mRNA vaccines

Abstract

The present specification relates to a vaccine comprising an mRNA polynucleotide encoding an antigen derived from an infectious microorganism and an amphipathic cell membrane-penetrating RALA peptide.

Description

[Technical field]

[0001] The present disclosure relates to vaccines. In particular, the present disclosure relates to vaccines comprising an mRNA polynucleotide encoding an antigen derived from an infectious microorganism, in particular an HPV antigen, and an amphipathic cell membrane penetrating peptide derived from the RALA family of peptides. The present disclosure further relates to methods for preparing such vaccines and their use in therapy. [Background technology]

[0002] A vaccine involves introducing an antigen, a substance that the immune system attacks, into the body either directly or indirectly, typically by injection. Vaccines fall into two broad categories: preventative and therapeutic. Prophylactic vaccines are designed to build immunity - the antigen stimulates the body's immune system to produce antibodies, triggering a cellular response to protect against future infections and / or disease. Therapeutic vaccines are designed to help the body do a better job of fighting infections and / or disease that you already have.

[0003] To generate a therapeutic vaccine, a specific type of immune response, known as a CD8+ response, is required. CD8+ T cells are the cytotoxic T cells of the immune system. They are triggered upon encountering a pathogenic stimulus and kill cells presenting foreign immunogenic antigen fragments. CD8+ T cells are a key component of the cellular immune response and play a key role in controlling viral infections. CD8+ T cells can not only recognize and destroy infected cells, but also inhibit viral binding and transcription. CD8+-mediated responses are believed to be essential to treat active, latent, and persistent viral pathogens (e.g., Cosma et al., 2018 Apr. 27;7:F1000 Faculty Rev-508; Beura et al., Immunity, 2018 Feb. 20;48(2):327-338.e5; and McMichael, Cold Spring Harb. Perspect. Biol., 2018 Sep. 4;10(9):a029124) and require specific delivery of antigen to antigen-presenting cells (Gessani et al., Toxins (Basel), 2014 May 26;6(6):1696-723). Current antigen protein vaccines delivered by intramuscular injection utilize a CD4+-mediated approach with adjuvants to generate either a cytolytic CD4+ immune response, or antigen cross-presentation to elicit a CD8+ response. CD4+ T cells are "helper" cells that regulate immune responses to specific antigens and act more indirectly than CD8+ T cells. Vaccines that induce CD8+-dominant responses remain elusive due to the inability to induce cell-mediated innate immunity and deliver antigens to the appropriate location within the appropriate immune cells.

[0004] There are several types of vaccines, including killed vaccines, live attenuated vaccines, messenger ribonucleic acid (mRNA) vaccines, subunit, recombinant, polysaccharide, and conjugate vaccines, toxoid vaccines, and viral vector vaccines. These different vaccines use different methods to introduce antigens into the body.

[0005] mRNA vaccines convey instructions to cells to make antigens, which in turn trigger an immune response. The selected mRNA codes for the antigen of the infection and / or disease to be treated. mRNA vaccines have several advantages compared to other types of vaccines, including shorter production times, and because the mRNA cannot be integrated into the cellular genome, they carry no risk of causing disease in the vaccinated person. However, unmodified synthetic mRNA is not stable and the mRNA itself is undesirable (e.g., CD4 + ) may stimulate activation of the immune response. Thus, chemical modifications at the ribose, RNA termini, and nucleobases may be required to improve stability and reduce immunogenicity (see, e.g., Gao et al., Acta. Biomater. 2021 Sep. 1; 131: 1-15).

[0006] Human papillomavirus (HPV) is the most common sexually transmitted disease, infecting an estimated 200 million people worldwide (WHO Global HPV Market Study 2018). Infection with HPV can result in genital warts, precancerous lesions, and cancer. Most HPV infections are subclinical, and over 90% of all new HPV infections are undetectable within two years, even without treatment. Unfortunately, some infections persist and lead to complications, and as a pathogen, HPV is responsible for 5% of all cancers worldwide, including 99% of all cervical cancers and 60% of all cutaneous non-melanoma cancers, such as basal and squamous cell (de Martel et al., New Microbiol. 2017 Apr;40(2):80-85 and Brianti et al., Int J Cancer. 2017 Aug 15;141(4):664-670). HPV-induced cervical cancer is the second most common cause of death in women, after breast cancer.

[0007] Of the more than 150 strains of HPV, 40 affect the genital area, but most do not pose a serious risk to health. A person can be infected with more than one strain of HPV at a time, and the strains are identified by a number. HPV strains are classified as low-risk or high-risk HPV. Infection with most low-risk HPV strains is asymptomatic, and these strains are not associated with cancer. Infection with high-risk HPV is more problematic and can result in cervical dysplasia and certain types of cancer. There are at least 12 high-risk strains of HPV, including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59, and two of these, HPV16 and HPV18, cause the majority of HPV-related cancers.

[0008] Current HPV vaccines are prophylactic and are designed to be administered at ages 11-14 to prevent infection (Cook et al., Pediatrics. 2018 Sep;142(3)). These vaccines are ineffective in people already infected with HPV, and even with universal prophylactic coverage, it will take at least another 20 years to achieve a significant reduction in HPV-related morbidity (Kumar et al., Med J Armed Forces India, 2015 Apr;71(2):171-7). Meanwhile, the incidence of cervical cancer is predicted to rise by 43% in the UK alone, with 17 new cases per 100,000 women by 2035 (British Gynaecological Cancer Society (BGCS) Cervical Cancer Guidelines: Recommendations for Practice). There is clearly a need for therapeutic vaccines, or vaccines that can act both therapeutically and preventatively, to address existing HPV, particularly HPV16 and HPV18 infections.

[0009] There are several known HPV proteins (L1, L2, E1, E2, E4, E5, E6 and E7) that can serve as antigens in vaccination approaches for the treatment of HPV infection. Prophylactic vaccines on the market utilize the major capsid (viral surface) protein L1 (Kirnbauer et al., Proc. Natl. Acad. Sci. USA, Dec. 15, 1992; 89(24), and Schiller et al., Vaccine, Aug. 6, 2018; 36(32 Pt A):4768-4773). However, L1 (and L2) proteins are inactivated or deleted during integration of the HPV genome into the cellular genome, and as a result, vaccines using L1 and L2 antigens are ineffective against HPV infection once HPV infection is established. The early proteins E1, E2, E4, E5, E6 and E7 are expressed for a longer period during the HPV life cycle and represent better targets for therapeutic vaccines, even against advanced HPV infections (Pal et al., Front Microbiol. 2019; 10: 3116).

[0010] The antigen delivery system used in mRNA vaccination plays a key role in determining the type of immune response. The carrier:mRNA ratio also plays a role - mRNA molecules are negatively charged polynucleotides and the carrier is required not only to protect the cargo from degradation but also to deliver the cargo to the correct target cells. Optimization of the carrier:mRNA ratio facilitates the generation of nanoparticles with suitable size and charge characteristics to ensure uptake by antigen presenting cells.

[0011] The RALA family of peptides are amphipathic peptides composed of repeating RALA units that can overcome biological barriers to gene delivery both in vitro and in vivo. The term "RALA" is used inconsistently in the literature, but typically refers to an amphipathic peptide or group of peptides composed of repeating RALA units, generally less than about 50 amino acid residues. Cohen-Avrahami et al. (J. Phys. Chem. B 2011, 115:10 189-1 097, and Colloids and Surfaces B: Biointerfaces 77 (201 0) 131-138) disclose an amphipathic 16-mer peptide called "RALA". Similar to McCarthy et al. (Journal of Controlled Release 189 (2014) 141-149), Faranack et al. (Biomacromolecules 2013, 14, 2033-2040) use the term "RALA" to describe the 30-mer RALA peptide, but for different 30-mer peptides. WO 2014 / 087023 and WO 2015 / 189205 have defined the term "RALA" as a general term for the group of peptides that are within the scope of the inventions described in these documents.

[0012] Peptides of the RALA family have been shown to be effective in binding genetic material such as plasmid DNA (McCarthy HO et al., J Control Release, 2014 Sep 10;189:141-9; and Ali AA et al., Nanomedicine, 2017 Apr;13(3):921-932), mRNA (Udhayakumar et al., Adv. Healthc. Mater. 2017 Jul;6(13)), siRNA (Mulholland EJ et al., J Control Release, 2019 Dec 28;316:53-65), and small molecules such as bisphosphonates (Jena LN et al., J. Nanobiotechnology. 2021 May 4;19(1):127), and calcium phosphate (Sathy BN et al., J. Mater. Chem. B. 2017 Mar 7;5(9):1753-1764).

[0013] The specific RALA peptide WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) is a 30 amino acid non-toxic peptide with a charge of +6 at physiological pH, which converts to a helical cell membrane-permeabilizing conformation of +8 in the acidic conditions found inside the endosomes of cells. When complexed in water with certain payloads, such as DNA and mRNA, it has been shown to be capable of spontaneous self-assembly into nanoparticles (McCarthy et al., 2014 and Udhayakumar et al., 2017). This pH-dependent change allows escape of the peptide and cargo inside the cell, resulting in highly efficient cell entry and cargo delivery at a physiological pH of 7.4 outside the cell without any toxicity. The nanoparticles have been shown to be very stable over a wide range of temperatures and over time, and the RALA peptide does not provoke an immunological response by itself.

[0014] Udhayakumar et al. (Udhayakumar et al., 2017) assessed the ability of this RALA peptide in an mRNA nanocomplex to prime CD8+ T cells by immunizing mice with RALA-complexed mRNA encoding the model antigen ovalbumin, the major protein found in egg white. Udhayakumar et al. found that multiple different mRNA nucleotide modifications and a very high N / P ratio (i.e., the ratio of positively charged nitrogen atoms in the peptide to negatively charged phosphates in the mRNA backbone) were required for optimal complexation of Ova mRNA and efficient CD8 T cell priming. + We conclude that it is required to enable T cell priming.

[0015] Ali et al. (Ali et al., 2017) investigated DNA vaccination against cervical cancer, delivering RALA-condensed E6 / E7 plasmid DNA nanoparticles by polymer microneedles. However, for DNA vaccines to work, the cargo must reach the nucleus, be transcribed into mRNA, and the protein translated. mRNA vaccines remove the transcription step and the need for the nucleic acid to reach the target cell nucleus.

[0016] Coolen et al. (Biomaterials, Vol. 195, 2019 al., pp. 23-37) investigated the ability of poly(lactic acid) nanoparticles conjugated with cell membrane-penetrating peptides to enhance mRNA-based vaccine expression in dendritic cells and induce their activation. However, the RALA peptide complexed with eGFP (green fluorescent protein) could not be efficiently transfected into DC2.4 dendritic cells and was not selected for further evaluation. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] WO 2014 / 087023 [Patent Document 2] WO 2015 / 189205 [Non-patent literature]

[0018] [Non-licensed document 1] Cosmaら, April 27, 2018;7:F1000 Faculty Rev-508,

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[0019] Described herein are vaccines that contain mRNA polynucleotides encoding antigens derived from infectious microorganisms, particularly HPV antigens, and specific RALA peptides. These vaccines provide intracellular delivery of the mRNA cargo, protecting it from degradation and promoting natural (CD4 + ) do not provoke an immune response. Furthermore, RALA-complexed mRNA nanoparticles can be easily lyophilized (while retaining functionality), are stable, are easily reconstituted, do not require cold chain storage, and are relatively inexpensive to manufacture.

[0020] overview This specification is presented in part as follows: i) an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; and ii) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; The present invention describes a vaccine comprising:

[0021] This specification also describes, in part, the vaccines described herein for use in therapy, particularly for use in the treatment of viral infections and in the treatment of cancer. [Brief description of the drawings]

[0022] [Figure 1] Figure 1 shows particle size (Z-average) plots and zeta potentials for RALA / E7 mRNA nanoparticles produced by manual formulation (compounds 001, 002, 003, 004 and 005) and a representative size distribution spectrum of nanoparticles containing 10 μg E6 and 10 μg E7 in N:P7 (compound 021). Results from Example 4. [Diagram 2]Figure 2 shows the accumulation of fluorescent signal in the skin-draining lymph nodes of C57BL / 6 mice vaccinated intradermally via the ear. The fluorescent signal from Cy5-EGFP mRNA complexed in N:P7 (compound 010) nanoparticles migrated out of the ear faster and was evident to a greater extent in the draining lymph nodes than the fluorescent signals from the other N:P ratios tested (compounds 006, 007, 008 and 009). Results from Example 5. [Diagram 3] Figure 3 shows Cy5 content from immune cells (CD11c+ dendritic cells and F4 / 80+ macrophages) from disaggregated lymph nodes. Results from Example 5. [Figure 4] Figure 4 shows that dendritic cells that take up composite 010 nanoparticles are of a migratory phenotype. Results from Example 5. [Diagram 5] Figure 5 shows E7 protein production by compounds 011, 012, 013, 014 and 015 after transfection into HEK-293 cells (analyzed by in-cell ELISA). Results from Example 6. [Figure 6] Figure 6 shows the results of an in vivo study to evaluate the IFNγ response of each of compounds 011, 012, 013, 014 and 015. Results from Example 6. [Figure 7] Figure 7 shows the number of IFNγ and IL-2 spot forming units (SFU) 48 hours after vaccination of C57BL / 6 mice with conjugates 013, 016 or 017 lyophilized using trehalose alone, mannose alone or a trehalose-mannose mixture (85% / 15%) in a two dose regimen. Results from Example 7. [Figure 8] Figure 8 shows intracellular IFNγ, IL-2 and TNFα content by flow cytometry. Results from Example 7. [Figure 9] Figure 9 shows IFNγ (Th1) and interleukin 4 (IL-4; Th2) content. Results from Example 7. [Figure 10]Figure 10 shows the number of IFNγ and IL-2 spot forming units (SFU). Results from Example 8. [Figure 11] Figure 11 shows IFNγ content. Results from Example 8. [Figure 12] Figure 12 shows tumor volumes. Results from Example 9. [Figure 13] Figure 13 shows the survival rate of mice treated with compounds 019, 020, 021, 022, 023 or 024. Results from Example 9. [Figure 14] Figure 14 shows tumor volumes. Results from Example 9. [Figure 15] Figure 15 shows the survival rate of mice treated with compounds 019, 020, 021, 022, 023 or 024. Results from Example 9. [Figure 16] Figure 16 shows the number of IFNγ spot forming units counted. Results from Example 10. [Figure 17] Figure 17 shows CD62L+ cells and E7 Dex+ cells. Results from Example 11. [Figure 18] Figure 18 shows the tumor volume of mice treated with compound 031. Results from Example 12. [Figure 19] Figure 19 shows the survival rate of mice treated with compound 031. Results from Example 12. [Figure 20] Figure 20 shows the immunogenicity of compounds 031, 033, 034, 035, 036 and 037 in vaccinated mice as determined using IFNγ ELISpot. Results from Example 13. [Figure 21] Figure 21 shows the immunogenicity of compound 035 in vaccinated mice as determined using IFNγ ELISpot. Results from Example 14. [Figure 22] Figure 22 shows the tumor volume of mice treated with compound 035. Results from Example 15. [Figure 23] Figure 23 shows the survival rate of mice treated with compound 035. Results from Example 15. [Figure 24]Figure 24 shows the tumor volume of mice treated with compound 035. Results from Example 16. [Diagram 25] Figure 25 shows the survival rate of mice treated with compound 035. Results from Example 16. [Figure 26] Figure 26 shows the tumor volume of mice treated with compounds 035 or 039. Results from Example 17. [Figure 27] Figure 27 shows the survival rate of mice treated with compounds 035 or 039. Results from Example 17. [Figure 28] Figure 28 shows the immunogenicity of compounds 035 or 040 in vaccinated mice as determined using IFNγ ELISpot. Results from Example 18. [Figure 29] Figure 29 shows the immunogenicity of compound 035 in vaccinated mice as determined using IFNγ ELISpot. Results from Example 19. [Diagram 30] FIG. 30 shows the uptake of compound 041 after intravenous delivery in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Asterisks in the figures indicate statistical significance.

[0024] Many embodiments of the present invention are detailed throughout this specification and will be apparent to those of skill in the art, and the present invention should not be construed as being limited to any of the embodiments listed.

[0025] "A" (or "an") means "at least one." In any embodiment where "a" is used to refer to a given material or element, "a" can mean one.

[0026] "Comprising" means that a given material or element may contain other materials or elements. In any embodiment where "comprising" is referred to, a given material or element may be formed of at least 10% w / w, at least 20% w / w, at least 30% w / w, or at least 40% w / w of that material or element. In any embodiment where "comprising" is referred to, "comprising" can also mean "consisting of" (or "consists of") or "consisting essentially of" (or "consists essentially of") a given material or element.

[0027] "Consisting of" or "consists of" means that a given material or element is formed entirely from that material or element. In any embodiment where "consisting of" or "consists of" is referred to, the given material or element may be formed from 100% w / w of that material or element.

[0028] "Consisting essentially of" or "consists essentially of" means that a given material or element consists almost entirely of that material or element. In any embodiment where "consisting essentially of" or "consists essentially of" is referred to, the given material or element may be formed from at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, at least 95% w / w, or at least 99% w / w of that material or element.

[0029] In any embodiment where "is" or "may be" is used to define a material or element, "is" or "may be" can mean that the material or element "consists of" or "consists essentially of" that material or element.

[0030] The embodiments may be combined.

[0031] The claims are embodiments.

[0032] mRNA polynucleotide A polynucleotide is a compound and / or substance that comprises a polymer of nucleotides (nucleotide monomers). The polynucleotides of the present disclosure function as messenger RNA (mRNA). "Messenger RNA" refers to any polynucleotide that encodes a polypeptide, both naturally occurring (wild-type) and non-naturally occurring, and that can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo.

[0033] The basic components of an mRNA molecule typically include at least one open reading frame, a 5' untranslated region (UTR), a 3'UTR, a 5' cap, and a polyA tail. The mRNA polynucleotides described herein function as mRNAs, but may differ from wild-type mRNAs in both functional and / or structural design features.

[0034] "5' untranslated region" (5'UTR) refers to the region of an mRNA immediately upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript that is translated by ribosomes) that does not encode a polypeptide.

[0035] "3' untranslated region" (3'UTR) refers to the region of an mRNA immediately downstream (ie, 3') from a stop codon (ie, a codon in the mRNA transcript that signals the end of translation) that does not encode a polypeptide.

[0036] A "poly-A tail" is a region of an mRNA downstream, e.g., immediately downstream (i.e., 3') from the 3'UTR, that contains multiple, consecutive adenosine monophosphates. A poly-A tail can contain 10-150 adenosine monophosphates. In one embodiment, a poly-A tail contains 50-150 adenosine monophosphates. In one embodiment, a poly-A tail contains 75-125 adenosine monophosphates. In one embodiment, a poly-A tail contains about 120 adenosine monophosphates. In one embodiment, a poly-A tail contains 120 adenosine monophosphates. In one embodiment, a poly-A tail contains about 80 adenosine monophosphates. In one embodiment, a poly-A tail contains 80 adenosine monophosphates. In relevant biological contexts (e.g., within a cell, in vivo), the poly-A tail functions to protect the mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, transport of the mRNA from the nucleus, and translation.

[0037] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, wherein the mRNA polynucleotide comprises between 200 and 5,000 nucleotides.

[0038] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, wherein the mRNA polynucleotide comprises between 200 and 4,000 nucleotides.

[0039] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, wherein the mRNA polynucleotide comprises between 200 and 3,000 nucleotides.

[0040] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, wherein the mRNA polynucleotide comprises between 200 and 1,000 nucleotides.

[0041] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, wherein the mRNA polynucleotide comprises between 500 and 1000 nucleotides.

[0042] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, wherein the mRNA polynucleotide comprises between 500 and 800 nucleotides.

[0043] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, wherein the mRNA polynucleotide comprises 753 nucleotides.

[0044] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises about 753 nucleotides.

[0045] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises 573 nucleotides.

[0046] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises about 573 nucleotides.

[0047] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises 703 nucleotides.

[0048] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises about 703 nucleotides.

[0049] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises 522 nucleotides.

[0050] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, the mRNA polynucleotide comprising about 522 nucleotides.

[0051] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises 2175 nucleotides.

[0052] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises about 2175 nucleotides.

[0053] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises 2211 nucleotides.

[0054] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, the mRNA polynucleotide comprising about 2211 nucleotides.

[0055] In one embodiment, the vaccine comprises an mRNA polynucleotide encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, comprising an open reading frame, a 5'UTR, a 3'UTR, a 5'cap, and a polyA tail.

[0056] In one embodiment, the vaccine comprises an mRNA polynucleotide encoding an antigen or immunogenic fragment thereof from an infectious microorganism, the polynucleotide comprising an open reading frame, a 5'UTR, a 3'UTR, a 5' cap and a polyA tail, the 5'UTR, 3'UTR and polyA tail comprising approximately 250-300 nucleotides.

[0057] In one embodiment, the vaccine comprises an mRNA polynucleotide encoding an antigen or immunogenic fragment thereof from an infectious microorganism, comprising an open reading frame, a 5'UTR, a 3'UTR, a 5' cap and a polyA tail, wherein the 5'UTR, 3'UTR and polyA tail comprise approximately 270-280 nucleotides.

[0058] In one embodiment, the vaccine comprises an mRNA polynucleotide encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, comprising an open reading frame, a 5'UTR, a 3'UTR, a 5' cap and a polyA tail, wherein the 5'UTR, 3'UTR and polyA tail comprise 276 nucleotides.

[0059] In one embodiment, the vaccine comprises an mRNA polynucleotide encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, comprising an open reading frame, a 5'UTR, a 3'UTR, a 5' cap and a polyA tail, wherein the 5'UTR, 3'UTR and polyA tail comprise approximately 276 nucleotides.

[0060] In one embodiment, the vaccine comprises an mRNA polynucleotide encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, comprising an open reading frame, a 5'UTR, a 3'UTR, a 5' cap and a polyA tail, wherein the 5'UTR, 3'UTR and polyA tail consist of 276 nucleotides.

[0061] In one embodiment, the vaccine comprises an mRNA polynucleotide encoding an antigen or immunogenic fragment thereof from an infectious microorganism, the polynucleotide comprising an open reading frame, a 5'UTR, a 3'UTR, a 5' cap and a polyA tail, the 5'UTR, 3'UTR and polyA tail comprising approximately 200-300 nucleotides.

[0062] In one embodiment, the vaccine comprises an mRNA polynucleotide encoding an antigen or immunogenic fragment thereof from an infectious microorganism, the polynucleotide comprising an open reading frame, a 5'UTR, a 3'UTR, a 5' cap and a polyA tail, the 5'UTR, 3'UTR and polyA tail comprising approximately 210-280 nucleotides.

[0063] In one embodiment, the vaccine comprises an mRNA polynucleotide encoding an antigen or immunogenic fragment thereof from an infectious microorganism, comprising an open reading frame, a 5'UTR, a 3'UTR, a 5' cap and a polyA tail, wherein the 5'UTR, 3'UTR and polyA tail comprise between 210 and 230 nucleotides.

[0064] In one embodiment, the vaccine comprises an mRNA polynucleotide encoding an antigen or immunogenic fragment thereof from an infectious microorganism, the polynucleotide comprising an open reading frame, a 5'UTR, a 3'UTR, a 5' cap and a polyA tail, the 5'UTR, 3'UTR and polyA tail comprising approximately 210-230 nucleotides.

[0065] In one embodiment, the vaccine comprises an mRNA polynucleotide encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, the polynucleotide comprising an open reading frame, a 5'UTR, a 3'UTR, a 5' cap and a polyA tail, the 5'UTR, 3'UTR and polyA tail consisting of 210 to 230 nucleotides.

[0066] Open reading frame An "open reading frame" (ORF) is a contiguous stretch of polynucleotides that begins with a start codon (e.g., methionine (AUG)) and ends with a stop codon (e.g., UAA, UAG, or UGA). An open reading frame encodes a polypeptide (in this case an antigen or an immunogenic fragment thereof) and has the potential to be translated into a polypeptide. An open reading frame is also known as a coding region.

[0067] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, the open reading frame comprising between 250 and 2500 nucleotides.

[0068] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, the open reading frame comprising between 250 and 500 nucleotides.

[0069] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, the open reading frame comprising 1950 nucleotides.

[0070] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, the open reading frame comprising about 1950 nucleotides.

[0071] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, the open reading frame comprising 477 nucleotides.

[0072] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, the open reading frame comprising about 477 nucleotides.

[0073] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, the open reading frame comprising 297 nucleotides.

[0074] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, the open reading frame comprising about 297 nucleotides.

[0075] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the open reading frame constitutes 45-90% of the total nucleotides of the mRNA.

[0076] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the open reading frame constitutes 45-70% of the total nucleotides of the mRNA.

[0077] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, the open reading frame comprising 50-65% of the total nucleotides of the mRNA.

[0078] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, the open reading frame making up 90% of the total nucleotides of the mRNA.

[0079] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, the open reading frame comprising about 90% of the total nucleotides of the mRNA.

[0080] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the open reading frame comprises 63% of the total nucleotides of the mRNA.

[0081] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, the open reading frame comprising about 63% of the total nucleotides of the mRNA.

[0082] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, the open reading frame making up 52% ​​of the total nucleotides of the mRNA.

[0083] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, the open reading frame comprising about 52% of the total nucleotides of the mRNA.

[0084] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof.

[0085] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E1, E2, E4, E5, E6, E7, L1, or L2 HPV antigen or an immunogenic fragment thereof.

[0086] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E1, E2, E4, E5, E6, or E7 HPV antigen, or an immunogenic fragment thereof.

[0087] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E6 or E7 HPV antigen or an immunogenic fragment thereof.

[0088] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E1 or E6 or E7 HPV antigen, or an immunogenic fragment thereof.

[0089] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E1 HPV antigen or an immunogenic fragment thereof.

[0090] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding the E6 HPV antigen or an immunogenic fragment thereof.

[0091] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding the E7 HPV antigen or an immunogenic fragment thereof.

[0092] In one embodiment, the vaccine does not comprise an mRNA polynucleotide comprising an open reading frame encoding the E5 HPV antigen or an immunogenic fragment thereof.

[0093] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an L1 or L2 HPV antigen or an immunogenic fragment thereof.

[0094] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an L1 HPV antigen or an immunogenic fragment thereof.

[0095] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an L2 HPV antigen or an immunogenic fragment thereof.

[0096] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding a high-risk HPV antigen or an immunogenic fragment thereof.

[0097] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68 antigens or immunogenic fragments thereof.

[0098] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV 6, 8, 11, 16, and / or 18 antigen or an immunogenic fragment thereof.

[0099] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV 16 and / or 18 antigen or an immunogenic fragment thereof.

[0100] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding a low-risk HPV antigen or an immunogenic fragment thereof.

[0101] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV6 and / or 11 antigen or an immunogenic fragment thereof.

[0102] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding the E1 HPV16 antigen or an immunogenic fragment thereof.

[0103] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding the E1 HPV18 antigen or an immunogenic fragment thereof.

[0104] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding the E6 HPV16 antigen or an immunogenic fragment thereof.

[0105] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding the E6 HPV18 antigen or an immunogenic fragment thereof.

[0106] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding the E7 HPV16 antigen or an immunogenic fragment thereof.

[0107] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding the E7 HPV18 antigen or an immunogenic fragment thereof.

[0108] Table 1 lists certain mRNA open reading frame sequences encoding antigens from infectious microorganisms that may be incorporated into the mRNA polynucleotides in the vaccines described herein.

[0109] [Table 1] TIFF2025506695000002.tif241162TIFF2025506695000003.tif34162

[0110] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 2, or a sequence having at least 80% sequence identity or homology.

[0111] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 2, or a sequence having at least 90% sequence identity or homology.

[0112] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 2, or a sequence having at least 95% sequence identity or homology.

[0113] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 2, or a sequence having at least 98% sequence identity or homology.

[0114] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:2.

[0115] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame consisting essentially of a sequence selected from SEQ ID NO:2.

[0116] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame consisting of a sequence selected from SEQ ID NO:2.

[0117] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 3, or a sequence having at least 80% sequence identity or homology.

[0118] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 3, or a sequence having at least 90% sequence identity or homology.

[0119] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 3, or a sequence having at least 95% sequence identity or homology.

[0120] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 3, or a sequence having at least 98% sequence identity or homology.

[0121] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:3.

[0122] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame consisting essentially of a sequence selected from SEQ ID NO:3.

[0123] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame consisting of a sequence selected from SEQ ID NO:3.

[0124] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 4, or a sequence having at least 80% sequence identity or homology.

[0125] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 4, or a sequence having at least 90% sequence identity or homology.

[0126] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 4, or a sequence having at least 95% sequence identity or homology.

[0127] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 4, or a sequence having at least 98% sequence identity or homology.

[0128] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:4.

[0129] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame consisting essentially of a sequence selected from SEQ ID NO:4.

[0130] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame consisting of a sequence selected from SEQ ID NO:4.

[0131] Encoding the antigen Antigens are proteins or polypeptides derived from infectious microorganisms that bind to specific antibodies or T-cell receptors and elicit an immune response.

[0132] The antigens derived from infectious microorganisms encoded by the mRNA polynucleotides in the vaccines described herein may be "wild-type", i.e., naturally occurring, antigens, or modified naturally occurring antigens. Non-naturally occurring antigens that may be encoded by the mRNA polynucleotides in the vaccines herein are sufficiently similar (e.g., 80% sequence identity or homology) to elicit the same immunogenic response as the equivalent wild-type antigen.

[0133] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the antigen or immunogenic fragment thereof has at least 80% sequence identity or homology to the wild-type antigen or fragment thereof.

[0134] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the antigen or immunogenic fragment thereof has at least 90% sequence identity or homology to the wild-type antigen or fragment thereof.

[0135] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the antigen or immunogenic fragment thereof has at least 95% sequence identity or homology to the wild-type antigen or fragment thereof.

[0136] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the antigen or immunogenic fragment thereof has at least 98% sequence identity or homology to the wild-type antigen or fragment thereof.

[0137] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the antigen or immunogenic fragment thereof is identical to a wild-type antigen or fragment thereof.

[0138] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the antigen or the immunogenic fragment thereof is a polypeptide.

[0139] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism, wherein the antigen or the immunogenic fragment thereof is a polypeptide consisting of 80 to 700 amino acid residues.

[0140] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism, wherein the antigen or the immunogenic fragment thereof is a polypeptide consisting of 80 to 180 amino acid residues.

[0141] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism, wherein the antigen or the immunogenic fragment thereof is a polypeptide consisting of 600 to 700 amino acid residues.

[0142] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of approximately 649 amino acid residues.

[0143] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of 649 amino acid residues.

[0144] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism, wherein the antigen or the immunogenic fragment thereof is a polypeptide consisting of 140 to 170 amino acid residues.

[0145] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of approximately 158 amino acid residues.

[0146] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of 158 amino acid residues.

[0147] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism, wherein the antigen or the immunogenic fragment thereof is a polypeptide consisting of 90 to 110 amino acid residues.

[0148] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of approximately 98 amino acid residues.

[0149] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of 98 amino acid residues.

[0150] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof.

[0151] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof that forms part or all of the HPV viral capsid.

[0152] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV antigen, or an immunogenic fragment thereof, that is responsible for binding of HPV to a cell to be infected.

[0153] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen, or an immunogenic fragment thereof, derived from an infectious microorganism that interacts with the retinoblastoma protein (pRb).

[0154] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen, or an immunogenic fragment thereof, derived from an infectious microorganism that interacts with p53.

[0155] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism that attaches to a cellular receptor.

[0156] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism that causes fusion of viral and cellular membranes.

[0157] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen, or an immunogenic fragment thereof, derived from an infectious microorganism that is responsible for binding of the virus to the cell to be infected.

[0158] Table 2 lists the sequences of antigens from infectious microorganisms that may be encoded by the mRNA polynucleotides of the vaccines described herein:

[0159] [Table 2] TIFF2025506695000005.tif35160

[0160] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:5, or a sequence having at least 80% sequence identity or homology.

[0161] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:5, or a sequence having at least 90% sequence identity or homology.

[0162] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:5, or a sequence having at least 95% sequence identity or homology.

[0163] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:5, or a sequence having at least 98% sequence identity or homology.

[0164] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:5.

[0165] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting essentially of a sequence selected from SEQ ID NO:5.

[0166] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting of SEQ ID NO:5.

[0167] In one embodiment, an mRNA polynucleotide is provided comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:5.

[0168] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:6, or a sequence having at least 80% sequence identity or homology.

[0169] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:6, or a sequence having at least 90% sequence identity or homology.

[0170] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:6, or a sequence having at least 95% sequence identity or homology.

[0171] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:6, or a sequence having at least 98% sequence identity or homology.

[0172] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:6.

[0173] In one embodiment, an mRNA polynucleotide is provided comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:6.

[0174] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting essentially of a sequence selected from SEQ ID NO:6.

[0175] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting of SEQ ID NO:6.

[0176] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO: 7, or a sequence having at least 80% sequence identity or homology.

[0177] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO: 7, or a sequence having at least 90% sequence identity or homology.

[0178] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO: 7, or a sequence having at least 95% sequence identity or homology.

[0179] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO: 7, or a sequence having at least 98% sequence identity or homology.

[0180] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:7.

[0181] In one embodiment, an mRNA polynucleotide is provided comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID NO:7.

[0182] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting essentially of a sequence selected from SEQ ID NO:7.

[0183] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting of SEQ ID NO:7.

[0184] Table 3 shows the GenBank accession numbers for the sequences of certain HPV antigens from infectious microorganisms that can be encoded by the mRNA polynucleotides of the vaccines described herein:

[0185] [Table 3] TIFF2025506695000007.tif230146TIFF2025506695000008.tif227145TIFF2025506695000009.tif98146

[0186] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from Table 3, or a sequence having at least 80% sequence identity or homology.

[0187] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from Table 3, or a sequence having at least 90% sequence identity or homology.

[0188] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from Table 3, or a sequence having at least 95% sequence identity or homology.

[0189] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from Table 3, or a sequence having at least 98% sequence identity or homology.

[0190] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from Table 3.

[0191] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting essentially of a sequence selected from Table 3.

[0192] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting of a sequence selected from Table 3.

[0193] Immunogenic fragments thereof An immunogenic fragment of an antigen (also known as an epitope or antigenic determinant) is a fragment of an antigen that is recognized by the immune system, specifically antibodies, B cells, or T cells, and that induces an immune response. + Immunogenic fragments that elicit a response are typically 8-10 amino acids in length.

[0194] infectious microorganisms Infectious microorganisms are classified into five main categories: bacteria, parasites (e.g., protozoa), fungi, viruses, and prions. These pathogenic microorganisms can be transmitted by animals, humans, insects, or other vectors causing disease in the host organism.

[0195] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from a bacterium, a parasite, a fungus, a virus, or a prion.

[0196] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from the bacteria.

[0197] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from a parasite.

[0198] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from a fungus.

[0199] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from a virus.

[0200] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding a prion-derived antigen or an immunogenic fragment thereof.

[0201] Multicomponent vaccines In one embodiment, the vaccine comprises a plurality, e.g., 2-10, mRNA polynucleotides each comprising an open reading frame encoding a single antigen or immunogenic fragment thereof from an infectious microorganism. The two or more mRNA polynucleotides may encode the same or different antigens, or sequences from different portions of the same antigen, additionally or alternatively, from different strains of the infectious microorganism.

[0202] In one embodiment, the vaccine comprises an mRNA polynucleotide that includes an open reading frame encoding multiple, e.g., 2-10, antigens or immunogenic fragments thereof from an infectious microorganism (e.g., as a fusion polypeptide). The open reading frames encoding multiple antigens or immunogenic fragments thereof from an infectious microorganism may encode sequences from the same or different antigens, or different portions of the same antigen, additionally or alternatively, from different strains of the infectious microorganism.

[0203] In one embodiment, the vaccines described herein are multivalent. Multivalent vaccines combine antigens from different strains of a single pathogen to immunize against a single disease.

[0204] In one embodiment, the vaccine comprises a first mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism, and a second mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism.

[0205] In one embodiment, the vaccine comprises a first mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism, a second mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism, and a third mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism.

[0206] In one embodiment, the vaccine comprises one mRNA polynucleotide that contains open reading frames encoding two antigens or immunogenic fragments thereof from an infectious microorganism.

[0207] In one embodiment, the vaccine comprises one mRNA polynucleotide that contains open reading frames encoding three antigens or immunogenic fragments thereof from an infectious microorganism.

[0208] In one embodiment, the vaccine comprises: i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, and iii) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; Includes.

[0209] In one embodiment, the vaccine comprises: i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; iii) a third mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; and iv) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; Includes.

[0210] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, the two mRNA polynucleotides being present in a ratio of about 50:50% w / w.

[0211] In one embodiment, the vaccine comprises three mRNA polynucleotides each comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, wherein the three mRNA polynucleotides are present in an approximately equal % w / w ratio.

[0212] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism, the two mRNA polynucleotides being present in a ratio of 50:50% w / w.

[0213] In one embodiment, the vaccine comprises three mRNA polynucleotides each comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the three mRNA polynucleotides are present in an equal % w / w ratio.

[0214] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, the two mRNA polynucleotides being present in a ratio of about 90:10% w / w.

[0215] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, the two mRNA polynucleotides being present in a ratio of 90:10% w / w.

[0216] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, the two mRNA polynucleotides being present in a ratio of about 75:25% w / w.

[0217] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, the two mRNA polynucleotides being present in a ratio of 75:25% w / w.

[0218] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism, wherein the two mRNA polynucleotides are present in a 1:1 copy number ratio.

[0219] "Copy number" refers to the number of copies of an mRNA that are present. To determine the copy number of an mRNA, the molecular weight (MW) of one copy of the mRNA is calculated by adding up the molecular weights of all constituent nucleotides in the entire nucleotide sequence, where: A = 329.2 g / mol C = 305.2 g / mol G = 345.2 g / mol T = 306.2 g / mol It is.

[0220] Any proprietary sequences (non-ORF) where the exact nucleotides are unknown are described by using an average molecular weight of A / G / C / T = 321.45 g / mol.

[0221] The number of copies present in an mRNA of known mass (m) is then calculated as follows: Number of copies = (m / MW)*NA (where NA = Avogadro's number = 6.0221409 x 10^23)

[0222] In one embodiment, the vaccine comprises three mRNA polynucleotides each comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, wherein the three mRNA polynucleotides are present in a copy number ratio of 1:1:1.

[0223] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising two or more open reading frames, each encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the open reading frames are present in an equal copy number ratio.

[0224] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism, wherein the two mRNA polynucleotides are present in a copy number ratio of about 1:1.

[0225] In one embodiment, the vaccine comprises three mRNA polynucleotides each comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, wherein the three mRNA polynucleotides are present in a copy number ratio of about 1:1:1.

[0226] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising two or more open reading frames, each encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the open reading frames are present in approximately equal copy number ratios.

[0227] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, the two mRNA polynucleotides being present in a copy number ratio of 3:1.

[0228] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, the two mRNA polynucleotides being present in a copy number ratio of about 3:1.

[0229] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an antigen or immunogenic fragment thereof derived from an infectious microorganism, the two mRNA polynucleotides being present in a copy number ratio of 9:1.

[0230] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism, the two mRNA polynucleotides being present in a copy number ratio of about 9:1.

[0231] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising an open reading frame, each encoding an HPV antigen selected from E1, E6 and E7, or an immunogenic fragment thereof, wherein the three mRNA polynucleotides are present in a copy number ratio of approximately 1 (E1):3 (E6):4 (E7).

[0232] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising an open reading frame, each encoding an HPV antigen selected from E1, E6 and E7, or an immunogenic fragment thereof, wherein the three mRNA polynucleotides are present in a copy number ratio of 1 (E1):3 (E6):4 (E7).

[0233] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising an open reading frame, each encoding an HPV antigen selected from E1, E6 and E7, or an immunogenic fragment thereof, wherein the three mRNA polynucleotides are present in a copy number ratio of approximately 1 (E1):3.2 (E6):4.2 (E7).

[0234] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising an open reading frame, each encoding an HPV antigen selected from E1, E6 and E7, or an immunogenic fragment thereof, wherein the three mRNA polynucleotides are present in a copy number ratio of 1 (E1):3.2 (E6):4.2 (E7).

[0235] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an HPV antigen selected from E1, E2, E4, E5, E6, E7, L1, or L2, or an immunogenic fragment thereof.

[0236] In one embodiment, the vaccine comprises three mRNA polynucleotides each comprising an open reading frame encoding an HPV antigen selected from E1, E2, E4, E5, E6, E7, L1, or L2, or an immunogenic fragment thereof.

[0237] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an HPV antigen selected from E1, E2, E4, E5, E6 or E7, or an immunogenic fragment thereof.

[0238] In one embodiment, the vaccine comprises three mRNA polynucleotides each comprising an open reading frame encoding an HPV antigen selected from E1, E2, E4, E5, E6, or E7, or an immunogenic fragment thereof.

[0239] In one embodiment, the vaccine comprises two mRNA polynucleotides containing open reading frames, one encoding the E6 HPV antigen or an immunogenic fragment thereof and the other encoding the E7 HPV antigen or an immunogenic fragment thereof.

[0240] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising open reading frames, one encoding an E6 HPV antigen or an immunogenic fragment thereof, one encoding an E7 HPV antigen or an immunogenic fragment thereof, and one encoding an E1 HPV antigen or an immunogenic fragment thereof.

[0241] In one embodiment, the vaccine comprises two mRNA polynucleotides comprising an open reading frame, one encoding an L1 HPV antigen or an immunogenic fragment thereof and the other encoding an L2 HPV antigen or an immunogenic fragment thereof.

[0242] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding a high-risk HPV antigen or an immunogenic fragment thereof.

[0243] In one embodiment, the vaccine comprises three mRNA polynucleotides each comprising an open reading frame encoding a high-risk HPV antigen or an immunogenic fragment thereof.

[0244] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68 antigen or an immunogenic fragment thereof.

[0245] In one embodiment, the vaccine comprises three mRNA polynucleotides each comprising an open reading frame encoding an HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68 antigen or an immunogenic fragment thereof.

[0246] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an HPV 6, 8, 11, 16 and / or 18 antigen or an immunogenic fragment thereof.

[0247] In one embodiment, the vaccine comprises three mRNA polynucleotides each comprising an open reading frame encoding an HPV 6, 8, 11, 16 and / or 18 antigen or an immunogenic fragment thereof.

[0248] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an HPV 16 and / or 18 antigen or an immunogenic fragment thereof.

[0249] In one embodiment, the vaccine comprises three mRNA polynucleotides each comprising an open reading frame encoding an HPV 16 and / or 18 antigen or an immunogenic fragment thereof.

[0250] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding a low-risk HPV antigen or an immunogenic fragment thereof.

[0251] In one embodiment, the vaccine comprises three mRNA polynucleotides each comprising an open reading frame encoding a low-risk HPV antigen or an immunogenic fragment thereof.

[0252] In one embodiment, the vaccine comprises two mRNA polynucleotides each comprising an open reading frame encoding an HPV6 and / or 11 antigen or an immunogenic fragment thereof.

[0253] In one embodiment, the vaccine comprises three mRNA polynucleotides each comprising an open reading frame encoding an HPV6 and / or 11 antigen or an immunogenic fragment thereof.

[0254] In one embodiment, the vaccine comprises two mRNA polynucleotides comprising an open reading frame, one encoding a high-risk HPV antigen or an immunogenic fragment thereof and the other encoding a low-risk HPV antigen or an immunogenic fragment thereof.

[0255] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising an open reading frame, at least one encoding a high-risk HPV antigen or an immunogenic fragment thereof and at least one encoding a low-risk HPV antigen or an immunogenic fragment thereof.

[0256] In one embodiment, the vaccine comprises two mRNA polynucleotides containing open reading frames, one encoding the E6 HPV16 antigen or an immunogenic fragment thereof and the other encoding the E7 HPV16 antigen or an immunogenic fragment thereof.

[0257] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising open reading frames, one encoding the E6 HPV16 antigen or an immunogenic fragment thereof, one encoding the E7 HPV16 antigen or an immunogenic fragment thereof, and one encoding the E1 HPV16 antigen or an immunogenic fragment thereof.

[0258] In one embodiment, the vaccine comprises two mRNA polynucleotides comprising an open reading frame, one encoding the E6 HPV18 antigen or an immunogenic fragment thereof and the other encoding the E7 HPV18 antigen or an immunogenic fragment thereof.

[0259] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising open reading frames, one encoding the E6 HPV18 antigen or an immunogenic fragment thereof, one encoding the E7 HPV18 antigen or an immunogenic fragment thereof, and one encoding the E1 HPV18 antigen or an immunogenic fragment thereof.

[0260] In one embodiment, the vaccine comprises two mRNA polynucleotides comprising an open reading frame, one encoding the E6 HPV16 antigen or an immunogenic fragment thereof and the other encoding the E7 HPV18 antigen or an immunogenic fragment thereof.

[0261] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising open reading frames, one encoding the E6 HPV16 antigen or an immunogenic fragment thereof, one encoding the E7 HPV18 antigen or an immunogenic fragment thereof, and one encoding the E1 HPV18 antigen or an immunogenic fragment thereof.

[0262] In one embodiment, the vaccine comprises two mRNA polynucleotides containing open reading frames, one encoding the E6 HPV18 antigen or an immunogenic fragment thereof and the other encoding the E7 HPV16 antigen or an immunogenic fragment thereof.

[0263] In one embodiment, the vaccine comprises two mRNA polynucleotides containing an open reading frame, one encoding an E6 HPV antigen or an immunogenic fragment thereof and the other encoding an E7 HPV antigen or an immunogenic fragment thereof, wherein the two mRNA polynucleotides are present in a ratio of about 50:50% w / w.

[0264] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising open reading frames, one encoding an E6 HPV antigen or an immunogenic fragment thereof, one encoding an E7 HPV antigen or an immunogenic fragment thereof, and one encoding an E1 HPV antigen or an immunogenic fragment thereof, wherein the three mRNA polynucleotides are present in approximately equal % w / w ratio.

[0265] In one embodiment, the vaccine comprises two mRNA polynucleotides containing an open reading frame, one encoding an E6 HPV antigen or an immunogenic fragment thereof and the other encoding an E7 HPV antigen or an immunogenic fragment thereof, wherein the two mRNA polynucleotides are present in a ratio of 50:50% w / w.

[0266] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising an open reading frame, one encoding an E6 HPV antigen or an immunogenic fragment thereof, one encoding an E7 HPV antigen or an immunogenic fragment thereof, and one encoding an E1 HPV antigen or an immunogenic fragment thereof, wherein the three mRNA polynucleotides are present in an equal % w / w ratio.

[0267] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 2, or a sequence with at least 80% sequence identity or homology, and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 3, or a sequence with at least 80% sequence identity or homology.

[0268] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:2 or a sequence with at least 80% sequence identity or homology; an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:3 or a sequence with at least 80% sequence identity or homology; and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:4 or a sequence with at least 80% sequence identity or homology.

[0269] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 2 or a sequence with at least 80% sequence identity or homology, and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 3 or a sequence with at least 80% sequence identity or homology, wherein the two mRNA polynucleotides are present in a ratio of about 50:50% w / w.

[0270] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 2 or a sequence with at least 80% sequence identity or homology, an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 3 or a sequence with at least 80% sequence identity or homology, and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 4 or a sequence with at least 80% sequence identity or homology, wherein the three mRNA polynucleotides are present in approximately equal % w / w ratio.

[0271] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 2 or a sequence with at least 80% sequence identity or homology, and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 3 or a sequence with at least 80% sequence identity or homology, wherein the two mRNA polynucleotides are present in a ratio of 50:50% w / w.

[0272] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 2 or a sequence with at least 80% sequence identity or homology, an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 3 or a sequence with at least 80% sequence identity or homology, and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO: 4 or a sequence with at least 80% sequence identity or homology, wherein the three mRNA polynucleotides are present in equal % w / w ratio.

[0273] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:2 and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:3, wherein the two mRNA polynucleotides are present in a ratio of about 50:50% w / w.

[0274] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:2, an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:3, and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:4, wherein the three mRNA polynucleotides are present in approximately equal % w / w ratios.

[0275] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:2 and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:3, wherein the two mRNA polynucleotides are present in a ratio of 50:50% w / w.

[0276] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:2, an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:3, and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:4, wherein the three mRNA polynucleotides are present in equal % w / w ratio.

[0277] In one embodiment, the vaccine comprises two mRNA polynucleotides comprising an open reading frame, one encoding an E6 HPV antigen selected from SEQ ID NO:5 or a sequence having at least 80% sequence identity or homology, and the other encoding an E7 HPV antigen selected from SEQ ID NO:6 or a sequence having at least 80% sequence identity or homology, wherein the two mRNA polynucleotides are present in a ratio of 50:50% w / w.

[0278] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising open reading frames, one encoding an E6 HPV antigen selected from SEQ ID NO:5 or a sequence having at least 80% sequence identity or homology, one encoding an E7 HPV antigen selected from SEQ ID NO:6 or a sequence having at least 80% sequence identity or homology, and one encoding an E1 HPV antigen selected from SEQ ID NO:7 or a sequence having at least 80% sequence identity or homology, wherein the three mRNA polynucleotides are present in equal % w / w ratio.

[0279] In one embodiment, the vaccine comprises two mRNA polynucleotides comprising an open reading frame, one encoding an E6 HPV antigen selected from SEQ ID NO:5 and the other encoding an E7 HPV antigen selected from SEQ ID NO:6, wherein the two mRNA polynucleotides are present in a ratio of 50:50% w / w.

[0280] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising open reading frames, one encoding an E6 HPV antigen selected from SEQ ID NO:5, one encoding an E7 HPV antigen selected from SEQ ID NO:6, and one encoding an E1 HPV antigen selected from SEQ ID NO:7, wherein the three mRNA polynucleotides are present in equal % w / w ratios.

[0281] In one embodiment, the vaccine comprises two mRNA polynucleotides containing an open reading frame, one encoding an E6 HPV antigen or an immunogenic fragment thereof and the other encoding an E7 HPV antigen or an immunogenic fragment thereof, wherein the two mRNA polynucleotides are present in a ratio of about 50:50% w / w.

[0282] In one embodiment, the vaccine comprises three mRNA polynucleotides comprising open reading frames, one encoding an E6 HPV antigen or an immunogenic fragment thereof, one encoding an E7 HPV antigen or an immunogenic fragment thereof, and one encoding an E1 HPV antigen or an immunogenic fragment thereof, wherein the three mRNA polynucleotides are present in approximately equal % w / w ratio.

[0283] Immune adjuvants In one embodiment, the vaccine further comprises an immune adjuvant, which is a substance that acts to accelerate, prolong, or enhance an antigen-specific immune response when used in combination with a particular vaccine antigen.

[0284] Suitable immune adjuvants include inorganic compounds such as potassium alum KAl(SO4)2, aluminum hydroxide, aluminum phosphate, and calcium hydroxide phosphate; oils such as paraffin oil, propolis, and peanut oil; and cytokines such as IL-1 (interleukin-1), IL-2, IL-7, IL-12, and IL-15. Other suitable immune adjuvants include granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0285] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant, hi one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof and an open reading frame encoding an immune adjuvant.

[0286] In one embodiment, the vaccine comprises: i) a first mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:2; ii) a second mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:3; iii) a third mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:8; and iv) an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); Includes.

[0287] In one embodiment, the vaccine comprises: i) a first mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:2; ii) a second mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:3; iii) a third mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:4; iv) a fourth mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:8; and v) an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); Includes.

[0288] In one embodiment, the vaccine comprises: i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; iii) a third mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant; and iv) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; Includes.

[0289] In one embodiment, the vaccine comprises: i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; iii) a third mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; iv) a fourth mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant; and v) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; Includes.

[0290] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding a cytokine.

[0291] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding IL-1, IL-2, IL-7, IL-12, or IL-15.

[0292] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding IL-15.

[0293] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

[0294] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding granulocyte-macrophage colony-stimulating factor.

[0295] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding human granulocyte-macrophage colony-stimulating factor.

[0296] In one embodiment, the vaccine comprises: i) a first mRNA polynucleotide comprising an open reading frame encoding an HPV E6 antigen or an immunogenic fragment thereof; ii) a second mRNA polynucleotide comprising an open reading frame encoding an HPV E7 antigen or an immunogenic fragment thereof; iii) a third mRNA polynucleotide comprising an open reading frame encoding human IL-15; and iv) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; Includes.

[0297] In one embodiment, the vaccine comprises: i) a first mRNA polynucleotide comprising an open reading frame encoding an HPV E6 antigen or an immunogenic fragment thereof; ii) a second mRNA polynucleotide comprising an open reading frame encoding an HPV E7 antigen or an immunogenic fragment thereof; iii) a third mRNA polynucleotide comprising an open reading frame encoding an HPV E1 antigen or an immunogenic fragment thereof; iv) a fourth mRNA polynucleotide comprising an open reading frame encoding human IL-15; and v) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; Includes.

[0298] In one embodiment, the vaccine comprises: i) a first mRNA polynucleotide comprising an open reading frame encoding the HPV16 E6 antigen or an immunogenic fragment thereof; ii) a second mRNA polynucleotide comprising an open reading frame encoding the HPV16 E7 antigen or an immunogenic fragment thereof; iii) a third mRNA polynucleotide comprising an open reading frame encoding human IL-15; and iv) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; Includes.

[0299] In one embodiment, the vaccine comprises: i) a first mRNA polynucleotide comprising an open reading frame encoding the HPV16 E6 antigen or an immunogenic fragment thereof; ii) a second mRNA polynucleotide comprising an open reading frame encoding the HPV16 E7 antigen or an immunogenic fragment thereof; iii) a third mRNA polynucleotide comprising an open reading frame encoding an HPV16 E1 antigen or an immunogenic fragment thereof; iv) a fourth mRNA polynucleotide comprising an open reading frame encoding human IL-15; and v) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; Includes.

[0300] In one embodiment, the vaccine comprises: i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID NO:5; ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID NO:6; iii) a third mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant selected from SEQ ID NO: 10; and iv) an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); Includes.

[0301] In one embodiment, the vaccine comprises: i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID NO:5; ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID NO:6; iii) a third mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID NO:7; iv) a fourth mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant selected from SEQ ID NO: 10; and v) an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); Includes.

[0302] In one embodiment, the vaccine further comprises up to 20% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant, meaning that the mRNA encoding the immune adjuvant constitutes 20% by weight of the total amount of mRNA in the vaccine.

[0303] In one embodiment, the vaccine further comprises up to 20% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

[0304] In one embodiment, the vaccine further comprises 5-20% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant.

[0305] In one embodiment, the vaccine further comprises 5-20% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

[0306] In one embodiment, the vaccine further comprises 5% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant.

[0307] In one embodiment, the vaccine further comprises about 5% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

[0308] In one embodiment, the vaccine further comprises 10% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant.

[0309] In one embodiment, the vaccine further comprises about 10% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

[0310] In one embodiment, the vaccine further comprises 15% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant.

[0311] In one embodiment, the vaccine further comprises about 15% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

[0312] In one embodiment, the vaccine further comprises 20% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant.

[0313] In one embodiment, the vaccine further comprises about 20% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

[0314] In one embodiment, the vaccine further comprises up to 20% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding GM-CSF.

[0315] In one embodiment, the vaccine further comprises 5-20% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding GM-CSF.

[0316] In one embodiment, the vaccine further comprises about 5% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding GM-CSF.

[0317] In one embodiment, the vaccine further comprises about 10% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding GM-CSF.

[0318] In one embodiment, the vaccine further comprises about 15% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding GM-CSF.

[0319] In one embodiment, the vaccine further comprises about 20% w / w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding GM-CSF.

[0320] Table 4 lists certain mRNA open reading frame sequences encoding immune adjuvants that can be incorporated into an mRNA polynucleotide and added to the vaccines described herein.

[0321] [Table 4] TIFF2025506695000011.tif95160

[0322] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant comprising a sequence selected from SEQ ID NO: 8, or a sequence having at least 80% sequence identity or homology.

[0323] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant comprising a sequence selected from SEQ ID NO: 8, or a sequence having at least 90% sequence identity or homology.

[0324] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant comprising a sequence selected from SEQ ID NO: 8, or a sequence having at least 95% sequence identity or homology.

[0325] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant comprising a sequence selected from SEQ ID NO: 8, or a sequence having at least 98% sequence identity or homology.

[0326] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant comprising a sequence selected from SEQ ID NO:8.

[0327] In one embodiment, an mRNA polynucleotide is provided that comprises an open reading frame encoding an immune adjuvant comprising a sequence selected from SEQ ID NO:8.

[0328] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant comprising a sequence selected from SEQ ID NO: 9, or a sequence having at least 80% sequence identity or homology.

[0329] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant comprising a sequence selected from SEQ ID NO: 9, or a sequence having at least 90% sequence identity or homology.

[0330] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant comprising a sequence selected from SEQ ID NO: 9, or a sequence having at least 95% sequence identity or homology.

[0331] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant comprising a sequence selected from SEQ ID NO: 9, or a sequence having at least 98% sequence identity or homology.

[0332] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant comprising a sequence selected from SEQ ID NO:9.

[0333] In one embodiment, an mRNA polynucleotide is provided that comprises an open reading frame encoding an immune adjuvant comprising a sequence selected from SEQ ID NO:9.

[0334] Table 5 lists the sequences of immune adjuvants that may be encoded by the mRNA polynucleotides of the vaccines described herein:

[0335] [Table 5]

[0336] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant selected from SEQ ID NO: 10, or a sequence having at least 80% sequence identity or homology.

[0337] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant selected from SEQ ID NO: 10, or a sequence having at least 90% sequence identity or homology.

[0338] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant selected from SEQ ID NO: 10, or a sequence having at least 95% sequence identity or homology.

[0339] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant selected from SEQ ID NO: 10, or a sequence having at least 98% sequence identity or homology.

[0340] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant selected from SEQ ID NO:10.

[0341] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant selected from SEQ ID NO: 11 or a sequence having at least 80% sequence identity or homology.

[0342] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant selected from SEQ ID NO: 11 or a sequence having at least 90% sequence identity or homology.

[0343] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant selected from SEQ ID NO: 11 or a sequence having at least 95% sequence identity or homology.

[0344] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant selected from SEQ ID NO: 11, or a sequence having at least 98% sequence identity or homology.

[0345] In one embodiment, the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant selected from SEQ ID NO:11.

[0346] mRNA modification In one embodiment, the mRNA polynucleotides described herein contain one or more modifications compared to naturally occurring "wild-type" mRNA. Modification refers to an alteration in at least one of the adenosine (A), guanosine (G), uridine (U), or cytidine (C) nucleotides in their position, pattern, percentage, and / or structure. Modifications may include naturally occurring modifications, non-naturally occurring modifications, or a combination of naturally occurring and non-naturally occurring modifications. Modifications may be modifications of the sugar, nucleobase, or internucleotide linkage (e.g., to the phosphate group). Modifications may be introduced during or after synthesis, by chemical synthesis or by polymerase enzymes. Any region of the mRNA polynucleotide may be modified.

[0347] In one embodiment, the vaccine comprises an mRNA polynucleotide that comprises one or more modifications that result in reduced degradation of the mRNA following administration, compared to an unmodified (ie, wild-type) polynucleotide.

[0348] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising one or more modifications that result in reduced immunogenicity (e.g., reduced innate response) following administration compared to an unmodified polynucleotide. Modifications of the mRNA polynucleotides described herein can include codon optimization, capping, e.g., 5 prime 5' cap, and uridine modifications.

[0349] i) Codon optimization In one embodiment, the mRNA polynucleotide can be codon-optimized. Codon optimization ensures the most efficient translation in humans. In one embodiment, codon optimization can be used to match codon frequencies in target and host organisms to ensure proper folding; bias G / C content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene assembly or expression; customize transcriptional and translational control regions; insert or remove protein transport sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translation rates to allow various domains of the protein to fold properly; or reduce or eliminate problematic secondary structures in the polynucleotide. Codon optimization tools, algorithms and services are known in the art - non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.), and / or proprietary methods. In one embodiment, open reading frame (ORF) sequences are optimized using an optimization algorithm.

[0350] In one embodiment, a codon-optimized mRNA can be, for example, an mRNA with enhanced levels of guanosine (G) and / or cytidine (C) residues. The G / C content of a nucleic acid molecule can affect the stability of the mRNA. An mRNA with an increased amount of guanosine (G) and / or cytidine (C) residues can be more functionally stable than a nucleic acid containing a large amount of adenosine (A) and uridine (T) nucleotides. Due to the degeneracy of the genetic code, the modification works by replacing existing codons with codons that promote higher RNA stability without changing the resulting amino acid. This approach is limited to the open reading frame(s) of the mRNA.

[0351] In one embodiment, the codon-optimized mRNA may contain a uridine deletion compared to an unmodified polynucleotide, which reduces the likelihood of a natural immune response against the mRNA.

[0352] ii) Capping In one embodiment, the mRNA polynucleotide comprises a 5 prime cap (5' cap). A 5' cap is a specifically modified nucleotide at the 5' end of an mRNA. mRNA capping can help generate the most biologically active, least immunogenic (less likely to provoke an innate immune response) mRNA, enhancing translation into protein.

[0353] In one embodiment, the mRNA polynucleotide comprises a 5 prime cap (5' cap) selected from CleanCap® available from TriLink Biotechnologies® (e.g., those described in WO 2017 / 053297). Suitable caps include: CleanCap® Reagent AG for co-transcriptional capping of mRNA: m7G(5')ppp(5')(2'OMeA)pG, or ARCA (Anti Reverse Cap Analog): 3'-O-Me-m 7 G(5')ppp(5')G.

[0354] 5'-capping prevents mRNA degradation in the cytoplasm and promotes ribosome recruitment and protein translation.

[0355] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises a 5' cap.

[0356] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, wherein the mRNA polynucleotide comprises a 5' cap.

[0357] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises a 5' cap selected from m7G(5')ppp(5')(2'OMeA)pG.

[0358] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises a 5' cap selected from m7G(5')ppp(5')(2'OMeA)pG.

[0359] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, the mRNA polynucleotide comprising a 3'-O-Me- 7 G(5')ppp(5')G.

[0360] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, the mRNA polynucleotide comprising a 3'-O-Me-m 7 G(5')ppp(5')G.

[0361] iii) Uridine modification In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, the uridine modifications reducing the likelihood of a natural immune response against the mRNA.

[0362] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, the uridine modifications reducing the likelihood of a natural immune response against the mRNA.

[0363] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, the mRNA polynucleotide comprising one or more modified uridine nucleotides selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine nucleotides.

[0364] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, wherein the mRNA polynucleotide comprises one or more modified uridine nucleotides selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine nucleotides.

[0365] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises one or more modified uridine nucleotides selected from N1-methylpseudouridine nucleotides.

[0366] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises one or more modified uridine nucleotides selected from N1-methylpseudouridine nucleotides.

[0367] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen derived from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, at least 80% of the uridine nucleotides are N1-methylpseudouridine nucleotides.

[0368] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein at least 80% of the uridine nucleotides are N1-methylpseudouridine nucleotides.

[0369] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen derived from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, at least 90% of the uridine nucleotides are N1-methylpseudouridine nucleotides.

[0370] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, at least 90% of the uridine nucleotides are N1-methylpseudouridine nucleotides.

[0371] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen derived from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, at least 95% of the uridine nucleotides are N1-methylpseudouridine nucleotides.

[0372] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, at least 95% of the uridine nucleotides are N1-methylpseudouridine nucleotides.

[0373] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen derived from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, at least 97% of the uridine nucleotides being N1-methylpseudouridine nucleotides.

[0374] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, at least 97% of the uridine nucleotides are N1-methylpseudouridine nucleotides.

[0375] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen derived from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, at least 99% of the uridine nucleotides are N1-methylpseudouridine nucleotides.

[0376] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, at least 99% of the uridine nucleotides are N1-methylpseudouridine nucleotides.

[0377] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen derived from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein all uridine nucleotides are N1-methylpseudouridine nucleotides.

[0378] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein all uridine nucleotides are N1-methylpseudouridine nucleotides.

[0379] iv) Cytidine Modifications In one embodiment, the vaccine comprises an mRNA polynucleotide that includes modified cytidine nucleotides. Cytidine modifications may be used to confer desirable properties, such as increased nuclease stability, increased translation, or reduced interaction of innate immune receptors with the in vitro transcribed RNA.

[0380] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising one or more modified cytidine nucleotides selected from N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), 5-methylcytidine-5'-triphosphate (5-methyl-CTP), and 2-thio-5-methyl-cytidine.

[0381] In one embodiment, the mRNA polynucleotide does not comprise a cytidine modification.

[0382] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein at least 80% of the uridine nucleotides are N1-methylpseudouridine nucleotides, and the cytidine nucleotides are unmodified.

[0383] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein at least 80% of the uridine nucleotides are N1-methylpseudouridine nucleotides, and the cytidine nucleotides are unmodified.

[0384] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein at least 90% of the uridine nucleotides are N1-methylpseudouridine nucleotides, and the cytidine nucleotides are unmodified.

[0385] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein at least 90% of the uridine nucleotides are N1-methylpseudouridine nucleotides, and the cytidine nucleotides are unmodified.

[0386] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein at least 95% of the uridine nucleotides are N1-methylpseudouridine nucleotides, and the cytidine nucleotides are unmodified.

[0387] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein at least 95% of the uridine nucleotides are N1-methylpseudouridine nucleotides, and the cytidine nucleotides are unmodified.

[0388] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein at least 97% of the uridine nucleotides are N1-methylpseudouridine nucleotides, and the cytidine nucleotides are unmodified.

[0389] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein at least 97% of the uridine nucleotides are N1-methylpseudouridine nucleotides, and the cytidine nucleotides are unmodified.

[0390] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein at least 99% of the uridine nucleotides are N1-methylpseudouridine nucleotides, and the cytidine nucleotides are unmodified.

[0391] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein at least 99% of the uridine nucleotides are N1-methylpseudouridine nucleotides, and the cytidine nucleotides are unmodified.

[0392] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein all uridine nucleotides are N1-methylpseudouridine nucleotides and the cytidine nucleotides are unmodified.

[0393] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein all uridine nucleotides are N1-methylpseudouridine nucleotides and the cytidine nucleotides are unmodified.

[0394] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising one or more modifications selected from a 5' cap and a uridine modification.

[0395] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising two modifications selected from a 5' cap and a uridine modification.

[0396] In one embodiment, the vaccine comprises an mRNA polynucleotide that is uniformly modified (eg, completely modified throughout the entire sequence) for a particular modification.

[0397] In one embodiment, the mRNA polynucleotides described herein comprise one or more modifications selected from a 5' cap and fully modified uridine modifications throughout the entire sequence.

[0398] In one embodiment, the vaccine comprises an mRNA polynucleotide comprising one or more modifications selected from a 5' cap and a uridine modification in a region of the polynucleotide.

[0399] Amphiphilic cell membrane penetrating peptides According to convention, peptides described herein are depicted with the "N-terminus" first, ie, on the left.

[0400] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology.

[0401] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence with at least 80% sequence identity or homology.

[0402] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology.

[0403] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 85% sequence identity or homology.

[0404] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence with at least 85% sequence identity or homology.

[0405] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 85% sequence identity or homology.

[0406] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 90% sequence identity or homology.

[0407] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 90% sequence identity or homology.

[0408] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 90% sequence identity or homology.

[0409] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 95% sequence identity or homology.

[0410] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence with at least 95% sequence identity or homology.

[0411] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 95% sequence identity or homology.

[0412] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1).

[0413] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1).

[0414] In one embodiment, the vaccine comprises an amphipathic cell membrane penetrating peptide consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1).

[0415] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) consists of 35 amino acid residues or less.

[0416] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) consists of 30 or fewer amino acid residues.

[0417] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) consists of 26 to 30 amino acid residues.

[0418] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises at least five arginine residues (R).

[0419] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises at least 6 arginine residues (R).

[0420] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises at least 10 alanine residues (A).

[0421] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises at least 12 alanine residues (A).

[0422] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) contains at least 5 leucine residues (L).

[0423] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) contains at least 6 leucine residues (L).

[0424] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) contains at least one cysteine ​​residue (C).

[0425] In one embodiment, a sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) contains at least two but no more than three glutamic acid (E) residues.

[0426] In one embodiment, a sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) contains at least 6 arginine residues (R), at least 12 alanine residues (A), at least 6 leucine residues (L), optionally at least one cysteine ​​residue (C) and at least 2 but not more than 3 glutamic acid residues (E).

[0427] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises the consensus sequence EARLARALARALAR and / or LARALARALRA.

[0428] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises the consensus sequence EARLARALARALAR.

[0429] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises the consensus sequence LARALARALRA.

[0430] In one embodiment, sequences having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) include the consensus sequences EARLARALARALAR and LARALARALRA.

[0431] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) does not contain a glycine (G).

[0432] Sequences with at least 80% sequence identity or homology Any of a variety of sequence alignment methods may be used to determine percent sequence identity, including, but not limited to, global, local, and hybrid methods, such as segment approach methods. Protocols for determining percent identity are routine procedures within the skill of the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up the scores of individual residue pairs and by imposing gap penalties. Traditional methods include Altschul et al., Bull. Math. Bio. 48: 603-16, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992, in which two amino acid sequences are aligned to optimize alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff.

[0433] "Sequence identity" between two or more sequences is expressed as a percentage and is related to the number of identical positions shared by the sequences. Thus, sequence identity can be calculated as the number of identical amino acids or nucleotides divided by the total number of amino acids or nucleotides, multiplied by 100. The calculation of sequence identity % can also take into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. The sequence comparison and identity percentage determination between two or more sequences can be performed using certain mathematical algorithms, such as BLAST, which are well known to those skilled in the art.

[0434] Homologous sequences may be characterized as having one or more amino acid or nucleotide substitutions, deletions or additions / insertions. These changes are of a minor nature that do not significantly affect the folding or activity of the peptide. They may be minor amino acid or nucleotide substitutions, minor deletions, and minor terminal extensions, or other minor additions / insertions. Algorithms (e.g., BLAST) look for the minimum number of editing operations (insertions, deletions, and substitutions) to convert one sequence into an exact copy of the other and assign a % similarity (homology).

[0435] In one embodiment, the term "sequence identity or homology" refers to sequence identity.

[0436] In one embodiment, the term "sequence identity or homology" refers to sequence homology.

[0437] Nanoparticles In one embodiment, the vaccine described herein comprises or consists of nanoparticles. The nanoparticles can be formed by self-assembly by adding both the mRNA polynucleotide and the amphiphilic cell membrane penetrating peptide in ultrapure water. The formulation can be lyophilized and then reconstituted for administration.

[0438] In one embodiment, i) an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; and ii) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; A nanoparticle formulation is provided comprising:

[0439] In one embodiment, i) an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; and ii) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; The vaccine described herein is provided comprising a nanoparticle formulation of

[0440] In one embodiment, there is provided a vaccine as described herein formulated in nanoparticles.

[0441] In one embodiment, nanoparticles comprising a vaccine described herein are provided.

[0442] In one embodiment, a nanoparticle formulation comprising a vaccine described herein is provided.

[0443] In one embodiment, there is provided a vaccine as described herein comprising nanoparticles.

[0444] In one embodiment, a vaccine as described herein is provided that comprises nanoparticles having a Z-average of 30-150 nm, where Z-average is the intensity-weighted average hydrodynamic size of a collection of particles as measured by dynamic light scattering (DLS).

[0445] In one embodiment there is provided a vaccine as described herein comprising nanoparticles having a Z-average of 60-100 nm.

[0446] In one embodiment, there is provided a vaccine as described herein comprising nanoparticles having a polydispersity index of < 0.3. The polydispersity index (PI) is a size-based measure of the heterogeneity of a sample.

[0447] In one embodiment there is provided a vaccine as described herein comprising nanoparticles having a polydispersity index of ≦0.5.

[0448] In one embodiment, there is provided a vaccine as described herein that comprises nanoparticles that have a net positive charge at neutral pH.

[0449] mRNA:N:P ratio of amphiphilic cell membrane penetrating peptides The amount of RALA peptide relative to mRNA is indicated by the N:P ratio, which represents the molar ratio of positively charged nitrogen atoms in the peptide to negatively charged phosphates in the mRNA. The mRNA quoted in the ratio may represent one mRNA or a mixture of mRNAs, depending on the product.

[0450] The N:P ratio is widely used to describe the content of peptide or protein-based nucleic acid nanoparticles because it is simple compared to mass. It is the molar ratio of the positively charged nitrogen atoms in amino acids to the negatively charged phosphates contributed by the nucleic acid backbone and can be described as the mass of protein or peptide required to neutralize the charge of 1 μg of nucleic acid. The N:P ratio can be calculated using the following equation:

[0451]

number

[0452] [In the formula, M RALA is the mass of protein in the nanoparticle, M mRNA is the mass of mRNA in the nanoparticles, and C NP is the N:P constant. The N:P constant is the ratio of the positive charge density of the amino acid chain to the negative charge density of the mRNA backbone, where the charge density is defined as the charge divided by the molecular mass. This N:P constant can be calculated based on the knowledge that arginine is the positively charged amino acid and that the mass and charge of the bases in the mRNA backbone are constant, leaving only the molecular mass of the protein and the charge of the protein as variables. Therefore, the calculation of the N:P constant can be simplified to:

[0453]

number

[0454] [In the formula, QRALA is the positive charge provided by the seven arginine residues in the sequence of RALA, and 340 is the average molecular weight of an mRNA nucleoside].

[0455] For example, a 1:1 N:P ratio (peptide to mRNA) is 1.40 μg peptide (SEQ ID NO:1):1 μg mRNA, a 7:1 N:P ratio is 9.79 μg peptide (SEQ ID NO:1):1 μg mRNA, and for a 9:1 N:P ratio it is 12.6 μg peptide (SEQ ID NO:1):1 μg mRNA.

[0456] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptides:mRNA polynucleotides in the vaccine can be varied, which can have beneficial effects on physiochemical properties (e.g., Z-average, Zeta potential (particle charge), and / or polydispersity index), cellular uptake, and / or treatment efficacy.

[0457] In one embodiment, the N:P ratio of the amphipathic cell membrane-penetrating peptide:mRNA polynucleotide in the vaccine is about 1 to 12:1.

[0458] In one embodiment, the N:P ratio of the amphipathic cell membrane-penetrating peptide:mRNA polynucleotide is 1 to 12:1.

[0459] In one embodiment, the N:P ratio of the amphipathic cell membrane-penetrating peptide:mRNA polynucleotide in the vaccine is about 5 to 12:1.

[0460] In one embodiment, the N:P ratio of the amphipathic cell membrane-penetrating peptide:mRNA polynucleotide in the vaccine is 5 to 12:1.

[0461] In one embodiment, the N:P ratio of amphipathic cell membrane-penetrating peptide:mRNA polynucleotide in the vaccine is about 7-10:1.

[0462] In one embodiment, the N:P ratio of the amphipathic cell membrane-penetrating peptide:mRNA polynucleotide in the vaccine is 7 to 10:1.

[0463] In one embodiment, the N:P ratio of the amphipathic cell membrane-penetrating peptide:mRNA polynucleotide in the vaccine is about 8.5 to 9.5:1.

[0464] In one embodiment, the N:P ratio of the amphipathic cell membrane-penetrating peptide:mRNA polynucleotide in the vaccine is 8.5 to 9.5:1.

[0465] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is 1:1.

[0466] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is about 1:1.

[0467] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is 2:1.

[0468] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is about 2:1.

[0469] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is 3:1.

[0470] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is about 3:1.

[0471] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is 4:1.

[0472] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is about 4:1.

[0473] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is 5:1.

[0474] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is about 5:1.

[0475] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is 6:1.

[0476] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is about 6:1.

[0477] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is 7:1.

[0478] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is about 7:1.

[0479] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is 8:1.

[0480] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is about 8:1.

[0481] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is 9:1.

[0482] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is about 9:1.

[0483] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is 10:1.

[0484] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is about 10:1.

[0485] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is 11:1.

[0486] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is about 11:1.

[0487] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is 12:1.

[0488] In one embodiment, the N:P ratio of amphipathic cell membrane penetrating peptide:mRNA polynucleotide in the vaccine is about 12:1.

[0489] Bulking agents, cryoprotectants and tonicity agents In one embodiment, bulking agents can be added prior to lyophilization of the nanoparticles for shipping and storage. Bulking agents are additives that increase the bulk volume of the product without affecting the properties of the product.

[0490] In one embodiment, a cryoprotectant may be added prior to freeze-drying of the nanoparticles. Cryoprotectants are substances used to protect biological materials from freeze damage.

[0491] In one embodiment, solutes may be added to impart tonicity when water is added to the formulation, e.g., to create an isotonic formulation, which has the same solute concentration as blood, i.e., 290-310 mOsmol / kg.

[0492] In one embodiment, the osmolality of a solution of a vaccine described herein in water is between 10 and 1000 mOsmol / kg.

[0493] In one embodiment, the osmolality of a solution of a vaccine described herein in water is between 100 and 500 mOsmol / kg.

[0494] In one embodiment, the osmolality of a solution of a vaccine described herein in water is between 200 and 400 mOsmol / kg.

[0495] In one embodiment, the osmolality of a solution of a vaccine described herein in water is between 290 and 310 mOsmol / kg.

[0496] In one embodiment, the osmolality of a solution of a vaccine described herein in water is about 300 mOsmol / kg.

[0497] In one embodiment, the osmolality of a solution of a vaccine described herein in water is 300 mOsmol / kg.

[0498] Suitable bulking agents include trehalose, sucrose, mannose, dextrose or any mixture of such agents. These agents may also be used as cryoprotectants and / or tonicity agents.

[0499] In one embodiment, the vaccines described herein further comprise trehalose, sucrose, mannose, dextrose, or any mixture of such agents.

[0500] In one embodiment, the vaccine described herein further comprises >85% w / w trehalose, sucrose, mannose, dextrose or any mixture of such agents.

[0501] In one embodiment, the vaccine described herein further comprises >90% w / w trehalose, sucrose, mannose, dextrose or any mixture of such agents.

[0502] In one embodiment, the vaccine described herein further comprises >95% w / w trehalose, sucrose, mannose, dextrose or any mixture of such agents.

[0503] In one embodiment, the vaccine described herein comprises a bulking agent.

[0504] In one embodiment, the vaccine described herein comprises a bulking agent selected from trehalose, sucrose, mannose, and dextrose.

[0505] In one embodiment, the vaccine described herein comprises trehalose.

[0506] In one embodiment, the vaccine described herein further comprises trehalose.

[0507] In one embodiment, the vaccine described herein further comprises >85% w / w trehalose.

[0508] In one embodiment, the vaccine described herein further comprises >90% w / w trehalose.

[0509] In one embodiment, the vaccine described herein further comprises >95% w / w trehalose.

[0510] In one embodiment, the vaccine described herein further comprises >98% w / w trehalose.

[0511] In one embodiment, the vaccine described herein further comprises about 99% w / w trehalose.

[0512] In one embodiment, the vaccine described herein further comprises 99% w / w trehalose.

[0513] In one embodiment, the vaccine described herein comprises sucrose.

[0514] In one embodiment, the vaccine described herein further comprises sucrose.

[0515] In one embodiment, the vaccine described herein further comprises >85% w / w sucrose.

[0516] In one embodiment, the vaccine described herein further comprises >90% w / w sucrose.

[0517] In one embodiment, the vaccine described herein further comprises >95% w / w sucrose.

[0518] In one embodiment, the vaccine described herein comprises mannose.

[0519] In one embodiment, the vaccine described herein further comprises mannose.

[0520] In one embodiment, the vaccine described herein further comprises >85% w / w mannose.

[0521] In one embodiment, the vaccine described herein further comprises >90% w / w mannose.

[0522] In one embodiment, the vaccine described herein further comprises >95% w / w mannose.

[0523] In one embodiment, the vaccine described herein comprises dextrose.

[0524] In one embodiment, the vaccine described herein comprises mannose and trehalose.

[0525] In one embodiment, the vaccine described herein comprises 5-20% w / v mannose and trehalose, which means 5-20% w / v (mannose + trehalose).

[0526] In one embodiment, the vaccine described herein comprises about 10% w / v mannose and trehalose.

[0527] In one embodiment, the vaccine described herein comprises 10% w / v mannose and trehalose.

[0528] In one embodiment, the vaccine described herein comprises trehalose and mannose in a ratio of 85:15 w / v.

[0529] In one embodiment, the vaccine described herein comprises trehalose and mannose in a ratio of about 85:15 w / v.

[0530] In one embodiment, the vaccine described herein comprises trehalose and mannose in a ratio of 70:30 w / v.

[0531] In one embodiment, the vaccine described herein comprises trehalose and mannose in a ratio of about 70:30 w / v.

[0532] In one embodiment, the vaccine described herein comprises 5-20% w / v of trehalose and mannose in a ratio of about 85:15 w / v.

[0533] In one embodiment, the vaccine described herein comprises 5-20% w / v of trehalose and mannose in a ratio of about 70:30 w / v.

[0534] In one embodiment, the vaccine described herein comprises about 10% w / v of trehalose and mannose in a ratio of about 85:15 w / v.

[0535] In one embodiment, the vaccine described herein comprises about 10% w / v of trehalose and mannose in a ratio of about 70:30 w / v.

[0536] In one embodiment, the vaccine described herein comprises: (i) 0.01 to 1% w / w of one or more mRNA polynucleotides comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; (ii) 0.5 to 5% w / w of an amphipathic cell membrane-penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 89 to 99% w / w of trehalose, sucrose, mannose and / or dextrose Includes.

[0537] In one embodiment, the vaccine described herein comprises: (i) 0.01 to 1% w / w of one or more mRNA polynucleotides comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof; (ii) 0.5 to 2% w / w of an amphipathic cell membrane-penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 95-99% w / w trehalose Includes.

[0538] In one embodiment, the vaccine described herein comprises: (i) 0.1% w / w of one or more mRNA polynucleotides comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof; (ii) 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 99% w / w trehalose Includes.

[0539] In one embodiment, the vaccine described herein comprises: (i) about 0.08% w / w of one or more mRNA polynucleotides comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof; (ii) about 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) about 99% w / w trehalose Includes.

[0540] In one embodiment, the vaccine described herein comprises: (i) 0.08% w / w of one or more mRNA polynucleotides comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof; (ii) 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 99% w / w trehalose Includes.

[0541] In one embodiment, the vaccine described herein comprises: (i) two or more mRNA polynucleotides comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof, in approximately equal % w / w, about 0.08% w / w; (ii) about 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) about 99% w / w trehalose Includes.

[0542] In one embodiment, the vaccine described herein comprises: (i) 0.08% w / w, two or more mRNA polynucleotides containing an open reading frame encoding an HPV antigen or an immunogenic fragment thereof, in equal % w / w; (ii) 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 99% w / w trehalose Includes.

[0543] In one embodiment, the vaccine described herein comprises: (i) three mRNA polynucleotides containing open reading frames encoding HPV antigens or immunogenic fragments thereof, in approximately equal % w / w, about 0.08% w / w; (ii) about 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) about 99% w / w trehalose Includes.

[0544] In one embodiment, the vaccine described herein comprises: (i) 0.08% w / w, three mRNA polynucleotides containing an open reading frame encoding an HPV antigen or an immunogenic fragment thereof, in equal % w / w, (ii) 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 99% w / w trehalose Includes.

[0545] Preparations in water The vaccines described herein may be conveniently formulated in water, especially ultrapure water, for ease of administration, especially by intravenous injection.

[0546] In one embodiment, the vaccine described herein comprises: (i) 0.01 to 1% w / w of one or more mRNA polynucleotides comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; (ii) 0.5 to 5% w / w of an amphipathic cell membrane-penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 89 to 99% w / w of trehalose, sucrose, mannose and / or dextrose and is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0547] In one embodiment, the vaccine described herein comprises: (i) 0.01 to 1% w / w of one or more mRNA polynucleotides comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof; (ii) 0.5 to 2% w / w of an amphipathic cell membrane-penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 95-99% w / w trehalose and is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0548] In one embodiment, the vaccine described herein comprises: (i) 0.1% w / w of one or more mRNA polynucleotides comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof; (ii) 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 99% w / w trehalose and is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0549] In one embodiment, the vaccine described herein comprises: (i) about 0.08% w / w of one or more mRNA polynucleotides comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof; (ii) about 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) about 99% w / w trehalose and is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0550] In one embodiment, the vaccine described herein comprises: (i) 0.08% w / w of one or more mRNA polynucleotides comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof; (ii) 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 99% w / w trehalose and is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0551] In one embodiment, the vaccine described herein comprises: (i) two or more mRNA polynucleotides comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof, in approximately equal % w / w, about 0.08% w / w; (ii) about 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) about 99% w / w trehalose and is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0552] In one embodiment, the vaccine described herein comprises: (i) 0.08% w / w, two or more mRNA polynucleotides containing an open reading frame encoding an HPV antigen or an immunogenic fragment thereof, in equal % w / w; (ii) 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 99% w / w trehalose and is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0553] In one embodiment, the vaccine described herein comprises: (i) three mRNA polynucleotides containing open reading frames encoding HPV antigens or immunogenic fragments thereof, in approximately equal % w / w, about 0.08% w / w; (ii) about 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) about 99% w / w trehalose and is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0554] In one embodiment, the vaccine described herein comprises: (i) 0.08% w / w, three mRNA polynucleotides containing an open reading frame encoding an HPV antigen or an immunogenic fragment thereof, in equal % w / w, (ii) 1% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 99% w / w trehalose and is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0555] Automated Formulation In one embodiment, the vaccine is prepared by an automated controllable mixing system, such as an automated microfluidics system, such as Precision Nanosystems Ignite NanoAssemblr. This technology controls both the mixing speed and the mixing ratio during nanoparticle formulation, resulting in a reduction in Z-average particle size and potentially a further reduction in polydispersity index when compared to manual formulation methods.

[0556] Microfluidics refers to the behavior, precise control, and manipulation of fluids geometrically constrained to small scales (typically sub-millimeter) where surface forces dominate over volume forces.

[0557] In one embodiment, the vaccines described herein are prepared by an automated controlled mixing system.

[0558] In one embodiment, a method for preparing a nanoparticle formulation is provided, comprising formulating a solution of an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, together with an amphiphilic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology thereto, in an automated controlled mixing system, in particular an automated microfluidics system.

[0559] In one embodiment, a method is provided for preparing a nanoparticle formulation comprising formulating a solution of an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof together with an amphiphilic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology thereto in an automated controlled mixing system, wherein the flow rate ratio of mRNA:peptide is 1:3.

[0560] In one embodiment, a method is provided for preparing a nanoparticle formulation comprising formulating a solution of an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof together with an amphiphilic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology thereto in an automated controlled mixing system, wherein the flow rate ratio of mRNA:peptide is about 1:3.

[0561] In one embodiment, there is provided a method for preparing a nanoparticle formulation comprising formulating in an automated controlled mixing system a solution of an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, together with an amphiphilic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology thereto, wherein the flow rate ratio of mRNA:peptide is between 1:5 and 5:1.

[0562] In one embodiment, there is provided a method for preparing a nanoparticle formulation comprising formulating in an automated controlled mixing system a solution of an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, together with an amphiphilic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology thereto, wherein the flow rate ratio of mRNA:peptide is about 1:5 to 5:1.

[0563] formulation Nanoparticles can be formed by self-assembly by adding mRNA and amphiphilic cell membrane penetrating peptides together in ultrapure water, resulting in immediate formulation. The resulting vaccine can be lyophilized for transportation and storage, and then rehydrated in water for use.

[0564] The vaccines described herein may be used by a variety of routes of administration, including oral, nasal, inhalation, rectal, topical, transdermal, intravitreal, intravenous, intramuscular, or intradermal, particularly intradermal or intramuscular administration, which may maximize delivery to dendritic cells, which are most commonly found near the surface of the skin.

[0565] In one embodiment, the vaccine of the present disclosure may be used in an injectable formulation, for example an intradermal injection.

[0566] In one embodiment, the vaccine of the present disclosure may be used in an injectable formulation, for example, intramuscular injection.

[0567] In one embodiment, the vaccine of the present disclosure may be used in an injectable formulation, for example, intravenous injection.

[0568] In one embodiment, the vaccine of the present disclosure can be used in an injectable formulation, such as an intradermal patch. The surface of the intradermal patch is covered with small microneedles that dissolve in the body. The patch can be applied painlessly like a bandage, allowing the vaccine to quickly overcome the epidermal barrier and be delivered directly to the area with the most dendritic cells.

[0569] dose The exact amount required for effective vaccination varies from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the specific composition, its mode of administration, its mode of activity, the desired prophylactic and / or therapeutic effect, etc. Vaccine compositions are typically formulated in dosage unit form for ease of administration and uniformity of dosage. However, it is understood that the total dose of the vaccine composition can be determined by the attending physician within the scope of sound medical judgment. The vaccine can be administered more than once, for example, two or three times on separate occasions, to maximize effectiveness.

[0570] In one embodiment, the vaccine may be administered to a subject at a dosage of between 0.1 μg / kg and 500 μg / kg.

[0571] In one embodiment, the vaccine may be administered to a subject at a dosage of between 1 μg / kg and 500 μg / kg.

[0572] In one embodiment, the vaccine described herein comprises between 10 and 150 μg of an mRNA polynucleotide.

[0573] In one embodiment, the vaccine described herein comprises about 100 μg of an mRNA polynucleotide.

[0574] In one embodiment, the vaccine described herein comprises 100 μg of an mRNA polynucleotide.

[0575] In one embodiment, the vaccine is administered twice.

[0576] In one embodiment, the vaccine is administered three times.

[0577] In one embodiment, the vaccine described herein comprises 10-150 μg of mRNA polynucleotide and is administered in three doses.

[0578] In one embodiment, the vaccine described herein comprises about 100 μg of mRNA polynucleotide and is administered three times.

[0579] In one embodiment, the vaccine described herein comprises 100 μg of mRNA polynucleotide and is administered three times.

[0580] In one embodiment, a vaccine described herein comprises at least two mRNA polynucleotides that comprise an open reading frame and encode an antigen or immunogenic fragment thereof from an infectious microorganism, wherein the dosage for the vaccine is a combined therapeutic dosage and the dosage of each individual mRNA polynucleotide is a sub-therapeutic dosage.

[0581] age In one embodiment, the vaccines described herein may be administered to subjects under the age of 5.

[0582] In one embodiment, the vaccines described herein may be administered to subjects between 5 and 11 years of age.

[0583] In one embodiment, the vaccines described herein may be administered to subjects between the ages of 9 and 45.

[0584] In one embodiment, the vaccines described herein may be administered to subjects between 12 and 15 years of age.

[0585] In one embodiment, the vaccines described herein may be administered to subjects aged 16 or 17 years.

[0586] In one embodiment, the vaccines described herein may be administered to subjects 17 years of age or younger.

[0587] In one embodiment, the vaccines described herein may be administered to subjects aged 18 years or older.

[0588] In one embodiment, the vaccines described herein may be administered to subjects aged 60 years or older.

[0589] Vaccine effectiveness Vaccine efficacy can be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1:201(11):1607-10). For example, vaccine efficacy can be measured by double-blind, randomized, controlled clinical trials. Vaccine efficacy may be expressed as the proportional reduction in disease incidence (AR) between unvaccinated (ARU) and vaccinated (ARV) study cohorts, and can be calculated from the relative risk (RR) of disease in the vaccinated group using the following formula: Efficacy = (ARU-ARV) / ARU x 100, and Efficacy = (1-RR) x 100.

[0590] Similarly, vaccine effectiveness may be assessed using standard analyses (see, for example, Weinberg et al., J Infect Dis. 2010 Jun 1; 201(11):1607-10). Vaccine effectiveness is an assessment of the extent to which a vaccine (which may already be proven to have high vaccine efficacy) reduces disease in a population. This measure allows the assessment of the net balance of benefits and adverse effects of the vaccination program as well as the vaccine itself under natural field conditions, rather than in a controlled clinical trial. Vaccine effectiveness is proportional to the efficacy (potency) of the vaccine, but is also influenced by how well target groups are immunized in the population, as well as by other non-vaccine related factors that affect the "real world" outcomes of hospitalization, outpatient visits, or costs. For example, retrospective case-control analyses comparing vaccination rates among a series of infected cases and appropriate controls may be used. Vaccine effectiveness may be expressed as a rate difference, using the odds ratio (OR) of developing an infection despite vaccination: Efficacy = (1-OR) x 100.

[0591] In one embodiment, the vaccine immunizes a subject for up to two years.

[0592] In one embodiment, the vaccine immunizes a subject for more than 2 years, more than 3 years, more than 4 years, or 5-10 years.

[0593] An "effective amount" of a vaccine may depend on the target tissue, the type of target cell, the means of administration, the physical characteristics of the vaccine components, and other determinants. In general, an effective amount of a vaccine composition provides an induced or enhanced immune response in response to antigen production in cells. Increased antigen production may be demonstrated by increased cell transfection, increased protein translation, decreased nucleic acid degradation, or altered antigen-specific immune response of host cells. In one embodiment, an "effective amount" is a therapeutically effective amount.

[0594] charge density The average surface charge density of nanoparticles may be a contributing factor to nanoparticle toxicity by promoting oxidative stress mechanisms, which in turn may promote mitochondrial dysfunction and reduced viability.

[0595] Charge density can be measured by polyelectrolyte titration using the methods described in Ritz et al., Biomacromolecules. 2015 Apr 13;16(4):1311-21. doi: 10.1021 / acs.biomac.5b00108. Epub 2015 Apr 3, and Weiss et al., J Nanobiotechnology. 2021 Jan 6;19(1):5. doi: 10.1186 / s12951-020-00747-7.

[0596] Polyelectrolyte titration may be performed using 0.01 M poly(acrylic acid) (PAA) at pH 7.4; addition of PAA to the nanoparticles and measurement of the charge produces a sigmoidal curve, and the volume (V) of this curve can be obtained from the equivalence point. Together, the mass of peptide in the system (w) and the concentration of PAA used (0.01 M) can be used to calculate the average charge density (Q ek ) to calculate

[0597] In one embodiment, the nanoparticles comprising a vaccine described herein have an average surface charge density of ≦2 μmol / mg at 20° C. The average surface charge density values ​​were measured using an Orion STAR® multiparameter bench meter.

[0598] In one embodiment, the nanoparticles comprising a vaccine described herein have an average surface charge density of ≦1.5 μmol / mg at 20° C.

[0599] In one embodiment, the average surface charge density of the nanoparticles comprising a vaccine described herein is about 1 μmol / mg at 20°C.

[0600] In one embodiment, the nanoparticles comprising a vaccine described herein have an average surface charge density of 1 μmol / mg at 20°C.

[0601] use In one embodiment there is provided a vaccine as described herein for use as a medicament.

[0602] In one embodiment there is provided the use of a vaccine as described herein as a medicament.

[0603] In one embodiment there is provided a vaccine as described herein for use in therapy.

[0604] As used herein, the terms "treatment" and "treating" refer to the prevention, reversal, alleviation, delay of onset, or inhibition of progression of a disease or disorder, or one or more symptoms thereof, as described herein. In one embodiment, treatment may be performed before an infection occurs. In one embodiment, treatment may be performed in a subject exposed to an infection. In one embodiment, treatment may be performed in a subject at risk of infection. In one embodiment, treatment may be performed after one or more symptoms have developed. In other embodiments, treatment may be performed in the absence of symptoms. For example, treatment may be performed in a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors and / or suggestive diagnostic tests). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay recurrence of symptoms.

[0605] When the use in the treatment of cancer is described herein, this may be cancer in an early stage, cancer that is actively progressing, metastatic cancer and / or drug-resistant cancer. In one embodiment of cancer, the cancer is an early stage cancer. In one embodiment of cancer, the cancer is a locally advanced cancer. In one embodiment of cancer, the cancer is a locally advanced cancer and / or a metastatic cancer. In one embodiment of cancer, the cancer is a metastatic cancer. In one embodiment of cancer, the cancer is an invasive cancer.

[0606] In one embodiment, there is provided a vaccine described herein for use as a therapeutic vaccine.

[0607] In one embodiment, there is provided a vaccine as described herein for use as a prophylactic vaccine.

[0608] In one embodiment, there is provided a vaccine as described herein for use as a therapeutic or prophylactic vaccine.

[0609] In one embodiment there is provided a vaccine as described herein for use in generating, maintaining or restoring antigenic memory against a viral strain.

[0610] In one embodiment, the antigen-specific immune response in a subject, particularly CD8 + Provided is a vaccine as described herein for use in eliciting a T cell response.

[0611] In one embodiment, a vaccine as described herein is provided for use in delivering an mRNA comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism to a cell.

[0612] In one embodiment, a vaccine as described herein is provided for use in delivering an mRNA comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism to target tissues of the immune system, in particular lymph nodes, spleen and / or bone marrow.

[0613] In one embodiment there is provided a vaccine as described herein for use in producing an antigen, or an immunogenic fragment thereof, from an infectious microorganism in a cell, tissue or organism.

[0614] In one embodiment, there is provided a vaccine as described herein for use in the treatment of cancer.

[0615] In one embodiment, there is provided a vaccine as described herein for use in treating HPV-associated cancer.

[0616] In one embodiment there is provided a vaccine as described herein for use in the treatment of squamous cell carcinoma and adenocarcinoma.

[0617] In one embodiment there is provided a vaccine as described herein for use in the treatment of cervical cancer, oropharyngeal and other head and neck cancers, anal cancer, penile cancer, vaginal cancer and / or vulvar cancer.

[0618] In one embodiment there is provided a vaccine as described herein for use in the treatment of a precancerous or dysplastic lesion.

[0619] In one embodiment there is provided a vaccine as described herein for use in the treatment of cervical, vulvar, vaginal and / or anal intraepithelial neoplasia.

[0620] In one embodiment there is provided a vaccine as described herein for use in treating a sexually transmitted disease.

[0621] In one embodiment there is provided a vaccine as described herein for use in treating genital warts.

[0622] In one embodiment, there is provided a vaccine as described herein for use in treating an HPV infection.

[0623] In one embodiment, there is provided a vaccine as described herein for use in treating a mucosal HPV infection.

[0624] In one embodiment there is provided a vaccine as described herein for use in treating a cutaneous HPV infection.

[0625] In one embodiment there is provided a vaccine as described herein for use in treating HPV 6, 11, 8, 16 and / or 18 infection.

[0626] Pharmaceutical Compositions In one embodiment, there is provided a pharmaceutical composition comprising a vaccine as described herein for use in intradermal injection.

[0627] In one embodiment, a pharmaceutical composition comprising a vaccine as described herein is provided for use in intramuscular injection.

[0628] In one embodiment, a pharmaceutical composition comprising a vaccine described herein for use as a therapeutic vaccine is provided.

[0629] In one embodiment, there is provided a pharmaceutical composition comprising a vaccine described herein for use as a prophylactic vaccine.

[0630] In one embodiment, pharmaceutical compositions comprising the vaccines described herein are provided for use as therapeutic and prophylactic vaccines.

[0631] In one embodiment there is provided a pharmaceutical composition comprising a vaccine as described herein for use in generating, maintaining or restoring antigenic memory against a viral strain.

[0632] In one embodiment, the antigen-specific immune response in a subject, particularly CD8 + Pharmaceutical compositions comprising the vaccines described herein for use in eliciting a T cell response are provided.

[0633] In one embodiment, a pharmaceutical composition is provided comprising the vaccine described herein for use in delivering an mRNA comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism to a cell.

[0634] In one embodiment, a pharmaceutical composition is provided comprising the vaccine described herein for use in delivering an mRNA comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism to target tissues of the immune system, in particular lymph nodes, spleen and / or bone marrow.

[0635] In one embodiment, there is provided a pharmaceutical composition comprising a vaccine as described herein for use in producing an antigen, or an immunogenic fragment thereof, from an infectious microorganism in a cell, tissue or organism.

[0636] In one embodiment, there is provided a pharmaceutical composition comprising a vaccine described herein for use in the treatment of cancer.

[0637] In one embodiment, there is provided a pharmaceutical composition comprising a vaccine described herein for use in treating HPV-associated cancer.

[0638] In one embodiment, there is provided a pharmaceutical composition comprising a vaccine as described herein for use in the treatment of squamous cell carcinoma and adenocarcinoma.

[0639] In one embodiment there is provided a pharmaceutical composition comprising a vaccine as described herein for use in the treatment of cervical cancer, oropharyngeal and other head and neck cancers, anal cancer, penile cancer, vaginal cancer and / or vulvar cancer.

[0640] In one embodiment there is provided a pharmaceutical composition comprising a vaccine as described herein for use in the treatment of a precancerous or dysplastic lesion.

[0641] In one embodiment there is provided a pharmaceutical composition comprising a vaccine as described herein for use in the treatment of cervical, vulvar, vaginal and / or anal intraepithelial neoplasia.

[0642] In one embodiment, there is provided a pharmaceutical composition comprising a vaccine described herein for use in treating a sexually transmitted disease.

[0643] In one embodiment there is provided a pharmaceutical composition comprising a vaccine as described herein for use in the treatment of genital warts.

[0644] In one embodiment, there is provided a pharmaceutical composition comprising a vaccine as described herein for use in treating HPV infection.

[0645] In one embodiment, there is provided a pharmaceutical composition comprising a vaccine as described herein for use in treating a mucosal HPV infection.

[0646] In one embodiment there is provided a pharmaceutical composition comprising a vaccine as described herein for use in treating a cutaneous HPV infection.

[0647] In one embodiment there is provided a pharmaceutical composition comprising a vaccine as described herein for use in the treatment of HPV 6, 11, 8, 16 and / or 18 infection.

[0648] Treatment In one embodiment, a method of intradermal injection is provided that includes administering a vaccine described herein.

[0649] In one embodiment, a method of intravenous injection is provided that includes administering a vaccine described herein.

[0650] In one embodiment there is provided a method of therapeutic vaccination in a warm-blooded animal, such as a human, comprising administering to said animal an effective amount of a vaccine described herein.

[0651] In one embodiment there is provided a method of prophylactic vaccination in a warm-blooded animal, such as a human, comprising administering to said animal an effective amount of a vaccine described herein.

[0652] In one embodiment, there is provided a method of therapeutic and prophylactic vaccination in a warm-blooded animal, such as a human, comprising administering to said animal an effective amount of a vaccine described herein.

[0653] In one embodiment, there is provided a method of generating, maintaining or restoring antigenic memory to a viral strain in a warm-blooded animal, such as a human, comprising administering to the animal an effective amount of a vaccine described herein.

[0654] In one embodiment, an antigen-specific immune response in a subject, particularly CD8 in a warm-blooded animal such as a human, is + A method of inducing a T cell response is provided, comprising administering to the animal an effective amount of a vaccine described herein.

[0655] In one embodiment, a method is provided for delivering an mRNA comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism to a cell in a warm-blooded animal, such as a human, comprising administering to the animal an effective amount of a vaccine described herein.

[0656] In one embodiment, there is provided a method for delivering an mRNA comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism to target tissues of the immune system, in particular lymph nodes, spleen and / or bone marrow, in a warm-blooded animal such as a human, comprising administering to the animal an effective amount of a vaccine described herein.

[0657] In one embodiment, there is provided a method for producing an antigen, or an immunogenic fragment thereof, derived from an infectious microorganism in a cell, tissue or organism in a warm-blooded animal, such as a human, comprising administering to the animal an effective amount of a vaccine described herein.

[0658] In one embodiment, a method of treating cancer in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a vaccine described herein.

[0659] In one embodiment, a method of treating HPV-associated cancer in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a vaccine described herein.

[0660] In one embodiment, a method of treating squamous cell carcinoma and adenocarcinoma in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a vaccine described herein.

[0661] In one embodiment, there is provided a method of treating cervical cancer, oropharyngeal and other head and neck cancer, anal cancer, penile cancer, vaginal cancer and / or vulvar cancer in a warm-blooded animal such as a human, comprising administering to the animal an effective amount of a vaccine described herein.

[0662] In one embodiment, a method of treating a precancerous or dysplastic lesion in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a vaccine described herein.

[0663] In one embodiment, a method is provided for treating cervical, vulvar, vaginal, and / or anal intraepithelial neoplasia in a warm-blooded animal, such as a human, comprising administering to the animal an effective amount of a vaccine described herein.

[0664] In one embodiment, a method of treating a sexually transmitted disease in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a vaccine described herein.

[0665] In one embodiment, a method of treating genital warts in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a vaccine described herein.

[0666] In one embodiment, a method of treating an HPV infection in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a vaccine described herein.

[0667] In one embodiment, a method of treating a mucosal HPV infection in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a vaccine described herein.

[0668] In one embodiment, a method of treating a cutaneous HPV infection in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a vaccine described herein.

[0669] In one embodiment, a method of treating HPV types 6, 11, 8, 16 and / or 18 infection in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a vaccine described herein.

[0670] Vaccine use In one embodiment there is provided the use of a vaccine as described herein for the manufacture of a medicament for intradermal injection.

[0671] In one embodiment there is provided a use of a vaccine as described herein for the manufacture of a medicament for intramuscular injection.

[0672] In one embodiment there is provided the use of a vaccine as described herein for the manufacture of a medicament for therapeutic vaccination.

[0673] In one embodiment there is provided the use of a vaccine as described herein for the manufacture of a medicament for prophylactic vaccination.

[0674] In one embodiment there is provided the use of a vaccine as described herein for the manufacture of a medicament for therapeutic and prophylactic vaccination.

[0675] In one embodiment there is provided a use of a vaccine as described herein for the manufacture of a medicament for generating, maintaining or restoring antigenic memory against a viral strain.

[0676] In one embodiment, the antigen-specific immune response in a subject, particularly CD8 + There is provided the use of a vaccine as described herein for the manufacture of a medicament for eliciting a T cell response.

[0677] In one embodiment there is provided a use of a vaccine as described herein for the manufacture of a medicament for delivering an mRNA comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism to a cell.

[0678] In one embodiment there is provided the use of a vaccine as described herein for the manufacture of a medicament for delivering an mRNA comprising an open reading frame encoding an antigen or an immunogenic fragment thereof derived from an infectious microorganism to a target tissue of the immune system, in particular the lymph nodes, spleen and / or bone marrow.

[0679] In one embodiment there is provided a use of a vaccine as described herein for the manufacture of a medicament for producing an antigen, or an immunogenic fragment thereof, from an infectious microorganism in a cell, tissue or organism.

[0680] In one embodiment there is provided a use of a vaccine described herein for the manufacture of a medicament for the treatment of cancer.

[0681] In one embodiment, there is provided a use of a vaccine described herein for the manufacture of a medicament for the treatment of an HPV-associated cancer.

[0682] In one embodiment there is provided the use of a vaccine as described herein for the manufacture of a medicament for the treatment of squamous cell carcinoma and adenocarcinoma.

[0683] In one embodiment there is provided a use of a vaccine described herein for the manufacture of a medicament for the treatment of cervical cancer, oropharyngeal and other head and neck cancers, anal cancer, penile cancer, vaginal cancer and / or vulvar cancer.

[0684] In one embodiment there is provided a use of a vaccine as described herein for the manufacture of a medicament for the treatment of a precancerous or dysplastic lesion.

[0685] In one embodiment there is provided a use of a vaccine as described herein for the manufacture of a medicament for the treatment of cervical, vulvar, vaginal and / or anal intraepithelial neoplasia.

[0686] In one embodiment there is provided a use of a vaccine described herein for the manufacture of a medicament for the treatment of a sexually transmitted disease.

[0687] In one embodiment there is provided a use of a vaccine as described herein for the manufacture of a medicament for the treatment of genital warts.

[0688] In one embodiment there is provided a use of a vaccine described herein for the manufacture of a medicament for the treatment of HPV infection.

[0689] In one embodiment there is provided a use of a vaccine as described herein for the manufacture of a medicament for the treatment of a mucosal HPV infection.

[0690] In one embodiment there is provided a use of a vaccine as described herein for the manufacture of a medicament for the treatment of a cutaneous HPV infection.

[0691] In one embodiment there is provided a use of a vaccine as described herein for the manufacture of a medicament for the treatment of HPV 6, 11, 8, 16 and / or 18 infection.

[0692] kit In one embodiment, a) a vaccine as described herein; b) container means for containing said vaccine A kit is provided comprising:

[0693] In one embodiment, a) a vaccine as described herein; b) a container means for containing said vaccine, and optionally c) Instructions for use A kit is provided comprising: EXAMPLES

[0694] General Experiment In the Examples section, the following applies: RALA refers to the peptide WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1), obtained in lyophilized powder form from Biomatik®, Canada or Bachem, Switzerland; · mRNA (E6) refers to an mRNA with 5' and 3' untranslated regions, a polyA tail containing 80 or 120 adenosines, and a basic open reading frame sequence of SEQ ID NO: 2 (uridine and cytidine modifications may vary and are described in the table below) and with 5' CleanCap® purchased from Trilink Biotechnologies®; mRNA (E7) refers to an mRNA with 5' and 3' untranslated regions, a polyA tail with 80 or 120 adenosines and a basic open reading frame sequence of SEQ ID NO: 3 (uridine and cytidine modifications may vary and are described in the table below) with 5'CleanCap® or ARCA cap purchased from Trilink Biotechnologies®; mRNA (E1) refers to an mRNA with 5' and 3' untranslated regions, a polyA tail with 80 or 120 adenosines and a basic open reading frame sequence of SEQ ID NO: 4 (uridine and cytidine modifications may vary and are described in the table below) with 5'CleanCap® purchased from Trilink Biotechnologies®; · mRNA (IL-15) refers to an mRNA with 5' and 3' untranslated regions, a polyA tail containing 120 adenosines, and a basic open reading frame sequence of SEQ ID NO: 8 (uridine and cytidine modifications may vary and are described in the table below) with 5' CleanCap® purchased from Trilink Biotechnologies®; · All mRNA was purchased from Trilink Biotechnologies and used without further modification; ·TC-1 cells were obtained from Johns Hopkins University Cell Repository, USA; ·C3.43 cells were obtained from The University of Southern California; · C57BL / 6 mice were obtained from Envigo®; ·HEK-293 cells were obtained from ATCC; · Cy5-EGFP refers to cyanine 5 fluorescent-tagged mRNA encoding enhanced green fluorescent protein obtained from Trilink Biotechnologies®; EGFP mRNA refers to the mRNA encoding enhanced green fluorescent protein obtained from Trilink Biotechnologies®; and ·NCTC-929 mouse fibroblast cells were obtained from ATCC.

[0695] Example 1: Preparation of RALA peptide RALA was reconstituted to the desired concentration with molecular grade water, aliquoted, and stored at -20°C until further use. Aliquots were taken as needed and thawed on ice. Aliquots were not refrozen once thawed.

[0696] Example 2: Nanoparticle Formulations 2.1: Manual formulation RALA / mRNA nanoparticles were formulated at various N:P ratios by first adding the required volume of ultrapure water to the mRNA in solution to achieve a concentration of 1 μg / μl. The corresponding volume of peptide solution at a concentration of 10 μg / μl (Table 6) was added to the diluted mRNA solution. The mixture was pipetted up and down approximately 5-10 times to ensure homogenous mixing. Nanoparticles formed spontaneously in the solution.

[0697] [Table 6]

[0698] 2.2: Automated Formulation RALA / mRNA nanoparticles were formulated at various N:P ratios by using an automated microfluidics system (e.g., Precision Nanosystems Ignite NanoAssemblr). The two solutions (Table 7) at appropriate concentrations were loaded into a syringe, which was then loaded into the microfluidics system. Nanoparticles were produced using a total flow rate (TFR) of 10 mL / min and a flow rate ratio (FRR) of 1:3 (mRNA:RALA). The resulting solution from the system contained nanoparticles at a concentration of 0.1 μg / μl of mRNA.

[0699] [Table 7]

[0700] The following nanoparticles were prepared (mRNA and trehalose:mannose ratios quoted are in % w / w):

[0701] [Table 8] TIFF2025506695000018.tif236165TIFF2025506695000019.tif204166

[0702] Example 3: Freeze-drying of RALA / mRNA nanoparticles 200 μL of RALA / mRNA nanoparticles prepared as in Example 2 were transferred to a 2 mL lyophilization vial. Trehalose (20% w / v), mannose (20% w / v), or a mixture of trehalose and mannose (85:15, final 20% w / v) was added to the nanoparticle solution in the vial as a cryoprotectant to ensure that the volume added was equal to half of the final reconstitution volume and that the resulting solution contained 10% w / v cryoprotectant when reconstituted in water after lyophilization. After addition of the cryoprotectant, the rubber stopper of the lyophilization vial was partially placed on the vial so that the airflow notch still functioned to facilitate sublimation during the lyophilization process. The vials were then loaded into a programmable lyophilizer (e.g., SP Scientific AdVantage Pro) according to the following lyophilization procedure (Table 9).

[0703] [Table 9]

[0704] Example 4: Size and charge analysis of nanoparticles Z-average particle size measurements and polydispersity (PdI) of RALA / mRNA nanoparticles prepared as in Example 2 were performed using dynamic light scattering (DLS) to obtain particle size and charge distribution. Measurements of the surface charge of RALA nanoparticles were determined by laser Doppler anemometry. The zeta potential of the particles was measured using a disposable collapsible zeta cuvette. The zeta cuvette for the measurement of zeta potential was first washed with 70% ethanol and then rinsed twice with double distilled H2O before loading the sample. The lyophilized samples were reconstituted by the addition of 50 μL of ultrapure water. Undiluted samples were used for size measurements, then diluted to 1 mL with ultrapure water, and then 700-800 μL of diluted sample was used for the determination of zeta potential. Nanoparticles were constructed at various N:P ratios using at least 1 μg of mRNA for each sample. The nanoparticles were analyzed on a Zetasizer-Nano-ZS (Malvern Instruments) using DTS software (Malvern Instruments, UK) and exemplary results for compounds 001, 002, 003, 004, 005 are shown in Figure 1. Figure 1 also contains an example of a size distribution plot for compound 021 nanoparticles.

[0705] Example 5: In vivo tracking of manually formulated RALA / Cy5 mRNA nanoparticles The nanoparticles were lyophilized according to Example 3 and then reconstituted in ultrapure water (0.5 mg mRNA / ml) prior to administration.

[0706] Nanoparticle tracking analysis was performed after intradermal administration of RALA / Cy5-EGFP mRNA nanoparticles (compounds 006, 007, 008, 009, or 010) in 5-6 week old female C57BL / 6 mice. Mice were anesthetized with intraperitoneal administration of ketamine HCl (Ketaset) and xylazine (Rompun) and administered compound 006, 007, 008, 009, or 010 nanoparticles equivalent to 10 μg mRNA by intradermal injection into the ear pinna. Mice were sacrificed at the indicated time points and fluorescence at the administration site and skin-draining lymph nodes was determined by imaging using a Bruker In Vivo Xtreme Imaging System (Figure 2).

[0707] Lymph nodes were excised from mice, disaggregated by mechanical passage through a 70 μm cell strainer, and cells were stained for dendritic cell and macrophage markers. Dendritic cells (CD11c+) and macrophages (F480), both antigen presenting cells, were found to associate with Cy5-mRNA ex vivo, and dendritic cells that had taken up RALA / Cy5-mRNA were migratory, suggesting that cells that had actively taken up nanoparticles migrated to lymph nodes (Figures 3 and 4).

[0708] The nanoparticles used in the following experiments were lyophilized according to Example 3 and then reconstituted in ultrapure water (1 mg mRNA / ml) before administration.

[0709] Example 6: Evaluation of immunogenicity of manually formulated RALA / E7 mRNA Initial studies were undertaken to determine a set of nucleotide modifications to drive E7 protein expression and immunogenicity. Compounds 011, 012, 013, 014 and 015 were assessed for their ability to drive E7 expression in NCTC-929 mouse fibroblasts by in-cell ELISA. Cells plated in 96-well plates were transfected with RALA / E7 mRNA and assessed for E7 expression 24 hours later. After fixation and permeabilization, cells were probed with anti-HPV16 E7 primary antibody (raised in rabbit, Abcam), excess antibody was removed by washing with PBS / 0.05% Tween® 20, and cells were probed with anti-rabbit secondary antibody conjugated to horseradish peroxidase. E7 expression was detected using TMB and stop solution (standard ELISA reagents) and optical density at 450 nm was measured using a Molecular Devices SpectraMax® iD3 plate reader (Figure 5).

[0710] C57BL / 6 mice were injected intradermally with 20 μg of compounds 011, 012, 013, 014 and 015 on days 0 and 21 and sacrificed on day 42. Spleens were removed and single cell splenocyte suspensions were seeded on FluoroSpot® plates (Mabtech) and stimulated with either E7 overlapping peptides (Miltenyi Biotec) or culture medium alone. FluoroSpot® analysis revealed that only compound 013 produced large amounts of IFNγ (**p<0.01 compared to naive, Kruskal-Wallis test with Dunn's multiple comparison test, FIG. 6).

[0711] Example 7: Effect of cryoprotectants on the immunogenicity of manually formulated RALA / E7 mRNA The effect of cryoprotectant on the immunogenicity of compounds 013, 016 and 017 was evaluated using nanoparticles lyophilized using either trehalose, mannose or an 85:15 mixture of trehalose:mannose as cryoprotectant. The assay and regimen were as used in Example 6. The 85:15 mixture of trehalose:mannose produced a slightly more potent response in terms of IFNγ and IL-2 production by splenocytes from compound 017 vaccinated mice stimulated with E7 overlapping peptide (Figure 7). Further analysis of splenocytes from these mice was performed by flow cytometry. Cells stimulated with E7 overlapping peptide (in the presence of brefeldin A to prevent cytokine secretion by stimulated cells) were stained for viability, CD3, CD4, CD8, IFNγ, IL-2 and TNFα. Viable T cells were gated according to their CD4 / CD8 expression status and assessed for their IFNγ, IL-2 and TNFα content using a FACSCanto II flow cytometer (Becton Dickinson) ( FIG. 8 ). Flow cytometry confirmed that the source of IFNγ and IL-2 in the FluoroSpot® analysis was CD8 + These were revealed to be cytotoxic T cells. Further analysis was performed on splenocytes cultured with the E7 overlapping peptide but in the absence of Brefeldin A, which meant that the cytokines produced as a result of stimulation were secreted into the culture medium. Analysis by IFNγ (characteristic of Th1 immune responses) and interleukin 4 (IL-4 - characteristic of Th2 responses) ELISA (both Abcam) revealed a strong bias towards Th1 responses in RALA / E7 mRNA vaccinated mice (Figure 9).

[0712] Example 8: Evaluation of immunogenicity of manually formulated RALA / E6 mRNA C57BL / 6 mice were injected intradermally with 20 μg of compound 018 on days 0 and 21 and sacrificed on day 42. Spleens were removed and single cell splenocyte suspensions were seeded on FluoroSpot® plates (Mabtech®) and stimulated with either E6 overlapping peptide (Miltenyi Biotec) or culture medium alone. Stimulation of splenocytes from vaccinated mice produced IFNγ and IL-2 (Figure 10), but not as potently as splenocytes from mice vaccinated with RALA / E7 mRNA and stimulated with E7 peptide. This is supported by literature stating that HPV16 E6 does not produce a potent epitope in mice. Splenocytes from vaccinated mice stimulated with E6 overlapping peptide also secreted IFNγ into the culture medium as assessed by ELISA (Figure 11).

[0713] Example 9: Evaluation of the prophylactic and therapeutic benefits of compounds 019, 020, 021, 022, 023 and 024 in a TC-1 tumor challenge study Two studies were designed to determine the vaccine's potential in therapeutic and preventive models of HPV16-associated cancer. The TC-1 cell line was derived from primary lung epithelial cells of C57BL / 6 mice. The cells were immortalized with the resulting amphotropic retroviral vector LXSN16E6E7 and then transformed with pVEJB plasmid expressing the activated human c-Ha-ras oncogene. Thus, the cells express HPV16 E6 and E7 and are a model for HPV16-associated malignancies.

[0714] In the prophylactic model, female C57BL / 6 mice were vaccinated with compounds 019, 020, 021, 022, 023 and 024 (a two-dose regimen with a three-week interval). On day 28, mice were vaccinated with 5×10 4 TC-1 cells were implanted and tumor development was monitored.

[0715] In the treatment model, female C57BL / 6 mice were injected with 5 × 10 4TC-1 cells were implanted and mice received compounds 019, 020, 021, 022, 023 or 024 on days 8, 15 (all mice) and 43 (mice in groups of compounds 019, 020, 021 and 022). Tumor development was monitored.

[0716] On day 108, tumor-free mice in both the prophylactic and therapeutic studies were re-challenged with TC-1 and an age-matched naive control group was added to the study.

[0717] Therapeutic vaccination with compounds 019, 020, 021 and 022 delayed the development of TC-1 xenografts, whereas compound 023 had no therapeutic benefit. This is not surprising given the relative lack of immunogenicity of compound 018 (containing RALA with E6 mRNA) in a mouse immunogenicity model (Figure 10). The majority of mice that were tumor-free at day 108 when the mice were rechallenged with TC-1 remained tumor-free until day 196 (Figures 12 and 13).

[0718] Prophylactic vaccination with compounds 019, 020, 021 and 022 prevented the engraftment of TC-1 xenografts in all mice in their respective groups. Again, compound 023, like compound 024, failed to provide protection against TC-1 tumor development. Of 30 mice rechallenged with TC-1 on day 108, only 2 developed tumors (Figures 14 and 15).

[0719] Example 10: Scaling up manual formulation of RALA / E7 mRNA nanoparticles to an automated formulation process The nanoparticle formulation process has been transferred to a bench-top automated microfluidics system where flow rates and flow rate ratios are carefully controlled and mixing within the microfluidics cartridge is controlled by the cartridge geometry. An immunogenicity study similar to that described in Example 6 was undertaken to determine the response to vaccination with compounds 025, 026, 027, 028, 029 and 030. Compound 027 elicited the strongest E7-specific response in terms of IFNγ and IL-2 secretion in the FluoroSpot® assay (Figure 16).

[0720] Example 11: Incorporation of IL-15 mRNA into the formulation induces a stronger memory phenotype in T cells C57BL / 6 mice were injected intradermally with compounds 031 or 032 on days 0, 4 and 7 and sacrificed on day 28. Spleens were removed and single cell splenocyte suspensions were cloned into E7-specific CD8 + Cells were stained with E7-specific dextramer, which binds to T cells. Cells were stained with CD3 (a pan-T cell marker) and CD62L (the presence of which indicates an effector phenotype (CD62L - ) T cells and the central memory phenotype (CD62L + We also stained for IL-15, which distinguishes between T cells with and without E7 dextramer. The presence of IL-15 did not affect the number of T cells positive for E7 dextramer, but inclusion of IL-15 mRNA in compound 032 increased the number of T cells with a central memory phenotype compared to compound 031, which lacks IL-15 (Figure 17).

[0721] Example 12: Evaluation of the optimal dose for therapeutic benefit of compound 031 in a TC-1 tumor challenge study Female C57BL / 6 mice (5–6 weeks old) were injected with 1 × 10 5 TC-1 cells were implanted subcutaneously with a 23-gauge needle. Tumor volumes were monitored for the following 245 days. All mice were vaccinated intradermally in the left ear with compound 031 on study days 4, 7, and 11. 3A maximum tumor volume threshold of 1×10 was set for this study ( FIG. 18 ). Tumor-free mice at day 100 received 1×10 5 TC-1 cells were re-challenged. Maximal efficacy was observed with 20 μg compound 031, with 3 out of 9 mice tumor-free at endpoint. In the 10 μg and 33.3 μg groups, 1 out of 10 mice remained tumor-free. Vertical dotted lines labeled 1, 2, and 3 represent treatment days. All doses of compound 031 improved survival of tumor-challenged mice, with only 20 μg improving survival significantly above that observed with 5 μg compound 031 (*p<0.05, Logrank Mantel-Cox test, FIG. 19).

[0722] Example 13: Evaluation of the immunogenicity of compound 031 compared to compound 033, formulated with RALA from Bachem This study was undertaken to determine the equivalence between the Bachem® RALA peptide supplier and the Biomatik® RALA peptide supplier.

[0723] C57BL / 6 mice were injected intradermally with 20 μg of compound 031 (Biomatik® RALA) or compounds 033, 034, 035, 036 or 037 on days 0 and 21 and sacrificed on day 42. Spleens were removed and single cell splenocyte suspensions were seeded in ELISpot plates (Mabtech®) and stimulated with E7 overlapping peptides (Jerini Peptide Technologies®) or culture medium alone. Vaccination of mice with all compounds resulted in IFNγ-responsive cells in the spleen on day 42 (***p<0.001, FIG. 20).

[0724] Example 14: Evaluation of the immunogenicity of compound 035 when administered via intradermal, intravenous, and intramuscular routes of administration Female C57BL / 6 mice (5-6 weeks old) received 20 μg / dose of compound 035 in a volume of 20 μl (ID and IM) or 100 μl (IV) and were treated on days 0 and 21. On day 42, mice were sacrificed by carbon dioxide asphyxiation and spleens were aseptically removed from each mouse and placed in MACS Tissue Storage Solution for downstream processing. Spleens were removed and single cell splenocyte suspensions were seeded in ELISpot plates (Mabtech®) and stimulated with E7 overlapping peptides (Jerini Peptide Technologies®) or culture medium alone. Vaccination by all routes of administration led to the accumulation of E7-responsive cells in the spleens of mice as determined by IFNγ ELISpot (****p<0.001 compared to control, Mann-Whitney test, FIG. 21).

[0725] Example 15: Evaluation of the therapeutic benefit of intravenous or intradermal administration of compound 035 in a TC-1 tumor challenge study Female C57BL / 6 mice (5–6 weeks old) were injected with 1 × 10 5 TC-1 cells were implanted subcutaneously with a 23-gauge needle. Tumor volumes were monitored for the following 240 days. All mice were vaccinated intravenously (lateral tail vein) or intradermally (left ear) with compound 035 on study days 4, 7, and 11. 3 A maximum tumor volume threshold of 1×10 was set for this study. Treatment with compound 035 via either route slowed tumor development, with higher efficacy observed in the intravenous group (FIG. 22). Tumor-free mice at day 123 received 1×10 5The mice were re-challenged with 10 TC-1 cells. The greatest efficacy was observed in the IV group, with 5 of 7 mice tumor-free at the endpoint. In the ID group, 0 of 10 mice remained tumor-free. The vertical dotted lines labeled 1, 2, and 3 represent treatment days. Treatment with compound 035 via either route also significantly improved survival in tumor-challenged mice. Importantly, survival in intravenously treated mice was significantly improved compared to intradermally treated mice (***p<0.01 compared to vehicle, Logrank Mantel-Cox test, FIG. 23).

[0726] Example 16: Further evaluation of the therapeutic benefit of intravenous or intradermal administration of compound 035 in a TC-1 tumor challenge study Female C57BL / 6 mice (5–6 weeks old) were injected with 1 × 10 5 TC-1 cells were implanted subcutaneously with a 23-gauge needle. Tumor volumes are monitored up to day 168 (study is still ongoing). All mice were vaccinated intravenously (lateral tail vein) or intradermally (left ear) with compound 035 on study days 4, 7, and 11. 3 A maximum tumor volume threshold of 1×10 was set for this study. Treatment with compound 035 via either route slowed tumor development, with higher efficacy observed in the intravenous group (FIG. 24). Tumor-free mice at day 111 received 1×10 5 On day 111, tumor-free mice were re-challenged with 1 × 10 TC-1 cells. 5 The mice were re-challenged with TC-1 cells. At this time, 7 of 9 mice in group IV and 4 of 10 mice in group ID are tumor free. The vertical dotted lines labeled 1, 2, and 3 represent the days of treatment.

[0727] Treatment with compound 035 via either route also improved survival in tumor-challenged mice (Figure 25). As this study is still ongoing, statistical analysis of survival rates is not appropriate.

[0728] Example 17: Evaluation of the therapeutic benefit of intravenous or intradermal administration of Compound 035 and Compound 039 in a C3.43 tumor challenge study Female C57BL / 6 mice (5–6 weeks old) were injected with 1 × 10 5 C3.43 cells were implanted subcutaneously with a 23 gauge needle. Tumor volumes are monitored up to day 168 (study is still ongoing). All mice were vaccinated intravenously (lateral tail vein) or intradermally (left ear) with compound 035 or compound 039 on study days 4, 7, and 11. 3 A maximum tumor volume threshold of 1×10 was set for this study. Treatment with compounds 035 or 039 via either route slowed tumor development, with higher efficacy observed in the intravenous group (FIG. 26). Tumor-free mice at day 111 received 1×10 5 On day 111, tumor-free mice were re-challenged with 1 × 10 C3.43 cells. 5 The mice were re-challenged with 10 C3.43 cells. At this time, 9 of 10 (Compound 035) and 6 of 7 (Compound 039) mice in the IV group and 1 of 10 (Compound 035) and 3 of 8 (Compound 039) mice in the ID group are tumor free. The vertical dotted lines labeled 1, 2, and 3 represent the days of treatment.

[0729] Treatment with compound 035 or compound 039 via either route also improved survival in tumor-challenged mice (Figure 27). As this study is still ongoing, statistical analysis of survival rates is not appropriate.

[0730] Example 18: Evaluation of the immunogenicity of RALA vaccine nanoparticles containing mRNA with an 80 nucleotide long poly(A) tail This study was undertaken to determine the equivalence between 120 nucleotide long poly(A) tailed mRNAs and 80 nucleotide poly(A) tailed mRNAs.

[0731] C57BL / 6 mice were injected intradermally with 20 μg of compound 035 (120 poly(A)) or compound 040 (80 poly(A)) on days 0 and 21 and sacrificed on day 42. Spleens were removed and single cell splenocyte suspensions were seeded in ELISpot plates (Mabtech®) and stimulated with E7 overlapping peptides (Jerini Peptide Technologies®) or culture medium alone. Vaccination of mice with compound 035 or 040 led to the accumulation of E7 antigen-responsive cells in the spleen on day 42 (demonstrated using IFNγ ELISpot) (Figure 28). *p<0.01 compared to naive (Kruskal-Wallis test with Dunn's multiple comparison test). There was no significant difference in the response between compound 035 and compound 040.

[0732] Example 19: Evaluation of the stability of Composite 035 at 4°C and 25°C and 60% relative humidity Conjugate 035 was formulated and stored for 3 months at either 4°C (refrigerator) or 25°C at 60% relative humidity (stability cabinet with controlled temperature and humidity). After 3 months of storage, the formulation was reconstituted. C57BL / 6 mice were injected intradermally with 20 μg of conjugate 035 (120 poly(A)) or conjugate 040 (80 poly(A)) on days 0 and 21 and sacrificed on day 42. Spleens were removed and single cell splenocyte suspensions were seeded in ELISpot plates (Mabtech®) and stimulated with E7 overlapping peptides (Jerini Peptide Technologies®) or culture medium alone. Vaccination of mice with conjugate 035 or 040 led to the accumulation of E7 antigen-responsive cells in the spleen on day 42 (demonstrated using IFNγ ELISpot). There was no significant difference in response between freshly formulated Composite 035 (T0) and Composite 035 stored at 60% relative humidity at either 4°C or 25°C (Kruskal-Wallis test with Dunn's multiple comparison test, Figure 29).

[0733] Example 20: Evaluation of antigen-presenting cell uptake of compound 041 after intravenous administration Female C57BL / 6 mice (5-6 weeks old) were treated intravenously with compound 041 (10 μg), and either 1 h or 24 h after treatment, the mice were sacrificed and the spleens were isolated and processed into single cell suspensions. To facilitate the identification of dendritic cells and macrophages, cells were stained with fluorescently conjugated antibodies (CD8 - Thermo Fisher_CD8a monoclonal antibody (53-6.7), Super Bright™ 600_[63-0081-82]; CD4 - ThermoFisher_CD4 monoclonal antibody (GK1.5), Super Bright™ 702_[67-0041-82]; cD11c - ThermoFisher_CD11c monoclonal antibody (N418), PE-Cyanine 7_[25-0114-82]; cD11b - ThermoFisher_CD11b monoclonal antibody (M1 / 70), PE-eFluor™ 610_[61-0112-82]; F4 / 80 - Staining was performed using ThermoFisher_F4 / 80 monoclonal antibody (BM8), PerCP-Cyanine 5.5_[45-4801-82]; CD103 - Thermo Fisher_CD103 monoclonal antibody (2E7), PE_ [12-1031-82]; Viability - eBioscience™ Fixable Viability Dye eFluor™ 450_[65-0863-14]). Cells that had taken up compound 041 were positive for Cy5 signal. At 1 hour after treatment, there was a significant uptake of compound 041 by dendritic cells and macrophages (2-way ANOVA with Dunnett's multiple comparison test, ****p<0.0001 compared to naive mice, FIG. 30).

[0734] statement Statement 1. i) an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; and ii) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; Including, vaccines.

[0735] Statement 2. The vaccine of Statement 1, further comprising a second mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism.

[0736] Statement 3. The vaccine of statement 2, wherein the two mRNA polynucleotides are present in a ratio of about 50:50% w / w.

[0737] Statement 4. The vaccine of any one of the preceding statements, wherein the open reading frame encodes an HPV antigen or an immunogenic fragment thereof.

[0738] Statement 5. The vaccine of any one of the preceding statements, wherein the open reading frame encodes an E6 or E7 HPV antigen or an immunogenic fragment thereof.

[0739] Statement 6. The vaccine of any one of the preceding statements, wherein the open reading frame encodes an HPV 6, 8, 11, 16 or 18 antigen or an immunogenic fragment thereof.

[0740] Statement 7. The vaccine of any one of the preceding statements, wherein the open reading frame encodes an HPV16 antigen or an immunogenic fragment thereof.

[0741] Statement 8. The vaccine of any one of the preceding statements, wherein the amphipathic cell membrane-penetrating peptide comprises the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1).

[0742] Statement 9. i) a first mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:2; ii) a second mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:3; iii) an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); 4. The vaccine according to any one of statements 1 to 3, comprising:

[0743] Statement 10. i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID NO:5; ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID NO:6; iii) an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); 4. The vaccine according to any one of statements 1 to 3, comprising:

[0744] Statement 11. The vaccine of any one of the preceding statements, wherein the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant.

[0745] Statement 12. The vaccine of statement 11, wherein the mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant encodes a cytokine.

[0746] Statement 13. The vaccine of statement 11 or statement 12, wherein the mRNA polynucleotide comprising an open reading frame encoding the immune adjuvant comprises a sequence selected from SEQ ID NO:8.

[0747] Statement 14. The vaccine of statement 11 or statement 12, wherein the mRNA polynucleotide comprising an open reading frame encoding an immune adjuvant encodes an immune adjuvant selected from SEQ ID NO:10.

[0748] Statement 15. The vaccine of any one of the preceding statements, wherein the mRNA polynucleotide comprises a 5 prime cap (5' cap).

[0749] Statement 16. The vaccine of any one of the preceding statements, wherein the mRNA polynucleotide comprises modified uridine nucleotides, wherein all uridine nucleotides are N1-methylpseudouridine nucleotides.

[0750] Statement 17. The vaccine of any one of the preceding statements, wherein the mRNA polynucleotide comprises unmodified cytidine nucleotides.

[0751] Statement 18. The vaccine of any one of the preceding statements, wherein the amphipathic cell membrane penetrating peptide consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1).

[0752] Statement 19. The vaccine of any one of the preceding statements, wherein the vaccine comprises nanoparticles.

[0753] Statement 20. The vaccine of any one of the preceding statements, wherein the N:P ratio of amphipathic cell membrane-penetrating peptide:mRNA polynucleotide in the vaccine is about 8.5-9.5:1.

[0754] Statement 21. The vaccine of any one of the preceding statements, further comprising mannose and trehalose.

[0755] Statement 22. A vaccine according to any one of the preceding statements for use as a therapeutic vaccine.

[0756] Statement 23. A vaccine according to statements 1 to 21 for use in the treatment of cancer.

[0757] Statement 24. A pharmaceutical composition comprising a vaccine according to statements 1 to 21 for use in treating a sexually transmitted disease.

[0758] Statement 25. A method of treating HPV infection in a warm-blooded animal, such as a human, comprising administering to said animal an effective amount of a vaccine according to statements 1-21.

Claims

1. i) an mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism; and ii) an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; Including, vaccines.

2. 2. The vaccine of claim 1, further comprising a second mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism.

3. The vaccine of claim 2, further comprising a third mRNA polynucleotide comprising an open reading frame encoding an antigen or an immunogenic fragment thereof from an infectious microorganism.

4. 4. The vaccine of claim 2 or 3, wherein the mRNA polynucleotides are present in approximately equal % w / w ratio.

5. The vaccine of any one of claims 1 to 4, wherein the open reading frame encodes an HPV antigen or an immunogenic fragment thereof.

6. The vaccine of any one of claims 1 to 5, wherein the open reading frame encodes E1 and / or E6 and / or E7 HPV antigens or immunogenic fragments thereof.

7. The vaccine of any one of claims 1 to 6, wherein the open reading frame encodes an HPV6, 8, 11, 16 or 18 antigen or an immunogenic fragment thereof.

8. The vaccine of any one of claims 1 to 7, wherein the open reading frame encodes an HPV16 antigen or an immunogenic fragment thereof.

9. The vaccine of any one of claims 1 to 8, wherein the amphipathic cell membrane penetrating peptide comprises the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1).

10. The vaccine according to any one of claims 1 to 9, wherein the amphipathic cell membrane penetrating peptide consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1).

11. i) a first mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:2; ii) a second mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:3; iii) a third mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID NO:4; iv) an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); The vaccine according to any one of claims 1 to 4, comprising:

12. i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID NO:5; ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID NO:6; iii) a third mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID NO:7; iv) an amphipathic cell membrane penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); The vaccine according to any one of claims 1 to 4, comprising:

13. The vaccine of any one of claims 1 to 12, wherein the mRNA polynucleotide comprises a 5 prime cap (5' cap).

14. The vaccine of any one of claims 1 to 13, wherein the mRNA polynucleotide comprises modified uridine nucleotides, and all uridine nucleotides are N1-methylpseudouridine nucleotides.

15. The vaccine of any one of claims 1 to 14, wherein the mRNA polynucleotide comprises unmodified cytidine nucleotides.

16. The vaccine of any one of claims 1 to 15, wherein the vaccine comprises nanoparticles.

17. The vaccine according to any one of claims 1 to 16, wherein the N:P ratio of amphipathic cell membrane-penetrating peptide:mRNA polynucleotide in the vaccine is about 8.5 to 9.5:

1.

18. The vaccine of any one of claims 1 to 17, further comprising trehalose.

19. (i) 0.01 to 1% w / w of one or more mRNA polynucleotides comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof; (ii) 0.5-2% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 95-99% w / w trehalose The vaccine of any one of claims 1 to 18, comprising:

20. An intravenous formulation comprising a vaccine according to any one of claims 1 to 19 formulated in water.

21. A vaccine according to any one of claims 1 to 20 for use as a therapeutic vaccine.

22. A vaccine according to any one of claims 1 to 20 for use in the treatment of cancer.

23. A pharmaceutical composition comprising a vaccine according to any one of claims 1 to 20 for use in the treatment of a sexually transmitted disease.

24. A method for treating HPV infection in a warm-blooded animal, such as a human, comprising administering to said animal an effective amount of a vaccine according to any one of claims 1 to 20.

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