, each encoding one or more coronavirus polypeptides (e.g., SARS-CoV-2 S protein, a variant thereof, or an immunogenic fragment of any of the above); one or more polypeptides of one or more influenza viruses; and One or more polypeptides of one or more RSV Includes:

[0167] In some embodiments, the composition comprises: RNA encoding a SARS-CoV-2 S protein of the omicron variant (e.g., RNA encoding an S protein of the omicron BA.1, BA.4 / 5, or XBB.1.5 variant described herein); recombinant influenza vaccines (e.g., those described herein (e.g., FluBlok vaccine)) or inactivated virus vaccines (e.g., those described herein (e.g., Fluzone)); and RSV vaccines comprising a prefusion stabilized F protein or immunogenic fragment thereof (e.g., RSV vaccines described herein (e.g., Arexvy™ or ABRYSVO™)) Includes:

[0168] In some embodiments, described herein are SARS-CoV-2 vaccines that are combination vaccines, the combination vaccines comprising one or more mRNAs encoding a prefusion stabilized SARS-CoV-2 spike protein or variants thereof; and (a) an influenza vaccine comprising (i) one or more mRNAs encoding the HA protein of an influenza virus, or (ii) one or more HA polypeptides; and / or (b) a RSV vaccine comprising one or more prefusion-stabilized RSV F proteins or immunogenic fragments thereof; The combination drug comprises:

[0169] In some embodiments, the combination comprises one or more mRNAs encoding a pre-fusion stabilized SARS-CoV-2 spike protein or variants thereof, wherein the one or more mRNAs are formulated as LNPs.

[0170] In some embodiments, the composition comprises one or more mRNAs encoding an HA protein of an influenza virus, wherein the one or more mRNAs are formulated as LNPs.

[0171] In some embodiments, the combination comprises (1) a SARS-CoV-2 vaccine comprising one or more mRNAs encoding a prefusion stabilized SARS-CoV-2 spike protein or variants thereof, and (2) an influenza vaccine or an RSV vaccine, wherein (1) the SARS-CoV-2 vaccine and (2) the influenza vaccine or RSV vaccine are provided in separate containers (e.g., vials or syringes). In some embodiments, the combination comprises (1) a SARS-CoV-2 vaccine comprising one or more mRNAs encoding a prefusion stabilized SARS-CoV-2 spike protein or variants thereof, and (2) an influenza vaccine or an RSV vaccine, wherein (1) the SARS-CoV-2 vaccine and (2) the influenza vaccine or RSV vaccine are provided in a single container (e.g., vial or syringe).

[0172] In some embodiments, the combination comprises a SARS-CoV-2 vaccine, an influenza vaccine, and an RSV vaccine. In some embodiments, the combination comprises a SARS-CoV-2 vaccine, an influenza vaccine, and an RSV vaccine, all of which are provided in a single container (e.g., a vial or syringe). In some embodiments, the combination comprises a SARS-CoV-2 vaccine, an influenza vaccine, and an RSV vaccine, each of which are provided in separate containers (e.g., separate vials and / or syringes).

[0173] In some embodiments, the combination drug comprises: (a) a SARS-CoV-2 vaccine and an influenza vaccine provided in a single container and an RSV vaccine provided in separate containers; or (b) SARS-CoV-2 vaccine and RSV vaccine provided in a single container and influenza vaccine provided in separate containers Includes:

[0174] In some embodiments, the combination comprises a SARS-CoV-2 vaccine that is BNT162b2 (e.g., a monovalent or bivalent vaccine described herein).

[0175] In some embodiments, the combination comprises an influenza vaccine that is a recombinant influenza vaccine (e.g., one described herein (e.g., FluBlok vaccine)) or comprises an inactivated influenza virus (e.g., Fluzone). In some embodiments, the combination comprises an RSV vaccine that comprises a pre-fusion stabilized F protein or immunogenic fragment thereof (e.g., an RSV vaccine described herein (e.g., RSVpreF or ABRYSVO™)).

[0176] In some embodiments, disclosed herein are methods of inducing an immune response to a first infectious agent and a second infectious agent, comprising: (i) a first nanoparticle-formulated RNA comprising a nucleotide sequence encoding an antigenic polypeptide associated with the first infectious agent; and (ii) a second LNP-formulated RNA comprising a nucleotide sequence encoding an antigenic polypeptide associated with the second infectious agent; the immune response induced against each of the first and second infectious agents is greater than the immune response induced when the LNPs are administered individually; This is the method in question.

[0177] In some embodiments, disclosed herein is a method of reducing the amount of a first LNP-formulated RNA required to generate an immune response to a first infectious agent, wherein the RNA of the first LNP-formulated RNA comprises a nucleotide sequence encoding one or more antigenic polypeptides associated with the first infectious agent; the method includes co-administering a second LNP-formulated RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent; The first infectious agent is different from the second infectious agent; This is the method in question.

[0178] Also provided herein are containers containing freshly admixed combinations including, inter alia, a SARS-CoV-2 vaccine and an influenza vaccine; a SARS-CoV-2 vaccine and an RSV vaccine; or a SARS-CoV-2 vaccine, an influenza vaccine, and an RSV vaccine.

[0179] In some embodiments, the container contains a freshly mixed combination, the combination comprising: (a) a SARS-CoV-2 vaccine; and (b) Influenza vaccine Including, The SARS-CoV-2 vaccine comprises one or more RNAs encoding an immunogenic portion of the SARS-CoV-2 spike (S) protein and formulated into a nanoparticle (e.g., lipid nanoparticle (LNA)); The influenza vaccine is either (i) a nanoparticle (eg, LNP) formulated RNA vaccine or (ii) comprises one or more antigenic polypeptides (eg, HA protein) of one or more influenza virus strains.

[0180] In some embodiments, the container contains a freshly mixed combination, the combination comprising: (a) a SARS-CoV-2 vaccine; and (b) RSV vaccine Including, The SARS-CoV-2 vaccine comprises one or more RNAs encoding an immunogenic portion of the SARS-CoV-2 spike (S) protein and formulated into a nanoparticle (e.g., lipid nanoparticle (LNA)); RSV vaccines comprise one or more antigenic polypeptides (eg, F protein or immunogenic fragments thereof) associated with one or more RSV strains.

[0181] In some embodiments, the container contains a freshly mixed combination, the combination comprising: (a) SARS-CoV-2 vaccine; (b) RSV vaccine; (c) Influenza vaccine Including, The SARS-CoV-2 vaccine comprises one or more RNAs encoding an immunogenic portion of the SARS-CoV-2 spike (S) protein and formulated into a nanoparticle (e.g., lipid nanoparticle (LNA)); The RSV vaccine comprises one or more antigenic polypeptides (e.g., F protein or immunogenic fragments thereof) associated with one or more RSV strains; The influenza vaccine is either (i) a nanoparticle (eg, LNP) formulated RNA vaccine or (ii) comprises one or more antigenic polypeptides (eg, HA proteins) of one or more influenza virus strains.

[0182] In some embodiments, the container contains a SARS-CoV-2 vaccine that is a monovalent or bivalent vaccine.

[0183] In some embodiments, the container comprises an influenza vaccine, and the influenza vaccine is a tetravalent vaccine.

[0184] In some embodiments, the container contains an influenza vaccine that is an inactivated influenza virus, a recombinant influenza vaccine, a live attenuated influenza vaccine, an unadjuvanted influenza vaccine, an adjuvanted influenza vaccine, or a subunit or split vaccine.

[0185] In some embodiments, the container contains a RSV vaccine comprising a pre-fusion stabilized F protein or immunogenic fragment thereof of one or more RSV strains.

[0186] In some embodiments, the container is a syringe or a vial.

[0187] Also provided herein are, inter alia, methods for simultaneously vaccinating a human subject against each of SARS-CoV-2 and influenza, each of SARS-CoV-2 and RSV, or each of SARS-CoV-2, influenza, and RSV.

[0188] In some embodiments, the method of simultaneously vaccinating a human subject against each of SARS-CoV-2 and influenza comprises: Co-administration of a SARS-CoV-2 vaccine composition and an influenza vaccine composition to the same site Includes; The SARS-CoV-2 vaccine comprises one or more RNAs encoding an immunogenic portion of the SARS-CoV-2 spike (S) protein and formulated into a nanoparticle (e.g., lipid nanoparticle (LNA)); The influenza vaccine is either (i) a nanoparticle (eg, LNP) formulated RNA vaccine or (ii) comprises one or more antigenic polypeptides (eg, HA proteins) of one or more influenza virus strains.

[0189] In some embodiments, the method of simultaneously vaccinating a human subject against each of SARS-CoV-2 and RSV comprises: Co-administration of a SARS-CoV-2 vaccine composition and an RSV vaccine composition at the same site Includes; The SARS-CoV-2 vaccine comprises one or more RNAs encoding an immunogenic portion of the SARS-CoV-2 spike (S) protein and formulated into a nanoparticle (e.g., lipid nanoparticle (LNA)); RSV vaccines comprise one or more antigenic polypeptides (eg, F protein or immunogenic fragments thereof) associated with one or more RSV strains.

[0190] In some embodiments, the method of simultaneously vaccinating a human subject against each of SARS-CoV-2, influenza, and RSV comprises: Co-administration of a SARS-CoV-2 vaccine composition, an influenza vaccine composition, and an RSV vaccine composition at the same site Includes; The SARS-CoV-2 vaccine comprises one or more RNAs encoding an immunogenic portion of the SARS-CoV-2 spike (S) protein and formulated into a nanoparticle (e.g., lipid nanoparticle (LNA)); The influenza vaccine (i) is a nanoparticle (e.g., LNP)-formulated RNA vaccine or (ii) comprises one or more antigenic polypeptides (e.g., HA proteins) of one or more influenza virus strains; RSV vaccines comprise one or more antigenic polypeptides (eg, F protein or immunogenic fragments thereof) associated with one or more RSV strains.

[0191] In some embodiments, the method of simultaneously vaccinating against each of SARS-CoV-2 and influenza comprises an administering step comprising injecting the composition via a needle or port; The composition to be injected comprises both a SARS-CoV-2 vaccine composition and an influenza vaccine composition; The SARS-CoV-2 vaccine composition and the influenza vaccine composition are optionally administered using a syringe (e.g., a dual-chamber syringe).

[0192] In some embodiments, the method of simultaneously vaccinating against each of SARS-CoV-2 and RSV comprises an administering step comprising injecting the composition via a needle or port; The composition to be injected comprises both a SARS-CoV-2 vaccine composition and a RSV vaccine composition; The SARS-CoV-2 vaccine composition and the RSV vaccine composition are optionally administered using a syringe (e.g., a dual-chamber syringe).

[0193] In some embodiments, the method of simultaneously vaccinating against each of SARS-CoV-2 and RSV comprises an administering step comprising injecting the composition via a needle or port; The compositions to be injected include each of a SARS-CoV-2 vaccine composition, an influenza vaccine composition, and an RSV vaccine composition; The SARS-CoV-2 vaccine composition, the RSV vaccine composition, and the influenza vaccine composition are optionally administered using a syringe (e.g., a dual-chamber syringe).

[0194] In some embodiments, the method of simultaneously vaccinating against each of SARS-CoV-2 and influenza further comprises the step of combining the SARS-CoV-2 vaccine composition and the influenza vaccine composition prior to the administering step.

[0195] In some embodiments, the method of simultaneously vaccinating against both SARS-CoV-2 and RSV includes combining the SARS-CoV-2 vaccine composition and the RSV vaccine composition prior to the administering step.

[0196] In some embodiments, the method of simultaneously vaccinating against each of SARS-CoV-2, influenza, and RSV comprises combining a SARS-CoV-2 vaccine composition, an influenza vaccine composition, and a RSV vaccine composition prior to the administering step.

[0197] In some embodiments, the combining step occurs within a period of time prior to administration, the period of time being 2 hours or less (e.g., 1 hour, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes or less).

[0198] In some embodiments, the SARS-CoV-2 vaccine is contained in a container or used in a simultaneous vaccination method, where the SARS-CoV-2 vaccine composition comprises two or more RNAs, each encoding the S protein of a different SARS-CoV-2 strain or variant, and the two or more RNAs are encapsulated in separate LNP populations.

[0199] In some embodiments, an influenza vaccine comprising two or more RNAs (e.g., four RNAs), each encoding an antigenic polypeptide (e.g., HA protein) of a different influenza strain, is contained in a container or used in a simultaneous vaccination method, and the two or more RNAs are encapsulated in separate LNP populations.

[0200] In some embodiments, the SARS-CoV-2 vaccine is contained in a container or used in a co-vaccination method, and the SARS-CoV-2 vaccine comprises: (a) (i) RNA comprising a nucleotide sequence encoding a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, the RNA comprising a modified uridine and encoding a polypeptide comprising an amino acid sequence at least 85% identical to SEQ ID NO:7, and / or comprising a nucleotide sequence at least 85% identical to SEQ ID NO:20 and / or comprising a nucleotide sequence at least 85% identical to SEQ ID NO:9, and (ii) RNA comprising a nucleotide sequence encoding an S polypeptide from the Omicron BA.4 / 5 SARS-CoV-2 variant, the RNA comprising a nucleotide sequence encoding a polypeptide comprising a sequence at least 85% identical to SEQ ID NO:69, and / or comprising a nucleotide sequence at least 85% identical to SEQ ID NO:72 and / or comprising a nucleotide sequence at least 85% identical to SEQ ID NO:70; or (b) RNA comprising a modified uridine and a nucleotide sequence encoding a SARS-CoV-2 spike (S) polypeptide, wherein the RNA comprises a nucleotide sequence encoding a polypeptide comprising a sequence at least 85% identical to SEQ ID NO: 129, and / or a nucleotide sequence at least 85% identical to SEQ ID NO: 132 and / or a nucleotide sequence at least 85% identical to SEQ ID NO: 130. Includes:

[0201] In some embodiments, the SARS-CoV-2 vaccine is contained in a container or used in a co-administration method, and the SARS-CoV-2 vaccine comprises an influenza vaccine, and the influenza vaccine comprises: (a) (i) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from an influenza A H1N1 strain, the nucleotide sequence being at least 85% identical to SEQ ID NO: 94 and / or at least 85% identical to SEQ ID NO: 92; (ii) RNA comprising a modified uridine and encoding an influenza A hemagglutinin antigen from an influenza A H1N1 strain, the nucleotide sequence being at least 85% identical to SEQ ID NO: 94 and / or at least 85% identical to SEQ ID NO: 92; (iii) RNA comprising a nucleotide sequence encoding an influenza hemagglutinin antigen from the influenza B Victoria strain, said RNA comprising a modified uridine and said nucleotide sequence being at least 85% identical to SEQ ID NO: 104 and / or at least 85% identical to SEQ ID NO: 102; and (iv) RNA comprising a nucleotide sequence encoding an influenza hemagglutinin antigen from the influenza B Yamagata strain, said RNA comprising a modified uridine and said nucleotide sequence being at least 85% identical to SEQ ID NO: 109 and / or at least 85% identical to SEQ ID NO: 107; or (b) (i) an RNA comprising a modified uridine and a nucleotide sequence encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the nucleotide sequence is at least 85% identical to SEQ ID NO: 92 and / or at least 85% identical to SEQ ID NO: 94; (ii) an RNA comprising a modified uridine and an influenza A (iii) RNA comprising a nucleotide sequence encoding an influenza hemagglutinin antigen from the influenza B Victoria strain, which comprises a modified uridine, and which comprises a nucleotide sequence encoding an influenza hemagglutinin antigen from the influenza B Yamagata strain, which comprises a nucleotide sequence at least 85% identical to SEQ ID NO: 87 and / or at least 85% identical to SEQ ID NO: 89; and (iv) RNA comprising a nucleotide sequence encoding an influenza hemagglutinin antigen from the influenza B Yamagata strain, which comprises a modified uridine, and which comprises a nucleotide sequence encoding an influenza hemagglutinin antigen from the influenza B Yamagata strain, which comprises a nucleotide sequence at least 85% identical to SEQ ID NO: 107 and / or at least 85% identical to SEQ ID NO: 109. Includes:

[0202] SARS-CoV-2 is an RNA virus with four structural proteins. One of these, the spike protein, is a surface protein that binds to angiotensin-converting enzyme 2 (ACE-2) present on host cells. Therefore, the spike protein is considered to be an important antigen in vaccine development.

[0203] BNT162b2 (which contains RNA containing SEQ ID NO: 20) is an mRNA vaccine for the prevention of COVID-19 and has demonstrated greater than 95% efficacy in preventing COVID-19. The vaccine contains a 5'-capped mRNA encoding the full-length SARS-CoV-2 spike glycoprotein (S) encapsulated in lipid nanoparticles (LNPs). The finished product is provided as a concentrate for injectable dispersion containing BNT162b2 as the active substance. Other ingredients include ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and in some embodiments, potassium chloride, potassium dihydrogen phosphate, sodium chloride, disodium phosphate dihydrate, sucrose, and water for injection.

[0204] In some embodiments, a different buffer may be used instead of PBS. In some embodiments, BNT162b2 is formulated in a Tris buffer solution, optionally containing sucrose. In some embodiments, the formulation includes ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride, and water.

[0205] In some embodiments, the concentration of RNA in the pharmaceutical RNA formulation is about 0.1 to 0.2 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA formulation is about 0.1 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA formulation is about 0.12 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA formulation is about 0.14 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA formulation is about 0.16 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA formulation is about 0.18 mg / ml. In some embodiments, about 30 μg of RNA is administered by administering about 200 μL of the RNA formulation. In some embodiments, the RNA in the pharmaceutical RNA formulation is diluted (e.g., diluted to a concentration of about 0.05 mg / ml) prior to administration. In some embodiments, the administration volume is about 200 μl to about 300 μl. In some embodiments, the RNA in the pharmaceutical RNA formulation is formulated in about 10 mM Tris buffer and about 10% sucrose.

[0206] In some embodiments, the pharmaceutical RNA formulation comprises RNA at a concentration of about 0.1 mg / ml, formulated in about 10 mM Tris buffer, and about 10% sucrose. In some embodiments, the pharmaceutical RNA formulation comprises RNA at a concentration of about 0.12 mg / ml, formulated in about 10 mM Tris buffer, and about 10% sucrose. In some embodiments, the pharmaceutical RNA formulation comprises RNA at a concentration of about 0.14 mg / ml, formulated in about 10 mM Tris buffer, and about 10% sucrose. In some embodiments, the pharmaceutical RNA formulation comprises RNA at a concentration of about 0.16 mg / ml, formulated in about 10 mM Tris buffer, and about 10% sucrose. In some embodiments, the pharmaceutical RNA formulation comprises RNA at a concentration of about 0.18 mg / ml, formulated in about 10 mM Tris buffer and about 10% sucrose.

[0207] In some embodiments, the formulations provided herein (e.g., formulations comprising about 10 mM Tris buffer and about 10% sucrose) can be diluted as needed prior to administration to administer different doses of RNA while keeping the total injection volume relatively constant. For example, a dose of about 10 μg of RNA can be administered by diluting a pharmaceutical formulation comprising RNA at a concentration of about 0.1 mg / ml about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA formulation.

[0208] In some embodiments, the vaccine is formulated in a vial (e.g., a glass vial), which in some embodiments is sealed with a bromobutyl elastomer stopper and an aluminum closure with a flip-off plastic cap.

[0209] In some embodiments, the composition comprises RNA comprising a nucleotide sequence encoding an antigenic polypeptide associated with a coronavirus. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the betacoronavirus is SARS-CoV-2. In some embodiments, the antigenic polypeptide associated with a coronavirus is a spike (S) protein (e.g., a SARS-CoV-2 S protein). The SARS-CoV-2 S protein encoded by BNT162b2 was selected based on the sequence published in "SARS-CoV-2 isolate Wuhan-Hu-1": GenBank: MN908947.3 (complete genome) and GenBank: QHD43416.1 (spike surface glycoprotein). In some embodiments, the RNA comprising a sequence encoding the SARS-CoV-2 S protein is a single-stranded, 5'-capped, codon-optimized mRNA that translates into a spike antigen of SARS-CoV-2. In some embodiments, the encoded spike antigen protein sequence contains two proline mutations (P2 S) that stabilize the pre-fusion confirmation with improved antigenicity. In some embodiments, the RNA containing a nucleotide sequence encoding the SARS-CoV-2 S protein does not contain any uridines. In some embodiments, N1-methylpseudouridine is used instead of uridine in RNA synthesis. The RNA encoding the SARS-CoV-2 S protein is translated into the SARS-CoV-2 S protein within the host cell. The S protein is then expressed on the cell surface, where it induces an adaptive immune response. The S protein has been identified as a target for neutralizing antibodies against the virus and is considered an important vaccine component.

[0210] The emergence of novel circulating variants of SARS-CoV-2 has raised serious concerns about the geographic and temporal effectiveness of vaccine interventions. One of the earliest variants to emerge and rapidly become dominant globally was D614G.

[0211] The alpha variant (also known as B.1.1.7, VOC202012 / 01, 501Y.V1, or GRY) was first detected in the United Kingdom. The alpha variant has numerous mutations, including several in the S gene. It has been shown to be inherently more transmissible, with growth rates estimated to be 40–70% higher than other SARS-CoV-2 strains in multiple countries (Volz et al., 2021, Nature, https: / / doi.org / 10.1038 / s41586-021-03470-x; Washington et al., 2021, Cell https: / / doi.org / 10.1016 / j.cell.2021.03.052).

[0212] The beta variant (also known as B.1.351 or GH / 501Y.V2) was first detected in South Africa. The beta variant carries several mutations in the S gene. Three of these mutations are in sites within the RBD associated with immune evasion: N501Y (shared with alpha), E484K, and K417N.

[0213] The gamma variant (also known as P.1 or GR / 501Y.V3), first detected in Brazil, carries several mutations affecting the spike protein, including two shared with the beta variant (N501Y and E484K), as well as another mutation at position 417 (K417T).

[0214] The delta variant (also known as B.1.617.2 or G / 478K.V1) was first reported in India. The delta variant has several point mutations affecting the spike protein, including P681R (a mutation position shared with alpha and adjacent to the furin cleavage site) and L452R, which is within the RBD and is associated with enhanced binding to ACE2 and resistance to neutralizing antibodies. There is also a deletion in the spike protein at positions 156 / 157.

[0215] These four VOCs are prevalent worldwide and represent the dominant species in the regions where they were first identified.

[0216] The Omicron (B.1.1.529) variant was first reported to WHO from South Africa on November 24, 2021. SARS-CoV-2 Omicron and its sublineages have had a major impact on the epidemiological landscape of the COVID-19 pandemic since their initial emergence in November 2021 (WHO Technical Advisory Group on SARS-CoV-2 Virus Evolution (TAG-VE): Classification of Omicron (B.1.1.259): SARS-CoV-2 Variant of Concern (2021), WHO Headquarters (HQ), WHO Health Emergencies Programme, Enhancing Response to the Omicron SARS-CoV-2 variant: Technical brief and priority actions for Member States (2022)).The first omicron variant, BA.1, had significant changes in its spike (S) glycoprotein that led to the loss of many neutralizing antibody epitopes (M. Hoffmann et al., "The Omicron variant is highly resistant against antibody-mediated neutralization: Implications for control of the COVID-19 pandemic", Cell 185, 447-456.e11 (2022)), allowing BA.1 to partially evade immunity based on the previously established SARS-CoV-2 wild-type strain (Wuhan-Hu-1) (V. Servellita, et al., "Neutralizing immunity in vaccine breakthrough infections from the SARS-CoV-2 Omicron and Delta variants", Cell 185, 1539-1548.e5 (2022); Y. Cao et al., "Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies", Nature 602, 657-663 (2022)). Therefore, breakthrough infections in individuals vaccinated with omicron are more common than with previous variants of concern (VOCs).While the omicron BA.1 has been replaced by the BA.2 variant in many countries around the world, other variants, such as BA.1.1 and BA.3, have gained traction temporarily and / or locally but have not become globally dominant (S. Xia et al., "Origin, virological features, immune evasion and intervention of SARS-CoV-2 Omicron sublineages. Signal Transduct. Target. Ther. 7, 241 (2022); H. Gruell et al., "SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns, Cell Host Microbe 7, 241 (2022)"). Omicron BA.2.12.1 subsequently replaced BA.2 and became dominant in the United States, while BA.4 and BA.5 replaced BA.2 in Europe, parts of Africa, and Asia / Pacific (H. Gruell et al., “SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns,” Cell Host Microbe 7, 241 (2022); European Centre for Disease Prevention and Control, Weekly COVID-19 country overview - Country overview report: Week 31 2022 (2022); J. Hadfield et al., “Nextstrain: Real-time tracking of pathogen evolution,” Bioinformatics 34, 4121-4123 (2018)).Omicron BA.5 is currently dominant worldwide, including in the United States (Centers for Disease Control and Prevention. COVID Data Tracker. Atlanta, GA: US Department of Health and Human Services, CDC; 2022, August 12. https: / / covid.cdc.gov / coviddata-tracker(2022)).

[0217] Omicron has acquired many mutations in the S glycoprotein (amino acid exchanges, insertions, or deletions); some of these are common among all Omicron VOCs, while others are unique to one or more Omicron sublineages. Antigenically, BA.2.12.1 exhibits high similarity to BA.2 but not to BA.1, whereas BA.4 and BA.5 are significantly different from their ancestor BA.2 and even more different from BA.1, consistent with their lineage (AZMykytyn et al., “Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.1 and BA.2 are antigenically distinct,” Sci. Immunol. 7, Eabq4450 (2022)). Significant differences between BA.1 and other Omicron VOCs include Δ143-145, L212I, or ins214EPE in the N-terminal domain of the S glycoprotein, and G446S or G496S in the receptor-binding domain (RBD). Furthermore, amino acid changes T376A, D405N, and R408S in the RBD are shared between BA.2 and its descendants but absent in BA.1. Additionally, several mutations are unique to individual descendant VOCs of BA.2, such as L452Q in BA.2.12.1 or L452R and F486V in BA.4 and BA.5 (which encode the same S sequence). Most of these shared and VOC-specific mutations have been shown to play important roles in immune escape from monoclonal antibodies and polyclonal sera raised against the wild-type S glycoprotein.In particular, BA.4 / BA.5-specific mutations have been strongly implicated in the immune evasion of these VOCs (P. Wang et al., "Antibody evasion of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature 593, 130-135 (2021); Q. Wang et al., "Antibody evasion by SARS-CoV-2 Omicron subvariants BA.2.12.1, BA.4, & BA.5. Nature 608, 603-608 (2022)"). As of the filing date of this application, the XBB group variants, resulting from the recombination of BA.10.1 and BA.2.75, are the most prevalent SARS-CoV-2 variants of concern, accounting for the top three most prevalent strains in the United States between May 28, 2023 and June 10, 2023.

[0218] Similarly, the ability of influenza to mutate and evade pre-existing immune responses is well-known. Influenza viruses are part of the Orthomyxoviridae family and are classified into three genera or types (A, B, and C) based on antigenic differences in the nucleoprotein and matrix protein. Influenza A viruses are further classified into subtypes based on the membrane glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (Cox NJ, Subbarao K. Influenza. Lancet. 1999;354(9186):1277-82). Influenza A subtypes H1N1 (also designated A(H1N1)pdm09) and H3N2 are currently circulating in humans. H1N1 caused the 2009 pandemic and replaced the influenza A(H1N1) virus that circulated prior to 2009. All influenza pandemics to date have been caused by influenza A viruses.

[0219] The influenza RNA genome is segmented, allowing genetic reassortment among viruses of the same species. This genetic instability can lead to a phenomenon called antigenic shift, involving large changes in either or both the HA and NA, which, if efficiently transmitted, can lead to pandemics. More common are multiple point mutations in the genome, leading to smaller changes in the HA and NA, known as antigenic drift (Hall E. Influenza. Chapter 12. In: Centers for Disease Control and Prevention. Hall E, Wodi AP, Hamborsky J, et al., eds. Epidemiology and Prevention of Vaccine-Preventable Diseases. 14th ed. Washington, DC: Public Health Foundation; 2021:179-92). Currently, influenza B is circulating in two lineages, Victoria (B / Victoria) and Yamagata (B / Yamagata), based on differences in the HA (Rota PA, Hemphill ML, Whistler T, Regnery HL, Kendal APJ, JoGV. Antigenic and genetic characterization of the hemagglutinins of recent cocirculating strains of influenza B virus. J General Virology. 1992;73(10):2737-42). Both influenza A and B undergo genetic mutation, which leads to drift under selective pressure from the human immune response. Because of this genetic instability, current vaccines must be adjusted annually to match the influenza strains currently circulating or predicted to be circulating.

[0220] Effective vaccine strategies against SARS-CoV-2 and influenza remain needed.

[0221] The present disclosure discloses techniques (e.g., compositions and methods) for inducing an immune response against multiple infectious agents. In particular, the present disclosure provides techniques for inducing an immune response against coronaviruses and one or more additional respiratory diseases (e.g., influenza). In some embodiments, the compositions disclosed herein comprise RNA comprising a nucleotide sequence encoding amino acids of the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof. In some embodiments, the compositions disclosed herein comprise RNA comprising a nucleotide sequence encoding an HA protein from influenza virus or an immunogenic variant thereof, or encoding an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof. The RNA encoding the antigenic polypeptide is administered to provide the antigen (after expression of the polynucleotide by the appropriate target cell) for induction, i.e., stimulation, priming, and / or amplification, of an immune response, e.g., antibodies and / or immune effector cells, that target the target antigen (e.g., coronavirus S protein, particularly SARS-CoV-2 S protein, and influenza virus HA protein) or its processing products. In one embodiment, the immune response to be induced in accordance with the present disclosure is a B cell-mediated immune response, i.e., an antibody-mediated immune response. Additionally or alternatively, in one embodiment, the immune response to be induced in accordance with the present disclosure is a T cell-mediated immune response. In one embodiment, the immune response is an anti-coronavirus, particularly an anti-SARS-CoV-2, immune response. In one embodiment, the immune response is an anti-influenza virus immune response, particularly an anti-subtype A and / or anti-subtype B immune response.

[0222] In some embodiments, the vaccines described herein comprise, as active ingredients, one or more single-stranded RNAs that can be translated into respective proteins upon entry into the recipient's cells. In addition to wild-type or codon-optimized sequences encoding antigen sequences, the RNAs may contain one or more structural elements (e.g., a 5' cap, a 5' UTR, a 3' UTR, a poly(A) tail, or a combination thereof) that are optimized for maximum efficacy with respect to stability and translation efficiency. In one embodiment, the RNAs described herein contain all of these elements. In one embodiment, a cap1 structure may be utilized as a specific capping structure at the 5' end of the RNA drug substance. In one embodiment, a beta-S-ARCA(D1)(m2) tail may be utilized at the 5' end of the RNA drug substance. 7,2’-O GppSpG), or m2 7,3’-O Gppp(m1 2’-O) ApG may be used as a specific capping structure. The 5'-UTR sequence may be the 5'-UTR sequence of human alpha globin mRNA (e.g., SEQ ID NO: 12), optionally with an optimized "Kozak sequence" to enhance translation efficiency. The 3'-UTR sequence may be a combination of two sequence elements (FI element) (e.g., SEQ ID NO: 13)—an "amino terminal enhancer of split" (AES) mRNA (designated F) and a mitochondrially encoded 12S ribosomal RNA (designated I)—located between the coding sequence and the poly(A) tail to ensure higher maximum protein levels and extended mRNA duration. These sequences were identified using an in vitro selection process for sequences that confer RNA stability and increase total protein expression (see WO 2017 / 060314, incorporated herein by reference). Alternatively, the 3'-UTR may be two repeat 3'-UTRs of human beta globin mRNA. Additionally or alternatively, in some embodiments, the poly(A) tail can comprise a length of at least 100 adenosine residues (SEQ ID NO: 180) (e.g., at least 110 adenosine residues, at least 120 adenosine residues, 130 adenosine residues, or longer). In some embodiments, the poly(A) tail can comprise a length of about 100 to about 150 adenosine residues. In some embodiments, the poly(A) tail can comprise a truncated poly(A) tail. For example, in some such embodiments, a 110-nucleotide long poly(A) tail (e.g., SEQ ID NO: 14) can be used, consisting of a stretch of 30 adenosine residues (SEQ ID NO: 174) followed by a 10-nucleotide linker sequence (of miscellaneous nucleotides) and another 70 adenosine residues (SEQ ID NO: 175). This poly(A) tail sequence is designed to enhance RNA stability and translation efficiency.

[0223] In some embodiments, the antigens described herein may be fused to a secretory signal peptide (sec) (e.g., as an N-terminal tag), or RNA may contain such an antigen fused to sec. In one embodiment, sec corresponds to the secretory signal peptide of the S protein and is fused to the N-terminus of the S protein. A sequence encoding a short linker peptide consisting mainly of the amino acids glycine (G) and serine (S), as commonly used in fusion proteins, may be used as the GS / linker between sec and the antigen domain.

[0224] In some embodiments, the RNAs described herein can be complexed with proteins and / or lipids, preferably lipids, to form RNA particles for administration. When a combination of different RNAs is used, the RNAs can be complexed together or separately with proteins and / or lipids to form RNA particles for administration.

[0225] In one aspect, the present disclosure relates to a composition or medical formulation comprising RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0226] In one embodiment, the immunogenic fragment of the SARS-CoV-2 S protein comprises the S1 subunit of the SARS-CoV-2 S protein or the receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 S protein.

[0227] In one embodiment, an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof can form a multimeric complex, particularly a trimeric complex. To this end, the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof can comprise a domain that allows for the formation of a multimeric complex, particularly a trimeric complex, of the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof. In one embodiment, the domain that allows for the formation of a multimeric complex comprises a trimerization domain, e.g., a trimerization domain described herein, e.g., the SARS-CoV-2 S protein trimerization domain. In one embodiment, trimerization is achieved by the addition of a trimerization domain, such as the "foldon" trimerization domain from T4 fibritin (e.g., SEQ ID NO: 10), particularly when the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof corresponds to a portion of the SARS-CoV-2 S protein that does not include the trimerization domain of the SARS-CoV-2 S protein.

[0228] In one embodiment, an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to the wild-type coding sequence, and the codon optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence. Those skilled in the art will appreciate that codon optimization requires selection from among alternative codons that encode the same amino acid residue. Codon optimization typically involves consideration of the codon(s) preferred by the particular host in which the sequence is to be expressed. According to the present disclosure, in many embodiments, the preferred host is a human. In some embodiments, the preferred host may be a domestic animal. Alternatively, or in addition, in some embodiments, the selection from among possible codons encoding the same amino acid may take into account one or more other characteristics, such as overall G / C content (as described above) and / or similarity to a particular reference. For example, in some embodiments of the present disclosure, the provided coding sequences encoding a SARS-CoV-2 S protein or immunogenic variant thereof that differ in amino acid sequence compared to that encoded by the BNT162b2 constructs described herein utilize a codon at at least one position of such difference that retains greater similarity to the BNT162b2 construct sequence than at least one alternative codon that encodes the same amino acid at such position of difference.

[0229] In one embodiment, the RNA is modified RNA, particularly stabilized mRNA. In one embodiment, the RNA comprises a modified nucleoside in place of at least one uridine. In one embodiment, the RNA comprises a modified nucleoside in place of each uridine. In one embodiment, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0230] In one embodiment, the RNA includes modified nucleosides in place of uridine.

[0231] In one embodiment, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0232] In one embodiment, the RNA comprises a 5' cap.

[0233] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9 or the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and / or (ii) The SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1.

[0234] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30 or the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30; and / or (ii) The SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof comprises the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29.

[0235] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9 or the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9; and / or (ii) The SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof, comprises the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1.

[0236] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9 or the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9; and / or (ii) The SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7 or the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

[0237] In one embodiment, the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a secretory signal peptide.

[0238] In one embodiment, the secretory signal peptide is preferably fused to the N-terminus of the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0239] In one embodiment, (i) the RNA encoding the secretory signal peptide comprises the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9 or the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9; and / or (ii) The secretory signal peptide comprises the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.

[0240] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO: 6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 6, or a fragment of the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 6; and / or (ii) The SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof comprises the amino acid sequence of SEQ ID NO:5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5.

[0241] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30 or the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30; and / or (ii) The SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof comprises the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29.

[0242] In one embodiment, each RNA in the composition is a modified RNA, particularly a stabilized mRNA. In one embodiment, each RNA in the composition comprises a modified nucleoside in place of at least one uridine. In one embodiment, each RNA in the composition comprises a modified nucleoside in place of each uridine. In one embodiment, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0243] In one embodiment, each RNA in the composition comprises a modified nucleoside in place of a uridine.

[0244] In one embodiment, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0245] In one embodiment, each RNA in the composition comprises a 5' cap.

[0246] In one embodiment, each RNA in the composition comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:12.

[0247] In one embodiment, each RNA in the composition comprises a 3'UTR comprising the nucleotide sequence of SEQ ID NO:13 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:13.

[0248] In one embodiment, each RNA in the composition comprises a polyA sequence.

[0249] In one embodiment, the polyA sequence comprises at least 100 nucleotides.

[0250] In one embodiment, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:14.

[0251] In one embodiment, each RNA in the composition is or will be formulated as a liquid, a solid, or a combination thereof.

[0252] In one embodiment, each RNA in the composition is or will be formulated for injection.

[0253] In one embodiment, each RNA in the composition is or will be formulated for intramuscular administration.

[0254] In one embodiment, each RNA in the composition is or will be formulated as a particle.

[0255] In one embodiment, the particle is a lipid nanoparticle (LNP) or lipoplex (LPX) particle.

[0256] In one embodiment, the LNP particles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol. In one embodiment, the RNA lipoplex particles are obtained by mixing RNA with liposomes. In one embodiment, the RNA lipoplex particles are obtained by mixing RNA with lipids.

[0257] In one embodiment, the RNA is or will be formulated as a colloid. In one embodiment, the RNA is or will be formulated as particles forming the dispersed phase of the colloid. In one embodiment, at least 50%, at least 75%, or at least 85% of the RNA is in the dispersed phase. In one embodiment, the RNA is or will be formulated as particles comprising RNA and lipids. In one embodiment, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase. In one embodiment, the organic phase comprises ethanol. In one embodiment, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in the organic phase. In one embodiment, the lipids dispersed in the aqueous phase form liposomes.

[0258] In one embodiment, each RNA in the composition is an mRNA or a saRNA.

[0259] In one embodiment, the composition or medical formulation is a pharmaceutical composition.

[0260] In one embodiment, the composition or medical formulation is a vaccine.

[0261] In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0262] In one embodiment, the composition or medical preparation is a kit.

[0263] In one embodiment, the RNA and, optionally, the particle-forming components are contained in separate vials.

[0264] In one embodiment, the kit further comprises instructions for use of the composition or medical formulation to induce an immune response against coronavirus in a subject.

[0265] In one aspect, the present disclosure relates to a composition or medicinal formulation described herein for pharmaceutical use.

[0266] In one embodiment, the medical use comprises inducing an immune response against coronavirus in a subject.

[0267] In one embodiment, the medical use includes the therapeutic or prophylactic treatment of coronavirus infection.

[0268] In one embodiment, the compositions or medical formulations described herein are for administration to a human.

[0269] In one embodiment, the coronavirus is a betacoronavirus.

[0270] In one embodiment, the coronavirus is a sarbecovirus.

[0271] In one embodiment, the coronavirus is SARS-CoV-2.

[0272] In one aspect, the disclosure relates to a method of inducing an immune response to coronavirus and influenza in a subject, the method comprising administering to the subject a composition comprising RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, and RNA encoding an amino acid sequence comprising the influenza HA protein, an immunogenic variant thereof, or an immunogenic fragment of the influenza HA protein or an immunogenic variant thereof.

[0273] In one embodiment, the immunogenic fragment of the SARS-CoV-2 S protein comprises the S1 subunit of the SARS-CoV-2 S protein or the receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 S protein.

[0274] Both the N-terminal domain (NTD) and RBD of the coronavirus S protein are known to be binding sites for antibodies that neutralize viral activity. In the case of SARS-CoV-2, the RBD is part of the S protein, which binds to angiotensin-converting enzyme 2 (ACE2) on the surface of host cells. Although the function of the NTD in the SARS-CoV-2 S protein is not fully understood, this domain is thought to play a role in binding to carbohydrate moieties and facilitating the conformational change of the S protein from the pre-fusion to the post-fusion conformation. Both the NTD and RBD can induce high binding and neutralizing antibody titers.

[0275] In some embodiments, the disclosure provides a method, comprising administering to a human subject a therapeutic dose of a composition comprising RNA (e.g., mRNA) comprising an open reading frame (ORF) encoding a fusion protein comprising at least two domains of the SARS-CoV-2 spike (S) protein, but less than the full-length spike protein, wherein the RNA is present in a lipid nanoparticle.

[0276] In one embodiment, an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof can form a multimeric complex, particularly a trimeric complex. To this end, the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof can comprise a domain that allows the formation of a multimeric complex, particularly a trimeric complex, of the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof. In one embodiment, the domain that allows the formation of a multimeric complex comprises a trimerization domain, e.g., a trimerization domain described herein.

[0277] In one embodiment, the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to the wild-type coding sequence, and the codon-optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence.

[0278] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9 or the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and / or (ii) The SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1.

[0279] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30 or the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30; and / or (ii) The SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof comprises the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29.

[0280] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9 or the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9; and / or (ii) The SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof, comprises the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1.

[0281] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9 or the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9; and / or (ii) The SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7 or the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7. In one embodiment, the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a secretory signal peptide.

[0282] In one embodiment, the secretory signal peptide is preferably fused to the N-terminus of the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0283] In one embodiment, (i) the RNA encoding the secretory signal peptide comprises the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9 or the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9; and / or (ii) The secretory signal peptide comprises the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.

[0284] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO: 6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 6, or a fragment of the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 6; and / or (ii) The SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof comprises the amino acid sequence of SEQ ID NO:5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5.

[0285] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30 or the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30; and / or (ii) The SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof comprises the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29.

[0286] In one embodiment, each RNA in the composition is a modified RNA, particularly a stabilized mRNA. In one embodiment, each RNA in the composition comprises a modified nucleoside in place of at least one uridine. In one embodiment, each RNA in the composition comprises a modified nucleoside in place of each uridine. In one embodiment, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0287] In one embodiment, each RNA in the composition comprises a modified nucleoside in place of a uridine.

[0288] In one embodiment, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0289] In one embodiment, each RNA in the composition comprises a cap.

[0290] In one embodiment, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO:12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:12.

[0291] In one embodiment, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a 3'UTR comprising the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 13.

[0292] In one embodiment, each RNA in the composition comprises a polyA sequence.

[0293] In one embodiment, the polyA sequence comprises at least 100 nucleotides.

[0294] In one embodiment, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:14.

[0295] In one embodiment, each RNA in the composition is formulated as a liquid, a solid, or a combination thereof.

[0296] In one embodiment, each RNA in the composition is administered by injection.

[0297] In one embodiment, each RNA in the composition is administered by intramuscular administration.

[0298] In one embodiment, each RNA in the composition is formulated as a particle.

[0299] In one embodiment, the particle is a lipid nanoparticle (LNP) or lipoplex (LPX) particle.

[0300] In one embodiment, the LNP particles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol. In one embodiment, the RNA lipoplex particles are obtained by mixing RNA with liposomes. In one embodiment, the RNA lipoplex particles are obtained by mixing RNA with lipids.

[0301] In one embodiment, the RNA is formulated as a colloid. In one embodiment, the RNA is formulated as particles that form the dispersed phase of the colloid. In one embodiment, at least 50%, at least 75%, or at least 85% of the RNA in the composition is present in the dispersed phase. In one embodiment, the RNA is formulated as particles comprising RNA and lipids. In one embodiment, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase. In one embodiment, the organic phase comprises ethanol. In one embodiment, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dispersed in the aqueous phase. In one embodiment, the lipids dispersed in the aqueous phase form liposomes.

[0302] In one embodiment, each RNA in the composition is an mRNA or a saRNA.

[0303] In one embodiment, the method is a method for vaccination against coronavirus.

[0304] In one embodiment, the method is for the therapeutic or prophylactic treatment of a coronavirus infection.

[0305] In one embodiment, the subject is a human.

[0306] In one embodiment, the coronavirus is a betacoronavirus.

[0307] In one embodiment, the coronavirus is a sarbecovirus.

[0308] In one embodiment, the coronavirus is SARS-CoV-2.

[0309] In one embodiment of the methods described herein, the composition is a composition described herein.

[0310] In one aspect, the disclosure relates to a composition or pharmaceutical formulation described herein for use in a method described herein.

[0311] Among other things, the present disclosure teaches that a composition comprising (i) lipid nanoparticle-encapsulated RNA encoding at least a portion (e.g., an epitope or comprising thereof) of a polypeptide encoded by SARS-CoV-2 (e.g., an S protein encoded by SARS-CoV-2) and (ii) lipid nanoparticle-encapsulated RNA encoding at least a portion (e.g., an epitope or comprising thereof) of a polypeptide encoded by influenza virus (e.g., an HA protein encoded by influenza virus) can result in detectable antibody titers against each epitope in serum within seven days after administration to a population of adult subjects according to a regimen comprising at least one dose of the vaccine composition. In such a composition, the mRNA encoding at least a portion of the polypeptide encoded by SARS-CoV-2 and the mRNA encoding at least a portion of the polypeptide encoded by influenza virus may be formulated in the same or separate lipid nanoparticle formulations. Furthermore, the present disclosure teaches the durability of such antibody titers. In some embodiments, the present disclosure teaches that the use of modified mRNA increases such antibody titers compared to those produced using the corresponding unmodified mRNA.

[0312] In some embodiments, the provided regimen comprises at least one dose. In some embodiments, the provided regimen comprises a first dose and at least one subsequent dose. In some embodiments, the first dose is the same amount as at least one subsequent dose. In some embodiments, the first dose is the same amount as all subsequent doses. In some embodiments, the first dose is a different amount than at least one subsequent dose. In some embodiments, the first dose is a different amount than all subsequent doses. In some embodiments, the provided regimen comprises two doses. In some embodiments, the provided regimen consists of two doses.

[0313] In detailed embodiments, the immunogenic composition is formulated as a single dose in a container, e.g., a vial. In some embodiments, the immunogenic composition is formulated as a multi-dose formulation in a vial. In some embodiments, the multi-dose formulation contains at least two doses per vial. In some embodiments, the multi-dose formulation contains a total of 2-20 doses per vial, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses per vial. In some embodiments, each dose in a vial is equal in volume. In some embodiments, the first dose is a different volume from subsequent doses.

[0314] A "stable" multi-dose formulation does not exhibit unacceptable levels of microbial growth and exhibits substantial or no degradation or deterioration of the active biological molecular component(s). As used herein, a "stable" immunogenic composition includes a formulation that remains capable of eliciting a desired immunological response when administered to a subject.

[0315] In some embodiments, the multi-dose formulation remains stable for a specified period of time even after multiple or repeated inoculations / insertion into a multi-dose container. For example, in some embodiments, the multi-dose formulation may be stable for at least 3 days when contained in a multi-dose container, even after up to 10 uses. In some embodiments, the multi-dose formulation remains stable after 2-20 inoculations / insertion.

[0316] In some embodiments, administration of a composition comprising lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., an epitope or comprising thereof) of a SARS-CoV-2-encoded polypeptide (e.g., a SARS-CoV-2-encoded S protein), e.g., according to a regimen described herein, may result in lymphopenia in some subjects (e.g., all subjects, most subjects, less than about 50%, less than about 40%, less than about 40%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, etc.). Among other things, the present disclosure teaches that such lymphopenia may resolve over time. For example, in some embodiments, lymphopenia resolves within about 14 days, about 10 days, about 9 days, about 8 days, about 7 days, or less. In some embodiments, lymphopenia is Grade 3, Grade 2, or less.

[0317] Thus, among other things, the present disclosure provides compositions comprising lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., an epitope or a portion thereof) of a polypeptide encoded by SARS-CoV-2 (e.g., the S protein encoded by SARS-CoV-2) and lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., an epitope or a portion thereof) of a polypeptide encoded by influenza virus (e.g., the HA protein encoded by influenza virus), wherein the compositions exhibit specific properties (e.g., produce specific effects) described herein when administered to a relevant adult population. In such compositions, the mRNA encoding at least a portion of the polypeptide encoded by SARS-CoV-2 and the mRNA encoding at least a portion of the polypeptide encoded by influenza virus may be formulated in the same or separate lipid nanoparticle formulations. In some embodiments, the provided compositions may have been prepared, stored, transported, characterized, and / or used under conditions in which the temperature does not exceed a specific threshold. Alternatively, or in addition, in some embodiments, provided compositions can be protected from light (e.g., certain wavelengths) during some or all of their preparation, storage, transportation, characterization, and / or use. In some embodiments, one or more characteristics of provided compositions (e.g., mRNA stability, which can be assessed by one or more of, for example, size, presence of particular moieties or modifications, etc.; lipid nanoparticle stability or aggregation; pH, etc.) can be, or have been, assessed at one or more time points during preparation, storage, transportation, and / or use prior to administration.

[0318] Among other things, the present disclosure reports that certain provided compositions, in which the nucleotides in the mRNA are unmodified (e.g., naturally occurring A, U, C, G), and / or provided methods relating to such compositions, are characterized by an inherent adjuvant effect (e.g., when administered to a relevant population, which in some embodiments may include, or be an adult population). In some embodiments, such compositions and / or methods are capable of inducing antibody and / or T cell responses. In some embodiments, such compositions and / or methods are capable of inducing a higher T cell response compared to conventional vaccines (e.g., non-mRNA vaccines such as protein vaccines).

[0319] Alternatively, or in addition, the present disclosure provides compositions (e.g., compositions comprising (i) lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., an epitope, or comprising thereof) of a polypeptide encoded by SARS-CoV-2 (e.g., an S protein encoded by SARS-CoV-2), and (ii) lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., an epitope, or comprising thereof) of a polypeptide encoded by influenza virus (e.g., an HA protein encoded by influenza virus), wherein nucleotides in the mRNA are modified, and / or provided methods related to such compositions are characterized by either no inherent adjuvant effect or a reduced inherent adjuvant effect compared to an otherwise equivalent composition (or method) with unmodified results (e.g., when administered to a population of interest, which in some embodiments may be an adult population or a population of interest that includes the same). Alternatively, or in addition, in some embodiments, such compositions (or methods) are characterized by inducing an antibody response and / or a CD4+ T cell response (e.g., when administered to an adult population or a relevant population, which in some embodiments may include an adult population). Furthermore, alternatively or additionally, in some embodiments, such compositions (or methods) are characterized by inducing a CD4+ T cell response (e.g., when administered to an adult population or a relevant population, which in some embodiments may include an adult population) that is greater than that observed with another vaccine format (e.g., a peptide vaccine). In some embodiments involving modified nucleotides, such modified nucleotides may be present, for example, in the 3' UTR sequence, the antigen-encoding sequence, and / or the 5' UTR sequence. In some embodiments, the modified nucleotides are or include one or more modified uracil residues and / or one or more modified cytosine residues.

[0320] In particular, the present disclosure provides compositions (e.g., compositions and / or methods comprising (i) lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., an epitope, or a portion thereof) of a polypeptide encoded by SARS-CoV-2 (e.g., an S protein encoded by SARS-CoV-2), and (ii) lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., an epitope, or a portion thereof) of a polypeptide encoded by influenza virus (e.g., an HA protein encoded by influenza virus), when administered (e.g., to a population of interest, which in some embodiments may include an adult population) to produce the encoded polypeptide (e.g., (i) a protein [e.g., S protein], or a portion thereof, and (ii) an influenza virus-encoded protein (e.g., an HA protein), or portion thereof, which in some embodiments may include an epitope thereof. For example, in some embodiments, such compositions and / or methods, when administered to a human, result in detectable polypeptide expression in a biological sample (e.g., serum) from such a human, and in some embodiments, such expression is characterized by persistence of at least 36 hours or longer, e.g., at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 148 hours, or longer.

[0321] Those skilled in the art will understand, upon reading this disclosure, that various compositions are described that include one or more mRNA constructs that encode at least a portion (e.g., an epitope or comprising thereof) of a SARS-CoV-2-encoded polypeptide (e.g., a SARS-CoV-2-encoded S protein) and one or more mRNA constructs that encode at least a portion (e.g., an epitope or comprising thereof) of an influenza virus-encoded polypeptide (e.g., an influenza virus-encoded HA protein). Such skilled artisans will understand, upon reading this disclosure, that various compositions are described that include one or more of various mRNA constructs that encode at least a portion of a SARS-CoV-2 S protein, such as at least the RBD portion of the SARS-CoV-2 S protein. Moreover, such skilled artisans will understand, upon reading this disclosure, that certain properties and / or advantages are described for compositions comprising one or more mRNA constructs encoding at least a portion (e.g., an epitope or comprising thereof) of a polypeptide encoded by SARS-CoV-2 (e.g., the S protein encoded by SARS-CoV-2), and one or more mRNA constructs encoding at least a portion (e.g., an epitope or comprising thereof) of a polypeptide encoded by influenza virus. In some embodiments, the compositions may comprise one or more mRNA constructs encoding at least one domain of a SARS-CoV-2-encoded polypeptide (e.g., one or more domains of a SARS-CoV-2-encoded polypeptide described in WO2021 / 159040, e.g., the N-terminal domain (NTD) of the SARS-CoV-2 spike protein, the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, the heptapeptide repeat 1 (HR1) of the SARS-CoV-2 spike protein, the heptapeptide repeat 2 (HR1) of the SARS-CoV-2 spike protein, and / or combinations thereof).Among other things, this disclosure particularly reports surprising, useful properties and / or advantages of compositions comprising one or more RNAs comprising a nucleotide sequence encoding an antigenic polypeptide associated with influenza virus and a particular mRNA construct encoding a portion of the SARS-CoV-2 RBD, but in some embodiments not encoding the full-length SARS-CoV-2 S protein. Without wishing to be bound by any particular theory, this disclosure suggests that RNAs encoding less than the full-length SARS-CoV-2 S protein, particularly RNAs encoding at least the RBD portion of such a SARS-CoV-2 S protein, may be particularly useful and / or effective for use as or in immunogenic compositions (e.g., vaccines) and / or in producing the immunological effects described herein (e.g., generating SARS-CoV-2 neutralizing antibodies and / or T cell responses (e.g., CD4+ and / or CD8+ T cell responses)).

[0322] In some embodiments, the present disclosure provides compositions comprising RNA (e.g., mRNA) comprising an open reading frame encoding a polypeptide comprising a receptor-binding portion of the SARS-CoV-2 S protein, wherein the RNA is suitable for intracellular expression of the polypeptide. In some embodiments, such encoded polypeptide does not comprise the entire S protein. In some embodiments, the encoded polypeptide comprises a receptor-binding domain (RBD), e.g., as set forth in SEQ ID NO: 5. In some embodiments, the encoded polypeptide comprises a peptide according to SEQ ID NO: 29 or 31. In some embodiments, such RNA (e.g., mRNA) may form a complex with a (poly)cationic polymer, polyplex(es), protein(s), or peptide(s). In some embodiments, such RNA may be formulated into lipid nanoparticles (e.g., those described herein). In some embodiments, such RNA (e.g., mRNA) may be particularly useful and / or effective for use as or in immunogenic compositions (e.g., vaccines) and / or in producing the immunological effects described herein (e.g., generating SARS-CoV-2 neutralizing antibodies and / or T cell responses (e.g., CD4+ and / or CD8+ T cell responses)). In some embodiments, such RNA (e.g., mRNA) may be useful for vaccinating humans (e.g., including humans with known exposure and / or infection to SARS-CoV-2 and / or humans with no known exposure to SARS-CoV-2).

[0323] Those skilled in the art will further appreciate, upon reading this disclosure, that various mRNA constructs comprising nucleic acid sequences encoding full-length SARS-CoV-2 spike proteins (including, for example, embodiments in which such encoded SARS-CoV-2 spike proteins may include at least one or more amino acid substitutions, such as proline substitutions described herein, and / or in which the mRNA sequences have been codon-optimized, e.g., for a mammalian subject, e.g., a human subject) are described. In some embodiments, such full-length SARS-CoV-2 spike proteins may have an amino acid sequence that is or includes the amino acid sequence set forth in SEQ ID NO:7. Moreover, such skilled artisans will appreciate, upon reading this disclosure, that, among other things, detailed properties and / or advantages of particular mRNA constructs comprising nucleic acid sequences encoding full-length SARS-CoV-2 spike proteins are described.

[0324] Without wishing to be bound by any particular theory, the present disclosure suggests that provided compositions (e.g., compositions comprising one or more mRNA constructs encoding a full-length SARS-CoV-2 S protein and one or more mRNA constructs encoding an HA protein) may be particularly useful and / or effective for use as or included in immunogenic compositions (e.g., vaccines) in particular subject populations (e.g., particular age groups). For example, in some embodiments, such mRNA compositions may be particularly useful in younger subjects (e.g., under 25, 20, 18, 15, 10, or younger); alternatively, or in addition, in some embodiments, such mRNA compositions may be particularly useful in older subjects (e.g., over 55, 60, 65, 70, 75, 80, 85, or older). In particular embodiments, immunogenic compositions comprising such mRNA constructs provided herein exhibit minimal to moderate increases (e.g., no more than a 30% increase, no more than a 20% increase, or no more than a 10% or less increase) in systemic reactogenicity (e.g., fever, fatigue, headache, chills, diarrhea, muscle and / or joint pain, etc.) and / or local tolerance (e.g., pain, redness and / or swelling, etc.) that are dose-level and / or dose-frequency dependent in at least some subjects (e.g., some age groups of subjects); in some embodiments, such reactogenicity and / or local tolerance is particularly observed in younger subjects (e.g., under 25, 20, 18, or younger) and / or older (e.g., elderly) subjects (e.g., 65-85 years of age).In some embodiments, provided compositions comprising one or more mRNA constructs encoding a full-length SARS-CoV-2 S protein and one or more mRNA constructs encoding an HA protein may be particularly useful and / or effective for use as or in immunogenic compositions (e.g., vaccines) to induce SARS-CoV-2 and influenza virus neutralizing antibody response levels in subject populations at high risk for severe disease associated with SARS-CoV-2 infection and / or influenza virus infection (e.g., elderly populations, e.g., those aged 65-85 years).

[0325] In some embodiments, the methods, compositions, or combinations described herein may be administered to older adult subjects (e.g., subjects 50 years of age or older, 55 years of age or older, 60 years of age or older, or 65 years of age or older) who are at increased risk for severe disease caused by RSV infection (e.g., have one of the risk factors described herein). In some embodiments, the methods, compositions, or combinations described herein are administered to older adults (e.g., subjects 60 years of age or older or 65 years of age or older) who are at increased risk for severe disease caused by RSV infection (e.g., have one of the risk factors described herein).

[0326] In some embodiments, the methods, compositions, or combinations described herein can be administered to infants or children at high risk for severe disease caused by RSV infection (e.g., having one of the risk factors described herein). In some embodiments, the methods, compositions, or combinations described herein are administered to subjects with a condition that may be exacerbated by RSV infection (e.g., having one of the conditions described herein).

[0327] In some embodiments, the methods, compositions, or combinations described herein can be administered to pregnant subjects (e.g., subjects between about 32 and about 36 weeks of gestation) to prevent lower respiratory tract disease (LRTD) and severe LRTD caused by RSV, for example, in newborn infants (e.g., children from birth to about 6 months of age).

[0328] In some embodiments, a higher dose may be administered to elderly subjects (e.g., subjects 65 years of age or older) compared to younger patients. For example, in some embodiments, twice the dose administered to non-elderly patients (e.g., patients under 65 years of age) is administered to elderly patients (e.g., patients 65 years of age or older). In some embodiments, 60 μg of RNA encoding one or more antigens associated with an infectious agent (e.g., influenza) is administered to an elderly patient. In some embodiments, 60 μg of a tetravalent influenza vaccine (e.g., a vaccine comprising 15 μg of RNA encoding an HA polypeptide associated with an H1N1 influenza A virus, 15 μg of RNA encoding an HA polypeptide associated with an H3N2 influenza A virus, 15 μg of RNA encoding an HA polypeptide associated with the B / Yamagata lineage, and 15 μg of RNA encoding an HA polypeptide associated with the B / Yamagata lineage) is administered to an elderly patient.

[0329] In some embodiments, upon reading this disclosure, one of skill in the art will understand that provided compositions comprising one or more mRNA constructs encoding a full-length SARS-CoV-2 S protein and one or more RNA constructs encoding an HA protein that exert a favorable reactogenicity profile (e.g., as described herein), especially in younger and older populations, may be particularly useful and / or effective for use as or in immunogenic compositions (e.g., vaccines) to produce the immunological effects described herein (e.g., generating SARS-CoV-2 neutralizing antibodies, influenza virus neutralizing antibodies, and / or T cell responses (e.g., CD4+ and / or CD8+ T cell responses)). In some embodiments, the present disclosure also suggests that provided compositions comprising one or more mRNA constructs encoding a full-length SARS-CoV-2 S protein and one or more mRNA constructs encoding an HA protein may be particularly effective in protecting against SARS-CoV-2 and / or influenza infection, characterized by faster clearance of SARS-CoV-2 viral and / or influenza viral RNA in non-human mammalian subjects (e.g., rhesus macaques) immunized with an immunogenic composition comprising such mRNA constructs and subsequently infected with SARS-CoV-2 and / or influenza virus. In some embodiments, such faster clearance of SARS-CoV-2 viral RNA and / or influenza viral RNA may be observed in the noses of non-human mammalian subjects (e.g., rhesus macaques) immunized with an immunogenic composition comprising such mRNA constructs and subsequently challenged with SARS-CoV-2 and / or influenza virus.

[0330] In some embodiments, the present disclosure provides compositions comprising one or more RNAs (e.g., one or more mRNAs) each comprising an open reading frame encoding a full-length SARS-CoV-2 S protein (e.g., a full-length SARS-CoV-2 S protein with one or more amino acid substitutions) and one or more RNAs (e.g., one or more mRNAs) each comprising an open reading frame encoding an HA protein, wherein the RNAs are suitable for intracellular expression of the polypeptides. In some embodiments, the encoded SARS-CoV-2 proteins comprise the amino acid sequence of SEQ ID NO: 7, 50, or 69. In some embodiments, the encoded HA protein comprises the amino acid sequence of any one of SEQ ID NOs: 80, 85, 90, 95, 100, or 105. In some embodiments, such RNAs (e.g., mRNAs) may form complexes with (poly)cationic polymers, polyplex(es), protein(s), or peptide(s). In some embodiments, such RNAs may be formulated into lipid nanoparticles (e.g., those described herein).

[0331] In some embodiments, the immunogenic compositions provided herein may comprise multiple (e.g., at least two or more, e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, etc.) immunoreactive epitopes of a SARS-CoV-2 polypeptide or variant thereof. In some such embodiments, such multiple immunoreactive epitopes may be encoded by multiple RNAs (e.g., mRNAs). In some such embodiments, such multiple immunoreactive epitopes may be encoded by a single RNA (e.g., mRNA). In some embodiments, nucleic acid sequences encoding multiple immunoreactive epitopes may be separated from each other by linkers (in some embodiments, peptide linkers) in a single RNA (e.g., mRNA). Without wishing to be bound by any particular theory, in some embodiments, provided polyepitopic immunogenic compositions (including, for example, those encoding the full-length SARS-CoV-2 spike protein) may be particularly useful in providing protection against multiple viral variants, given the genetic diversity of SARS-CoV-2 variants, and / or in eliciting diverse and / or otherwise robust (e.g., sustained, e.g., detectable about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 days or more after administration of one or more doses) neutralizing antibody and / or T cell responses, particularly remarkably robust T H This may provide a greater opportunity for the development of type 1 T cell (e.g., CD4+ and / or CD8+ T cell) responses.

[0332] In some embodiments, the present disclosure reports that provided compositions and / or methods are characterized in that they produce one or more particular therapeutic results (e.g., an effective immune response as described herein and / or detectable expression of the encoded SARS-CoV-2 S protein and the encoded influenza HA protein(s) or immunogenic fragments thereof) upon administration (e.g., when administered to a relevant population, which in some embodiments may include an adult population), and in some such embodiments, the results may be evaluated, for example, relative to the results observed in the absence of an mRNA vaccine described herein. In some embodiments, the results may be evaluated relative to the results observed after administration of a monovalent vaccine (e.g., a composition comprising only one of the RNAs disclosed herein). In some embodiments, the particular results may be produced at lower doses than required for one or more alternative strategies.

[0333] In some embodiments, the present disclosure provides immunogenic compositions comprising (i) one or more messenger ribonucleic acid (mRNA) polynucleotides, each comprising an open reading frame encoding a polypeptide comprising a receptor-binding portion of the SARs-CoV-2 S protein, and (ii) one or more messenger ribonucleic acid (mRNA) polynucleotides, each comprising an open reading frame encoding a polypeptide comprising an HA protein, wherein the mRNA polynucleotides (i) and (ii) are each (together or separately) formulated into at least one lipid nanoparticle. For example, in some embodiments, such lipid nanoparticles may comprise, by molar ratio, 20-60% ionizable cationic lipid, 5-25% non-cationic lipid (e.g., neutral lipid), 25-55% sterol or steroid, and 0.5-15% polymer-conjugated lipid (e.g., PEG-modified lipid). In some embodiments, the sterol or steroid contained in the lipid nanoparticles may be or may include cholesterol. In some embodiments, the neutral lipid may be or may include 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC). In some embodiments, the polymer-bound lipid may be or may include PEG2000 DMG. In some embodiments, such immunogenic compositions may have a total lipid content of about 1 mg to 10 mg, or 3 mg to 8 mg, or 4 mg to 6 mg. In some embodiments, such immunogenic compositions may have a total lipid content of about 5 mg / mL to 15 mg / mL, or 7.5 mg / mL to 12.5 mg / mL, or 9 to 11 mg / mL. In some embodiments, such compositions are provided in an amount effective to induce an immune response in a subject administered at least one dose of the immunogenic composition. In some embodiments, the polypeptide encoded by the mRNA polynucleotide (i) does not include the complete S protein. In some embodiments, the mRNA polynucleotide in such immunogenic compositions is free of self-replicating RNA.

[0334] In some embodiments, the compositions disclosed herein can induce an immune response against a first infectious agent and a second infectious agent. In some embodiments, the compositions can induce an immune response against coronavirus, another respiratory disease. In some embodiments, the compositions can induce an immune response against SARS-CoV-2 and influenza virus.

[0335] In some embodiments, the immune response can include generating binding antibody titers against a SARS-CoV-2 protein (including, for example, in some embodiments, a stabilized prefusion spike trimer) and / or an influenza virus protein, or fragments thereof. In some embodiments, the immune response can include generating binding antibody titers against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. In some embodiments, provided immunogenic compositions are established to result in detectable binding antibody titers, e.g., by about two weeks, following administration of a first dose, with seroconversion in at least 70% (including, for example, at least 80%, at least 90%, at least 95%, and up to 100%) of a subject population receiving such provided immunogenic composition.

[0336] In some embodiments, the immune response may include generating neutralizing antibody titers against a SARS-CoV-2 protein (which may include, for example, in some embodiments, a stabilized prefusion spike trimer) and / or an influenza virus protein, or a fragment thereof. In some embodiments, the immune response may include generating neutralizing antibody titers against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. In some embodiments, the provided immunogenic compositions have been established to produce neutralizing antibody titers in an appropriate system (e.g., humans infected with SARS-CoV-2, influenza virus, and / or populations thereof, and / or systems that model them). For example, in some embodiments, such neutralizing antibody titers may be demonstrated in one or more of a human population, a non-human primate model (e.g., rhesus macaque), and / or a mouse model.

[0337] In some embodiments, the neutralizing antibody titer is sufficient (e.g., established to be sufficient) to reduce viral infection of B cells below that observed in an appropriate control (e.g., an unvaccinated control subject, or a subject vaccinated with a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine, or a combination thereof). In some such embodiments, such reduction is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

[0338] In some embodiments, the neutralizing antibody titer is sufficient (e.g., established to be sufficient) to reduce the rate of asymptomatic viral infection below that observed in an appropriate control (e.g., unvaccinated control subjects, or subjects vaccinated with a live-attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine, or a combination thereof). In some such embodiments, such a reduction is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, such a reduction may be characterized by assessment of SARS-CoV-2 N protein and / or influenza virus serology. Significant protection against asymptomatic infection may also be confirmed by practical findings (see, for example, the SARS-CoV-2-related results summarized in Dagan N. et al., N Engl J Med. 2021, doi:10.1056 / NEJMoa2101765. Epub ahead of print. PMID:33626250).

[0339] In some embodiments, the neutralizing antibody titer is sufficient (e.g., established to be sufficient) to reduce or block viral fusion with epithelial cells and / or B cells in a vaccinated subject relative to that observed in an appropriate control (e.g., an unvaccinated control subject, or a subject vaccinated with a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine, or a combination thereof). In some such embodiments, such a reduction is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

[0340] In some embodiments, the induction of neutralizing antibody titers may be characterized by an increase in the number of B cells, which in some embodiments may include plasma cells, class-switched IgG1- and IgG2-positive B cells, and / or germinal center B cells. In some embodiments, provided immunogenic compositions have been established to result in such an increase in the number of B cells in an appropriate system (e.g., humans infected with SARS-CoV-2 and / or populations thereof, and / or model systems). For example, in some embodiments, such an increase in the number of B cells may be demonstrated in one or more of a human population, a non-human primate model (e.g., rhesus macaque), and / or a mouse model. In some embodiments, such an increase in the number of B cells may be demonstrated in the draining lymph nodes and / or spleen of a mouse model (e.g., at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days) after immunization of such mouse model with a provided immunogenic composition.

[0341] In some embodiments, the induction of neutralizing antibody titers may be characterized by a reduction in the number of circulating B cells in the blood. In some embodiments, the provided immunogenic compositions have been established to result in such a reduction in the number of circulating B cells in the blood of an appropriate system (e.g., humans infected with SARS-CoV-2 and / or populations thereof, and / or model systems). For example, in some embodiments, such a reduction in the number of circulating B cells in the blood may be demonstrated in one or more of a human population, a non-human primate model (e.g., rhesus macaque), and / or a mouse model. In some embodiments, such a reduction in the number of circulating B cells in the blood may be demonstrated in a mouse model (e.g., at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days) after immunization of such mouse model with a provided immunogenic composition. Without wishing to be bound by theory, the reduction in circulating B cells in the blood may be due to homing of B cells to the lymphoid compartment.

[0342] In some embodiments, the immune response induced by the provided immunogenic compositions can include an increase in the number of T cells. In some embodiments, such an increase in the number of T cells can include one or more subsets of follicular helper T cells (T cells), which in some embodiments can include one or more subsets accompanied by upregulation of ICOS. FH Those skilled in the art will appreciate that the increase in the number of T FH The proliferation of T cells is essential for the generation of adaptive B cell responses, and in humans, the proliferation of T cells that appear in the circulation after vaccination is crucial. FH It will be appreciated that a high frequency of antigen-specific antibodies typically correlates with a high frequency of antigen-specific antibodies. In some embodiments, the provided immunogenic compositions are capable of inducing T cells (e.g., T) in an appropriate system (e.g., humans infected with SARS-CoV-2 and / or populations thereof, and / or model systems). FH It has been established that this results in such an increase in the number of T cells (e.g., T FHSuch an increase in the number of T cells (e.g., T cells) may be demonstrated in one or more of a human population, a non-human primate model (e.g., rhesus monkey), and / or a mouse model. In some embodiments, the increase in the number of T cells (e.g., T FH Such an increase in the number of IL-16 cells (IL-16 cells) can be demonstrated in the draining lymph nodes, spleen, and / or blood of the mouse model (e.g., at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days) following immunization of such mouse model with a provided immunogenic composition.

[0343] In some embodiments, a protective response against SARS-CoV-2 and / or influenza virus induced by a provided immunogenic composition is established in an appropriate model system for SARS-CoV-2 and / or influenza. For example, in some embodiments, such a protective response can be demonstrated in an animal model, e.g., a non-human primate model (e.g., rhesus macaque) and / or a mouse model. In some embodiments, a non-human primate (e.g., rhesus macaque), or population thereof, that has been immunized at least once with a provided immunogenic composition is challenged with SARS-CoV-2 and / or influenza virus, e.g., via the intranasal and / or intratracheal route. In some embodiments, such challenge can occur several weeks (e.g., 5-10 weeks) after at least one immunization (including, e.g., at least two immunizations) with a provided immunogenic composition. In some embodiments, such a challenge may occur when a non-human primate(s) (e.g., rhesus macaque(s)) that has received at least one immunization (including, e.g., at least two immunizations) with a provided immunogenic composition produces a detectable level of SARS-CoV-2 neutralizing titer and / or influenza neutralizing titer (e.g., an antibody response against the SARS-CoV-2 spike protein, influenza virus HA protein, and / or fragments thereof, including, but not limited to, the stabilized prefusion spike trimer, S-2P, RBD, and / or HA protein). In some embodiments, a protective response is characterized by the absence or reduction of detectable viral RNA in bronchoalveolar lavage fluid (BAL) and / or nasal swabs of the challenged non-human primate(s) (e.g., rhesus macaque(s)).In some embodiments, the immunogenic compositions described herein can be characterized by a greater proportion of animals in a challenged population, e.g., non-human primates (e.g., rhesus macaques), that have received at least one immunization (including, e.g., at least two immunizations) with a provided immunogenic composition, exhibiting an absence or reduction in detectable RNA in BAL and / or nasal swabs compared to a population of unimmunized animals, e.g., non-human primates (e.g., rhesus macaques). In some embodiments, the immunogenic compositions described herein can be characterized by a greater proportion of animals in a challenged population, e.g., non-humans (e.g., rhesus macaques), that have received at least one immunization (including, e.g., at least two immunizations) with a provided immunogenic composition, exhibiting clearance of viral RNA in nasal swabs within 10 days, e.g., within 8 days, within 6 days, within 4 days, etc., compared to a population of unimmunized animals, e.g., non-human primates (e.g., rhesus macaques).

[0344] In some embodiments, the immunogenic compositions described herein, when administered to a subject in need thereof, do not substantially increase the risk of vaccine-associated respiratory disease enhancement. In some embodiments, such vaccine-associated respiratory disease enhancement may be associated with antibody-dependent replication enhancement and / or associated with vaccine antigens that induced antibodies with poor neutralizing activity and a Th2-biased response. In some embodiments, the immunogenic compositions described herein, when administered to a subject in need thereof, do not substantially increase the risk of antibody-dependent replication enhancement.

[0345] In some embodiments, a single dose of an mRNA composition (e.g., formulated in lipid nanoparticles) can induce a therapeutic antibody response within 10 days of vaccination. In some embodiments, such a therapeutic antibody response can be characterized when such an mRNA vaccine can induce the production of approximately 10-100 μg / mL of IgG measured 10 days after vaccination in an animal model at a dose of 0.1-10 μg or 0.2-5 μg. In some embodiments, such a therapeutic antibody response can be characterized when such an mRNA vaccine can induce the production of approximately 100-1000 μg / mL of IgG measured 20 days after vaccination in an animal model at a dose of 0.1-10 μg or 0.2-5 μg. In some embodiments, a single dose can induce a pseudovirus neutralization titer of 10-200 pVN50 titers measured 15 days after vaccination in an animal model. In some embodiments, a single dose can induce pseudoviral neutralization titers of pVN50 titers of 50-500 15 days after vaccination as measured in animal models.

[0346] In some embodiments, a single dose of an mRNA composition can increase an antigen-specific CD8 and / or CD4 T cell response by at least 50% or more (including, for example, by at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more) compared to that observed in the absence of such mRNA composition. In some embodiments, a single dose of an mRNA composition can increase an antigen-specific CD8 and / or CD4 T cell response by at least 1.5-fold or more (including, for example, by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more) compared to that observed in the absence of such mRNA composition.

[0347] In some embodiments, a regimen (e.g., a single dose of an mRNA composition) may expand T cells exhibiting a Th1 phenotype (e.g., characterized by expression of IFN-gamma, IL-2, IL-4, and / or IL-5) by at least 50% or more (including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more) compared to those observed in the absence of such regimen. In some embodiments, a regimen (e.g., a single dose of an mRNA composition) may expand T cells exhibiting a Th1 phenotype (e.g., characterized by expression of IFN-gamma, IL-2, IL-4, and / or IL-5) by, for example, at least 1.5-fold (including, e.g., at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more) compared to those observed in the absence of such regimen. In some embodiments, the T cell phenotype may be or may include a predominantly Th1 cytokine profile (e.g., characterized by INF-gamma positivity and / or IL-2 positivity, and / or absent or biologically insignificant IL-4 secretion).

[0348] In some embodiments, the regimens described herein (e.g., one or more doses of an mRNA composition) induce and / or result in the production of RBD-specific CD4+ T cells. In particular, the present disclosure reports that mRNA compositions encoding an RBD-containing portion of the SARS-CoV-2 spike protein (e.g., a full-length SARS-CoV-2 spike protein) and one or more HA proteins can be particularly useful and / or effective in inducing and / or generating RBD-specific CD4+ T cells. In some embodiments, RBD-specific CD4+ T cells induced by the mRNA compositions described herein (e.g., mRNA compositions encoding an RBD-containing portion of the SARS-CoV-2 spike protein and an HA protein) demonstrate a predominantly Th1 cytokine profile (e.g., characterized by INF-gamma positivity and / or IL-2 positivity, and / or absent or biologically insignificant IL-4 secretion).

[0349] In some embodiments, assessment of CD4+ and / or CD8+ T cell responses (e.g., as described herein) in a subject receiving an mRNA composition (e.g., as described herein) can be performed using an in vitro assay using PBMCs collected from the subject.

[0350] In some embodiments, the immunogenicity of the mRNA compositions described herein may be assessed by one or more of the following serological immunogenicity assays: detection of IgG, IgM, and / or IgA against the SARS-CoV-2 S protein and / or HA protein present in a blood sample of a subject receiving a provided mRNA composition, and / or a neutralization assay using a SARS-CoV-2 pseudovirus, an influenza pseudovirus, and / or a wild-type SARS-CoV-2 virus or a wild-type influenza virus.

[0351] In some embodiments, an mRNA composition (e.g., as described herein) results in relatively mild side effects (e.g., Grade 1 to Grade 2 pain, redness, and / or swelling) within 7 days after vaccination at a dose of 10 μg to 100 μg or 1 μg to 50 μg. In some embodiments, an mRNA composition (e.g., as described herein) results in relatively mild systemic event symptoms (e.g., Grade 1 to Grade 2 fever, fatigue, headache, chills, vomiting, diarrhea, muscle pain, joint pain, medication use, and combinations thereof) within 7 days after vaccination at a dose of 10 μg to 100 μg.

[0352] In some embodiments, the mRNA composition is characterized in that, when administered to a subject at a dose of 10-100 μg or 1 μg-50 μg, IgG directed against SARS-CoV-2 immunogenic proteins, influenza virus immunogenic proteins, and / or fragments thereof (e.g., spike protein receptor binding domain, and / or HA protein) can be produced at levels of 100-100,000 U / mL or 500-50,000 U / mL 21 days after vaccination.

[0353] In some embodiments, the mRNA encodes a naturally folded trimeric receptor binding protein of SARS-CoV-2. In some embodiments, the mRNA encodes a variant of such receptor binding protein such that the encoded variant binds to ACE2 with a Kd of 10 pM or less, e.g., 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM or less. In some embodiments, the mRNA encodes a variant of such receptor binding protein such that the encoded variant binds to ACE2 with a Kd of 5 pM. In some embodiments, the mRNA encodes a trimeric receptor binding portion of SARS-CoV-2 that comprises the ACE2 receptor binding site. In some embodiments, the mRNA includes a coding sequence for a SARS-CoV-2 receptor-binding portion and a trimerization domain (e.g., the native trimerization domain (foldon) of T4 fibritin) such that the coding sequence directs expression of a trimeric protein that has an ACE2 receptor-binding site and binds to ACE2. In some embodiments, the mRNA encodes a SARS-CoV-2 trimeric receptor-binding portion or a variant thereof such that the Kd is reduced compared to the monomeric form of the SARS-CoV-2 receptor-binding domain (RBD). For example, in some embodiments, the mRNA encodes a SARS-CoV-2 trimeric receptor-binding portion or a variant thereof such that the Kd is reduced by at least 10-fold (including, e.g., at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, etc.) compared to the SARS-CoV-2 RBD.

[0354] In some embodiments, the trimeric receptor binding portion of SARS-CoV-2 encoded by the mRNA (e.g., as described herein) binds to ACE2 and B 0When complexed with the AT1 neutral amino acid transporter in a closed conformation, they can be determined to have a particle size of approximately 3-4 angstroms as assessed by electron cryomicroscopy (cryoEM). In some embodiments, the geometric mean SARS-CoV-2 neutralizing titers characterized and / or produced by the mRNA compositions or methods described herein can be at least 1.5-fold, e.g., at least 2-fold, at least 2.5-fold, at least 3-fold, or more, greater than that of a COVID-19 convalescent human panel (e.g., a panel of sera from humans recovering from COVID-19 obtained 20-40 days after the onset of symptoms and at least 14 days after the start of the asymptomatic convalescent phase).

[0355] In some embodiments, the mRNA compositions provided herein may be characterized in that subjects treated with such compositions (e.g., at least one dose, at least two doses, etc.) may exhibit reduced and / or shorter duration of viral RNA presence in the relevant site(s) (e.g., nose and / or lungs, and / or other tissues susceptible to infection) compared to appropriate controls (e.g., expected levels established for comparable subjects or populations not receiving such treatment and exposed to the virus under reasonably comparable exposure conditions).

[0356] In some embodiments, the RBD antigen expressed by an mRNA construct (e.g., as described herein) may be modified by the addition of the "foldon" trimerization domain from T4-fibritin, e.g., to increase its immunogenicity.

[0357] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that certain local reactions (e.g., pain, redness, and / or swelling, etc.) and / or systemic events (e.g., fever, fatigue, headache, etc.) may appear and / or peak on day 2 after vaccination. In some embodiments, the mRNA compositions described herein are characterized in that certain local reactions (e.g., pain, redness, and / or swelling, etc.) and / or systemic events (e.g., fever, fatigue, headache, etc.) may resolve by day 7 after vaccination.

[0358] In some embodiments, the mRNA compositions and / or methods described herein are characterized by the absence of Grade 1 or higher changes or test abnormalities in routine clinical laboratory tests in subjects receiving an mRNA composition (e.g., as described herein). Examples of such clinical laboratory assays may include lymphocyte counts, hematological changes, etc.

[0359] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that by 21 days after the first dose (e.g., 10-100 μg, or 1 μg-50 μg, inclusive), the geometric mean concentration (GMC) of IgG directed against a SARS-CoV-2 S polypeptide, influenza virus HA protein, or immunogenic fragment thereof (e.g., RBD) can reach 200-3000 Units / mL, or 500-3000 Units / mL, or 500-2000 Units / mL, compared to 602 Units / mL in a panel of COVID-19 convalescent human sera. In some embodiments, the mRNA compositions described herein are characterized by at least an 8-fold or greater increase in geometric mean concentration (GMC) of IgG against a SARS-CoV-2 spike polypeptide, HA polypeptide, or immunogenic fragment thereof (e.g., RBD) by 7 days after the second dose (e.g., 10-30 μg, or 1 μg-50 μg, inclusive). In some embodiments, the mRNA compositions described herein are characterized in that by 7 days after the second dose (e.g., 10-30 μg, or 1 μg-50 μg, inclusive), the geometric mean concentration (GMC) of IgG directed against a SARS-CoV-2 S polypeptide, an influenza HA polypeptide, or an immunogenic fragment thereof (e.g., RBD) can increase from 1500 Units / mL to 40,000 Units / mL, or from 4000 Units / mL to 40,000 Units / mL. In some embodiments, the antibody concentrations described herein can persist for at least 20 days or more, e.g., at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, or at least 50 days, after the first dose, or for at least 10 days or more, e.g., at least 15 days, at least 20 days, at least 25 days, or longer, after the second dose. In some embodiments, antibody concentrations may persist for 35 days after the first dose or at least 14 days after the second dose.

[0360] In some embodiments, the mRNA compositions described herein are characterized by a GMC of IgG directed against a SARS-CoV-2 S polypeptide, an influenza virus HA polypeptide, or an immunogenic fragment thereof (e.g., RBD) that is at least 30% higher (e.g., at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 95% higher) compared to antibody concentrations observed in a panel of COVID-19 convalescent human sera or influenza convalescent human sera, when measured 7 days after the second dose (e.g., 1-50 μg, inclusive). In many embodiments, the geometric mean concentration (GMC) of IgG described herein is the GMC of RBD-binding IgG.

[0361] In some embodiments, the mRNA compositions described herein are characterized by a GMC of IgG directed against a SARS-CoV-2 S polypeptide, an influenza virus HA polypeptide, or an immunogenic fragment thereof (e.g., RBD) that is at least 1.1-fold higher (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 15-fold higher, at least 20-fold higher, at least 25-fold higher, at least 30-fold higher) when measured 7 days after the second dose (e.g., 10-50 μg, inclusive). In many embodiments, the geometric mean concentration (GMC) of IgG described herein is the GMC of RBD-binding IgG.

[0362] In some embodiments, the mRNA compositions described herein are characterized by a GMC of IgG directed against a SARS-CoV-2 S polypeptide, an influenza virus HA polypeptide, or an immunogenic fragment thereof (e.g., RBD) that is at least 5-fold higher (e.g., at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 15-fold higher, at least 20-fold higher, at least 25-fold higher, at least 30-fold higher) compared to antibody concentrations observed in a panel of COVID-19 or influenza convalescent human sera, when measured 21 days after the second dose. In many embodiments, the geometric mean concentration (GMC) of IgG described herein is the GMC of RBD-binding IgG.

[0363] In some embodiments, the mRNA compositions and / or methods described herein are characterized by an increase (e.g., at least 30%, at least 40%, at least 50%, or more) in SARS-CoV-2 and / or influenza neutralization geometric mean titer (GMT) 21 days after the first dose. In some embodiments, the mRNA compositions described herein are characterized by a serum neutralization GMT of 150-300, which is substantially higher 7 days after a subject receives the second dose (e.g., 10 μg-30 μg, inclusive), compared to a serum neutralization GMT of 94 for a COVID-19 convalescent serum panel.

[0364] In some embodiments, the mRNA compositions and / or methods described herein are characterized by a protective efficacy of at least 60%, e.g., at least 70%, at least 80%, at least 90%, or at least 95%, 7 days after administration of the second dose. In one embodiment, the mRNA compositions and / or methods described herein are characterized by a protective efficacy of at least 70% 7 days after administration of the second dose. In one embodiment, the mRNA compositions and / or methods described herein are characterized by a protective efficacy of at least 80% 7 days after administration of the second dose. In one embodiment, the mRNA compositions and / or methods described herein are characterized by a protective efficacy of at least 90% 7 days after administration of the second dose. In one embodiment, the mRNA compositions and / or methods described herein are characterized by a protective efficacy of at least 95% 7 days after administration of the second dose.

[0365] In some embodiments, the RNA compositions provided herein are characterized by inducing an immune response against SARS-CoV-2 and / or influenza virus at least 7 days after dosing (e.g., after the second dose). In some embodiments, the RNA compositions provided herein are characterized by inducing an immune response against SARS-CoV-2 and / or influenza virus earlier than 14 days after dosing (e.g., after the second dose). In some embodiments, the RNA compositions provided herein are characterized by inducing an immune response against SARS-CoV-2 and / or influenza virus at least 7 days after a vaccination regimen. In some embodiments, the vaccination regimen includes a first dose and a second dose. In some embodiments, the first and second doses are administered at least 21 days apart. In some such embodiments, the immune response against SARS-CoV-2 and / or influenza virus is induced at least 28 days after the first dose.

[0366] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that the geometric mean concentration (GMC) of antibodies directed against a SARS-CoV-2 spike polypeptide, influenza virus HA polypeptide, or immunogenic fragment thereof (e.g., RBD) measured in serum from subjects receiving an mRNA composition of the disclosure (e.g., at a dose of 10-30 μg, inclusive) is substantially higher than that measured in a convalescent serum panel (e.g., as described herein). In some embodiments in which a subject may receive a second dose (e.g., 21 days after the first dose), the geometric mean concentration (GMC) of antibodies directed against a SARS-CoV-2 spike polypeptide, influenza virus HA polypeptide, or immunogenic fragment thereof (e.g., RBD) measured in serum from the subject may be 8.0- to 50-fold higher than the GMC of a convalescent serum panel. In some embodiments in which a subject may receive a second dose (e.g., 21 days after the first dose), the geometric mean concentration (GMC) of antibodies directed against a SARS-CoV-2 spike polypeptide, an influenza virus HA polypeptide, or an immunogenic fragment thereof (e.g., RBD) measured in serum from the subject may be at least 8.0-fold or more higher than the convalescent serum panel GMC, e.g., at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold or more.

[0367] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that the SARS-CoV-2 neutralization geometric mean titer and / or influenza virus neutralization geometric mean titer measured 28 days after the first dose or 7 days after the second dose can be at least 1.5-fold or greater (including, for example, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, or more) compared to the neutralization GMT of a convalescent serum panel.

[0368] In some embodiments, the regimen administered to a subject may be or may include a single dose. In some embodiments, the regimen administered to a subject may include multiple doses (e.g., at least two doses, at least three doses, or more doses). In some embodiments, the regimen administered to a subject may include a first dose and a second dose, where the doses are separated by at least two weeks, at least three weeks, at least four weeks, or longer. In some embodiments, the doses may be separated by at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, or longer. In some embodiments, the doses may be administered several days apart, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more days apart. In some embodiments, doses may be administered about 1 to about 3 weeks apart, or about 1 to about 4 weeks apart, or about 1 to about 5 weeks apart, or about 1 to about 6 weeks apart, or about 1 week to more than 6 weeks apart. In some embodiments, doses may be separated by a period of about 7 to about 60 days, such as about 14 to about 48 days. In some embodiments, the minimum number of days between doses may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more days.In some embodiments, the maximum number of days between doses can be about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 days or less. In some embodiments, doses can be separated by about 21 to about 28 days. In some embodiments, doses can be separated by about 19 to about 42 days. In some embodiments, doses can be separated by about 7 to about 28 days. In some embodiments, doses can be separated by about 14 to about 24 days. In some embodiments, doses can be separated by about 21 to about 42 days.

[0369] In some embodiments, particularly in the case of compositions established to provide elevated antibody and / or T cell titers over a period of greater than about three weeks, for example, in some embodiments, provided compositions are established to provide elevated antibody and / or T cell titers (e.g., specific for the SARS-CoV-2 spike protein or relevant portions of the influenza virus HA protein) over a period of greater than about three weeks, and in some such embodiments, the dosing regimen may include only a single dose, or may include two or more doses, which, in some embodiments, may be separated from each other by a period of greater than about 21 days or three weeks. For example, in some such embodiments, such a period may be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or in some embodiments, about 1 year or more.

[0370] In some embodiments, the first and second doses (and / or other subsequent doses) may be administered by intramuscular injection. In some embodiments, the first and second doses may be administered in the deltoid muscle. In some embodiments, the first and second doses may be administered in the same arm. In some embodiments, the mRNA compositions described herein are administered (e.g., by intramuscular injection) as a series of two doses (e.g., 0.3 mL each) 21 days apart. In some embodiments, each dose is about 30 μg. In some embodiments, each dose may be greater than 30 μg, e.g., about 40 μg, about 50 μg, about 60 μg. In some embodiments, each dose may be less than 30 μg, e.g., about 20 μg, about 10 μg, about 5 μg, etc. In some embodiments, each dose is about 3 μg or less, e.g., about 1 μg. In some such embodiments, the mRNA compositions described herein are administered to subjects 16 years of age or older (including, for example, between the ages of 16 and 85). In some such embodiments, the mRNA compositions described herein are administered to subjects between the ages of 18 and 55. In some such embodiments, the mRNA compositions described herein are administered to subjects between the ages of 56 and 85. In some embodiments, the mRNA compositions described herein are administered as a single dose (e.g., by intramuscular injection).

[0371] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that the RBD-specific IgG (e.g., polyclonal response) induced by such mRNA compositions and / or methods exhibits a higher binding affinity for the RBD compared to a reference human monoclonal antibody with SARS-CoV-2 RBD-binding affinity (e.g., CR3022, described in J. ter Meulen et al., PLOS Med. 3, e237 (2006)). In some embodiments, the mRNA compositions and / or methods described herein are characterized in that the HA-specific IgG (e.g., polyclonal response) induced by such mRNA compositions and / or methods exhibits a higher binding affinity for HA compared to a reference human monoclonal antibody with HA-binding affinity.

[0372] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity across a panel of SARS-CoV-2 spike variants and / or influenza virus HA variants (e.g., at least 10, at least 15, or more). In some embodiments, such SARS-CoV-2 spike variants include mutations in the RBD (e.g., but not limited to, Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P, etc., compared to SEQ ID NO: 1) and / or mutations in the spike protein (e.g., but not limited to, D614G, etc., compared to SEQ ID NO: 1). Those skilled in the art will be aware of the various spike variants and / or resources reporting them (e.g., the table of spike mutation sites maintained by the COVID-19 Viral Genome Analysis Pipeline and found at https: / / cov.lanl.gov / components / sequence / COV / int_sites_tbls.comp) (last accessed August 24, 2020), and will understand from reading this specification that the mRNA compositions and / or methods described herein may be characterized by their ability to induce sera that exhibit neutralizing activity against any or all of such variants and / or combinations thereof in vaccinated subjects.

[0373] In particular embodiments, the mRNA composition encoding the RBD of the SARS-CoV-2 spike protein is characterized in that sera from vaccinated subjects exhibit neutralizing activity across a panel (e.g., at least 10, at least 15, or more) of SARs-CoV-2 spike variants, including RBD variants (e.g., but not limited to, Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P, etc. compared to SEQ ID NO: 1) and spike protein variants (e.g., but not limited to, D614G, etc. compared to SEQ ID NO: 1).

[0374] In particular embodiments, mRNA compositions encoding SARS-CoV-2 spike protein variants comprising two consecutive proline substitutions at amino acid positions 986 and 987 at the apex of the central helix in the S2 subunit are characterized in that sera from vaccinated subjects exhibit neutralizing activity across a panel (e.g., at least 10, at least 15, or more) of SARs-CoV-2 spike variants, including RBD variants (e.g., but not limited to, Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P, etc., compared to SEQ ID NO: 1) and spike protein variants (e.g., but not limited to, D614G, etc., compared to SEQ ID NO: 1). For example, in some embodiments, an mRNA composition encoding SEQ ID NO:7 (SP2) elicits an immune response against any one of SARs-CoV-2 spike variants, including RBD variants (e.g., but not limited to, Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P, etc., compared to SEQ ID NO:1) and spike protein variants (e.g., but not limited to, D614G, compared to SEQ ID NO:1).

[0375] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising a mutation at position 501 of the spike protein compared to SEQ ID NO: 1. In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising a N501Y mutation in the spike protein compared to SEQ ID NO: 1.

[0376] The one or more SARs-CoV-2 spike variants comprising a mutation at position 501 of the spike protein compared to SEQ ID NO:1, or the one or more SARs-CoV-2 spike variants comprising a N501Y mutation in the spike protein compared to SEQ ID NO:1, may comprise one or more additional mutations compared to SEQ ID NO:1 (such as, but not limited to, an H69 / V70 deletion, a Y144 deletion, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, an L242 / A243 / L244 deletion, etc. compared to SEQ ID NO:1).

[0377] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARS-CoV-2 spike variant "Variant of Concern 202012 / 01" (VOC-202012 / 01, also known as lineage B.1.1.7). This variant was previously designated by Public Health England as Variant Under Investigation (VUI-202012 / 01) first in December 2020 but has since been reclassified as Variant of Concern (VOC-202012 / 01). VOC-202012 / 01 is a SARS-CoV-2 variant first detected in October 2020 during the COVID-19 pandemic in the UK in samples collected the previous month, and by mid-December it began to spread rapidly. This correlates with a significant increase in COVID-19 infection rates in the UK, which is thought to be at least in part due to the N501Y mutation within the receptor-binding domain of the spike glycoprotein, which is required for binding to ACE2 on human cells. The VOC-202012 / 0 variant is characterized by 23 mutations, including 13 nonsynonymous mutations, 4 deletions, and 6 synonymous mutations (i.e., 17 mutations that alter the protein and 6 that do not). Spike protein changes in VOC 202012 / 01 include deletions 69-70, 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H. One of the most significant changes in VOC-202012 / 01 appears to be N501Y, an asparagine (N) to tyrosine (Y) change at amino acid position 501. This mutation, alone or in combination with a deletion at positions 69 / 70 within the N-terminal domain (NTD), may enhance viral transmissibility.

[0378] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against SARs-CoV-2 spike variants comprising the following mutations compared to SEQ ID NO: 1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0379] In detailed embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "501.V2." This variant was first observed in samples in October 2020, and since then, over 300 cases of the 501.V2 variant have been confirmed by whole genome sequencing (WGS) in South Africa, becoming the dominant form of the virus by December 2020. Preliminary results indicate that this variant may have increased transmissibility. The 501.V2 variant is characterized by multiple spike protein changes, including D80A, D215G, E484K, N501Y, and A701V, with more recently isolated viruses harboring additional changes: L18F, R246I, K417N, and deletion 242-244.

[0380] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO: 1: D80A, D215G, E484K, N501Y, and A701V, and optionally L18F, R246I, K417N, and deletions 242-244, compared to SEQ ID NO: 1. The SARs-CoV-2 spike variant may also comprise a D614G mutation compared to SEQ ID NO: 1.

[0381] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising an H69 / V70 deletion in the spike protein compared to SEQ ID NO:1.

[0382] In some embodiments, one or more SARs-CoV-2 spike variants comprising an H69 / V70 deletion in the spike protein compared to SEQ ID NO:1 may comprise one or more additional mutations compared to SEQ ID NO:1 (e.g., but not limited to, a Y144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, an L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, etc. compared to SEQ ID NO:1).

[0383] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "Variant of Concern 202012 / 01" (VOC-202012 / 01, also known as lineage B.1.1.7).

[0384] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against SARs-CoV-2 spike variants comprising the following mutations compared to SEQ ID NO: 1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0385] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "Cluster 5," also referred to as the ΔFVI spike by the Danish Serum Institute (SSI). It was discovered in the North Jutland region of Denmark and is believed to have spread from mink to humans via mink farms. Several different mutations in the viral spike protein have been identified in Cluster 5. Specific mutations include 69-70deltaHV (deletion of histidine and valine residues at positions 69 and 70 of the protein), Y453F (a tyrosine to phenylalanine change at position 453), I692V (an isoleucine to valine at position 692), M1229I (a methionine to isoleucine at position 1229), and, optionally, S1147L (a serine to leucine change at position 1147).

[0386] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO: 1: deletion 69-70, Y453F, I692V, M1229I, and optionally S1147L.

[0387] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants that comprise a mutation at position 614 of the spike protein compared to SEQ ID NO: 1. In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants that comprise a D614G mutation in the spike protein compared to SEQ ID NO: 1.

[0388] In some embodiments, the one or more SARs-CoV-2 spike variants comprising a mutation at position 614 of the spike protein compared to SEQ ID NO:1, or the one or more SARs-CoV-2 spike variants comprising a D614G mutation in the spike protein compared to SEQ ID NO:1, may comprise one or more additional mutations compared to SEQ ID NO:1 (such as, but not limited to, an H69 / V70 deletion, a Y144 deletion, N501Y, A570D, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, an L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, etc., compared to SEQ ID NO:1).

[0389] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "Variant of Concern 202012 / 01" (VOC-202012 / 01, also known as lineage B.1.1.7).

[0390] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against SARs-CoV-2 spike variants comprising the following mutations compared to SEQ ID NO: 1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0391] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO:1: D80A, D215G, E484K, N501Y, A701V, and D614G, and optionally, L18F, R246I, K417N, and deletions 242-244 compared to SEQ ID NO:1.

[0392] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions 501 and 614 of the spike protein compared to SEQ ID NO: 1. In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising N501Y and D614G mutations in the spike protein compared to SEQ ID NO: 1.

[0393] In some embodiments, one or more SARs-CoV-2 spike variants comprising mutations at positions 501 and 614 of the spike protein compared to SEQ ID NO:1, or the one or more SARs-CoV-2 spike variants comprising the N501Y and D614G mutations in the spike protein compared to SEQ ID NO:1, may comprise one or more additional mutations compared to SEQ ID NO:1 (such as, but not limited to, an H69 / V70 deletion, a Y144 deletion, A570D, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, an L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, etc., compared to SEQ ID NO:1).

[0394] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "Variant of Concern 202012 / 01" (VOC-202012 / 01, also known as lineage B.1.1.7).

[0395] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against SARs-CoV-2 spike variants comprising the following mutations compared to SEQ ID NO: 1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0396] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO:1: D80A, D215G, E484K, N501Y, A701V, and D614G, and optionally, L18F, R246I, K417N, and deletions 242-244 compared to SEQ ID NO:1.

[0397] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants that comprise a mutation at position 484 of the spike protein compared to SEQ ID NO: 1. In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants that comprise an E484K mutation in the spike protein compared to SEQ ID NO: 1.

[0398] In some embodiments, the one or more SARs-CoV-2 spike variants comprising a mutation at position 484 of the spike protein compared to SEQ ID NO:1, or the one or more SARs-CoV-2 spike variants comprising an E484K mutation in the spike protein compared to SEQ ID NO:1, further comprise one or more additional mutations compared to SEQ ID NO:1 (such as, but not limited to, an H69 / V70 deletion compared to SEQ ID NO:1). deletion, Y144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, A701V, L18F, R246I, K417N, L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, K417T, H655Y, T1027I, V1176F, etc.

[0399] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "501.V2."

[0400] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO: 1: D80A, D215G, E484K, N501Y, and A701V, and optionally L18F, R246I, K417N, and deletions 242-244, compared to SEQ ID NO: 1. The SARs-CoV-2 spike variant may also comprise a D614G mutation compared to SEQ ID NO: 1.

[0401] Lineage B.1.1.248, also known as the Brazilian (Brazilian) variant, is one of a group of SARS-CoV-2 variants designated the P.1 lineage. It contains 17 unique amino acid changes, 10 of which are in the spike protein, including N501Y and E484K. B.1.1.248 descended from B.1.1.28. E484K is present in both B.1.1.28 and B.1.1.248. B.1.1.248 has multiple S protein polymorphisms [L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, V1176F] and is similar to variants said to have originated in South Africa at certain critical RBD positions (K417, E484, N501).

[0402] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "B.1.1.28."

[0403] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "B.1.1.248."

[0404] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

[0405] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions 501 and 484 of the spike protein compared to SEQ ID NO: 1. In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising N501Y and E484K mutations in the spike protein compared to SEQ ID NO: 1.

[0406] In some embodiments, one or more SARs-CoV-2 spike variants comprising mutations at positions 501 and 484 of the spike protein compared to SEQ ID NO:1, or the one or more SARs-CoV-2 spike variants comprising a N501Y mutation and an E484K mutation in the spike protein compared to SEQ ID NO:1, may further comprise one or more additional mutations compared to SEQ ID NO:1 (such as, but not limited to, one or more additional mutations at positions 501 and 484 of the spike protein compared to SEQ ID NO:1). and the like).

[0407] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "501.V2."

[0408] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO: 1: D80A, D215G, E484K, N501Y, and A701V, and optionally L18F, R246I, K417N, and deletions 242-244, compared to SEQ ID NO: 1. The SARs-CoV-2 spike variant may also comprise a D614G mutation compared to SEQ ID NO: 1.

[0409] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "B.1.1.248."

[0410] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

[0411] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions 501, 484, and 614 of the spike protein compared to SEQ ID NO: 1. In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions N501Y, E484K, and D614G in the spike protein compared to SEQ ID NO: 1.

[0412] In some embodiments, one or more SARs-CoV-2 spike variants comprising mutations at positions 501, 484, and 614 of the spike protein compared to SEQ ID NO:1, or the one or more SARs-CoV-2 spike variants comprising N501Y, E484K, and D614G mutations in the spike protein compared to SEQ ID NO:1, may further comprise one or more additional mutations compared to SEQ ID NO:1, such as, but not limited to, Compared to SEQ ID NO: 1, it may include the following: H69 / V70 deletion, Y144 deletion, A570D, P681H, T716I, S982A, D1118H, D80A, D215G, A701V, L18F, R246I, K417N, L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, K417T, H655Y, T1027I, V1176F, etc.

[0413] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO:1: D80A, D215G, E484K, N501Y, A701V, and D614G, and optionally, L18F, R246I, K417N, and deletions 242-244 compared to SEQ ID NO:1.

[0414] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising an L242 / A243 / L244 deletion in the spike protein compared to SEQ ID NO:1.

[0415] In some embodiments, one or more SARs-CoV-2 spike variants comprising an L242 / A243 / L244 deletion in the spike protein compared to SEQ ID NO:1 may comprise one or more additional mutations compared to SEQ ID NO:1 (for example, but not limited to, an H69 / V70 deletion, a Y144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, K417T, H655Y, T1027I, V1176F, etc. compared to SEQ ID NO:1).

[0416] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "501.V2."

[0417] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations: D80A, D215G, E484K, N501Y, A701V, and deletions 242-244 relative to SEQ ID NO: 1, and optionally L18F, R246I, and K417N relative to SEQ ID NO: 1. The SARs-CoV-2 spike variant may also comprise a D614G mutation relative to SEQ ID NO: 1.

[0418] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants that comprise a mutation at position 417 of the spike protein compared to SEQ ID NO: 1. In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants that comprise a K417N or K417T mutation in the spike protein compared to SEQ ID NO: 1.

[0419] In some embodiments, one or more SARs-CoV-2 spike variants comprising a mutation at position 417 of the spike protein compared to SEQ ID NO:1, or said one or more SARs-CoV-2 spike variants comprising a K417N or K417T mutation in the spike protein compared to SEQ ID NO:1, further comprise one or more additional mutations compared to SEQ ID NO:1 (for example, but not limited to, H69 / V70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, H655Y, T1027I, V1176F, etc.

[0420] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "501.V2."

[0421] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations relative to SEQ ID NO: 1: D80A, D215G, E484K, N501Y, A701V, and K417N, and optionally L18F, R246I, and deletions 242-244, relative to SEQ ID NO: 1. The SARs-CoV-2 spike variant may also comprise a D614G mutation relative to SEQ ID NO: 1.

[0422] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "B.1.1.248."

[0423] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

[0424] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions 417 and 484 and / or 501 of the spike protein compared to SEQ ID NO: 1. In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising K417N or K417T mutations and E484K and / or N501Y mutations in the spike protein compared to SEQ ID NO: 1.

[0425] In some embodiments, one or more SARs-CoV-2 spike variants comprising mutations at positions 417 and 484 and / or 501 of the spike protein compared to SEQ ID NO:1, or the one or more SARs-CoV-2 spike variants comprising a K417N or K417T mutation and an E484K and / or N501Y mutation in the spike protein compared to SEQ ID NO:1, further comprise one or more further mutations (e.g., For example, but not limited to, compared to SEQ ID NO: 1, the amino acid sequence may include an H69 / V70 deletion, a Y144 deletion, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, A701V, L18F, R246I, an L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, H655Y, T1027I, V1176F, etc.

[0426] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "501.V2."

[0427] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations relative to SEQ ID NO: 1: D80A, D215G, E484K, N501Y, A701V, and K417N, and optionally L18F, R246I, and deletions 242-244, relative to SEQ ID NO: 1. The SARs-CoV-2 spike variant may also comprise a D614G mutation relative to SEQ ID NO: 1.

[0428] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARs-CoV-2 spike variant "B.1.1.248."

[0429] In particular embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

[0430] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against the SARS-CoV-2 spike variant of the Omicron (B.1.1.529) variant. The Omicron (B.1.1.529) variant is a variant of SARS-CoV-2 detected in South Africa. Multiple Omicron sublineages have emerged, including the BA.1, BA.2, BA.2.12.1, BA.3, BA.4, BA.5, and BA.2.75 sublineages. As used herein, unless otherwise specified, "Omicron variant" refers to a SARS-CoV-2 variant having one or more mutations characteristic of BA.1, or any subsequently arising variant thereof (including, but not limited to, for example, BA.2, BA.2.12.1, BA.2.12.1, BA.4 or BA.5, BA.2.75, BA.2.75.2, BJ.1, BA.4.6 or BF.7, XBB, XBB.1, XBB.2, XBB.1.3, BA.2.3.20, BQ.1.1, as described herein). In some embodiments, the spike protein changes in the Omicron (B.1.1.529) BA.1 variant are A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE (insertion of EPE after amino acid 214), G339D, S371L, S373P , S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F.In some embodiments, spike protein changes in Omicron (B.1.1.529) variants include A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE (insertion of EPE after amino acid 214), G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F. In some embodiments, spike changes in the Omicron BA.2 variant include T19I, A24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K. In some embodiments, BA.4 and BA.5 have the same spike protein amino acid sequence, in which case "BA.4 / 5" is used to refer to either Omicron variant. In some embodiments, spike alterations in Omicron BA.4 / 5 include T19I, Δ24-26, A27S, Δ69 / 70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.In some embodiments, spike alterations in Omicron BA.2.75 include T19I, Δ24-26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, N354D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.

[0431] In some embodiments, the mRNA compositions and / or methods described herein detect a SARs-CoV-2 spike variant in sera of vaccinated subjects that comprises at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, or at least 37 of the following mutations compared to SEQ ID NO:1: Characterized by exhibiting neutralizing activity: T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S371L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H, T95I, A67V, L981F, S477N, G496 S, T478K, Q498R, Q493R, E484A, N501Y, S375F, Y505H, V143del, H69del, V70del, N211 del, L212I, ins214EPE, G142D, Y144del, Y145del, L141del, Y144F, Y145D, G142del.

[0432] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant that includes at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, or all of the following mutations compared to SEQ ID NO:1: T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S371L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H, T95I, A67V, L981F, S477N, G496S, T478K, Q498R, Q493R, E484A. The SARs-CoV-2 spike variant may include at least one, at least two, at least three, at least four, at least five, or all of the following mutations relative to SEQ ID NO: 1: N501Y, S375F, Y505H, V143del, H69del, V70del, and / or may include at least one, at least two, at least three, at least four, at least five, or all of the following mutations relative to SEQ ID NO: 1: N211del, L212I, ins214EPE, G142D, Y144del, Y145del. In some embodiments, the SARs-CoV-2 spike variant may include at least one, at least two, at least three, or all of the following mutations relative to SEQ ID NO: 1: L141del, Y144F, Y145D, G142del.

[0433] In some embodiments, the mRNA compositions and / or methods described herein provide for a method for detecting a SARs-CoV-2 spike polynucleotide in the serum of a vaccinated subject that contains at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33 of the following mutations compared to SEQ ID NO:1: and the following variants: A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F.

[0434] In some embodiments, the mRNA compositions and / or methods described herein detect a SARS-CoV-2 variant in which serum from a vaccinated subject detects at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or at least 31 of the following mutations compared to SEQ ID NO:1: - Characterized by neutralizing activity against the following CoV-2 spike variants: T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K.

[0435] In some embodiments, the mRNA compositions and / or methods described herein are adapted to detect a vaccinated subject's serum containing at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, or at least 34 of the following mutations compared to SEQ ID NO:1: It is characterized by exhibiting neutralizing activity against SARS-CoV-2 spike variants, including: T19I, Δ24-26, A27S, Δ69 / 70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.

[0436] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against SARs-CoV-2 spike variants that include the following mutations compared to SEQ ID NO:1: A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214E. PE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R , N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F.

[0437] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralization against SARS-CoV-2 spike variants containing the following mutations: T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K. In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralization against SARS-CoV2 spike variants comprising the following mutations compared to SEQ ID NO:1: T19I, Δ24-26, A27S, Δ69 / 70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.

[0438] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against SARs-CoV-2 spike variants comprising the following mutations compared to SEQ ID NO:1: A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F.

[0439] In some embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO:1: T19I, Δ24-26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, N354D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y, Y505H D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. The SARs-CoV-2 spike variants described herein may or may not include the D614G mutation compared to SEQ ID NO:1.

[0440] In some embodiments, the SARS-CoV-2 spike variants described herein comprise a mutation in the furin cleavage site (e.g., in some embodiments, residues 682-685 of SEQ ID NO: 1). In some embodiments, the SARS-CoV-2 spike variants comprise a mutation in the furin cleavage site that prevents cleavage by a furin protease (e.g., human furin protease). In some embodiments, the SARS-CoV-2 variants described herein comprise a furin mutation (e.g., a GSAS mutation) disclosed in WO2021163365 or WO2021243122, the contents of both of which are incorporated herein by reference in their entireties.

[0441] In some embodiments, the mRNA compositions and / or methods described herein may provide protection against SARS-CoV-2 and / or influenza virus (e.g., influenza A and / or B virus) and / or reduce the severity of SARS-CoV-2 infection and / or influenza virus infection (e.g., influenza A and / or B virus infection) in at least 50% of subjects receiving such mRNA compositions and / or methods. In some embodiments, the compositions disclosed herein may be used for active immunization to prevent both SARS-CoV-2 infection and influenza subtype A and subtype B infection in individuals (e.g., pediatric patients, pregnant patients, and patients 18 years of age and older).

[0442] In some embodiments, a population treated with an mRNA composition described herein includes subjects aged 18 or older. In some embodiments, a population treated with an mRNA composition described herein includes subjects aged 18-55. In some embodiments, a population treated with an mRNA composition described herein includes subjects aged 56-85. In some embodiments, a population treated with an mRNA composition described herein includes older subjects (e.g., greater than 60, 65, 70, 75, 80, 85, etc., e.g., subjects aged 65-85). In some embodiments, a population treated with an mRNA composition described herein includes subjects aged 18-85. In some embodiments, a population treated with an mRNA composition described herein includes subjects aged 18 or younger. In some embodiments, a population treated with an mRNA composition described herein includes subjects aged 12 or younger. In some embodiments, a population treated with an mRNA composition described herein includes subjects aged 10 or younger. In some embodiments, a population treated with an mRNA composition described herein may include an adolescent population (e.g., individuals between the ages of approximately 12 and approximately 17). In some embodiments, a population treated with an mRNA composition described herein may include a pediatric population (e.g., as described herein). In some embodiments, a population treated with an mRNA composition described herein includes infants (e.g., under 1 year of age). In some embodiments, a population treated with an mRNA composition described herein does not include infants (e.g., under 1 year of age) whose mothers received such mRNA compositions described herein during pregnancy. Without wishing to be bound by any particular theory, rat studies suggest that SARS-CoV-2 neutralizing antibody responses induced in female rats fed such mRNA compositions during pregnancy may be passed on to the fetus. In some embodiments, a population treated with an mRNA composition described herein includes infants (e.g., under 1 year of age) whose mothers did not receive such mRNA compositions described herein during pregnancy.In some embodiments, populations treated with the mRNA compositions described herein include pregnant women; in some embodiments, infants whose mothers were vaccinated during pregnancy (e.g., those who received at least one dose, or only those who received both doses) are not vaccinated for the first weeks, months, or years of life (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, or 1, 2, 3, 4, 5 years or more). Alternatively, or in addition, in some embodiments, infants whose mothers were vaccinated during pregnancy (e.g., those who received at least one dose, or only those who received both doses) may receive or require reduced vaccination (e.g., fewer doses, and / or fewer administrations—e.g., boosters—and / or lower total exposure over a given period of time) after birth, e.g., during the first weeks, months, or even years of life (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, or 1, 2, 3, 4, 5 years or more). In some embodiments, the compositions provided herein are administered to a population that does not include pregnant women.

[0443] In some detailed embodiments, the compositions provided herein are administered to a pregnant woman according to a regimen that includes a first dose administered after about 24 weeks of gestation (e.g., after about 22, 23, 24, 25, 26, 27, 28, or more weeks of gestation); in some embodiments, the compositions provided herein are administered to a pregnant woman according to a regimen that includes a first dose administered before about 34 weeks of gestation (e.g., before about 30, 31, 32, 33, 34, 35, 36, 37, 38 weeks of gestation). In some embodiments, the compositions provided herein are administered to a pregnant woman according to a regimen that includes a first dose administered after about 24 weeks of gestation (e.g., after about 27 weeks of gestation, e.g., between about 24 and 34 weeks, or between about 27 and 34 weeks), and a second dose administered about 21 days later; in some embodiments, both doses are administered before birth. Without wishing to be bound by any particular theory, it is proposed that such a regimen (e.g., a regimen involving administration of a first dose after about 24 or 27 weeks of gestation, and optionally before about 34 weeks of gestation, and optionally administration of a second dose within about 21 days, ideally before birth) may have several advantages in terms of safety (e.g., reduced risk of preterm birth or fetal morbidity or mortality) and / or efficacy (e.g., carryover of vaccinations dispensed to the infant) compared to alternative dosing regimens (e.g., dosing at any time during pregnancy, withholding dosing during pregnancy, and / or dosing late in pregnancy, e.g., so that only a single dose is administered during pregnancy). In some embodiments, infants born to mothers vaccinated during pregnancy, e.g., according to certain regimens described herein, may not require further vaccinations or may require reduced vaccinations (e.g., fewer doses, and / or fewer administrations—e.g., boosters—and / or lower total exposure over a given period of time) for a certain period after birth (e.g., as described herein).

[0444] In some embodiments, the compositions provided herein are administered to a population in which women are advised not to become pregnant for a period of time after receiving the vaccine (e.g., after receiving the first dose of the vaccine, after receiving the last dose of the vaccine, etc.); in some such embodiments, the period of time can be at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks or more, or at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months or more.

[0445] In some embodiments, populations treated with the mRNA compositions described herein may include one or more populations with a particularly high-risk condition or history, such as one or more of those described herein. For example, in some embodiments, populations treated with the mRNA compositions described herein may include subjects whose occupational and / or environmental exposures may dramatically increase their risk of SARS-CoV-2 infection and / or influenza virus infection (including, but not limited to, transportation workers, prisoners, grocery store employees, long-term care facility residents, slaughterhouses or other meat processing workers, healthcare workers, and / or first responders, e.g., emergency responders). In particular embodiments, populations treated with the mRNA compositions described herein may include healthcare workers and / or first responders, e.g., emergency responders. In some embodiments, populations treated with the mRNA compositions described herein may include individuals with a history of smoking or vaping (e.g., within 6 months, 12 months, or more, e.g., a history of chronic smoking or vaping). In some embodiments, the populations treated with the mRNA compositions described herein may include certain ethnic groups determined to be more susceptible to SARS-CoV-2 infection and / or influenza virus infection.

[0446] In some embodiments, populations treated with the mRNA compositions described herein may include some populations with blood types that may be determined to be more susceptible to SARS-CoV-2 infection and / or influenza virus infection. In some embodiments, populations treated with the mRNA compositions described herein may include immunocompromised subjects (e.g., those with HIV / AIDS; cancer patients (e.g., undergoing anti-tumor treatment); patients taking some immunosuppressive medications (e.g., transplant patients, cancer patients, etc.); patients with autoimmune diseases or other physiological conditions predicted to warrant immunosuppressive therapy (e.g., within three months, six months, or more); and patients with genetic diseases affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency)). In some embodiments, populations treated with the mRNA compositions described herein may include those with infectious diseases. For example, in some embodiments, populations treated with the mRNA compositions described herein may include those infected with human immunodeficiency virus (HIV) and / or hepatitis viruses (e.g., HBV, HCV). In some embodiments, populations treated with the mRNA compositions described herein may include those with underlying diseases. Examples of such underlying diseases include hypertension, cardiovascular disease, diabetes, chronic respiratory diseases such as chronic lung disease, asthma, cancer, and other chronic diseases such as lupus, rheumatoid arthritis, chronic liver disease, and chronic kidney disease (e.g., stage 3 or greater, e.g., in some embodiments, a glomerular filtration rate (GFR) of 60 mL / min / 1.73 m). 2 In some embodiments, populations treated with the mRNA compositions described herein may include, but are not limited to, overweight or obese subjects, including, for example, those with a body mass index (BMI) of about 30 kg / m 2In some embodiments, the population treated with the mRNA compositions described herein may include subjects who have been previously diagnosed with COVID-19 or have evidence of current or past SARS-CoV-2 infection, e.g., based on serology or nasal swabs. In some embodiments, the population treated includes white and / or non-Hispanic / non-Latino individuals.

[0447] In some embodiments, certain mRNA compositions described herein may be selected for administration to Asian populations (e.g., Chinese populations), or in particular embodiments, older Asian populations (e.g., 60 years of age or older, e.g., 60-85 years of age or 65-85 years of age).

[0448] In some embodiments, the mRNA compositions described herein are administered and / or evaluated in subject(s) determined prior to administration to not exhibit evidence of prior and / or current infection, and in some embodiments, evidence of prior and / or current infection can be or can include evidence of intact virus or any viral nucleic acid, protein, lipid, etc. present in the subject (e.g., their biological sample, e.g., blood, cells, mucus, and / or tissue), and / or evidence of the subject's immune response thereto. In some embodiments, the mRNA compositions described herein are administered and / or evaluated in subject(s) determined prior to administration to exhibit evidence of prior and / or current infection, and in some embodiments, evidence of prior and / or current infection can be or can include evidence of intact virus or any viral nucleic acid, protein, lipid, etc. present in the subject (e.g., their biological sample, e.g., blood, cells, mucus, and / or tissue), and / or evidence of the subject's immune response thereto. In some embodiments, a subject is considered to have been previously infected based on having a positive N-linked antibody test result or a positive nucleic acid amplification test (NAAT) result on Dose 1 day.

[0449] In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to subjects who have been informed of their risk of side effects, which may include, for example, one or more of chills, fever, headache, pain at the injection site, muscle aches, and fatigue; in some embodiments, the RNA (e.g., mRNA) compositions are administered to subjects who have been asked to inform their healthcare provider if one or more of such side effects occur, are experienced as more than mild or moderate, or persist for a period of more than one day or several days, or if any serious or unexpected event that the subject reasonably believes may be related to the administration of the composition is experienced. In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to subjects who have been asked to inform their healthcare provider of a particular medical condition, which may include, for example, one or more of allergies, bleeding disorders, or taking anticoagulants, breastfeeding, fever, being immunocompromised or taking medications that affect the immune system, pregnancy, or planning a pregnancy, etc. In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to a subject who is asked to inform their healthcare provider that they have received another COVID-19 vaccine and / or another influenza vaccine (e.g., a COVID-19 vaccine or an influenza vaccine described herein). In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to a subject who does not have one of the following medical conditions: experiencing a febrile illness, receiving immunosuppressant therapy (e.g., receiving a known immunosuppressant or radiation therapy within the past 60 days), receiving anticoagulant therapy, suffering from a bleeding disorder (e.g., one that contraindicates intramuscular injection), a history of cardiac disease, an abnormal troponin I screening laboratory test, probable or possible myocarditis or pericarditis (e.g., a subject with a mean QTcF interval greater than 450 milliseconds, complete left bundle branch block, signs of acute or cryptic myocardial infarction, ST-T interval changes suggestive of myocardial ischemia, second- or third-degree AV block, and / or a 12-lead electrocardiogram showing severe bradyarrhythmias or tachyarrhythmias), or is pregnant and / or lactating.In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to a subject who has not received another COVID-19 vaccine and / or another influenza vaccine. In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to a subject who has not previously experienced an allergic reaction to any component of the RNA (e.g., mRNA) composition. Examples of such allergic reactions include, but are not limited to, difficulty breathing, swelling of the face and / or throat, increased heart rate, rash, dizziness, and / or weakness. In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to a subject who has received a first dose and has not had an allergic reaction to the first dose (e.g., as described herein). In some embodiments, in which a subject(s) experiences an allergic reaction after receiving a dose of an RNA (e.g., mRNA) composition provided herein, such subject may be administered one or more interventions, e.g., treatments, e.g., antipyretics and / or anti-inflammatory agents, to manage and / or alleviate the symptoms of such allergic reaction.

[0450] In some embodiments, subjects who have received at least one dose of an RNA (e.g., mRNA) composition provided herein are advised to avoid exposure to coronavirus (e.g., SARS-CoV-2) and / or influenza virus unless and until several days (e.g., at least 7 days, at least 8 days, 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, etc.) have passed since the administration of the second dose. For example, subjects who have received at least one dose of an RNA (e.g., mRNA) composition provided herein are advised to take precautions against SARS-CoV-2 infection and / or influenza infection (e.g., practicing social distancing, wearing a mask, washing their hands frequently, etc.) unless and until several days (e.g., at least 7 days, at least 8 days, 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, etc.) have passed since the administration of the second dose. Thus, in some embodiments, methods of administering an RNA (e.g., mRNA) composition provided herein include administering a second dose of such an RNA (e.g., mRNA) composition provided herein to a subject who has received a first dose and has taken precautions to avoid exposure to coronavirus (e.g., SARS-CoV-2) and / or influenza virus. In some embodiments, the compositions described herein (e.g., RNA (e.g., mRNA) compositions) can be delivered to the draining lymph nodes of a subject in need thereof, e.g., for vaccine priming. In some embodiments, such delivery can be by intramuscular administration of the provided mRNA compositions.

[0451] In some embodiments, different specific mRNA compositions may be administered to different subject population(s); alternatively, or in addition, in some embodiments, different dosing regimens may be administered to different subject populations. For example, in some embodiments, mRNA compositions administered to specific subject population(s) may be characterized by one or more specific effects (e.g., incidence and / or magnitude of effect) in those subject populations. In some embodiments, such effect(s) may be, for example, an increase in neutralizing antibodies and / or T cells (e.g., T H Type 1 T cells, e.g., CD4 + and / or CD8 + The parameters may be or may include the titer and / or persistence of immune responses (e.g., immune response to a given antigen), the titer and / or persistence of immune responses (e.g., immune T cells), protection against challenge (e.g., by injection and / or intranasal exposure), the incidence, severity and / or persistence of side effects (e.g., reactogenicity), etc.

[0452] In some embodiments, one or more mRNA compositions described herein can be administered according to a regimen established to reduce the incidence of COVID-19 and / or influenza per 1000 person-years, e.g., based on clinical testing, e.g., nucleic acid amplification tests (NAATs). In some embodiments, one or more mRNA compositions described herein can be administered according to a regimen established to reduce the incidence of COVID-19 and / or influenza per 1000 person-years, based on clinical testing, e.g., nucleic acid amplification tests (NAATs), in subjects who have received at least one dose of a provided mRNA composition and who have no serological or virological evidence of SARS-CoV-2 and / or influenza virus infection in the past (e.g., up to 7 days after receiving their last dose). In some embodiments, one or more mRNA compositions described herein can be administered according to a regimen established to reduce the incidence of confirmed severe COVID-19 and / or influenza per 1000 person-years. In some embodiments, one or more mRNA compositions described herein can be administered according to a regimen established to reduce the incidence of confirmed severe COVID-19 and / or influenza per 1000 person-years in subjects without serological or virological evidence of prior SARS-CoV-2 and / or prior influenza virus infection who have received at least one dose of a provided mRNA composition.

[0453] In some embodiments, one or more mRNA compositions described herein may be administered according to an established regimen to produce neutralizing antibodies directed against a SARS-CoV-2 spike polypeptide, an influenza virus HA polypeptide, and / or an immunogenic fragment thereof (e.g., RBD) as measured in serum from a subject that achieves or exceeds a reference level (e.g., a reference level determined based on human SARS-CoV-2 infection / COVID-19 convalescent serum and / or human influenza convalescent serum) over a period of time, and / or induce a cell-mediated immune response (e.g., a T cell response against SARS-CoV-2 and / or influenza virus), e.g., in some embodiments, induces T cells that recognize at least one or more MHC-restricted (e.g., MHC class I-restricted) epitopes in a SARS-CoV-2 spike polypeptide, an influenza virus HA polypeptide, and / or an immunogenic fragment thereof (e.g., RBD) over a period of time. In some such embodiments, the period of time may be at least 2 months, 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more. In some embodiments, one or more epitopes recognized by vaccine-induced T cells (e.g., CD8+ T cells) may be presented on an MHC class I allele present in at least 50%, e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the subjects in the population; in some such embodiments, the MHC class I allele is ... * 0702, HLA-A * 2402, HLA-B * 3501, HLA-B * 4401, or HLA-A * In some embodiments, the epitope may be HLA-A * 0201 YLQPRTFLL (SEQ ID NO: 35), HLA-A * 0201 RLQSLQTYV (SEQ ID NO: 36), HLA-A* 2402 QYIKWPWYI (SEQ ID NO: 37), HLA-A * 2402 NYNYLYRLF (SEQ ID NO: 38), HLA-A * 2402 KWPWYIWLGF (SEQ ID NO: 39), HLA-B * 3501 QPTESIVRF (SEQ ID NO: 40), HLA-B * 3501 IPFAMQMAY (SEQ ID NO: 41), or HLA-B * 3501 LPFNDGVYF (SEQ ID NO: 42).

[0454] In some embodiments, efficacy is assessed as the incidence rate of COVID-19 and / or influenza per 1000 person-years in individuals without serological or virological evidence of previous SARS-CoV-2 infection and / or previous influenza virus infection before and during the vaccination regimen; alternatively, or in addition, in some embodiments, efficacy is assessed as the incidence rate of COVID-19 and / or influenza per 1000 person-years in subjects with and without evidence of previous SARS-CoV-2 infection and / or influenza virus infection before and during the vaccination regimen. In some such embodiments, such incidence rates are for confirmed COVID-19 and / or influenza cases within a specified time period after the final vaccination dose (e.g., the first dose in a single-dose regimen; the second dose in a two-dose regimen, etc.); in some embodiments, such time period may be within (i.e., up to and including) a specified number of days (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more). In some embodiments, such time period may be within 7 days, or within 14 days, or within 21 days, or within 28 days. In some embodiments, such time period may be within 7 days. In some embodiments, such time period may be within 14 days.

[0455] In some embodiments (e.g., some embodiments evaluating efficacy), a subject is determined to have experienced a COVID-19 or influenza virus infection if one or more of the following is demonstrated: detection of SARS-CoV-2 nucleic acid or influenza virus in a sample from the subject, detection of antibodies that specifically recognize SARS-CoV-2 or influenza virus (e.g., SARS-CoV-2 spike protein or HA polypeptide), one or more symptoms of COVID-19 or influenza virus infection, and combinations thereof. In some such embodiments, detection of SARS-CoV-2 or influenza virus nucleic acid may involve, for example, NAAT testing on a middle turbinate swab sample. In some such embodiments, detection of relevant antibodies may involve serological testing of a blood sample or portion thereof. In some such embodiments, symptoms of COVID-19 infection may be or may include fever, onset or increase in cough, onset or increase in shortness of breath, chills, onset or increase in muscle pain, onset of loss of taste or smell, sore throat, diarrhea, vomiting, and combinations thereof. In some such embodiments, symptoms of COVID-19 infection may be fever, onset or increase in cough, onset or increase in shortness of breath, chills, onset or increase in muscle aches, onset of loss of taste or smell, sore throat, diarrhea, vomiting, fatigue, headache, stuffy or runny nose, nausea, and combinations thereof. In some such embodiments, a subject is determined to have experienced a COVID-19 infection if they experience one such symptom and also receive a positive test result for SARS-CoV-2 nucleic acid or antibody, or both. In some such embodiments, a subject is determined to have experienced a COVID-19 infection if they experience one such symptom and also receive a positive test result for SARS-CoV-2 nucleic acid. In some such embodiments, a subject is determined to have experienced a COVID-19 infection if they experience one such symptom and also receive a positive test result for SARS-CoV-2 antibody.

[0456] In some embodiments (e.g., some embodiments assessing efficacy), a subject is determined to have experienced severe COVID-19 infection if such subject experiences one or more of the following: suggestive or severe clinical signs of systemic illness at rest (e.g., one or more of respiratory rate ≥ 30 breaths per minute, heart rate ≥ 125 beats per minute, SpO2 ≤ 93% on room air at sea level or PaO2 / FiO2 < 300 mHg), respiratory failure (e.g., one or more of requiring high-flow oxygen, non-invasive ventilation, mechanical ventilation, ECMO), evidence of shock (systolic blood pressure < 90 mmHg, diastolic blood pressure < 60 mmHg, requiring vasopressors), significant acute renal, hepatic, or neurological dysfunction, admission to an intensive care unit, death, and combinations thereof.

[0457] In some embodiments, one or more mRNA compositions described herein may be administered according to a regimen established to reduce the proportion of subjects reporting at least one of the following: (i) one or more local reactions (e.g., those described herein) up to 7 days after each dose; (ii) one or more systemic events up to 7 days after each dose; (iii) an adverse event (e.g., those described herein) from the first dose up to 1 month after the final dose; and / or (iv) a serious adverse event (e.g., those described herein) from the first dose up to 6 months after the final dose.

[0458] In some embodiments, one or more subjects who receive an RNA (e.g., mRNA) composition described herein can be monitored (e.g., over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more) to assess, e.g., the presence of an immune response to a component(s) of the administered composition, evidence of exposure to and / or immune response to SARS-CoV-2 or another coronavirus, evidence of any adverse events, etc. In some embodiments, monitoring can be performed by remote visit. Alternatively, or additionally, in some embodiments, monitoring may be performed in person.

[0459] In some embodiments, the therapeutic effect conferred by one or more mRNA compositions described herein may be characterized by (i) a SARS-CoV-2 anti-S1-binding antibody level above a predetermined threshold, (ii) a SARS-CoV-2 anti-RBD-binding antibody level above a predetermined threshold, (iii) a SARS-CoV-2 serum neutralizing titer above a threshold level, (iv) an anti-HA-binding antibody level above a predetermined threshold, and / or (v) an influenza virus serum neutralizing titer above a threshold at baseline, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and / or 24 months after completed vaccination. In some embodiments, the anti-S1-binding antibody and / or anti-RBD-binding antibody level, and / or anti-HA-binding antibody level, and / or serum neutralizing titer may be expressed by geometric mean concentration (GMC), geometric mean titer (GMT), or geometric mean fold increase (GMFR).

[0460] In some embodiments, the therapeutic effect conferred by one or more mRNA compositions described herein may be characterized by a higher proportion of treated subjects exhibiting SARS-CoV-2 serum neutralizing titers and / or influenza virus neutralizing titers above a predetermined threshold, e.g., at baseline, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and / or 24 ...

Claims

1. A container containing a freshly mixed combination, the combination comprising: (a) a SARS-CoV-2 vaccine; and (b) influenza vaccine Including, The SARS-CoV-2 vaccine comprises: One or more RNAs encoding an immunogenic portion of the SARS-CoV-2 spike (S) protein and formulated into a nanoparticle (e.g., a lipid nanoparticle (LNA)). Including; The influenza vaccine (i) a nanoparticle (e.g., LNP)-formulated RNA vaccine; or (ii) comprises one or more antigenic polypeptides (e.g., HA proteins) of one or more influenza virus strains; The container.

2. A container containing a freshly mixed combination, the combination comprising: (a) a SARS-CoV-2 vaccine; and (b) RSV vaccine Including, The SARS-CoV-2 vaccine comprises: One or more RNAs encoding an immunogenic portion of the SARS-CoV-2 spike (S) protein and formulated into a nanoparticle (e.g., a lipid nanoparticle (LNA)). Including; the RSV vaccine comprises one or more antigenic polypeptides (e.g., F protein or immunogenic fragments thereof) associated with one or more RSV strains; The container.

3. A container containing a freshly mixed combination, the combination comprising: (a) SARS-CoV-2 vaccine; (b) RSV vaccine; (c) influenza vaccine Including, The SARS-CoV-2 vaccine comprises: One or more RNAs encoding an immunogenic portion of the SARS-CoV-2 spike (S) protein and formulated into a nanoparticle (e.g., a lipid nanoparticle (LNA)). Including; the RSV vaccine comprises one or more antigenic polypeptides (e.g., F protein or immunogenic fragments thereof) associated with one or more RSV strains; The influenza vaccine (i) a nanoparticle (e.g., LNP)-formulated RNA vaccine; or (ii) comprises one or more antigenic polypeptides (e.g., HA proteins) of one or more influenza virus strains; The container.

4. The container of any one of claims 1 to 3, wherein the SARS-CoV-2 vaccine is a monovalent or bivalent vaccine.

5. 5. The container of claim 1, 3 or 4, wherein the influenza vaccine is a tetravalent vaccine.

6. 6. The container of claim 1, 3, 4 or 5, wherein the influenza vaccine is an inactivated influenza virus, a recombinant influenza vaccine, a live attenuated influenza vaccine, an unadjuvanted influenza vaccine, an adjuvanted influenza vaccine, or a subunit or split vaccine.

7. 7. The container of any one of claims 2 to 6, wherein the RSV vaccine comprises a pre-fusion stabilized F protein or immunogenic fragment thereof of one or more RSV strains.

8. The container according to any one of claims 1 to 7, wherein the container is a syringe or a vial.

9. 1. A method for simultaneously vaccinating a human subject against each of SARS-CoV-2 and influenza, comprising: Co-administration of a SARS-CoV-2 vaccine composition and an influenza vaccine composition to the same site Including; The SARS-CoV-2 vaccine comprises: One or more RNAs encoding an immunogenic portion of the SARS-CoV-2 spike (S) protein and formulated into a nanoparticle (e.g., a lipid nanoparticle (LNA)). Including; The influenza vaccine (i) a nanoparticle (e.g., LNP)-formulated RNA vaccine; or (ii) comprises one or more antigenic polypeptides (e.g., HA proteins) of one or more influenza virus strains; The method.

10. 1. A method of simultaneously vaccinating a human subject against each of SARS-CoV-2 and RSV, comprising: Co-administration of a SARS-CoV-2 vaccine composition and an RSV vaccine composition at the same site Including; The SARS-CoV-2 vaccine comprises: One or more RNAs encoding an immunogenic portion of the SARS-CoV-2 spike (S) protein and formulated into a nanoparticle (e.g., a lipid nanoparticle (LNA)). Including; the RSV vaccine comprises one or more antigenic polypeptides (e.g., F protein or immunogenic fragments thereof) associated with one or more RSV strains; The method.

11. 1. A method for simultaneously vaccinating a human subject against each of SARS-CoV-2, influenza, and RSV, comprising: Co-administration of a SARS-CoV-2 vaccine composition, an influenza vaccine composition, and an RSV vaccine composition at the same site Including; The SARS-CoV-2 vaccine comprises: One or more RNAs encoding an immunogenic portion of the SARS-CoV-2 spike (S) protein and formulated into a nanoparticle (e.g., a lipid nanoparticle (LNA)). Including; The influenza vaccine (i) a nanoparticle (e.g., LNP)-formulated RNA vaccine; or (ii) comprises one or more antigenic polypeptides (e.g., HA proteins) of one or more influenza virus strains; the RSV vaccine comprises one or more antigenic polypeptides (e.g., F protein or immunogenic fragments thereof) associated with one or more RSV strains; The method.

12. the administering step comprises injecting the composition through a needle or port; the injected composition comprises both the SARS-CoV-2 vaccine composition and the influenza vaccine composition; The SARS-CoV-2 vaccine composition and the influenza vaccine composition are optionally administered using a syringe (e.g., a dual-chamber syringe).

10. The method of claim 9.

13. the administering step comprises injecting the composition through a needle or port; the injected composition comprises both the SARS-CoV-2 vaccine composition and the RSV vaccine composition; The SARS-CoV-2 vaccine composition and the RSV vaccine composition are optionally administered using a syringe (e.g., a dual-chamber syringe). The method of claim 10.

14. the administering step comprises injecting the composition through a needle or port; the injected composition comprises each of the SARS-CoV-2 vaccine composition, the influenza vaccine composition, and the RSV vaccine composition; The SARS-CoV-2 vaccine composition, the RSV vaccine composition, and the influenza vaccine composition are optionally administered using a syringe (e.g., a dual-chamber syringe). The method of claim 11.

15. 13. The method of claim 9 or 12, further comprising the step of combining the SARS-CoV-2 vaccine composition and the influenza vaccine composition prior to said administering step.

16. 14. The method of claim 10 or 13, further comprising the step of combining the SARS-CoV-2 vaccine composition and the RSV vaccine composition prior to said administering step.

17. 15. The method of claim 11 or 14, further comprising the step of combining the SARS-CoV-2 vaccine composition, the influenza vaccine composition, and the RSV vaccine composition prior to said administering step.

18. 18. The method of any one of claims 15 to 17, wherein the combining step is performed within a period of time of the administering step, and the period of time is 2 hours or less (e.g., 1 hour, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes or less).

19. The container of any one of claims 1 to 8 or the method of any one of claims 9 to 18, wherein the SARS-CoV-2 vaccine composition comprises two or more RNAs, each encoding an S protein of a different SARS-CoV-2 strain or variant, and the two or more RNAs are encapsulated in separate populations of LNPs.

20. The container described in any one of claims 1 and 3 to 8, or the method described in any one of claims 9 and 11 to 18, or the container or method described in claim 19, wherein the influenza vaccine comprises two or more RNAs (e.g., four RNAs), each encoding an antigenic polypeptide (e.g., HA protein) of a different influenza strain, and the two or more RNAs are encapsulated in separate LNP populations.

21. The SARS-CoV-2 vaccine comprises: (a) (i) an RNA comprising a nucleotide sequence that comprises a modified uridine and encodes a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein the RNA encodes a polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:7, and / or comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:20 and / or a nucleotide sequence that is at least 85% identical to SEQ ID NO:9; and (ii) an RNA comprising a nucleotide sequence that contains a modified uridine and encodes an S polypeptide from the Omicron BA.4 / 5 SARS-CoV-2 variant, wherein the RNA comprises a nucleotide sequence that encodes a polypeptide comprising a sequence that is at least 85% identical to SEQ ID NO:69, and / or comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:72 and / or a nucleotide sequence that is at least 85% identical to SEQ ID NO:70; or (b) an RNA comprising a nucleotide sequence that comprises a modified uridine and encodes a SARS-CoV-2 spike (S) polypeptide, wherein the RNA comprises a nucleotide sequence that encodes a polypeptide comprising a sequence that is at least 85% identical to SEQ ID NO: 129, and / or comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 132 and / or a nucleotide sequence that is at least 85% identical to SEQ ID NO:

130. A container according to any one of claims 1 to 8, or a method according to any one of claims 9 to 18, or a container or method according to claim 19 or 20, comprising:

22. The influenza vaccine (a) (i) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the RNA comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:94 and / or a nucleotide sequence that is at least 85% identical to SEQ ID NO:92; (ii) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein the nucleotide sequence is at least 85% identical to SEQ ID NO:99 and / or at least 85% identical to SEQ ID NO:97; (iii) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from influenza B Victoria strain, said RNA comprising a nucleotide sequence that is at least 85% identical to SEQ ID NO: 104 and / or a nucleotide sequence that is at least 85% identical to SEQ ID NO: 102; and (iv) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the nucleotide sequence is at least 85% identical to SEQ ID NO: 109 and / or at least 85% identical to SEQ ID NO: 107; or (b) (i) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the nucleotide sequence is at least 85% identical to SEQ ID NO: 92 and / or at least 85% identical to SEQ ID NO: 94; (ii) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein the nucleotide sequence is at least 85% identical to SEQ ID NO: 82 and / or at least 85% identical to SEQ ID NO: 84; (iii) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the nucleotide sequence is at least 85% identical to SEQ ID NO: 87 and / or at least 85% identical to SEQ ID NO: 89; and (iv) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the nucleotide sequence is at least 85% identical to SEQ ID NO: 107 and / or at least 85% identical to SEQ ID NO:

109. A container according to any one of claims 1 to 8, or a method according to any one of claims 9 to 18, or a container or method according to claim 19 or 20, comprising:

23. 1. A composition comprising: (i) an RNA comprising a first nucleotide sequence comprising a modified uridine and encoding a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein said first nucleotide sequence is at least 85% identical to SEQ ID NO:9; (ii) a RNA comprising a second nucleotide sequence comprising a modified uridine and encoding a second SARS-CoV-2 spike (S) polypeptide from a variant of said SARS-CoV-2 strain, wherein said second nucleotide sequence is at least 85% identical to SEQ ID NO:70; (iii) RNA comprising a third nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein said third nucleotide sequence is at least 85% identical to SEQ ID NO:92; (iv) RNA comprising a fourth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein said fourth nucleotide sequence is at least 85% identical to SEQ ID NO:97; (v) RNA comprising a fifth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the fifth nucleotide sequence is at least 85% identical to SEQ ID NO: 102; and (vi) an RNA comprising a sixth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the sixth nucleotide sequence is at least 85% identical to SEQ ID NO:

107. The composition comprising:

24. 1. A composition comprising: (i) an RNA comprising a nucleotide sequence that contains a modified uridine and encodes a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein the RNA comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:20; (ii) an RNA comprising a nucleotide sequence that comprises a modified uridine and encodes a second SARS-CoV-2 spike (S) polypeptide from a variant of said SARS-CoV-2 strain, said RNA comprising a nucleotide sequence that is at least 85% identical to SEQ ID NO:72; (iii) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the RNA comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:94; (iv) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from an influenza A H3N2 strain, said RNA comprising a nucleotide sequence that is at least 85% identical to SEQ ID NO:99; (v) RNA comprising a modified uridine and a nucleotide sequence encoding an influenza hemagglutinin antigen from influenza B Victoria strain, said RNA comprising a nucleotide sequence at least 85% identical to SEQ ID NO: 104; and (vi) RNA comprising a modified uridine and a nucleotide sequence encoding an influenza hemagglutinin antigen from influenza B Yamagata strain, said RNA comprising a nucleotide sequence that is at least 85% identical to SEQ ID NO:

109. The composition comprising:

25. 1. A composition comprising: (i) an RNA comprising a first nucleotide sequence comprising a modified uridine and encoding a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein said first nucleotide sequence is at least 85% identical to SEQ ID NO:9; (ii) a RNA comprising a second nucleotide sequence comprising a modified uridine and encoding a second SARS-CoV-2 spike (S) polypeptide from a variant of said SARS-CoV-2 strain, wherein said second nucleotide sequence is at least 85% identical to SEQ ID NO:70; (iii) RNA comprising a third nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein said third nucleotide sequence is at least 85% identical to SEQ ID NO:92; (iv) RNA comprising a fourth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein said fourth nucleotide sequence is at least 85% identical to SEQ ID NO:82; (v) RNA comprising a fifth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the fifth nucleotide sequence is at least 85% identical to SEQ ID NO:87; and (vi) an RNA comprising a sixth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the sixth nucleotide sequence is at least 85% identical to SEQ ID NO:

107. The composition comprising:

26. 1. A composition comprising: (i) an RNA comprising a first nucleotide sequence comprising a modified uridine and encoding a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein said first nucleotide sequence is at least 85% identical to SEQ ID NO:20; (ii) an RNA comprising a second nucleotide sequence comprising a modified uridine and encoding a second SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein said first nucleotide sequence is at least 85% identical to SEQ ID NO:72; (iii) RNA comprising a third nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein said third nucleotide sequence is at least 85% identical to SEQ ID NO:94; (iv) RNA comprising a fourth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein said fourth nucleotide sequence is at least 85% identical to SEQ ID NO:84; (v) RNA comprising a fifth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the fifth nucleotide sequence is at least 85% identical to SEQ ID NO:89; and (vi) an RNA comprising a sixth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the sixth nucleotide sequence is at least 85% identical to SEQ ID NO:

109. The composition comprising:

27. (a) the mass ratio of RNA (i) to (ii) to RNA (iii) to (vi) is 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:1; (b) the mass ratio of RNA (iii)-(iv) to RNA (v)-(vi) is 1:1 to 1:5; and / or (c) the mass ratio of RNA(i) to RNA(ii) is 1:1; The composition according to any one of claims 23 to 26.

28. 28. The composition of any one of claims 23 to 27, wherein the RNAs (iii), (iv), (v) and (vi) are present in a mass ratio of 1:1:1:1, or 1:1:5:

5.

29. The composition of any one of claims 23 to 28, wherein the total mass of RNA (i) to (vi) is from about 30 ug to about 100 ug.

30. the combined mass of RNA (i)-(ii) is between about 3 μg and about 60 μg (e.g., about 3 μg, about 10 μg, about 30 μg, or about 60 μg); and / or The total mass of RNA (iii) to (vi) is about 30 μg to about 60 μg (e.g., about 30 μg, or about 60 μg); The composition according to any one of claims 23 to 29.

31. (a) RNA (i) and (ii) are each present in an amount of about 15 μg, and RNA (iii)-(vi) are each present in an amount of about 7.5 μg; (b) RNA (i) and (ii) are each present in an amount of about 30 μg, and RNA (iii)-(vi) are each present in an amount of about 7.5 μg; (c) RNA (i) and (ii) are each present in an amount of about 15 μg, and RNA (iii)-(vi) are each present in an amount of about 11.25 μg; (d) RNA (i) and (ii) are each present in an amount of about 15 μg, RNA (iii) and (iv) are each present in an amount of about 5 μg, and RNA (v) and (vi) are each present in an amount of about 25 μg; (e) RNA (i) and (ii) are each present in an amount of about 15 μg, RNA (iii) and (iv) are each present in an amount of about 2.5 μg, and RNA (v) and (vi) are each present in an amount of about 12.5 μg; (f) RNA (i) and (ii) are each present in an amount of about 30 μg, RNA (iii) and (iv) are each present in an amount of about 2.5 μg, and RNA (v) and (vi) are each present in an amount of about 12.5 μg; or (g) RNAs (i)-(vi) are each present in an amount of about 15 μg; The composition according to any one of claims 23 to 29.

32. 1. A composition comprising: (i) an RNA comprising a first nucleotide sequence comprising a modified uridine and encoding a SARS-CoV-2 spike (S) polypeptide, wherein said first nucleotide sequence is at least 85% identical to SEQ ID NO: 129; (ii) an RNA comprising a second nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein said second nucleotide sequence is at least 85% identical to SEQ ID NO:92; (iii) RNA comprising a third nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein said third nucleotide sequence is at least 85% identical to SEQ ID NO:99; (iv) RNA comprising a fourth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the fourth nucleotide sequence is at least 85% identical to SEQ ID NO: 102; and (v) an RNA comprising a fifth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the fifth nucleotide sequence is at least 85% identical to SEQ ID NO:

107. The composition comprising:

33. 1. A composition comprising: (i) RNA comprising a nucleotide sequence that contains a modified uridine and encodes a SARS-CoV-2 spike (S) polypeptide, wherein the nucleotide sequence is at least 85% identical to SEQ ID NO: 132; (ii) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein the nucleotide sequence is at least 85% identical to SEQ ID NO:94; (iii) RNA comprising a modified uridine and a nucleotide sequence encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, said RNA comprising a nucleotide sequence that is at least 85% identical to SEQ ID NO:99; (iv) RNA comprising a nucleotide sequence that contains a modified uridine and encodes an influenza hemagglutinin antigen from the influenza B Victoria strain, said RNA comprising a nucleotide sequence that is at least 85% identical to SEQ ID NO: 104; and (v) RNA comprising a modified uridine and a nucleotide sequence encoding an influenza hemagglutinin antigen from influenza B Yamagata strain, said RNA comprising a nucleotide sequence at least 85% identical to SEQ ID NO:

109. The composition comprising:

34. 1. A composition comprising: (i) an RNA comprising a first nucleotide sequence comprising a modified uridine and encoding a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein said first nucleotide sequence is at least 85% identical to SEQ ID NO: 130; (ii) an RNA comprising a second nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein said second nucleotide sequence is at least 85% identical to SEQ ID NO:92; (iii) RNA comprising a third nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein said third nucleotide sequence is at least 85% identical to SEQ ID NO: 82; (iv) RNA comprising a fourth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the fourth nucleotide sequence is at least 85% identical to SEQ ID NO: 87; and (v) an RNA comprising a fifth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the fifth nucleotide sequence is at least 85% identical to SEQ ID NO:

107. The composition comprising:

35. 1. A composition comprising: (i) an RNA comprising a first nucleotide sequence comprising a modified uridine and encoding a first SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain, wherein said first nucleotide sequence is at least 85% identical to SEQ ID NO: 132; (ii) RNA comprising a second nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H1N1 strain, wherein said second nucleotide sequence is at least 85% identical to SEQ ID NO:94; (iii) RNA comprising a third nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from an influenza A H3N2 strain, wherein said third nucleotide sequence is at least 85% identical to SEQ ID NO:84; (iv) RNA comprising a fourth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Victoria strain, wherein the fourth nucleotide sequence is at least 85% identical to SEQ ID NO:89; and (v) an RNA comprising a fifth nucleotide sequence comprising a modified uridine and encoding an influenza hemagglutinin antigen from influenza B Yamagata strain, wherein the fifth nucleotide sequence is at least 85% identical to SEQ ID NO:

109. The composition comprising:

36. RNA (i) and RNAs (ii)-(v) are present in a mass ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:1; RNA (ii) and (iii) and RNA (iv) and (v) are present in a mass ratio of 1:1 to 1:5; RNAs (ii), (iii), (iv) and (v) are present in a mass ratio of 1:1:1:1 or 1:1:5:5; The composition according to any one of claims 32 to 35.

37. The composition according to any one of claims 24 to 36, wherein the total mass of RNAs (i) to (v) is 30 μg to 100 μg.

38. 38. The composition of any one of claims 32 to 37, wherein the mass of RNA(i) is from about 3 μg to about 60 μg (e.g., about 3 μg, about 10 μg, about 30 μg, or about 60 μg), and / or the combined mass of RNA(ii) to (v) is from about 30 μg to about 60 μg (e.g., about 30 μg, or about 60 μg).

39. (a) RNA(i) is present in an amount of about 30 μg, and RNAs(ii)-(v) are each present in an amount of about 7.5 μg; (b) RNA(i) is present in an amount of about 60 μg, and RNAs(ii)-(v) are each present in an amount of about 7.5 μg; (c) RNA(i) is present in an amount of about 30 μg, and RNAs (ii)-(v) are each present in an amount of about 11.25 μg; (d) RNA(i) is present in an amount of about 30 μg, RNA(ii) and (iii) are each present in an amount of about 5 μg, and RNA(iv) and (v) are each present in an amount of about 25 μg; (e) RNA(i) is present in an amount of about 30 μg, RNA(ii) and (iii) are each present in an amount of about 2.5 μg, and RNA(iv) and (v) are each present in an amount of about 12.5 μg; (f) RNA(i) is present in an amount of about 30 μg, RNA(ii) and (iii) are each present in an amount of about 2.5 μg, and RNA(iv) and (v) are each present in an amount of about 12.5 μg; or (g) RNA(i) is present in an amount of about 30 μg, and RNAs (ii)-(v) are each present in an amount of about 15 μg; The composition according to any one of claims 32 to 38.

40. 40. The composition of any one of claims 23 to 39, wherein the influenza A H1N1 strain is influenza A / Wisconsin / 588 / 2019 and the influenza B Yamagata strain is influenza B / Phuket / 3073 / 2013.

41. 41. The composition of any one of claims 23, 24, 27-31, 33, 34 and 37-40, wherein the influenza A H3N2 strain is influenza A / Cambodia / e0826360 / 2020 and the influenza B Victoria strain is influenza B / Washington / 02 / 2019.

42. 40. The composition of any one of claims 25 to 31 and 34 to 39, wherein the influenza A H3N2 strain is influenza A / Darwin / 6 / 2021.

43. 41. The composition of any one of claims 25 to 31 and 34 to 40, wherein the influenza A H3N2 strain is influenza A / Darwin / 6 / 2021 and / or the influenza B Victoria strain is influenza B / Austria / 1359417 / 2021.

44. 32. The composition of any one of claims 23-31, wherein the first SARS-CoV-2 spike (S) polypeptide is from the Wuhan strain and the second SARS-CoV-2 S polypeptide is from the Omicron BA.4 / 5 variant.

45. 44. The composition of any one of claims 32-43, wherein the SARS-CoV-2 spike (S) polypeptide is from the XBB.1.5 variant.

46. 46. ​​The composition of any one of claims 23 to 45, wherein each of the RNAs in the composition comprises the same non-coding elements, including the same 5' cap, cap adjacent sequence, 5' UTR sequence, 3' UTR sequence and poly A sequence.

47. 1. A composition comprising: (i) a coronavirus RNA vaccine comprising one or more RNAs, each comprising a nucleotide sequence encoding a SARS-CoV-2 antigen; and (ii) An influenza RNA vaccine comprising one or more RNAs each comprising one or more nucleotide sequences encoding influenza antigens, wherein the influenza RNA vaccine encodes at least four influenza antigens, each influenza antigen being from a distinct influenza virus predicted to be prevalent during a particular hemisphere flu season. Including, each of the RNAs in the composition contains the same non-coding elements, including the same 5' cap, cap adjacent sequence, 5' UTR sequence, 3' UTR sequence, and poly A sequence; The composition.

48. 48. The composition of claim 47, wherein each of the one or more RNAs in the coronavirus RNA vaccine and each of the one or more RNAs in the influenza RNA vaccine comprises one or more modified uridines.

49. 49. The composition of claim 47 or 48, wherein the at least four influenza antigens each are or comprise hemagglutinin antigens from distinct influenza virus strains predicted to be prevalent during a particular hemisphere's influenza season.

50. 50. The composition of claim 49, wherein the distinct influenza viruses are predicted to circulate during influenza season based on human serology data from the Northern or Southern Hemisphere.

51. 51. The composition of any one of claims 47 to 50, wherein the at least four influenza antigens are each encoded by a separate RNA.

52. 52. The composition of any one of claims 47-51, wherein the coronavirus RNA vaccine encodes at least two SARS-CoV-2 antigens, each from a distinct SARS-CoV-2 strain or variant.

53. 53. The composition of claim 52, wherein the at least two SARS-CoV-2 antigens are or comprise a SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 strain and a SARS-CoV-2 S polypeptide from a variant of the SARS-CoV-2 strain.

54. 54. The composition of claim 52 or 53, wherein the at least two SARS-CoV-2 antigens are each encoded by a separate RNA.

55. 55. The composition of any one of claims 47 to 54, wherein the RNA in the coronavirus vaccine and the RNA in the influenza vaccine are present in a mass ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:

1.

56. 56. The composition of any one of claims 47 to 55, wherein the at least four influenza antigens comprise at least two hemagglutinin antigens from influenza A virus and at least two hemagglutinin antigens from influenza B virus.

57. 57. The composition of claim 56, wherein the RNA encoding a hemagglutinin antigen from influenza A virus and the RNA encoding a hemagglutinin antigen from influenza B virus are present in a mass ratio of 1:1 to 1:5 (e.g., 1:1 or 1:5).

58. 58. The composition of any one of claims 54 to 57, wherein the at least two RNAs in the coronavirus vaccine are in a 1:1 mass ratio.

59. 59. The composition of any one of claims 51 to 58, wherein the at least four RNAs in the influenza vaccine are present in a mass ratio of 1:1:1:

1.

60. 60. The composition of any one of claims 47-59, wherein the total amount of RNA in the composition is about 30ug to about 100ug (e.g., about 30ug, about 45ug, about 60ug, about 75ug, or about 90ug).

61. 1. A composition comprising: one or more first RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a first infectious agent; one or more second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent; Including, the second infectious agent is different from the first infectious agent; each of the first and second RNAs in the composition comprises the same non-coding elements, including the same 5' cap, cap proximal sequence, 5' UTR sequence, 3' UTR sequence, and poly A sequence; At least one of the same non-coding elements (i) a 5'-UTR sequence that is or includes a modified human alpha globin 5'-UTR; (ii) a 3′-UTR sequence that is or includes a first sequence from an amino terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; (iii) a polyA sequence comprising at least 100 A nucleotides, wherein the first RNA and the second RNA each do not comprise a stretch of at least 30 consecutive C nucleotides between the 3′ UTR and the polyA sequence; (iv) a polyA sequence comprising a staggered sequence of A nucleotides, optionally comprising 30 adenine nucleotides (SEQ ID NO:174) followed by 70 adenine nucleotides (SEQ ID NO:175), wherein the 30 adenine nucleotides (SEQ ID NO:174) and the 70 adenine nucleotides (SEQ ID NO:175) are separated by a linker sequence; or (v) a 5′ cap comprising a Cap1 structure and a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA; is or contains; (a) The Cap1 structure is m7(3'OMeG)(5')ppp(5')(2'OMeA １ ) pG ２ where A １ is the +1 position of the RNA, and G ２ is the +2 position of the RNA; (b) the cap adjacent sequence is A of the Cap1 structure １ and G ２ and A at positions +3, +4 and +5 of said RNA ３ N ４ N ５ and wherein N ４ and N ５ are each independently selected from A, G, C, and U; The composition.

62. 1. A composition comprising: one or more first RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a first infectious agent; one or more second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent different from said first infectious agent. Including; each of the first and second RNAs in the composition comprises the same non-coding elements, including the same 5' cap, cap proximal sequence, 5' UTR sequence, 3' UTR sequence, and poly A sequence; Each of the first and second RNAs is (i) the level of immune response induced by the RNA in the composition is at least 80% of the level of immune response induced by the same RNA when administered alone; and / or (ii) the level of immune response induced by the RNA in the composition is at least 80% of the level of immune response induced by the same RNA when administered separately from other RNAs to a different location in the subject's body; and / or (iii) the level of immune response induced by the RNA in the composition is at least 80% of the level of immune response induced by the respective reference composition. The composition, characterized by:

63. 63. The composition of claim 62, wherein each of the reference compositions is an inactivated viral vaccine.

64. 64. The composition of claim 62 or 63, wherein the immune response induced by the one or more first RNA(s) and the one or more second RNA(s) is each at least 100% of the level of the immune response induced by the same RNA(s) when the one or more first RNA(s) and the one or more second RNA(s) are administered separately.

65. 65. The composition of any one of claims 62-64, wherein the immune response induced by the one or more first RNA(s) and the one or more second RNA(s) is each greater than the immune response induced by the same RNA(s) administered individually.

66. 65. The composition of any one of claims 62-64, wherein the one or more first RNA(s) and the one or more second RNA(s) are each present at a lower dose compared to a dose of the same RNA administered individually, and wherein an immune response induced by the lower doses of the one or more first RNA(s) and the one or more second RNA(s) is each substantially similar to or greater than an immune response induced by a larger dose of the same RNA administered individually.

67. 1. A composition comprising: one or more first RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a first infectious agent; one or more second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent different from said first infectious agent. Including; each of the first and second RNAs in the composition comprises the same non-coding elements, including the same 5' cap, cap proximal sequence, 5' UTR sequence, 3' UTR sequence, and poly A sequence; each of the first and second RNAs is encapsulated in a nanoparticle, either separately or together (e.g., each of the first RNAs is encapsulated in a first population of nanoparticles and each of the second RNAs is encapsulated in a second population of nanoparticles; or each of the first RNAs and each of the second RNAs are encapsulated in the same population of nanoparticles); The composition comprises: (i) the RNA content of the composition is at least 95% of the initial RNA content after storage for 24 hours; (ii) RNA encapsulation remains at least 95% of the initial RNA encapsulation after 24 hours of storage; (iii) the nanoparticles encapsulating the first and second RNAs maintain substantially the same particle size after storage for 24 hours; (iv) the nanoparticles encapsulating the first and second RNAs maintain a polydispersity of 0.3 or less after 24 hours; and / or (v) the mass ratio of the first RNA to the second RNA remains substantially the same after 24 hours of storage. The composition, characterized by:

68. 68. The composition of claim 67, wherein the nanoparticle comprises a lipid nanoparticle, a polyplex (PLX), a lipidated polyplex (LPLX), a liposome, or a polysaccharide nanoparticle.

69. 69. The composition of claim 68, wherein the nanoparticles comprise lipid nanoparticles.

70. 70. The composition of claim 69, wherein the lipid nanoparticles comprise a cationizable lipid, one or more neutral lipids, and a polymer-conjugated lipid.

71. 71. The composition of claim 70, wherein the polymer-conjugated lipid comprises a PEG-conjugated lipid.

72. 72. The composition of any one of claims 67 to 71, wherein the nanoparticles have an average diameter of about 50 to 150 nm.

73. 73. The composition of any one of claims 67-72, wherein for each of (i)-(v), the first 12 hours of storage is at 30°C, and the remaining 12 hours of storage is at 2-8°C.

74. 74. The composition of any one of claims 67-73, wherein the one or more first RNAs comprise at least two first RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with different strains or variants of the first infectious agent.

75. 75. The composition of any one of claims 67 to 74, wherein the one or more second RNAs comprise at least two second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with different strains or variants of the second infectious agent.

76. 76. The composition of any one of claims 67-75, wherein the one or more second RNAs comprise at least three second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with different strains or variants of the second infectious agent.

77. 77. The composition of any one of claims 67-76, wherein the one or more second RNAs comprise at least four second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with different variants or strains of the second infectious agent.

78. 1. A composition comprising: a plurality of first RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a first infectious agent of different strains and / or variants thereof; one or more second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent different from said first infectious agent. Including; each of the first and second RNAs is formulated separately or together in the same nanoparticle formulation; (i) the first RNA and the second RNA are present in a mass ratio of 1:2 to 2:1, and / or (ii) the first RNA and the second RNA are present in a total amount of about 10 ug to about 100 ug per dose; one or more of said first RNAs each comprise a nucleotide sequence encoding one or more antigenic polypeptides associated with said first infectious agent; one or more of the second RNAs each comprise a nucleotide sequence encoding one or more antigenic polypeptides associated with the second infectious agent, the second infectious agent being different from the first infectious agent; each of the first and second RNAs in the composition comprises the same non-coding elements, including the same 5' cap, cap proximal sequence, 5' UTR sequence, 3' UTR sequence, and poly A sequence; At least one of the same non-coding elements (i) a 5'-UTR sequence that is or includes a modified human alpha globin 5'-UTR; (ii) a 3′-UTR sequence that is or includes a first sequence from an amino terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; (iii) a polyA sequence comprising at least 100 A nucleotides, wherein the first RNA and the second RNA each do not comprise a stretch of at least 30 consecutive C nucleotides between the 3′ UTR and the polyA sequence; (iv) a polyA sequence comprising a staggered sequence of A nucleotides, optionally comprising 30 adenine nucleotides (SEQ ID NO:174) followed by 70 adenine nucleotides (SEQ ID NO:175), wherein the 30 adenine nucleotides (SEQ ID NO:174) and the 70 adenine nucleotides (SEQ ID NO:175) are separated by a linker sequence; or (v) a 5′ cap comprising a Cap1 structure and a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA; is or contains; (a) The Cap1 structure is m7(3'OMeG)(5')ppp(5')(2'OMeA １ ) pG ２ where A １ is the +1 position of the RNA, and G ２ is the +2 position of the RNA; (b) the cap adjacent sequence is A of the Cap1 structure １ and G ２ and A at positions +3, +4 and +5 of said RNA ３ N ４ N ５ and wherein N ４ and N ５ are each independently selected from A, G, C, and U; The composition.

79. 79. The composition of any one of claims 61-78, wherein each of the first RNAs is co-formulated in the same nanoparticle formulation, or each of the first RNAs is formulated in a separate nanoparticle formulation.

80. 80. The composition of any one of claims 61-79, wherein each of the second RNAs is co-formulated in the same nanoparticle formulation, or each of the second RNAs is formulated in a separate nanoparticle formulation.

81. 81. The composition of any one of claims 61 to 80, wherein the first RNA and the second RNA are formulated in separate populations of nanoparticles.

82. 82. The composition of any one of claims 61-81, wherein the first RNA and the second RNA are all co-formulated in the same nanoparticle formulation.

83. 83. The composition of any one of claims 61 to 82, wherein the first infectious agent is or comprises a coronavirus.

84. the one or more first RNAs (i) RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with the first coronavirus; and (ii) RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with the second coronavirus.

84. The composition of claim 83, comprising:

85. 85. The composition of any one of claims 61 to 84, wherein the one or more second RNAs comprise a plurality of second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent of different strains and / or variants thereof.

86. 86. The composition of any one of claims 61 to 85, wherein the one or more second RNAs comprise at least two second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent of a different strain and / or variant thereof.

87. 87. The composition of any one of claims 61 to 86, wherein the one or more second RNAs comprise at least three second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent of a different strain and / or variant thereof.

88. 88. The composition of any one of claims 61 to 87, wherein the one or more second RNAs comprise at least four second RNAs each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent of different strains and / or variants thereof.

89. 89. The composition of any one of claims 61 to 88, wherein the second infectious agent is or comprises a bacterial infectious agent.

90. 90. The composition of claim 89, wherein the bacterial infectious agent is Streptococcus pneumoniae.

91. 89. The composition of any one of claims 61 to 88, wherein the second infectious agent is or comprises a viral infectious agent.

92. 92. The composition of claim 91, wherein the second infectious agent is a viral infectious agent that causes an infectious respiratory disease.

93. 93. The composition of claim 92, wherein the viral infectious agent is or comprises an influenza virus, a pneumoviridae virus, or a Paramyxoviridae virus.

94. 94. The composition of claim 93, wherein the Pneumoviridae virus is respiratory syncytial virus (RSV).

95. 94. The composition of claim 93, wherein the infectious respiratory disease is or comprises influenza A, B and / or C viruses.

96. 96. The composition of claim 95, wherein the infectious respiratory disease is or comprises influenza A and / or B viruses.

97. the one or more second RNAs (i) at least one RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with influenza A virus; and (ii) at least one RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with influenza B virus; 97. The composition of claim 96, comprising:

98. the one or more second RNAs (i) at least two RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with different strains of influenza A virus; and (ii) at least two RNAs each containing a nucleotide sequence encoding one or more antigenic polypeptides associated with different strains of influenza B virus; 98. The composition of claim 96 or 97, comprising:

99. 99. The composition of any one of claims 95-98, wherein the antigenic polypeptide(s) associated with each influenza virus are independently a hemagglutinin (HA) polypeptide, a neuraminidase (NA) polypeptide, or a combination thereof or an immunogenic fragment thereof.

100. 100. The composition of any one of claims 96 to 99, wherein the influenza A and influenza B virus strain(s) are each predicted to be or are pandemic strains in the upcoming flu season, e.g., based on human serology data.

101. 101. The composition of any one of claims 96 to 100, wherein the strain(s) of influenza A virus is selected from H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N7 and H10N8 viruses.

102. 102. The composition of claim 101, wherein the strain(s) of influenza A virus is selected from H1N1, H3N2, H5N1, and H5N8 viruses.

103. 103. The composition of any one of claims 98-102, wherein the one or more second RNAs comprise an RNA comprising a nucleotide sequence encoding an antigenic polypeptide associated with an H1N1 virus.

104. 104. The composition of claim 103, wherein the H1N1 virus is A / Wisconsin / 588 / 2019.

105. The composition of claim 104, wherein the antigenic polypeptide associated with A / Wisconsin / 588 / 2019 is an HA polypeptide and comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

90.

106. The composition of claim 104 or 105, wherein the antigenic polypeptide associated with A / Wisconsin / 588 / 2019 is an HA polypeptide and the RNA encoding the HA polypeptide comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:

92.

107. 107. The composition of any one of claims 98-106, wherein the one or more second RNAs comprise an RNA comprising a nucleotide sequence encoding an antigenic polypeptide associated with an H3N2 virus.

108. 108. The composition of claim 107, wherein the H3N2 virus is A / Cambodia / e0826360 / 2020.

109. The composition of claim 108, wherein the antigenic polypeptide associated with A / Cambodia / e0826360 / 2020 is an HA polypeptide and comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

95.

110. 110. The composition of claim 108 or 109, wherein the antigenic polypeptide associated with A / Cambodia / e0826360 / 2020 is an HA polypeptide, and the RNA encoding the HA polypeptide comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:

92.

111. The composition of claim 107, wherein the H3N2 virus is A / Darwin / 6 / 2021.

112. The composition of claim 111, wherein the antigenic polypeptide associated with A / Darwin / 6 / 2021 is an HA polypeptide and comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

80.

113. The composition of claim 111 or 112, wherein the antigenic polypeptide associated with A / Darwin / 6 / 2021 is an HA polypeptide and the RNA encoding the HA polypeptide comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:

82.

114. The composition of any one of claims 98 to 113, wherein the one or more second RNAs comprise an RNA comprising a nucleotide sequence encoding an antigenic polypeptide associated with a B / Yamagata or B / Victoria lineage virus.

115. 115. The composition of claim 114, wherein the B / Victoria lineage influenza virus is B / Washington / 02 / 2019.

116. The composition of claim 115, wherein the antigenic polypeptide associated with B / Washington / 02 / 2019 is an HA polypeptide and comprises a sequence that is at least 85% identical to SEQ ID NO:

100.

117. The composition of claim 115 or 116, wherein the antigenic polypeptide associated with B / Washington / 02 / 2019 is an HA polypeptide, and the RNA encoding the HA polypeptide comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:

102.

118. 115. The composition of claim 114, wherein the B / Victoria lineage influenza virus is B / Austria / 1359417 / 2021.

119. The composition of claim 118, wherein the antigenic polypeptide associated with B / Austria / 1359417 / 2021 is an HA polypeptide and comprises a sequence that is at least 85% identical to SEQ ID NO:

85.

120. The composition of claim 118 or 119, wherein the antigenic polypeptide associated with B / Austria / 1359417 / 2021 is an HA polypeptide, and the RNA encoding the HA polypeptide comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:

87.

121. The composition of claim 114, wherein the B / Yamagata lineage influenza virus is B / Phuket / 3073 / 2013.

122. The composition of claim 121, wherein the antigenic polypeptide associated with B / Phuket / 3073 / 2013 is an HA polypeptide and comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

105.

123. The composition of claim 121 or 122, wherein the antigenic polypeptide associated with B / Phuket / 3073 / 2013 is an HA polypeptide, and the RNA encoding the HA polypeptide comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:

107.

124. 124. The composition of any one of claims 61 to 123, wherein the first infectious agent is a coronavirus.

125. 125. The composition of claim 124, wherein the coronavirus is an alphacoronavirus, betacoronavirus, gammacoronavirus, or deltacoronavirus.

126. The composition of claim 125, wherein the coronavirus is a betacoronavirus.

127. The composition of claim 126, wherein the betacoronavirus is a sarbecovirus, merbecovirus, enbecovirus, nobecovirus, or hibecovirus.

128. 128. The composition of claim 127, wherein the sarbecovirus is SARS-CoV-1 or SARS-CoV-2.

129. The composition of claim 128, wherein the sarbecovirus is SARS-CoV-2.

130. The composition of claim 127, wherein the Merbecovirus is MERS-CoV.

131. 130. The composition of claim 129, wherein said one or more first RNAs comprise RNAs comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a SARS-CoV-2 variant that is prevalent in a relevant population at the time of administration or that has been identified as a variant of concern.

132. 130. The composition of claim 129, wherein the one or more first RNAs comprise RNAs comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with an Omicron SARS-CoV-2 variant (e.g., a BA.1, BA.2, BA.4 / 5, or XBB.1.5 variant).

133. the one or more first RNAs (i) RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a first SARS-CoV-2 strain, wherein the first SARS-CoV-2 strain is a SARS-CoV-2 ancestral strain (Wuhan strain); and (ii) RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second SARS-CoV-2 variant, wherein the second SARS-CoV-2 is a variant of the SARS-CoV-2 ancestral strain and is circulating in a relevant population at the time of administration or has been identified as a variant of concern.

130. The composition of claim 129, comprising:

134. the one or more first RNAs (i) an RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with the first SARS-CoV-2 variant; and (ii) an RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second SARS-CoV-2 variant; wherein said first and said second SARS-CoV-2 variants are each identified as prevalent or variants of concern in a relevant population at the time of administration.

135. 135. The composition of claim 133 or 134, wherein the second SARS-CoV-2 variant is an Omicron variant of SARS-CoV-2.

136. 136. The composition of claim 135, wherein the Omicron variant of SARS-CoV-2 is or comprises an Omicron BA.1, BA.2, BA.4 / 5, or XBB.1.5 variant.

137. 137. The composition of any one of claims 124-136, wherein the antigenic polypeptide(s) associated with the coronavirus is a spike (S) polypeptide, or an immunogenic fragment or variant thereof.

138. 138. The composition of claim 137, wherein the S polypeptide is a pre-fusion stabilized S polypeptide.

139. 139. The composition of claim 138, wherein the pre-fusion stabilized S polypeptide comprises at least two proline substitutions.

140. 140. The composition of claim 139, wherein the two proline substitutions comprise proline residues at positions corresponding to residues 986 and 987 of SEQ ID NO:

1.

141. 141. The composition of any one of claims 138-140, wherein the pre-fusion stabilized S polypeptide comprises at least six proline substitutions.

142. 142. The composition of claim 141, wherein four of the at least six substitutions include proline residues at positions corresponding to residues 817, 892, 899 and 942 of SEQ ID NO:

1.

143. 133. The composition of claim 132, wherein the RNA encoding the one or more antigenic polypeptides associated with the Omicron SARS-CoV-2 variant encodes an S protein associated with the XBB.1.5 strain and comprising an amino acid sequence at least 85% identical to SEQ ID NO:

129.

144. 143. The composition of any one of claims 133-142, wherein the RNA encoding the one or more antigenic polypeptides associated with the SARS-CoV-2 ancestral strain encodes an S protein associated with the Wuhan strain and comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:

7.

145. The composition of claim 144, wherein the RNA encoding SEQ ID NO:7 comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:

9.

146. 146. The composition of any one of claims 133-145, wherein the RNA encoding the one or more antigenic polypeptides associated with the second SARS-CoV-2 variant encodes an S protein associated with the BA.4 / 5 variant and comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:

69.

147. The composition of claim 146, wherein the RNA encoding SEQ ID NO:69 comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:

70.

148. the one or more first RNAs (a) an RNA comprising a nucleotide sequence encoding a SARS-CoV-2 spike (S) polypeptide from an omicron variant of SARS-CoV-2 (e.g., a BA.1, BA.2, BA.4 / 5, or XBB.1.5 variant); or (b) RNA comprising a nucleotide sequence encoding a SARS-CoV-2 spike (S) polypeptide from a SARS-CoV-2 ancestral strain (Wuhan strain) and RNA comprising a nucleotide sequence encoding a SARS-CoV-2 spike (S) polypeptide from an omicron variant of SARS-CoV-2 (e.g., a BA.1, BA.2, BA.4 / 5, or XBB.1.5 variant). Including; the one or more second RNAs (i) RNA comprising a nucleotide sequence encoding an HA polypeptide from an influenza A / H1N1 virus; (ii) RNA comprising a nucleotide sequence encoding an HA polypeptide from an influenza A / H3N2 virus; (iii) RNA comprising a nucleotide sequence encoding an HA polypeptide from an influenza B / Victoria lineage virus; and (iv) RNA comprising a nucleotide sequence encoding an HA polypeptide from influenza B / Yamagata virus. The composition of any one of claims 61 to 147, comprising:

149. 149. The composition of claim 148, wherein the H1N1 virus is A / Wisconsin / 588 / 2019.

150. The composition of claim 149, wherein the HA polypeptide associated with A / Wisconsin / 588 / 2019 comprises an array that is at least 85% identical to SEQ ID NO:

90.

151. The composition of claim 149 or 150, wherein the RNA comprising a nucleotide sequence encoding an HA polypeptide associated with A / Wisconsin / 588 / 2019 comprises a sequence that is at least 85% identical to SEQ ID NO:

92.

152. The composition of any one of claims 148 to 151, wherein the H3N2 virus is A / Cambodia / e0826360 / 2020.

153. The composition of claim 152, wherein the HA polypeptide associated with A / Cambodia / e0826360 / 2020 comprises an sequence that is at least 85% identical to SEQ ID NO:

95.

154. The composition of claim 152 or 153, wherein the first RNA comprising a sequence encoding an HA polypeptide associated with A / Cambodia / e0826360 / 2020 comprises a sequence that is at least 85% identical to SEQ ID NO:

97.

155. The composition of any one of claims 148 to 154, wherein the B / Victoria lineage influenza virus is B / Washington / 02 / 2019.

156. The composition of claim 155, wherein the HA polypeptide associated with B / Washington / 02 / 2019 comprises an array that is at least 85% identical to SEQ ID NO:

100.

157. The composition of claim 155 or 156, wherein the RNA comprising a nucleotide sequence encoding an HA polypeptide associated with B / Washington / 02 / 2019 comprises a sequence that is at least 85% identical to SEQ ID NO:

102.

158. The composition of any one of claims 148 to 157, wherein the B / Yamagata lineage influenza virus is B / Phuket / 3073 / 2013.

159. The composition of claim 158, wherein the HA polypeptide associated with B / Phuket / 3073 / 2013 comprises an array that is at least 85% identical to SEQ ID NO:

105.

160. The composition of claim 158 or 159, wherein the RNA comprising a sequence encoding an HA polypeptide associated with B / Phuket / 3073 / 2013 comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:

107.

161. 161. The composition of any one of claims 148-160, wherein the S polypeptide associated with the Wuhan strain comprises a sequence that is at least 85% identical to SEQ ID NO:

7.

162. The composition of any one of claims 148-161, wherein the RNA comprising a nucleotide sequence encoding an S polypeptide associated with the Wuhan strain comprises a sequence that is at least 85% identical to SEQ ID NO:

70.

163. 163. The composition of any one of claims 148-162, wherein the Omicron variant is the BA.4 / 5 variant.

164. 164. The composition of claim 163, wherein the S polypeptide associated with the BA.4 / 5 Omicron variant comprises a sequence at least 85% identical to SEQ ID NO:

69.

165. 165. The composition of claim 163 or 164, wherein the RNA comprising a sequence encoding the S polypeptide associated with the BA.4 / 5 Omicron variant comprises a sequence at least 85% identical to SEQ ID NO:

70.

166. 161. The composition of any one of claims 148-160, wherein the Omicron variant is an XBB.1.5 variant.

167. 167. The composition of claim 166, wherein the S polypeptide associated with the XBB.1.5 omicron variant comprises a sequence at least 85% identical to SEQ ID NO:

129.

168. The composition of claim 166 or 167, wherein the RNA comprising a sequence encoding the S polypeptide associated with the XBB.1.5 omicron variant comprises a sequence at least 85% identical to SEQ ID NO:

130.

169. At least one of the non-coding elements (i) a 5'-UTR sequence that is or includes a modified human alpha globin 5'-UTR; (ii) a 3′-UTR sequence that is or includes a first sequence from an amino terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; (iii) a polyA sequence comprising at least 100 A nucleotides, wherein the first RNA and the second RNA each do not comprise a stretch of at least 30 consecutive C nucleotides between the 3′ UTR and the polyA sequence; (iv) a polyA sequence comprising a staggered sequence of A nucleotides, optionally comprising 30 adenine nucleotides (SEQ ID NO:174) followed by 70 adenine nucleotides (SEQ ID NO:175), wherein the 30 adenine nucleotides (SEQ ID NO:174) and the 70 adenine nucleotides (SEQ ID NO:175) are separated by a linker sequence; or (v) a 5′ cap comprising a Cap1 structure and a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA; is or contains; (a) The Cap1 structure is m7(3'OMeG)(5')ppp(5')(2'OMeA １ ) pG ２ where A １ is the +1 position of the RNA, and G ２ is the +2 position of the RNA; (b) the cap adjacent sequence is A of the Cap1 structure １ and G ２ and A at positions +3, +4 and +5 of said RNA ３ N ４ N ５ and wherein N ４ and N ５ are each independently selected from A, G, C, and U; The composition according to any one of claims 47 to 60 and 62 to 77.

170. At least one of the same non-coding elements (i) a 5'-UTR sequence that is or includes a modified human alpha globin 5'-UTR; (ii) a 3′-UTR sequence that is or includes a first sequence from an amino terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; (iii) a polyA sequence comprising at least 100 A nucleotides, wherein the first RNA and the second RNA each do not comprise a stretch of at least 30 consecutive C nucleotides between the 3′ UTR and the polyA sequence; (iv) a polyA sequence comprising a staggered sequence of A nucleotides, optionally comprising 30 adenine nucleotides (SEQ ID NO:174) followed by 70 adenine nucleotides (SEQ ID NO:175), wherein the 30 adenine nucleotides (SEQ ID NO:174) and the 70 adenine nucleotides (SEQ ID NO:175) are separated by a linker sequence; and (v) a 5′ cap comprising a Cap1 structure and a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA; is or contains; (a) The Cap1 structure is m7(3'OMeG)(5')ppp(5')(2'OMeA １ ) pG ２ where A １ is the +1 position of the RNA, and G ２ is the +2 position of the RNA; (b) the cap adjacent sequence is A of the Cap1 structure １ and G ２ and A at positions +3, +4 and +5 of said RNA ３ N ４ N ５ and wherein N ４ and N ５ are each independently selected from A, G, C, and U; The composition of any one of claims 47 to 169.

171. Each of the RNAs is (i) a 5' cap, optionally comprising a cap1 structure; (ii) cap-proximal sequence; (iii) a 5'UTR sequence, optionally a modified human alpha globin 5'-UTR; (iv) a 3′UTR sequence, optionally comprising a first sequence from an amino terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; and / or (v) a polyA sequence optionally comprising 30 adenine nucleotides (SEQ ID NO:174) followed by 70 adenine nucleotides (SEQ ID NO:175), wherein the 30 adenine nucleotides (SEQ ID NO:174) and the 70 adenine nucleotides (SEQ ID NO:175) are separated by a linker sequence. The composition of any one of claims 1 to 46, comprising:

172. The composition of any one of claims 47 to 171, wherein the 5' cap, the cap proximal sequence, the 5' UTR sequence, the 3' UTR sequence, and the poly A sequence are in 5' to 3' order.

173. Each of the RNAs is ２ ７，３’－Ｏ Gppp (m １ ２’－Ｏ 173. The composition of any one of claims 1 to 172, comprising a 5'-cap that is or comprises ApG.

174. 174. The composition of any one of claims 47-169 and 171-173, wherein the 5'UTR comprises or consists of a human alpha globin 5'-UTR.

175. The composition of claim 174, wherein the human alpha globin 5'-UTR comprises SEQ ID NO:

12.

176. 176. The composition of any one of claims 47-169 and 171-175, wherein the 3'UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrially encoded 12S ribosomal RNA.

177. 177. The composition of claim 176, wherein the 3'UTR comprises or consists of a sequence according to SEQ ID NO:

13.

178. The composition of any one of claims 47 to 177, wherein the polyA tail sequence is a truncated polyA tail sequence.

179. The composition of claim 178, wherein the interrupted poly-A tail sequence comprises 30 adenine nucleotides (SEQ ID NO: 174) followed by 70 adenine nucleotides (SEQ ID NO: 175), and the 30 adenine nucleotides (SEQ ID NO: 174) and the 70 adenine nucleotides (SEQ ID NO: 175) are separated by a linker sequence.

180. 180. The composition of claim 179, wherein the interrupted poly-A tail sequence comprises or consists of a ribonucleic acid sequence according to SEQ ID NO:

14.

181. The composition of any one of claims 47 to 180, wherein the sequence at the 3' end of the 3'UTR (e.g., the sequence immediately adjacent to the sequence encoding the antigenic polypeptide) is CUCGAG or GGAUUCCGAU.

182. 182. The composition of any one of claims 1-181, wherein each said RNA in said composition comprises modified uridines in place of all uridines.

183. 183. The composition of claim 182, wherein each of said modified uridines is an N1-methyl-pseudouridine.

184. 184. The composition of any one of claims 47-183, wherein the first RNA and the second RNA are present in a mass ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:

1.

185. The composition of any one of claims 1 to 184, wherein each of the RNAs in the composition is formulated in a nanoparticle.

186. 186. The composition of any one of claims 47-185, wherein all of the first RNAs are co-formulated together in the same nanoparticle population, all of the second RNAs are co-formulated together in the same nanoparticle population, and the first RNA and the second RNA are formulated in separate nanoparticle populations.

187. 187. The composition of any one of claims 47-186, wherein said first RNA and said second RNA are all co-formulated together in the same population of nanoparticles.

188. (a) each of said RNAs encoding an antigenic polypeptide of influenza A virus is co-formulated in a first population of nanoparticles, and each of said RNAs encoding an antigenic polypeptide of influenza B virus is co-formulated in a second population of nanoparticles; (b) each of said RNAs encoding an influenza virus antigenic polypeptide is formulated into a separate population of nanoparticles; or (c) each of the RNAs encoding an antigenic polypeptide of influenza A virus is co-formulated in a first population of nanoparticles, and each of the RNAs encoding an antigenic polypeptide of influenza B virus is formulated in a separate nanoparticle; The composition of any one of claims 1 to 60 and 96 to 186.

189. 189. The composition of any one of claims 185-188, wherein the nanoparticle comprises a lipid nanoparticle, a polyplex (PLX), a lipidated polyplex (LPLX), a liposome, or a polysaccharide nanoparticle.

190. 190. The composition of claim 189, wherein the nanoparticles comprise lipid nanoparticles.

191. The composition of claim 190, wherein each of the lipid nanoparticles comprises a cationizable lipid, one or more neutral lipids, and a polymer-bound lipid.

192. The composition of claim 191, wherein the polymer-conjugated lipid comprises a PEG-conjugated lipid.

193. The composition of any one of claims 186 to 192, wherein the nanoparticles have an average diameter of about 50 to 150 nm.

194. (a) one or more third RNAs, each comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a third infectious agent that is different from the first infectious agent and the second infectious agent; or (b) one or more polypeptides of a third infectious agent The composition of any one of claims 1 to 193, further comprising:

195. 195. The composition of claim 194, wherein the third infectious agent is a respiratory virus (e.g., a respiratory virus that is not SARS-CoV-2 or an influenza virus).

196. 196. The composition of claim 195, wherein the third infectious agent is respiratory syncytial virus (RSV).

197. (i) the composition comprises one or more RNAs, each encoding an RSV polypeptide; or (ii) the composition comprises one or more RSV polypeptides; The composition of claim 196.

198. (i) the composition comprises one or more RNAs, each encoding an RSV F protein, a variant thereof, or an immunogenic fragment of an RSV F protein or a variant thereof; or (ii) the composition comprises one or more RSV F proteins, immunogenic variants thereof, or immunogenic fragments of the RSV F protein or variants thereof; The composition of claim 197.

199. (i) the composition comprises one or more RNAs, each encoding a polypeptide of an RSV subtype A virus (e.g., an F protein of an RSV subtype A virus, a variant thereof, or an immunogenic fragment of an F protein or variant thereof of an RSV subtype B virus), and one or more RNAs, each encoding a polypeptide of an RSV subtype B virus (e.g., an F protein of an RSV subtype B virus, a variant thereof, or an immunogenic fragment of an F protein or variant thereof of an RSV subtype B virus); or (ii) the composition comprises one or more polypeptides of an RSV subtype A virus (e.g., an F protein, a variant thereof, or an immunogenic fragment of an F protein or a variant thereof), and one or more polypeptides of an RSV subtype B virus (e.g., an F protein, a variant thereof, or an immunogenic fragment of an F protein or a variant thereof); 199. The composition of claim 197 or 198.

200. 200. The composition of claim 198 or 199, wherein the RSV F protein, the variant, or the immunogenic fragment is stabilized in pre-fusion confirmation.

201. 194-200. The composition of any one of items 194-200, comprising Arexvy™ or Abrysvo™.

202. A pharmaceutical composition comprising the composition of any one of claims 1 to 201 and at least one pharmaceutically acceptable excipient.

203. 203. The pharmaceutical composition of claim 202, comprising a cryoprotectant, optionally wherein the cryoprotectant is or comprises sucrose.

204. The medicament comprises an aqueous buffer solution, optionally the aqueous buffer solution comprises Tris base, Tris HCl, NaCl, KCl, Na ２ HPO ４ , and K.H. ２ P.O. ４ 204. The pharmaceutical composition of claim 202 or 203, comprising one or more of:

205. 205. The pharmaceutical composition of any one of claims 202 to 204, formulated to provide a dose of 100 μg or less of total RNA.

206. 206. The pharmaceutical composition of claim 205, formulated to provide a dose of 90 μg of total RNA.

207. 206. The pharmaceutical composition of claim 205, formulated to provide a dose of 60 μg of total RNA.

208. 207. The pharmaceutical composition of claim 206, formulated to provide a 30 μg dose of said one or more first RNAs and a 60 μg dose of said one or more second RNAs.

209. 207. The pharmaceutical composition of claim 206, formulated to provide a 60 μg dose of said one or more first RNAs and a 30 μg dose of said one or more second RNAs.

210. 208. The pharmaceutical composition of claim 207, formulated to provide a 30 μg dose of said one or more first RNAs and a 30 μg dose of said one or more second RNAs.

211. 209. The pharmaceutical composition of claim 206 or 208, comprising four second RNAs each comprising a nucleotide sequence encoding an antigenic polypeptide associated with a different influenza virus, wherein the pharmaceutical composition is formulated to provide a dose of 15 μg of each second RNA.

212. 210. The pharmaceutical composition of claim 207 or 209, comprising four second RNAs each comprising a nucleotide sequence encoding an antigenic polypeptide associated with a different influenza virus, wherein the pharmaceutical composition is formulated to provide a dose of 7.5 μg of each second RNA.

213. 213. The pharmaceutical composition of any one of claims 206-208 and 210-212, comprising two first RNAs each comprising a nucleotide sequence encoding an antigenic polypeptide associated with a different coronavirus virus, wherein the pharmaceutical composition is formulated to provide a dose of 15 μg of each of the first RNAs.

214. 210. The pharmaceutical composition of claim 209, comprising two first RNAs each comprising a nucleotide sequence encoding an antigenic polypeptide associated with a different coronavirus virus, wherein the pharmaceutical composition is formulated to provide a dose of 30 μg of each of the first RNAs.

215. 216. A method comprising administering to a subject one or more doses of the composition of any one of claims 1 to 201 or one or more doses of the pharmaceutical composition of any one of claims 202 to 214.

216. 216. The method of claim 215, wherein the method is a method for treating coronavirus and influenza diseases.

217. The method comprises: (i) Prevent coronavirus disease and influenza disease; or (ii) induce an immune response against coronavirus and / or influenza virus The method of claim 215,

218. 218. The method of any one of claims 215 to 217, wherein each of said one or more doses of said composition or each of said one or more doses of said pharmaceutical composition is co-administered with a vaccine against a third infectious agent.

219. 219. The method of claim 218, wherein the third infectious agent is a virus that can cause a respiratory disease.

220. 220. The method of claim 219, wherein the third infectious agent is RSV.

221. 221. The method of claim 220, wherein the vaccine against the third infectious agent is Arexvy™ or Abrysvo™.

222. the vaccine against the third infectious agent is mixed with the one or more doses of the composition or the one or more doses of the pharmaceutical composition immediately prior to administration to the subject; or the vaccine against the third infectious agent is administered separately from the one or more doses of the composition or the one or more doses of the pharmaceutical composition (e.g., the vaccine against the third infectious agent and the one or more doses of the composition or the one or more doses of the pharmaceutical composition are administered to the subject at separate injection sites); 222. The method of any one of claims 218 to 221.

223. Use in the treatment of coronavirus and influenza diseases, comprising administering to said subject one or more doses of said composition or said pharmaceutical composition. A composition according to any one of claims 1 to 201 or a pharmaceutical composition according to any one of claims 202 to 214 for:

224. (a) preventing coronavirus disease and influenza disease; or (b) Inducing an immune response against coronaviruses and influenza viruses comprising administering to said subject one or more doses of said composition or said pharmaceutical composition. A composition according to any one of claims 1 to 201 or a pharmaceutical composition according to any one of claims 202 to 214 for:

225. the method or use comprises administering to the subject two or more doses of the composition or pharmaceutical composition; 226. The method of any one of claims 215 to 222, or the composition or pharmaceutical composition for use according to claim 223 or 224.

226. the two doses are administered at least about 21 days apart; 226. The method of claim 225, or the composition or pharmaceutical composition for use.

227. 227. The method of any one of claims 215 to 222, or the composition or pharmaceutical composition for use of claim 225 or 226, wherein said method or said use comprises administering to said subject three or more doses of said composition or said pharmaceutical composition.

228. the subject has previously been exposed to a coronavirus and / or influenza virus (e.g., by vaccination and / or by infection); 226. The method of any one of claims 215 to 222, or the composition or pharmaceutical composition for use according to claim 223 or 224.

229. the method or use induces an immune response in the subject against one or more coronaviruses and one or more influenza viruses.

229. The method, or the composition or pharmaceutical composition for use according to any one of claims 215 to 228.

230. the immune response comprises a B cell response; 230. The method of claim 229, or the composition or pharmaceutical composition for use.

231. the B cell response comprises the production of antibodies directed against the one or more antigens.

231. The method of claim 230, or the composition or pharmaceutical composition for use.

232. 232. The method of claim 230 or 231, or the composition or pharmaceutical composition for use, wherein the immune response comprises a T cell response.

233. the T cell response is or comprises a CD4+ T cell response; 233. The method of claim 232, or the composition or pharmaceutical composition for use.

234. the T cell response is or comprises a CD8+ T cell response; 234. The method, or the composition or pharmaceutical composition for use according to claim 232 or 233.

235. Use of a composition according to any one of claims 1 to 201 or a pharmaceutical composition according to any one of claims 202 to 214 in the treatment of coronavirus and influenza diseases in a subject.

236. Use of a composition according to any one of claims 1 to 201 or a pharmaceutical composition according to any one of claims 202 to 215 in the prevention of coronavirus and influenza diseases in a subject.

237. 216. Use of a composition according to any one of claims 1 to 201 or a pharmaceutical composition according to any one of claims 202 to 215 in inducing an immune response against one or more coronaviruses and one or more influenza viruses in a subject.

238. 1. A method of inducing an immune response to a first infectious agent and a second infectious agent, comprising: (i) a first nanoparticle (e.g., LNP)-formulated RNA comprising a nucleotide sequence encoding an antigenic polypeptide associated with a first infectious agent; and (ii) a second nanoparticle (e.g., LNP)-formulated RNA comprising a nucleotide sequence encoding an antigenic polypeptide associated with a second infectious agent. administering the immune response induced against each of the first and second infectious agents is greater than the immune response induced when the nanoparticles are administered individually. The method.

239. 1. A method for reducing the amount of first nanoparticle (e.g., LNP)-formulated RNA required to generate an immune response to a first infectious agent, comprising: the RNA of the first nanoparticle-formulated RNA comprises a nucleotide sequence encoding one or more antigenic polypeptides associated with the first infectious agent; the method includes co-administering a second nanoparticle (e.g., LNP)-formulated RNA comprising a nucleotide sequence encoding one or more antigenic polypeptides associated with a second infectious agent; the first infectious agent is different from the second infectious agent; The method.

240. 1. A composition comprising: one or more RNAs, each encoding a polypeptide of a first infectious agent; and One or more polypeptides of a second infectious agent The composition comprising:

241. 241. The composition of claim 240, wherein the first infectious agent is a coronavirus.

242. The composition of claim 241, wherein the coronavirus is SARS-CoV-2 virus.

243. 243. The composition of claim 242, wherein said one or more RNAs each encode a SARS-CoV-2 S protein, a variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or a variant thereof.

244. 244. The composition of claim 243, comprising one or more RNAs, each encoding a SARS-CoV-2 S protein of the Wuhan strain or a SARS-CoV-2 variant (e.g., an Omicron variant (e.g., an Omicron BA.1, BA.2, BA.4 / 5, or XBB.1.5 variant (e.g., an RNA described herein))), a variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or variant thereof.

245. 245. The composition of any one of claims 240 to 244, wherein the second infectious agent is an influenza virus.

246. The composition of claim 245, wherein the composition comprises one or more polypeptides of one or more influenza viruses (e.g., one or more polypeptides of two or more influenza virus strains (e.g., one or more polypeptides of four or more influenza virus strains that are circulating or predicted to be circulating in the jurisdiction of interest)).

247. 247. The composition of claim 245 or 246, comprising a commercially available influenza virus (e.g., a recombinant commercially available influenza virus described herein).

248. The composition of claim 247, wherein the commercially available influenza virus is Flublok.

249. 245. The composition of any one of claims 239-244, wherein the second infectious agent is RSV.

250. 250. The composition of claim 249, comprising one or more polypeptides associated with a first RSV subtype and one or more polypeptides of a second RSV subtype.

251. 251. The composition of claim 249 or 250, wherein the polypeptide of the second infectious agent is an RSV F protein, a variant thereof, or an immunogenic fragment of either the RSV F protein or a variant thereof.

252. The composition of claim 251, wherein the RSV F protein, the variant thereof, or the immunogenic fragment thereof comprises one or more mutations that stabilize pre-fusion confirmation of the F protein.

253. The composition of claim 252, comprising Arexvy™ or ABRYSVO™.

254. The composition of any one of claims 239-253, further comprising one or more polypeptides of a third infectious agent.

255. one or more RNAs, each encoding one or more coronavirus polypeptides (e.g., SARS-CoV-2 S protein, variants thereof, or immunogenic fragments of any of the above); one or more polypeptides of one or more influenza viruses; and One or more polypeptides of one or more RSV The composition of claim 254, comprising:

256. RNA encoding a SARS-CoV-2 S protein of the Omicron variant (e.g., RNA encoding an S protein of the Omicron BA.1, BA.4 / 5, or XBB.1.5 variant described herein); Recombinant influenza vaccines (e.g., those described herein (e.g., FluBlok vaccines)); and RSV vaccines comprising a pre-fusion stabilized F protein or variants or immunogenic fragments thereof (e.g., RSV vaccines described herein (e.g., Arexvy™ or ABRYSVO™)) The composition of claim 255, comprising:

257. A combination drug, A SARS-CoV-2 vaccine comprising one or more mRNAs encoding a prefusion stabilized SARS-CoV-2 spike protein or variants thereof; and (a) (i) one or more mRNAs encoding the HA protein of an influenza virus; or (ii) one or more HA polypeptides an influenza vaccine, and / or (b) an RSV vaccine comprising one or more prefusion stabilized RSV F proteins or variants or immunogenic fragments thereof; The combination comprising:

258. The combination of claim 257, wherein the one or more mRNAs encoding a pre-fusion stabilized SARS-CoV-2 spike protein or variants thereof are formulated as LNPs.

259. The combination of claim 257 or 258, wherein the one or more mRNAs encoding the HA protein of an influenza virus are formulated as LNPs.

260. The combination of any one of claims 257 to 259, wherein (1) the SARS-CoV-2 vaccine and (2) the influenza vaccine or the RSV vaccine are provided in separate containers (e.g., vials or syringes).

261. The combination of any one of claims 257 to 259, wherein (1) the SARS-CoV-2 vaccine, and (2) the influenza vaccine or the RSV vaccine are provided in a single container (e.g., a vial or syringe).

262. The combination according to any one of claims 257 to 261, comprising the SARS-CoV-2 vaccine, the influenza vaccine, and the RSV vaccine.

263. The combination of claim 262, wherein the SARS-CoV-2 vaccine, the influenza vaccine, and the RSV vaccine are provided in a single container (e.g., a vial or syringe).

264. The combination of claim 262, wherein the SARS-CoV-2 vaccine, the influenza vaccine, and the RSV vaccine are provided in separate containers (e.g., separate vials and / or syringes).

265. (a) the SARS-CoV-2 vaccine and the influenza vaccine are provided in a single container and the RSV vaccine is provided in a separate container; or (b) the SARS-CoV-2 vaccine and the RSV vaccine are provided in a single container, and the influenza vaccine is provided in a separate container; The combination of claim 262.

266. 266. The combination of any one of claims 257-265, wherein the SARS-CoV-2 vaccine is BNT162b2 (e.g., a monovalent or bivalent vaccine described herein).

267. 267. The combination of any one of claims 257 to 266, wherein the influenza vaccine is a recombinant influenza vaccine (such as those described herein (e.g., FluBlok vaccine)); or comprises an inactivated influenza virus (e.g., Fluzone).

268. 268. The combination of any one of claims 257-267, wherein the RSV vaccine comprises a pre-fusion stabilized F protein or a variant or immunogenic fragment thereof (e.g., an RSV vaccine described herein (e.g., Arexvy™ or ABRYSVO™)).

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