Immunogenic compositions

Abstract

Disclosed herein are compositions comprising protein antigens and RNA encoding the same (e.g., compositions comprising protein antigens and RNA encoding antigens) for use in treating and / or preventing various infectious agents. Also disclosed herein are immunogenic compositions and medical preparations comprising the same, and methods of making and using the same. In some embodiments, the technologies provided herein can be used to address and / or overcome immune imprinting in various infectious diseases.

Description

IMMUNOGENIC COMPOSITIONSCross-Reference to Related Applications

[0001] The present application claims the benefit of U.S. Prov. Appln. No. 63 / 448,244 filed February 24, 2023, U.S. Prov. Appln. No. 63 / 486,953 filed February 24, 2023, U.S. Prov. Appln. No. 63 / 448,243 filed February 24, 2023, and U.S. Prov. Appln. No. 63 / 486,958 filed February 24, 2023, the contents of all of which are incorporated herein in their entireties.Background

[0002] Infectious diseases represent a major threat to human health and well-being. Caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi, infectious diseases, also known as communicable diseases, can be spread directly or indirectly from one person to another. Vaccines, which are pharmaceutical preparations that provide or improve immunity to a particular disease, are useful to protect human subjects from certain infectious diseases.

[0003] Since the initial discovery of SARS-CoV-2, a number of variants have arisen around the world. The emergence of these novel circulating variants of SARS-CoV-2 has raised significant concerns about the temporal efficacy of vaccine interventions. The emergence of Omicron (B.1.1.529) variants, which comprise a number of mutations in the S protein, has been of particular concern.Summary

[0004] In some embodiments, the present disclosure provides technologies (e.g., compositions and methods) that can be used to induce an immune response against an infectious agent (e.g., a virus (e.g., SARS-CoV-2), bacteria, or eukaryotic infectious agent). Technologies provided herein include immunogenic compositions (e.g., RNA compositions), methods of inducing an immune response, and methods of manufacturing immunogenic compositions, among others. In some embodiments, an immunogenic composition delivers an infectious agent antigen (e.g., comprises an infectious agent antigen or a nucleic acid encoding an infectious agent antigen). In some embodiments, an immunogenic composition delivers a SARS-CoV-2 antigen (e.g., comprises a SARS-CoV-2 antigen or a nucleic acid encoding a SARS-CoV-2antigen). In some embodiments, an immunogenic composition delivers an infectious agent antigen, or an immunogenic portion thereof. In some embodiments, an immunogenic composition delivers a SARS-CoV-2 S protein, or an immunogenic portion thereof. In some embodiments, technologies described herein can produce an immune response characterized by an increased naive immune response, a de novo immune response, and / or a decreased memory B cell response. In some embodiments, technologies provided herein can provide an improved immune response (e.g., higher neutralization antibody titers, increased naive B cell activation, and / or higher titers of antibodies recognizing an epitope unique to a variant of concern (relative to a reference antigen)) against one or more variants of concern (e.g., one or more SARS-CoV-2 variants of concern) (e.g., variants of concern against which current vaccine technologies produce a weak neutralization response). In some embodiments, technologies provided herein can partially or fully address and / or overcome an immune imprinting effect.

[0005] Immune imprinting is a phenomenon in which a previous (e.g., initial) exposure to a first strain or variant of an infectious agent (or one or more antigens thereof) impedes development of an immune response against subsequent strains or variants of an infectious agent (e.g., by interfering with generation of antibodies that bind epitopes unique to the subsequent strain or variant). Immune imprinting can be a particular concern for infectious agents that can acquire a high number or density of mutations in neutralization sensitive region (e.g., SARS- CoV-2).

[0006] A schematic illustrating the immune imprinting phenomenon is shown in Fig. 1. Subjects administered a vaccine that delivers a wild-type (WT) antigen produce antibodies and form memory B cells recognizing the WT antigen. As new Variants of Concern (VOC) arise that evade the immune response induced by the first vaccine, VOC-adapted booster shots are developed and administered to subjects. VOCs often evade the immune system by acquiring mutations at neutralization sensitive epitopes (regions prone to mutation shown in different colors in Fig. 1). Subjects exposed to a VOC-adapted vaccine have a predisposition to activate memory B cells that were formed in response to the initial WT vaccine rather than activate naive B cells. As a result, administering a VOC-adapted vaccine induces production of antibodies that recognize both the WT virus and the VOC but few or no antibodies that are specific to the VOC. So long as the VOC retains some neutralization epitopes from the WT virus, a neutralization response against the VOC can still be induced. As new VOCs continue to lose neutralizationepitopes from the WT strain, however, the immune response induced by a VOC-adapted vaccine become less and less effective. Further discussion of the imprinting phenomenon in the SARS- CoV-2 context can be found in Wheatley et al., Trends Immunol, 2021, the contents of which are incorporated by reference herein in their entirety. Immune imprinting is expected to be a particular concern for V OC-adapted vaccines that encode an antigen that comprises a number of mutations at neutralization sensitive sites, (e.g., variants that exhibit close to no conserved neutralizing epitopes).

[0007] Immune imprinting can have important implications for vaccine development. As shown in Fig. 2, a single exposure to Omicron BA.l, BA.2, or BA.4 / 5 variants has not been found to induce neutralization of Omicron XBB. Without wishing to be bound by theory, this failure to cross neutralize the XBB variant may be due to its low retention of neutralizing B-cell epitopes relative to the original Wuhan strain (see Fig. 5(B)). In short, exposure to BA.l, BA.2, and BA.4 / 5 may be activating memory B cells that recognize epitopes in both Wuhan and these Omicron variants, and not generating immune responses that recognize features that are unique to these variants. If true, these results suggest that variant adapted vaccines may not produce effective immune responses to variants that retain few neutralization epitopes from the original SARS-CoV-2 variant.

[0008] Among other things, the present disclosure provides important insights for addressing and overcoming immune imprinting in the context of various infectious agents (e.g., in SARS-CoV-2). Without wishing to be bound by theory, the present disclosure provides an insight that immune imprinting can be caused by the retention of memory B cell epitopes in a variant antigen relative to a reference antigen (e.g., an antigen that a subject was first or previously exposed to). Previous strategies have sought to overcome immune imprinting by identifying certain antigen regions that are conserved and neutralizing. The present disclosure provides an insight that a fundamentally different approach can be used to overcome immune imprinting. Specifically, rather than identifying and retaining certain conserved neutralization epitopes, the present disclosure provides an insight that immune imprinting can be addressed and a de novo response induced by removing all memory B cell epitopes from a reference antigen.

[0009] Among other things, the present disclosure also provides certain insights as to how to design antigens that avoid immune imprinting, induce less of a memory B cell response,and / or induce more of a de novo immune response. In particular, the present disclosure provides an insight that such effects can be achieved by administering immunogenic portions of an antigen, and also provides insights in regards to (i) which portion(s) of an antigen can be removed to provide an improved immune response and (ii) which portions of an antigen are more likely to induce a de novo response. In particular, the present disclosure provides an insight that receptor binding domains and / or regions (e.g., of a SARS-CoV-2 S protein) having a high frequency of mutation and a high number of neutralization epitopes can provide improved immune responses (e.g., when administered as a booster to a subject previously administered a vaccine (e.g., a SARS-CoV-2 S protein ) against a given infectious agent).

[0010] Among other things, the present disclosure provides technologies that are useful for increasing the breadth of immune response. In some embodiments, such an immune response is or comprises a B cell immune response. In some embodiments, a B cell immune response is or comprises an antibody response (e.g., neutralizing antibody response) to arisen epitopes in variant polypeptides.

[0011] Among other things, the present disclosure provides an insight that it may be particularly desirable, especially for circulating infectious diseases (e.g., for which variants can be expected to arise), to encourage immune responses, specifically including antibody responses (e.g., neutralizing responses) to arisen epitopes. In some embodiments, such circulating infectious disease is a bacterial infectious disease. In some embodiments, such circulating infectious disease is a parasitic infectious disease. An exemplary parasitic infectious disease is malaria. In some embodiments, such circulating infectious disease is a viral infectious disease. In some embodiments, a viral infectious disease is associated with an RNA virus. Exemplary viral infectious diseases include, but are not limited to coronavirus, ebolavirus, influenza viruses, norovirus, rotavirus, respiratory syncytial virus, alphaherpesvirus, etc. In particular, the present disclosure, among other things, provides an insight that it may be desirable for SARS-CoV-2 infection (e.g., for which variants can be expected to arise), to encourage immune responses, specifically including antibody responses (e.g., neutralizing responses) to arisen epitopes.

[0012] Among other things, and without wishing to be bound by any particular theory, the present disclosure provides an insight that, where an antigen (e.g., S protein of SARS-CoV-2) includes one or more “memory epitopes”, such presence may bias an immune response to theantigen toward activation of memory B cells, in at least some instances to the detriment of developing a sufficiently effective antibody response (e.g., a neutralizing antibody response) to arisen epitope(s).

[0013] In some embodiments, the present disclosure, among other things, provides technologies for modulating the balance of immune response toward de no priming response to arisen epitopes in variant polypeptides (e.g., in some embodiments XBB variant of SARS-CoV- 2). In some embodiments, the present disclosure, among other things, provides technologies for increasing activation of naive B cell immune response to at least one of the arisen epitopes. In some embodiments, such arisen epitopes are neutralizing epitopes. In some embodiments, the present disclosure, among other things, provides technologies for inducing a priming-favorable cytokine milieu, for example, in lymphoid tissues. In some embodiments, the present disclosure, among other things, provides technologies for inducing a priming-favorable cytokine milieu, for example, in lymphoid tissues. Without wishing to be bound by a particular theory, in some embodiments, induction of a priming-favorable cytokine milieu can be mediated through interferon alpha (IFNa). Without wishing to be bound by a particular theory, in some embodiments, induction of a priming-favorable cytokine milieu can be mediated through a CD4+ T cell immune response.

[0014] In some embodiments, technologies provided herein may be particularly useful to subjects who have been previously exposed (e.g., via infection and / or vaccination) to a reference antigen (e.g., SARS-CoV-2) of an infectious agent and are receiving an immunogenic composition that delivers a variant polypeptide of the reference antigen (e.g., polypeptide of a prior circulating SARS-CoV-2 strain), or an immunogenic portion thereof. In some embodiments, such a variant polypeptide comprises arisen epitopes. In some embodiments, such arisen epitopes are or comprise neutralizing epitopes (e.g., neutralizing antibody epitopes). In some embodiments, technologies provided herein may be particularly used to induce activation of naive B cell immune response (e.g., in some embodiments antibody response, e.g., neutralizing antibody response) to at least one of the arisen epitopes (e.g., in some embodiments at least one of the neutralizing epitopes).

[0015] The present disclosure exemplifies certain aspects of provided technologies through administering a combination of a modified RNA vaccine that delivers a variantpolypeptide of a reference antigen of an infectious agent, e.g., a vaccine that delivers a variant of a coronavirus S protein or an immunogenic portion thereof, and a particular interferon-alpha (IFNa)-inducing agent, e.g., a non-modified RNA. In some embodiments, such a non-modified RNA encodes at least one or more T cell epitopes. In some embodiments, such a non-modified RNA encodes at least one or more B cell epitopes. A skilled person, having read the disclosure, will appreciate that such strategies utilized in coronavirus vaccines may be also useful in other infectious diseases, e.g., circulating infectious diseases.

[0016] In some embodiments, disclosed herein is an RNA comprising a nucleotide sequence that encodes a polypeptide comprising or consisting of a variant polypeptide of a reference antigen of an infectious agent (e.g., a SARS-CoV-2 Spike (S) protein), or an immunogenic portion thereof, wherein a B cell memory immune response has been established to the reference antigen (e.g., SARS-CoV-2 S protein), and wherein the variant polypeptide (e.g., SARS-CoV-2 S protein variant) or immunogenic portion thereof has an amino acid sequence that differs from that of the reference antigen (e.g., reference SARS-CoV-2 S protein) in that it has been engineered to reduce the variant’s activation of the B cell memory immune response relative to the reference antigen (e.g., reference SARS-CoV-2 S protein).

[0017] In some embodiments, an antigen of an infectious agent (or portion thereof) comprises an engineered amino acid sequence so that at least one B cell memory epitope present in a reference antigen of the infectious agent (e.g., a SARS-CoV-2 S protein) is modified so that the memory activation potency of a reference antigen (or portion thereof) (e.g., a SARS-CoV-2 S protein protein) is reduced.

[0018] In some embodiments, an amino acid sequence encoded by an RNA is at least 80% identical to the corresponding portion of the reference antigen (e.g., a SARS-CoV-2 S protein).

[0019] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) has an amino acid sequence that is at least 80% identical to that of a reference SARS-CoV-2 S protein (or an amino acid sequence of the corresponding portion of a reference SARS-CoV-2 S protein).

[0020] In some embodiments, an amino acid sequence encoded by an RNA comprises no more than 50% of the B cell memory epitopes present in a reference antigen.

[0021] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises no more than 50% of the memory B cell epitopes present in a reference SARS-CoV-2 S protein.

[0022] In some embodiments, an RNA comprises a nucleotide sequence that encodes an antigen of an infectious agent (or a portion thereof), wherein the amino acid sequence of the antigen was engineered by a process comprising a step of removing memory B cell epitopes of a reference antigen or an immunogenic portion thereof.

[0023] In some embodiments, an RNA comprises a nucleotide sequence that encodes a SARS-CoV-2 Spike (S) protein variant (or an immunogenic portion thereof) whose amino acid sequence is engineered so that at least one memory B cell epitope present in a reference SARS- CoV-2 S protein has been modified so that memory B cell activation potency of the SARS-CoV- 2 S protein variant (or immunogenic portion thereof) has been reduced relative to the reference SARS-CoV-2 S protein.In some embodiments, an RNA comprises a nucleotide sequence that encodes a SARS-CoV-2 Spike (S) protein variant (or an immunogenic portion thereof), wherein the amino acid sequence of the S protein variant or immunogenic portion thereof was engineered by a process comprising a step of removing memory B cell epitopes present in a reference SARS-CoV-2 S protein.

[0024] In some embodiments, a variant SARS-CoV-2 S protein (or immunogenic portion thereof) comprises few memory B cell epitopes of a reference SARS-CoV-2 S protein.

[0025] In some embodiments, one or more memory B cell epitopes in a reference SARS- CoV-2 S protein have been identified by antibody-binding studies (e.g., studies characterizing antibodies produced by subjects administered a vaccine that delivers the reference SARS-CoV-2 S protein and / or infected with a virus comprises the reference SARS-CoV-2 S protein).

[0026] In some embodiments, an antigen of an infectious agent or immunogenic portion thereof is engineered so as to lack regions of a reference antigen comprising a high number (or density) of conserved B cell epitopes.

[0027] In some embodiments, conserved B cell epitopes are non-neutralizing epitopes.

[0028] In some embodiments, one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes and neutralizing epitopes.

[0029] In some embodiments, an antigen of an infectious agent or immunogenic portion thereof is engineered so as to lack conserved neutralizing B cell epitopes and conserved non- neutralizing B cell epitopes.

[0030] In some embodiments, an infectious agent has a high mutation rate.

[0031] In some embodiments, an infectious agent has a high number of variants or species.

[0032] In some embodiments, an infectious agent has a large number of variants or species, many of which are immune escaping.

[0033] In some embodiments, an infectious agent is a virus, bacteria, or Plasmodium.

[0034] In some embodiments, an infectious agent is a virus. In some embodiments, a virus is a respiratory virus. In some embodiments, a virus is an influenza virus, RSV, a norovirus, or HIV.

[0035] In some embodiments, an infectious agent is a coronavirus. In some embodiments, a coronavirus is a betacoronavirus. In some embodiments, a coronavirus is a MERS, SARS, or SARS-CoV-2 virus.

[0036] In some embodiments, a plasmodium is P. falciparum, P. vivax, P. ovale, or P. malariae.

[0037] In some embodiments, a variant polypeptide lacks regions that are not mutated frequently in immune-escaping variants of the infectious agent.

[0038] In some embodiments, a variant polypeptide comprises an immunogenic portion of a coronavirus S protein that lacks sequences corresponding to regions outside of the S 1 domain or the receptor binding domain (RBD).

[0039] In some embodiments, a SARS-CoV-2 S protein variant or immunogenic portion thereof is engineered so as to lack regions of a reference SARS-CoV-2 S protein that comprise a high number or density of conserved memory B cell epitopes.

[0040] In some embodiments, conserved memory B cell epitopes are non-neutralizing epitopes.

[0041] In some embodiments, a variant SARS-CoV-2 S protein or immunogenic portion thereof is engineered so as to lack regions that are not mutated frequently in immune-escaping SARS-CoV-2 variants.

[0042] In some embodiments, an immunogenic portion of a coronavirus S protein does not comprise an S2 domain.

[0043] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises or consists of the SI domain or the receptor binding domain (RBD).

[0044] In some embodiments, an immunogenic portion of a coronavirus S protein does not comprise an N-terminal domain (NTD).

[0045] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises or consists of the RBD.

[0046] In some embodiments, a reference SARS-CoV-2 S protein is from a strain or variant that was previously prevalent or is currently prevalent in a relevant population of subjects.

[0047] In some embodiments, a reference SARS-CoV-2 S protein was previously delivered by a vaccine. In some embodiment, the vaccine is a commercially approved vaccine, a protein-based vaccine, an RNA vaccine, or any combination thereof.

[0048] In some embodiments, a reference SARS-CoV-2 S protein is a Wuhan S protein.

[0049] In some embodiments, a reference SARS-CoV-2 S protein is an Omicron BA.4 / 5S protein.

[0050] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises one or more mutations associated with a SARS-CoV-2 variant that has a high immune escape potential (e.g., a variant of concern).

[0051] In some embodiments, a SARS-CoV-2 variant has been determined to have a high immune escape potential using an in vitro assay (e.g., a viral neutralization assay), in silico analysis (e.g., sequence analysis and / or molecular dynamic simulations), and / or based on infection rates and / or growth rates.

[0052] In some embodiments, a SARS-CoV-2 variant with a high immune escape potential is an Omicron variant.

[0053] In some embodiments, a SARS-CoV-2 variant with a high immune escape potential is an XBB variant (e.g., an XBB.l or XBB.1.5 variant) or a BQ.l variant.

[0054] In some embodiments, one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0055] In some embodiments, one or more mutations associated with an XBB.1.5 RBD are G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, and Y505H, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0056] In some embodiments, one or more mutations associated with an XBB.1.5 SI domain are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, and P681H, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0057] In some embodiments, one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0058] In some embodiments, one or more mutations associated with an XBB.1.5 SI are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K,S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0059] In some embodiments, an RNA comprises a nucleotide sequence that encodes an immunogenic portion of a SARS-CoV-2 S protein variant comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 3.

[0060] In some embodiments, an RNA comprises a nucleotide sequence that encodes an immunogenic portion of the SARS-CoV-2 S protein variant comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 5.

[0061] In some embodiments, a variant polypeptide comprises a secretion signal. In some embodiments, a secretion signal is a homologous secretion signal. In some embodiments, a secretion signal is a heterologous secretion signal.

[0062] In some embodiments, a secretion signal is present in the N-terminal portion of a polypeptide (e.g., at the N-terminus).

[0063] In some embodiments, a secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gDl secretion signal, a gBl secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal.

[0064] In some embodiments, an antigen of an infectious agent or immunogenic portion thereof encoded comprises a hypervariable domain.

[0065] In some embodiments, a hypervariable domain is a region of an antigen that has a high mutation frequency.

[0066] In some embodiments, a hypervariable domain has a high density of neutralization epitopes.

[0067] In some embodiments, a hypervariable domain is a region that is frequently mutated in variants of the infectious agent that have a high immune escape potential.

[0068] In some embodiments, a hypervariable domain is a receptor binding domain (RBD).

[0069] In some embodiments, a hypervariable domain comprises or consists of an RBD or S 1 domain of a coronavirus S protein.

[0070] In some embodiments, a reference antigen is: (i) a surface protein or surface glycoprotein of an infectious agent strain or variant that was previously and / or is currently prevalent; and / or (ii) a surface protein or surface glycoprotein of an infectious agent that has been previously delivered in a vaccine (e.g., a commercially available vaccine, an RNA vaccine, or a protein-based vaccine).

[0071] In some embodiments, a surface protein or surface glycoprotein is a coronavirus S protein.

[0072] In some embodiments, a variant polypeptide has been engineered to eliminate one or more memory B cell epitopes of a reference antigen.

[0073] In some embodiments, one or more memory B cell epitopes have previously been determined to be bound by antibodies and / or B cells produced by a subject exposed to the reference antigen (e.g., via a vaccine that delivers the reference antigen and / or infection with a virus that comprises the reference antigen).

[0074] In some embodiments, one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes.

[0075] In some embodiments, one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes and neutralizing epitopes.

[0076] In some embodiments, a variant polypeptide comprises few intact memory B cell epitopes of the reference antigen.

[0077] In some embodiments, a variant polypeptide comprises one or more mutations associated with an infectious agent variant that has a high immune escape potential.

[0078] In some embodiments, an infectious agent variant has been determined to have a high immune escape potential using an in vitro assay (e.g., a viral neutralization assay), via in silico analysis (e.g., sequence analysis and / or molecular dynamic simulations), and / or based on infection rates in subjects in a relevant population.

[0079] In some embodiments, a variant polypeptide comprises few conserved memory B-cell epitopes relative to: (i) a reference antigen of a strain or variant that was previously or is currently prevalent in a relevant population, and / or (ii) one or more reference antigens that havepreviously been delivered in a vaccine (e.g., a commercially available vaccine and / or a vaccine previously administered to a subject).

[0080] In some embodiments, a variant polypeptide comprises 10 or fewer (e.g., 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, one or less, or no) conserved memory B cell epitopes.

[0081] In some embodiments, a variant polypeptide comprises a secretion signal.

[0082] In some embodiments, a secretion signal is a homologous secretion signal. In some embodiments, a secretion signal is a heterologous secretion signal.

[0083] In some embodiments, a secretion signal is present in the N-terminal portion of the polypeptide (e.g., at the N-terminus of the polypeptide).

[0084] In some embodiments, a secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gDl secretion signal, a gBl secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal.

[0085] In some embodiments, a SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 15.

[0086] In some embodiments, a SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9 or 16.

[0087] In some embodiments, a gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, a gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, a gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 33.

[0088] In some embodiments, a gDl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 12.

[0089] In some embodiments, a gBl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 37.

[0090] In some embodiments, a gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 35. In some embodiments, a gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 32.

[0091] In some embodiments, a gI2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 10 or 11.

[0092] In some embodiments, a gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 38.

[0093] In some embodiments, an EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39.

[0094] In some embodiments, an HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.

[0095] In some embodiments, a variant polypeptide (e.g., a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a multimerization domain.

[0096] In some embodiments a multimerization domain is in the C-terminal region of a SARS-CoV-2 variant protein or an immunogenic portion thereof (e.g., at the C-terminus).

[0097] In some embodiments, a polypeptide comprises a multimerization domain that is C-terminal to the variant polypeptide.

[0098] In some embodiments, a multimerization domain is a fibritin domain.

[0099] In some embodiments, a fibritin domain comprises a sequence that is at least80% identical to SEQ ID NO: 95. In some embodiments, a fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 96.

[0100] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a transmembrane (TM) domain.

[0101] In some embodiments, a variant polypeptide comprises a transmembrane (TM) domain. In some embodiments, a TM domain is a homologous TM domain. In some embodiments, a TM domain is a heterologous TM domain. In some embodiments, a TM domain is present in the C-terminal portion of the polypeptide (e.g., at the C-terminus).

[0102] In some embodiments, a variant polypeptide (e.g., SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a multimerization domain and a TM domain in the C-terminal portion of the polypeptide, wherein the TM domain is C-terminal to the multimerization domain (e.g., the TM domain is at the C-terminus of the variant polypeptide andthe multimerization domain is adjacent to the TM domain (e.g., directly adjacent to the TM domain and / or connected to the TM domain via a GS linker)).

[0103] In some embodiments, a TM domain is a SARS-CoV-2 S protein TM domain, or an influenza TM domain. In some embodiments, a SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 89. In some embodiments, a SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 90.

[0104] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 120.

[0105] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises a sequence that is at least 80% identical to SEQ ID NO: 130.

[0106] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 135.

[0107] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 145.

[0108] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 150.

[0109] In some embodiments, a nucleotide sequence that encodes a SARS-CoV-2 S protein variant (or immunogenic portion thereof) is codon-optimized for expression in mammalian subjects.

[0110] In some embodiments, a nucleotide sequence that encodes a SARS-CoV-2 S protein variant (or immunogenic portion thereof) is codon-optimized for expression in human subjects.

[0111] In some embodiments, a nucleotide sequence encoding a SARS-CoV-2 S protein variant (or immunogenic portion thereof) has an enriched G / C content relative to wild-type sequence.

[0112] In some embodiments, a nucleotide sequence that encodes a variant polypeptide or the polypeptide is codon-optimized for expression in mammalian subjects.

[0113] In some embodiments, a nucleotide sequence that encodes a variant polypeptide or an immunogenic portion thereof has been codon-optimized for expression in human subjects.

[0114] In some embodiments, a nucleotide sequence encoding a variant polypeptide or a portion thereof has an enriched G / C content relative to wild-type sequence.

[0115] In some embodiments, G / C content has been increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%.

[0116] In some embodiments, an RNA comprises a heterologous 3’ UTR or 5’UTR.

[0117] In some embodiments, a heterologous 5' UTR comprises or consists of a modified human alpha-globin 5 '-UTR.

[0118] In some embodiments, a heterologous 3’ UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

[0119] In some embodiments, an RNA comprises a poly(A) sequence.

[0120] In some embodiments, a poly(A) sequence has a length of about 100-150 nucleotides.

[0121] In some embodiments, a poly(A) sequence is a disrupted poly(A) sequence.

[0122] In some embodiments, an RNA comprises a 5' cap.

[0123] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 122 or 124.

[0124] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 131 or 133.

[0125] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 136 or 138.

[0126] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 146 or 148.

[0127] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 151 or 153.

[0128] In some embodiments, an RNA is unmodified RNA.

[0129] In some embodiments, an RNA comprises one or more modified nucleotides.

[0130] In some embodiments, a modified nucleotide is pseudouridine (e.g., N1 -methyl- pseudouridine).

[0131] In some embodiments, an RNA comprises a modified nucleotide in place of each uridine.

[0132] In some embodiments, an RNA is an mRNA, a self-amplifying RNA or a trans- amplifying RNA.

[0133] In some embodiments, a composition comprises an RNA described herein, wherein an RNA is fully or partially encapsulated within lipid nanoparticles (LNP), polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

[0134] In some embodiments, an RNA is fully or partially encapsulated within LNP.

[0135] In some embodiments, an LNP comprises a cationically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.

[0136] In some embodiments, an LNP comprises from about 40 to about 50 mol percent of the cationically ionizable lipid; from about 5 to about 15 mol percent of the neutral lipid; from about 35 to about 45 mol percent of the sterol; and from about 1 to about 10 mol percent of the PEG-lipid.

[0137] In some embodiments, provided herein is a method of inducing an immune response, comprising administering an RNA described herein or a composition described herein to a subject.

[0138] In some embodiments, described herein is a method of inducing an immune response in a subject who has previously been exposed to a reference antigen of an infectious agent (e.g., SARS-CoV-2), the method comprising: delivering a variant polypeptide of the reference antigen (e.g., SARS-CoV-2) or an immunogenic portion thereof to the subject, wherein a B cell memory immune response has been established to the reference antigen (e.g., SARS-CoV-2) , and wherein the variant polypeptide (e.g., SARS-CoV-2 variant) has an amino acid sequence that differs from that of the reference antigen (e.g., SARS-CoV-2) in that it has been engineered to reduce the variant polypeptide’s activation of the B cell memory immune response.

[0139] In some embodiments, an infectious agent is an influenza virus, RSV, norovirus, HIV, coronavirus, or a plasmodium.

[0140] In some embodiments, a subject has previously been administered one or more doses of one or more vaccines that deliver the reference antigen (e.g., SARS-CoV-2) .

[0141] In some embodiments, a reference SARS-CoV-2 S protein is a Wuhan SARS- CoV-2 S protein.

[0142] In some embodiments, an immune response comprises a naive B cell immune response.

[0143] In some embodiments, an immune response comprises a reduced memory B cell immune response or an immune response does not comprise a memory B cell immune response.

[0144] In some embodiments, provided herein is a method of manufacturing an immunogenic composition comprising: (a) providing a reference antigen of an infectious agent (e.g., SARS-CoV-2) , wherein the reference antigen is from a strain or variant (e.g., a strain or variant that has previously been prevalent and / or that has previously been delivered as a vaccine) of the infectious agent (e.g, SARS-CoV-2), (b) determining a variant polypeptide of the reference antigen (e.g, SARS-CoV-2 variant) that comprises fewer memory B cell epitopes relative to the reference antigen (e.g, SARS-CoV-2); and (c) producing an immunogenic composition that delivers the variant polypeptide (e.g, SARS-CoV-2 variant) .

[0145] In some embodiments, a variant polypeptide comprises a sequence that corresponds to an immunogenic portion of a reference antigen.

[0146] In some embodiments, a variant polypeptide (e.g, SARS-CoV-2 variant) comprises one or mutations at one or more B cell epitopes of the reference antigen (e.g, SARS- CoV-2) .

[0147] In some embodiments, provided herein is a method of assessing, predicting, or characterizing the ability of an immunogenic composition that delivers an antigen of an infectious agent (e.g, SARS-CoV-2) to induce activation of memory B cells in a subject or apopulation of subjects, the method comprising determining the number of memory B cell epitopes present in the antigen (e.g, SARS-CoV-2) relative to a reference antigen.

[0148] In some embodiments, a reference antigen (e.g, SARS-CoV-2) is from a strain or variant of an infectious agent that a subject was exposed to and / or that a large portion of a population was exposed to.

[0149] In some embodiments, provided herein is a method of producing a personalized vaccine (e.g, SARS-CoV-2 vaccine) for a subject against an infectious agent, the method comprising steps of: (a) determining a reference antigen of the infectious agent (e.g, SARS-CoV- 2) that a subject has previously been exposed to; (b) determining a variant polypeptide of the reference antigen (e.g, SARS-CoV-2 variant) that comprises fewer memory B cell epitopes relative to the reference antigen (e.g, SARS-CoV-2) ; and (c) producing an immunogenic composition that delivers the variant polypeptide (e.g, SARS-CoV-2 variant).

[0150] In some embodiments, a reference antigen is from a strain or variant of the infectious agent that the subject was first exposed to and / or that was first prevalent in the population of subjects.

[0151] In some embodiments, a reference SARS-CoV-2 S protein is a Wuhan SARS- CoV-2 S protein or an Omicron BA.4 / 5 SARS-CoV-2 S protein.

[0152] In some embodiments, a reference antigen is from an infectious agent (e.g, SARS-CoV-2) that a subject has previously been vaccinated against or is delivered by one or more vaccines that a significant proportion of the population (e.g., at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least abut 45%, at least about 50%, at least about 55%, or at least about 60%) has previously been administered.

[0153] In some embodiments, a vaccine previously administered to a subject or a large portion of a population was a first generation vaccine.

[0154] In some embodiments, a reference antigen is from an infectious agent (e.g, SARS-CoV-2) that was previously prevalent or is currently prevalent in a relevant geographic region.

[0155] In some embodiments, a reference antigen is from an infectious agent variant (e.g, SARS-CoV-2) that first became prevalent in a relevant jurisdiction.

[0156] In some embodiments, antigens (or immunogenic portions thereof) described herein can be engineered to comprise mutations or sequences from two or more antigens of a given infectious agent, where the two or more antigens are from different variants, strains, lineages, etc., of the infectious agent. For example, in some such embodiments, a mutation or sequence in an antigen of a first variant of an infectious agent can be introduced into the corresponding sequence of an antigen of a second variant (or an immunogenic portion thereof). This process can be repeated multiple times, and can be useful, e.g., for removing B cell epitopes (e.g., one or more conserved B cell epitopes). In certain embodiments, antigens described herein can be engineered to incorporate sequences and / or mutations from two or more SARS-COV-2 variants (e.g., epitopes from RBDs, S proteins, and / or SI domains from two or more SARS- CoV-2 variants). For example, in some such embodiments, mutations of a one or more SARS- CoV-2 variants can be introduced in conserved epitopes of a variant SARS-CoV-2 S protein, or an immunogenic portion thereof (e.g., an SI domain or an RBD). Exemplary approaches and methods for introducing mutations or sequences from an antigen of one or more infectious agent variants into a sequence of a first antigen (or an immunogenic portion thereof) are described, e.g., in the U.S. provisional application entitled “Systems and Methods for Engineering Antigens to Promote Tailored Immune Responses”, filed February 24, 2023, and having U.S. Provisional Application No. 63 / 448,215 (inter alia). Said application describes, among other things, technologies directed to in-silico design of custom, engineered, antigens (e.g., including engineered versions of SARS-CoV 2 variant proteins and portions thereof) for reducing an extent to which a memory immune response is triggered.

[0157] In one aspect, the present disclosure provides a combination comprising: (i) a modified RNA molecule encoding a polypeptide comprising or consisting of a variant polypeptide (e.g., SARS-CoV-2 variant) of a reference antigen of an infectious agent (e.g., SARS-CoV-2), or an immunogenic portion thereof, wherein the variant polypeptide (e.g., SARS- CoV-2 variant) comprises neutralizing epitopes that are absent in the reference antigen (e.g., SARS-CoV-2); and (ii) an agent that induces a priming-favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes.

[0158] In some embodiments, an agent that induces a priming-favorable cytokine milieu in lymphoid tissues is or comprises interferon alpha (IFN ^) or an IFN ^-inducing agent. In some embodiments, an agent that induces a priming-favorable cytokine milieu in lymphoid tissues is or comprises a CD4+ T cell response inducing agent.

[0159] In some embodiments, a reference antigen is: (i) a surface protein or surface glycoprotein of an infectious agent strain or variant (e.g., SARS-CoV-2 strain) that was previously and / or is currently prevalent; and / or (ii) a surface protein or surface glycoprotein of an infectious agent (e.g., SARS-CoV-2) that has been previously delivered in a vaccine (e.g., a commercially available vaccine, an RNA vaccine, or a protein-based vaccine). In some embodiments, a reference antigen is a SARS-CoV-2 S protein of a Wuhan strain or an Omicron BA.4 / 5 strain. In some embodiments, a reference antigen is a SARS-CoV-2 S protein of a XBB strain (e.g., XBB1, XBB1.5).

[0160] In some embodiments, a modified RNA molecule and an agent are co-delivered.

[0161] In some embodiments, an IFN ^-inducing agent is or comprises an unmodified RNA molecule. In some embodiments, the amount ratio of the modified RNA molecule to the unmodified RNA molecule is at least or greater than 1:1. In some embodiments, the ratio of modified ribonucleotides to unmodified ribonucleotides in the immunogenic composition is about 1:2 to about 1:10. In some embodiments, a modified ribonucleotide is 1- methylpseudouridine and an unmodified ribonucleotide is uridine.

[0162] In some embodiments, an unmodified RNA molecule encodes a polypeptide comprising an antigen of an infectious agent (e.g., SARS-CoV-2). In some embodiments, an antigen is a B-cell antigen. In some embodiments, an antigen is a T-cell antigen.

[0163] In some embodiments, an antigen is or comprises one or more T cell epitopes from at least one of an M protein, an N protein, and an ORF1ab protein of SARS-CoV-2. In some embodiments, an antigen is or comprises one or more T cell epitopes from at least two of an M protein, an N protein, and an ORF1ab protein of SARS-CoV-2.

[0164] In some embodiments, a modified RNA molecule and the unmodified RNA molecule are separately or co-formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

[0165] In some embodiments, an IFNoc-inducing agent is or comprises a self-amplifying RNA molecule or a trans-amplifying RNA molecule. In some embodiments, a self-amplifying RNA molecule or the trans-amplifying RNA molecule is an unmodified RNA molecule. In some embodiments, the amount ratio of the modified RNA molecule to the self-amplifying RNA molecule or the trans-amplifying RNA molecule is greater than 1:5. In some embodiments, a modified RNA molecule and the self- amplifying RNA molecule or trans-amplifying RNA molecule are separately or co-formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

[0166] In some embodiments, a modified RNA molecule comprises modified uridines. In some embodiments, a modified RNA molecule comprises a modified uridine in lieu of each uridine. In some embodiments, modified uridines are or comprise 1 -methyl pseudouridine.

[0167] In some embodiments, a modified RNA molecule encodes a polypeptide comprising an antigen of the infectious agent. In some embodiments, an antigen is a B-cell antigen. In some embodiments, an antigen is a T-cell antigen.

[0168] In another aspect, the present disclosure provides a combination comprising: (i) a composition that comprises or delivers polypeptide comprising or consisting of a variant polypeptide of a reference antigen of an infectious agent, or an immunogenic portion thereof, wherein the variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen; and (ii) an agent that induces a priming-favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes, and wherein the agent is or comprises (i) an unmodified RNA molecule or (ii) a self-amplifying RNA molecule or a trans- amplifying RNA molecule, and wherein the RNA molecule is formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes. In some embodiments, an agent encodes a polypeptide comprising an antigen of the infectious agent (e.g., SARS-CoV-2) . In some embodiments, an antigen is a B-cell antigen. In some embodiments, an antigen is a T-cell antigen.

[0169] In some embodiments, the amount ratio (by mass or moles) of the polypeptide to the agent is within a range of about 1 : 1 to about 20: 1.

[0170] In some embodiments, the present disclosure provides an RNA molecule comprising a nucleotide sequence that includes modified ribonucleotides and corresponding unmodified ribonucleotides, wherein the ratio of the modified ribonucleotides to the corresponding unmodified ribonucleotides is within a range of about 1: 10 to about 1: 1; and wherein the nucleotide sequence encodes an antigen of an infectious agent (e.g., SARS-CoV-2).

[0171] In some embodiments, a nucleotide sequence comprises a first domain and a second domain, wherein at least one of the first domain and the second domain comprises modified ribonucleotides and the other domain comprises no modified ribonucleotides. In some embodiments, modified ribonucleotides are 1 -methylpseudouridine and the corresponding unmodified ribonucleotides are uridine.

[0172] In one aspect, the present disclosure provides a method of inducing a priming immune response by: administering to a subject one or both of: (i) a composition that comprises or delivers a polypeptide antigen (e.g., SARS-CoV-2) ; and (ii) an agent that induces a priming- favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes.

[0173] In another aspect, the present disclosure provides a method of inducing or supporting a priming immune response to an antigen in a subject by exposing the subject to the antigen under immune priming conditions.

[0174] In some embodiments, a subject has previously been exposed to a variant of the antigen (e.g., SARS-CoV-2).

[0175] In some embodiments, a step of exposing comprises administering a composition that comprises or delivers the antigen. In some embodiments, an antigen is a polypeptide antigen. In some embodiments, a step of exposing comprises administering a “priming adjuvant” to a subject who is or will soon be exposed to the antigen.

[0176] In one aspect, the present disclosure provides a method of inducing an immune response in a subject in need thereof, comprising administering to the subject a first RNA molecule encoding a first antigen (e.g., SARS-CoV-2 antigen) and a second RNA molecule encoding a second antigen, wherein the first RNA molecule is a modified RNA molecule and thesecond RNA molecule (i) does not comprise a modified ribonucleotide or (ii) is a self-amplifying RNA molecule or a trans-amplifying RNA molecule.

[0177] In one aspect, the present disclosure provides a method of inducing an immune response in a subject in need thereof, comprising administering to the subject a composition comprising a first plurality of RNA molecules encoding first antigens (e.g., SARS-CoV-2) and a second plurality of RNA molecules encoding second antigens, wherein at least 10% (including, e.g., at least 20%, at least 30, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of the first plurality of RNA molecules are modified RNA molecules, and at least 10% (including, e.g., at least 20%, at least 30, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of the second plurality of RNA molecules (i) do not comprise a modified ribonucleotide or (ii) are self-amplifying RNA molecules or trans- amplifying RNA molecules.

[0178] In one aspect, the present disclosure provides a method of inducing an immune response in a subject in need thereof, comprising administering to the subject a first dose of a composition comprising a first RNA molecule encoding a first antigen (e.g., SARS-CoV-2) , and a second dose of a composition comprising a second RNA molecule encoding a second antigen, wherein the first RNA molecule is a modified RNA molecule, and the second RNA molecule (i) does not comprise a modified ribonucleotide or (ii) is a self-amplifying RNA molecule or trans- amplifying RNA molecule.

[0179] In some embodiments of various aspects described herein, a first RNA molecule comprises modified uridines. In some embodiments, modified uridines are in place of all uridines. In some embodiments, a second RNA molecule does not comprise a modified ribonucleotide. In some embodiments, a first antigen is or comprises a B cell antigen of an infectious agent and a second antigen is or comprises a T cell antigen. In some embodiments, a B cell antigen is a CoV-2 S antigen or immunogenic portion thereof. In some embodiments, a T cell antigen is from the same infectious agent. In some embodiments, a T cell antigen is a T string epitope. In some embodiments of certain aspects described herein, a T cell antigen is from SARS-CoV-2. In some embodiments, a B cell antigen of SARS-CoV-2 is SARS-CoV-2 S antigen or immunogenic portion thereof.

[0180] In some embodiments of various aspects described herein, a first RNA molecule and a second RNA molecule are co-administered. In some embodiments, a first RNA molecule and a second RNA molecule are separately or co-formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes. In some embodiments, a first RNA molecule and a second RNA molecule are separately administered. In some embodiments, a subject has previously been administered one or more doses of one or more vaccines directed to a reference antigen of an infectious agent, wherein the reference antigen is from an earlier strain or lineage of the infectious agent, and wherein a B cell memory immune response has been established to the reference antigen.

[0181] In some embodiments of certain aspects described herein, a reference antigen is a SARS-CoV-2 S protein of a Wuhan strain or an Omicron BA.4 / 5 strain. In some embodiments, a reference antigen is a SARS-CoV-2 S protein of a XBB strain. In some embodiments, a XBB strain is a XBBl or XBB 1.5.

[0182] In another aspect, the present disclosure provides a method of inducing an immune response in a subject who was previously exposed to a first SARS-CoV-2 Spike (S) protein, the method comprising a step of delivering a polypeptide comprising a fragment of a second SARS-CoV-2 S protein to the subject, wherein the fragment of the second SARS-CoV-2 S protein comprises or consists a Receptor Binding Domain (RBD) or an S 1 domain of the second SARS-CoV-2 S protein, and wherein the fragment of the second SARS-CoV-2 S protein comprises one or more mutations of one or more SARS-CoV-2 variants.

[0183] In some embodiments, the first SARS-CoV-2 S protein is from a strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction.

[0184] In some embodiments, the subject was previously exposed to the first SARS- CoV-2 S protein by: (a) administration of one or more doses of one or more vaccines that deliver the first SARS-CoV-2 S protein, previous infection by a SARS-CoV-2 virus comprising the first SARS-CoV-2 S protein, and / or presence in a jurisdiction where a SARS-CoV-2 strain or variant comprising the first SARS-CoV-2 S protein was prevalent.

[0185] In some embodiments, the fragment of the second SARS-CoV-2 S protein does not comprise one or more regions of a SARS-CoV-2 S protein that are infrequently mutated in SARS-CoV-2 variants. In some embodiments, the fragment of the second SARS-CoV-2 Sprotein does not comprise an S2 domain. In some embodiments, the fragment of the second SARS-CoV-2 S protein does not comprise an N-terminal domain (NTD). In some embodiments, the fragment of the second SARS-CoV-2 S protein comprises or consists of the RBD. In some embodiments, the fragment of the second SARS-CoV-2 S protein comprises or consists of the SI domain.

[0186] In some embodiments, the fragment of the second SARS-CoV-2 S protein comprises one or mutations associated with a SARS-CoV-2 variant that is prevalent, predicted to be prevalent, predicted to continue to be prevalent, and / or predicted to increase in prevalence in a relevant jurisdiction. In some embodiments, the fragment of the second SARS-CoV-2 S protein comprises one or more mutations associated with a SARS-CoV-2 variant that has a high immune escape potential. In some embodiments, the SARS-CoV-2 variant has been determined to have a high immune escape potential using an in vitro assay (e.g., a viral neutralization assay), in silico analysis (e.g., sequence analysis and / or molecular dynamic simulations), in vivo studies (e.g., mouse or rat studies), and / or based on an infection rate and / or growth rate in a human population.

[0187] In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, the Omicron variant is an XBB variant (e.g., an XBB.l or XBB.1.5 variant), a BQ.l variant, a BA.2.86 variant, or a JN variant. In some embodiments, the one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, or N969K, or a combination thereof, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0188] In some embodiments, the fragment of the second SARS-CoV-2 S protein comprises or consists of an RBD of an XBB.1.5 SARS-CoV-2 variant, and wherein the RBD comprises one or more of the following mutations relative to SEQ ID NO: 1: G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, or Y505H, or any combination thereof.

[0189] In some embodiments, the fragment of the second SARS-CoV-2 S protein comprises or consists of an S 1 domain, and wherein the one or more mutations associated with an XBB.1.5 variant are selected from: T19I, A24-26, A27S, V83A, G142D, Al 44, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, and P681H, or any combination thereof, wherein the positions of the one or more mutations are shown relative to SEQ ID NO: 1.

[0190] In some embodiments, the polypeptide comprising the fragment of the second SARS-CoV-2 S protein is delivered by administering an RNA that comprises a nucleotide sequence encoding the fragment of the second SARS-CoV-2 protein.

[0191] In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 3. In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 5.

[0192] In some embodiments, the polypeptide comprises a secretion signal. In some embodiments, the secretion signal is a homologous secretion signal. In some embodiments, the secretion signal is a heterologous secretion signal. In some embodiments, the secretion signal is present at or near the N-terminus of the polypeptide.

[0193] In some embodiments, the secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gDl secretion signal, a gBl secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal. In some embodiments, the SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 15. In some embodiments, the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 16. In some embodiments, the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, the gDl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 12. In someembodiments, the gBl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 37. In some embodiments, the gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 35. In some embodiments, the gI2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 38. In some embodiments, the EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39. In some embodiments, the HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.

[0194] In some embodiments, the polypeptide further a multimerization domain. In some embodiments, the multimerization domain in the C-terminal region (e.g., at the C- terminus). In some embodiments, the multimerization domain is a fibritin domain. In some embodiments, the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 95. In some embodiments, the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 96.

[0195] In some embodiments, the polypeptide comprises a transmembrane (TM) domain. In some embodiments, the TM domain is a homologous TM domain. In some embodiments, the TM domain is a heterologous TM domain.

[0196] In some embodiments, the TM domain is present in the C-terminal portion of the SARS-CoV-2 S protein variant or immunogenic portion thereof (e.g., at the C-terminus).

[0197] In some embodiments, the polypeptide comprises a multimerization domain and a TM domain at or near the C-terminus. In some embodiments, the TM domain is C-terminal to the multimerization domain. In some embodiments, the multimerization domain is directly adjacent to the fragment of the second SARS-CoV-2 protein or connected to the fragment of the second SARS-CoV-2 protein via a flexible linker, and / or the TM domain is directly adjacent to the multimerization domain or connected to the multimerization domain via a flexible linker.

[0198] In some embodiments, the TM domain is a SARS-CoV-2 S protein TM domain or an influenza TM domain. In some embodiments, the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 89. In some embodiments, the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 90.

[0199] In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 120. In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 130. In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 135. In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 145. In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 150.

[0200] In some embodiments, the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has been codon-optimized for expression in mammalian subjects. In some embodiments, the nucleotide sequence encoding the fragment of the second SARS- CoV-2 S protein has been codon-optimized for expression in human subjects.

[0201] In some embodiments, the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has an enriched G / C content relative to wild-type sequence. In some embodiments, the G / C content has been increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%.

[0202] In some embodiments, the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a heterologous 3’ UTR or 5’UTR. In some embodiments, the heterologous 5' UTR comprises or consists of a modified human alpha-globin 5'-UTR. In some embodiments, the heterologous 3’ UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

[0203] In some embodiments, the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a poly(A) sequence. In some embodiments, the poly(A) sequence has a length of about 100-150 nucleotides. In some embodiments, the poly(A) sequence is a disrupted poly(A) sequence.

[0204] In some embodiments, the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a 5' cap.

[0205] In some embodiments, the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 122 or 124. In some embodiments, the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 131 or 133. In some embodiments, the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 136 or 138. In some embodiments, the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 146 or 148. In some embodiments, the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 151 or 153.

[0206] In some embodiments, the RNA is unmodified RNA.

[0207] In some embodiments, the RNA comprises one or more modified nucleotides. In some embodiments, the modified nucleotide is pseudouridine (e.g., Nl-methyl-pseudouridine). In some embodiments, the RNA comprises a modified nucleotide in place of each uridine.

[0208] In some embodiments, the RNA is an self-amplifying RNA or trans-amplifying RNA.

[0209] In some embodiments, the RNA is fully or partially encapsulated within lipid nanoparticles (LNP), polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes. In some embodiments, the RNA is fully or partially encapsulated within LNP. In some embodiments, the LNP comprise a cationically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.

[0210] In some embodiments, the first SARS-CoV-2 S protein is from a strain or variant that the subject was first exposed to and / or that was first prevalent in a population of subjects.

[0211] In some embodiments, the first SARS-CoV-2 S protein is a Wuhan SARS-CoV-2 S protein or an Omicron BA.4 / 5 SARS-CoV-2 S protein.

[0212] In some embodiments, the first SARS-CoV-2 S protein is from a strain or variant that the subject has previously been vaccinated against or is delivered by one or more vaccines that a significant proportion of the population (e.g., at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%,at least abut 45%, at least about 50%, at least about 55%, or at least about 60%) has previously been administered.

[0213] In some embodiments, the vaccine previously administered to the subject or a significant proportion of the population was a first generation vaccine. In some embodiments, the first SARS-CoV-2 S protein is from a SARS-CoV-2 strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction. In some embodiments, the first SARS-CoV-2 S protein is from a variant that first became prevalent in a relevant jurisdiction.

[0214] In some embodiments, the immune response comprises a B cell immune response. In some embodiments, the immune response comprises a naive B cell immune response.

[0215] In some embodiments, (a) the immune response comprises a reduced memory B cell immune response as compared to an immune response induced by administering the full length sequence of the second SARS-CoV-2 S protein, (b) the immune response comprises an increased naive B cell immune response as compared to an immune response induced by administering the full length sequence of the second SARS-CoV-2 S protein, and / or (c) the ratio of the naive B cell immune response to the memory B cell immune response is increased. In some embodiments, (a) the memory B cell immune response is reduced by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein; (b) the memory B cell immune response is increased by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein; and / or (c) the ratio of the naive immune response to the memory B cell immune response is increased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein.Brief description of the Figures

[0216] Fig. 1 illustrates an immune imprinting phenomenon observed in various infectious diseases. SARS-CoV-2 is shown as a representative disease, but a similar process is thought to occur in a number of infectious diseases (in particular, in diseases caused byinfectious agents having high mutation rates in antigen regions that comprise a high number of neutralization epitopes). Subjects administered a vaccine that delivers a wild-type (WT) antigen produce antibodies and form memory B cells. As new Variants of Concern (VOC) arise, VOC- adapted booster shots are administered. Certain VOCs have high immune escape potential and comprise mutations at neutralization epitopes in hypervariable domains (represented by the portions of the antigen with different colors in the Figure). Subjects exposed to a VOC-adapted vaccine have a predisposition to activate memory B cells formed in response to the initial WT vaccine rather than produce de novo responses that recognize epitopes unique to the VOC (i.e., memory B cells that recognize conserved epitopes in the VOC antigen are more likely to be activated and naive B cells recognizing unique epitopes are less likely to be activated). So long as at least some of the neutralization epitopes in the WT antigen are preserved, administering a VOC-adapted vaccine will increase induction of neutralization antibodies against the VOC. As VOCs continue to evolve, however, and acquire further mutations at neutralization epitopes, neutralization responses induced by VOC-adapted vaccines become less efficacious. Further discussion of the imprinting phenomenon in the SARS-CoV-2 context can be found in Wheatley et al., Trends Immunol, 2021, the contents of which are incorporated by reference herein in their entirety.

[0217] Fig. 2: Exemplary characterization tools for assessing memory B cell response, which can be useful to evaluate immune imprinting phenomenon. (A), (B) Schematic of one- dimensial flow-cytometry analysis of memory B cell (BMEM) phenotyping using fluorochrome- labeled antigens (in Figure 2, a SARS-CoV-2 Spike protein is shown for illustrative purposes; in general any antigen delivered as part of a vaccine (or a subdomain of such an antigen) may be used as a label to characterize BMEM cells). BMEM specificity can be assessed by labelling with antigens (or subdomains) of different infectious agent variants. (C) Serological analysis after depletion of immune serum with antigen (or subdomain) bait (in the Figure, SARS-CoV-2 Spike protein is shown for illustrative purposes). Serum samples collected from a subject exposed to an antigen (e.g., via a prior infection and / or previous vaccination) are incubated with an antigen or subdomain thereof (e.g., RBD or SI domain in the case of a SARS-CoV-2 S protein) immobilized on a support (e.g., a magnetic bead as shown in the figure). Isolation of the bead removes antibodies that bind the bait, and the remaining serum is analyzed to determine the specificity of antibodies in the serum sample (e.g., in the Figure, sera samples are incubated withWild-Type (Wuhan) Spike immobilized on magnetic beads, the magenetic beads are removed, and any antibodies remaining in the sample that bind a variant are variant-specific antibodies). Beads lacking bait can be used as a negative control (e.g., as shown in the Figure).

[0218] Fig. 3. Experimental design for assessing impact of immune imprinting. For illustrative purposes, the Figure shows an experiment designed to assess the impact of immune imprinting in SARS-CoV-2, but one of skill in the art will recognize that the depicted experiment can be readily adpated to characterize immune imprinting in any infectious disease context. Sera samples were collected from subjects administered 2 or 3 doses of a vaccine delivering a SARS- CoV-2 S protein (e.g., BNT162b2) and (i) infected with an Omicron BA.l SARS-CoV-2 variant, (ii) infected with an Omicron BA.l variant and subsequently adminsitered an Omicron BA.l- adapted vaccine (BNT162b2(omi)), or (iii) two doses of an Omicron BA.l -adapted vaccine.

[0219] Fig. 4. Variant-induced broad neutralization can be mediated by expansion of responses against conserved epitopes (i.e. recall responses). Provided is data demonstrating that, while exposure to a new variant of a infectious agent (Omicron BA.l in the Figure) can induce a broad immune response, in some embodiments, that broad immune response is driven by the activation of memory B cells and recognition of conserved epitopes, rather than generation of new antibodies that recognize epitopes unique to the new variant. A person of skill in the art will recognize that while the data depicted in the Figure pertain to SARS-CoV-2, the results demonstrate that similar effects could be observed in other infectious diseasese and / or that similar experiments could be performed to characterize immune imprinting in other infectious dieases. Pseudovirus neutralization assays and FACS analysis of BMEM cells using fluorochrome-labeled Spike or RBD tetramers were performed on sera samples collected from the patient groups summarized in Figure 3. (A) Shows pseudo virus neutralization assay results. Pseudovirus neutralization titers (pVNso) were collected for pseudoviruses comprising S proteins of various coronavirus variants and strains (variants and strains indicated along X-axis). Assay results demonstrate that Omicron BA.l infection augments broadly neutralizing activity against Omicron variants, especially against BA.l. (B) Shows representative FACS plots, using flourescently labeled S proteins or RBDs of a full length S protein or RBD. FACS results show that a majority of memory B cells bind epitopes that are common to the Wuhan S protein and the Omicron BA.l S protein, or that are unique to the Wuhan S protein, but very few are specific to Omicron BA.l. These results suggest that a first exposure to wild-type S protein has imprintedagainst novel BMEM responses recognizing BA.l specific epitopes. BNT162b23corresponds to sera samples collected from subjects administered three doses of BNT162b2 and who showed no evidence of subsequent SARS-CoV-2 infection. BNT162b22+ Omi corresponds to sera samples collected from patients administered two doses of BNT162b2 and who subsequenctly experienced a breakthrough SARS-CoV-2 infection at a time of high Omicron BA.l prevalence. BNT162b23+ Omi corresponds to sera samples collected from patients administered three doses of BNT162b2 and who subsequenctly experineced a breakthrough infection at a time of high Omicron BA.l prevalence. Blood drawn 1 month after last vaccination (SARS-CoV-2 naive) or infection (BA.l breakthrough). Data also shown and described in Quandt and Muik et al., 2022, the contents of which are hereby incorporated by reference in their entirety.

[0220] Fig. 5. Immune imprinting can interfere with generation of a de novo response, resulting in poor cross-neutralization of new infectious agent variants. SARS-CoV-2 was chosen as an exemplary infectious agent. One of skill in the art will recognize that the data establishes that immune imprinting can interfere with the generation of effective immune responses in general, and in particular, for infectious diseases having a high concentration of mutations in neutralization sensitive regions of antigens. Sera samples were collected from subjects (i) administered three doses of BNT162b2 (“BNT162b23”) and showing no evidence of prior SARS-CoV-2 infection, (ii) administered four doses of BNT162b2 (“BNT162b24”) and showing no evidence of prior SARS-CoV-2 infection, (iii) administered three doses of an RNA vaccine and who experienced a subsequent Omicron BA.l breakthrough infection (“mRNA-Vax3 + BA.1”), (iv) administered three doses of an RNA vaccine and who experienced a subsequent Omicron BA.2 breakthrough infection (“mRNA-Vax3+ BA.2”), or (v) administered three doses of an RNA vaccine and who experienced a subsequent Omicron BA.4 / 5 breakthrough infection (“mRNA-Vax3+ BA.4 / 5”). (A) Shows pseudovirus neutralization titers. As shown in the figure, sera from mRNA-Vax experienced individuals with BA.l, BA.2 or BA.4 / 5 breakthrough infection showed limited neutralizing activity against the current, most immune-escaping variants like XBB (highlighted in red boxes). (B) Shows percent conservation of HLA class I and class II T-cell epitopes and neutralizing B-cell epitopes for a number of SARS-CoV-2 variants. XBB displayed the lowest conservation of neutralizing B-cell epitopes across VOCs characterized. Data reproduced from Muik, Alexander, et al. "Progressive loss of conserved spike protein neutralizing antibody sites in Omicron sublineages is balanced by preserved T-cellrecognition epitopes." bioRxiv (2022): 2022-12, the contents of which are incorporated by reference herein in their entirety

[0221] Fig. 6. Immune imprinting may not be effectively overcome by repeated exposures to an infectious disease variant. Data is shown from subjects exposed to an Omicron BA.l variant. Sera samples were depleted using the indicated bait protein (e.g., using an assay similar to that depicted in Fig.2(B)), and then screened in a pseudovirus neutralization assay comprising a Wuhan Spike protein (Wuhan-pVNT) or an Omicron BA.1 Spike protein (Omicron BA.l-pVNT). Of the 13 individuals screened, only one showed an Omicron BA.l -specific neutralization response (indicated in red).

[0222] Fig. 7. Imprinting limits build-up of unique epitope-specific B cell memory even after two subsequent exposures to a variant of an infectious agent (Omicron BA.l). Sera samples from subjects administered a booster dose of an RNA vaccine encoding a Omicron BA.l S protein (Omi BA.l Booster), an RNA vaccine encoding a SARS-CoV-2 Wuhan strain (BNT162b2 Booster), or no booster were collected, memory B cells isolated, and analyzed via depletion assays. Sera samples were collected on the day a booster dose was administered (VI), 7 days after a booster dose was administered (V2), and 1 month after a booster dose (V3). Memory B cells (BMEM) were stained for Spike binding, RBD binding, or NTD (N terminal domain) binding for each of Wuhan and Omicron BA.l. Indicated are the percent of screened B cells positive for the indicated probe. BMEM cells binding BA.l specific epitopes in the RBD were not observed. A small population of BMEM cells specific to the BA.1 NTD were observed 1 week after administering an Omicron BA.l -adapted booster. The slight increase of full-length BA.l Spike binding BMEM cells in the BA.l adapted vaccine group most likely represents NTD- binders.

[0223] Fig. 8. Exemplary strategies of addressing immune imprinting. An exemplary approach is to deliver a hypervariable domain of an antigen in an vaccine without other portions of the antigen that contain a large number of non-neutralization epitopes that are shared with a prior-exposure antigen. Shown are novel antigen designs (comprising the SI and RBD- subdomains of a SARS-CoV-2 S protein) for imprint-resistant SARS-CoV-2 vaccines. Mutation density in new SARS-CoV-2 variants of concern (e.g., XBB) is highest in the Sl-fragment and especially in the RBD. Hence, omitting the highly conserved S2 fragment can result in moreefficient priming (e.g., by removing conserved epitopes that can activate BMEM cells and / or prevent activation of naive B cells). Shown are certain exemplary antigen designs, including (1) an RBD of an VOC attached to a trimerization domain (e.g., an RBD of XBB.1.5 attached to a T4 foldon domain), (2) an SI domain of an VOC attached to a trimerization domain (e.g., an SI of XBB.1.5 attached to a T4 foldon domain), (3) an RBD of an VOC attached to a trimerization domain and a transmembrane (TM) domain (e.g., an RBD of XBB.1.5 attached to a T4 foldon domain and a TM domain of a SARS-CoV-2 S protein), and (4) an SI domain of an VOC attached to a trimerization domain and a transmembrane domain (e.g., an SI of XBB.1.5 attached to a T4 foldon domain and a TM domain of a SARS-CoV-2 S protein). Constructs (1) and (2) are soluble and secreted, whereas constructs (3) and (4) are TM-anchored. Similar strategies can be used to design imprint-resistant vaccines against other infectious diseases (in particular, diseases caused by infectious agents that comprise regions neutralization-sensitive regions with high rates of mutation).

[0224] Fig. 9. Immunogenicity study in vaccine-experienced mice. Mice are administered two doses of BNT162b2 (encoding an S protein of a Wuhan variant), or a composition comprising a first RNA that encodes a SARS-CoV-2 S protein of a Wuhan variant and a second RNA encoding a full length S protein of an Omicron BA.4 / 5 variant (Bivalent b2 + BA.4 / 5), followed by a third and fourth dose of a candidate vaccine. Third and fourth doses include RNA encoding full length Spike protein of a Wuhan strain (BNT162b2); RNA encoding a full length S protein of an XBB.1.5 variant (BNT162b2 (XBB.1.5)); RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal and a timerization domain (RBD (XBB.1.5)); RNA encoding an SI domain of an XBB.1.5 S protein comprising a timerization domain (SI (XBB.1.5)); RNA encoding an SI domain of an XBB.1.5 S protein comprising a timerization domain and a transmembrane domain (Sl-TM (XBB.1.5)); and RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal, a timerization domain, and a transmembrane domain (RBD-TM (XBB.1.5)). The third dose includes one of the vaccine candidate disclosed in Example 2 (Table 16). Yellow-filled cells indicate days on which sera sample will be collected, gray-filled cells indicate days on which vaccines will be administered, and green-filled cells indicate days on which mice are sacrificed and final samples collected.

[0225] Fig. 10. Immunogenicity study in vaccine-experienced mice - sample characterization. Summary of spleen sample, lymph nodes are collected and analyzed as shown in the Figure. Figure also summarizes analysis of blood samples collected throughout the study.

[0226] Fig. 11. Immunogenicity study in vaccine-naive mice. Mice are administered two doses of RNA encoding (i) a full length Spike protein of a Wuhan strain (BNT162b2); (ii) RNA encoding a full length S protein of an XBB.1.5 variant (BNT162b2 (XBB.1.5)); (iii) RNA encoding a full length S protein of an XBB.1.5 variant and comprising a 19 amino acid C- terminal truncation (BNT162b2 (XBB.1.5) Cdl9); (iv) RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP19) and a timerization domain (RBD (XBB.1.5) (SP19)); (v) RNA encoding an SI domain of an XBB.1.5 S protein comprising a trimerization domain (SI (XBB.1.5)); (vi) RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP19), a timerization domain, and a transmembrane domain (RBD-TM (XBB.1.5) (SP19)); (vii) RNA encoding an SI domain of an XBB.1.5 S protein comprising a timerization domain and a transmembrane domain (Sl-TM (XBB.1.5); and (viii) RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP16) and a timerization domain (RBD (XBB.1.5) (SP16)). Yellow-filled cells indicate days on which sera sample will be collected, gray-filled cells indicate days on which vaccines will be administered, and green-filled cells indicate days on which mice are sacrificed and final samples collected.

[0227] Fig. 12. Immunogenicity study in vaccine-naive mice - sample characterization. On the final day study day, spleen samples are collected and analyzed as shown in the figure.

[0228] Fig. 13. Design of an experiment to test immunogenecity of vaccine candidates in vaccine-experienced mice. Top row lists number of mice in each group ("size"), and days on which vaccines were administered (days 0, 21, 126, and 238 post dose 1), and samples were collected (days 0, 21, 35, 63, 91, 119. 126, 133, 154, 182, 210, 238, 245, 259, and 273. Subsequence rows indicate vaccines administered, where doses 1, 2, 3, and 4 are listed from left to right. Mice were split into 12 groups, 6 of which were administered a first dose and a second dose of a monovalent vaccine comprising RNA encoding a full-length S protein of a Wuhan strain ("BNT162b2"), and 6 of which were administered two doses of a bivalent vaccine comprising (i) an RNA encoding a SARS-CoV-2 S protein of a Wuhan strain and (ii) an RNA encoding a SARS-CoV-2 S protein of an Omicron BA.4 / 5 variant ("Bivalent b2 + BA.4 / 5"). Forall groups, the second dose was administered about 21 days after the first dose. Each group was administered a third dose and a fourth dose of a vaccine candidate. "BNT162b2" refers to a vaccine comprising RNA that encodes a full length S protein; "Bl RBD" refers to a vaccine comprising an RNA that encodes a soluble RBD (does not comprise a transmembrane domain); "B3-RBD-TM" refers to a vaccine comprising an RNA encoding a membrane-anchored RBD (comprises a transmembrane domain); "Bl -like SI" refers a vaccine comprising RNA encoding a soluble SI domain; "B3-like Sl-TM" refers to a vaccine comprising an RNA encoding a membrane- anchored SI domain; "T cell string" refers to RNA encoding T cell epitopes of a SARS-CoV-2 virus; "uRNA" refers to unmodified RNA (comprising unmodified nucleotides, aside from the 5' cap) and "modRNA" refers to RNA comprising modified uridines. If a group lists "XBB.1.5" the RBD, SI, or S protein comprises mutations characteristics of an XBB.1.5 variant; otherwise, encoded amino acid sequence corresponds to that of the original Wuhan strain.

[0229] Fig. 14. Neutralization titers prior to administering vaccine candidates. Shown are geometric mean neutralization titers in mice at day 63 (panel (A)) and 91 (panel (B)) post dose 1, where the mice were vaccinated per the protocol summarized in Fig. 10, and described in Example 4. Shown immediately below the x-axis of each plot is the strain against which neutralization titers were collected. Also below the strains against which neutralization titers were collected, and below the line, is the vaccines administered. "BNT162b22" refers to mice administered two doses of BNT162b2. "(Bivalent b2+BA.4 / 5)2" refers to mice administered two doses of a bivalent compisition comprising (i) RNA encoding a SARS-CoV-2 S protein of a Wuhan strain, and (ii) RNA encoding a SARS-CoV-2 S protein of an Omicron BA.4 / 5 variant. Indicated above each bar is the geometric mean of the neutralizing titers. "d42PD2" stands for 42 days post dose 2, and "d70PD2" stands for 70 days post-dose 2. "EEOD" stands for Eower Eimit of Detection. As shown in the Figure, mice administered two doses of a bivalent vaccine exhibited much higher neutralizing titers against SARS-CoV-2 variants than mice administered two doses of a monovalent composition delivering a full length S protein of a Wuhan strain. (C) Neutralization titers collected in an experiment in which mice were administered a first dose of a BNT162b2, and a second dose of a bivalent vaccine comprising RNA encoding an S protein of a Wuhan strain and RNA encoding an S protein of an Omicron BA.4 / 5 variant. As shown if the figure, and in contrast to (B), neutralization titers were much lower following dose 2, indicatingthat two doses of a bivalent vaccine induce higher neutralization titers as compared to a mixed dosing regimen in vaccine naive mice.

[0230] Fig. 15. Neutralization titers induced by vaccine candidates in vaccine- experienced mice. Vaccine candidate abbreviations are the same as those used in Fig. 10, except "B3-like" is used in place of "B3-like Sl-TM" and "B3-RBD" is used in place of "B3-RBD-TM". Shown are neutralization titers in mice administered BNT162b2 as a first and second dose. (A) shows geometric mean neutralization titers against an XBB.1.5-adapted pseudovirus, in mice administered the vaccine canididates indicated in the table, at the time points indicated in the table. Timing corresponds to that shown in Fig. 10. (B) is a plot of the data shown in (A). (C) shows the geometric fold increase for neutralization titers at day 154 vs day 126. (D) provides a bar chart summarizing the values provided in (C). (E) shows geometric mean neutralization titers against a Wuhan-adapted pseudovirus, in mice administered the vaccines indicated in each column, at the time points indicated in the table. (F) provides a plot of the values shown in (E). As shown in the figure, membrane-anchored RBD provided the highest neutralization titers after a single boost, with neturalization titers increased by about 64-fold on day 238 as compared to day 126. In general, shorter constructs delivering only the RBD (either membrane anchored or soluble) were more efficient in generating higher neutralization titers as compared to other constructs.

[0231] Figure 16: Representative FACS Data. Shown is representative FACS data, obtained using baits comprising (i) the full length S protein of the Wuhan strain, and (ii) the full length S protein of the XBB.1.5 SARS-CoV-2 variant attached to different fluorescent labels. Each point in the plot corresponds to a B cell. Indicated to the left of each row is the vaccine candidate administered. Each plot corresponds to cells obtained from individual mice. Each point in a plot corresponds to a B cell. Cells were classified into two groups: (i) Wuhan binders (Wuhan signal above background signal) and (ii) XBB.1.5 binders (XBB.1.5 signal above background signal). Wuhan-binders (black) and XBB.1.5 binders were overlayed on top of the entire B cell population (light grey). An increase in the number of cells located on the y-axis of each (medium grey events, not on the diagonal) indicating XBB.1.5-specific binding represents an improved naive response.

[0232] Figure 17: Transmembrane-anchored RBD induces a more-consistent immune response against SARS-CoV-2 variants in vaccine-experienced mice. Shown is a cumulative analysis of FACS data which was representatively show cased in Fig. 13. Spike specific B cell were categorized via a Boolean Gating approach into (i) Wuhan-specific, (ii) shared (binding both XBB.1.5 and Wuhan S protein), and (iii) XBB.1.5-specific B cells. Shown along the x-axis is the vaccine candidate administered. Shown along the y-axis is the percentage of CD19+cells positive for being labeled with the indicated S protein. As shown in the figure, constructs delivering a membrane anchored RBD (“b3”) provided the most consistent induction of B cell immune response, as indicated by the significantly reduced intragroup variability in neutralization titers.

[0233] Figure 18: Activation of memory B cells in vaccinated mice. B cells separated according to their ability to bind different S proteins were stained for CD95 (CD95+indicates B cell activation, serving as a proxy to show that they recently undergo Germinal Center (GM) reaction).

[0234] Figure 19: Example of an experimental protocol for sequencing individual B cells collected from splenocytes. As shown in the figure, splenocytes were harvested from each mouse at the end of the experiment, B -cells were isolated and blocked, and then all B cells were pooled and sorted by FACS using fluorescently labeled full length S protein (Wuhan or XBB.1.5, representative FACS plots for sorting are shown). After sorting cells were subjected to single cell BCR sequencing employing the 10X Genomics technology.

[0235] Figure 20: Sequencing summary statistics. Shown are summary statistics collected using the experimental protocol depicted in Figure 16. As shown in the figure, sequences of VH and VL regions were obtained, as well as a number of clonotypes for each sorted group.

[0236] Figure 21. Fraction and number of CD19+B cells binding a Wuhan S protein, an XBB.1.5 S protein, or both. (A) shows the number of CD19+B cells, and (B) shows the number of clonotypes of CD19+B cells that bind Wuhan Spike, XBB.1.5 Spike, or both. As shown in the figure, constructs encoding an RBD of an S protein produced the highest number of cells and clonotypes that are specific to XBB.1.5, which membrane anchored RBD in particular producing the highest numbers.

[0237] Figure 22. Ig isotypes in B cells with different binding specificities. As shown in the figure, following vaccination with different vaccine candidates, no significant difference was seen between them in the relative proportion of different Ig isotypes between B cells displaying different Ig isotypes.

[0238] Figure 23. Comparison of clonotypes across cohorts. Clonotypes that were shared in at least two cohorts were identified and compared across cohorts to determine whether certain clonotypes were characteristic of an individual construct design (e.g., if particular clonotypes were associated with RBD or full-length S protein constructs). B cell responses were found to be mostly private. Any shared clonotypes detected appeared to occur due to background binding, rather than S protein specific binding.Certain Definitions

[0239] In general, terminology used herein is in accordance with its understood meaning in the art, unless clearly indicated otherwise. Explicit definitions of certain terms are provided below; meanings of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from context.

[0240] In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0241] About. The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0242] Agent'. As used herein, the term “agent”, may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by a particular feature and / or effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term “agent” may refer toa compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety.

[0243] Amino acid: In its broadest sense, as used herein, the term “amino acid” refers to a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally- occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L- amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino- terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0244] Antibody agent. As used herein, the term “antibody agent” refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses a polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficientto confer specific binding. For example, in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1 -5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1 -5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent in or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art to correspond to CDRsl, 2, and 3 of an antibody variable domain; in some such embodiments, an antibody agent in orcomprises a polypeptide or set of polypeptides whose amino acid sequence(s) together include structural elements recognized by those skilled in the art to correspond to both heavy chain and light chain variable region CDRs, e.g., heavy chain CDRs 1, 2, and / or 3 and light chain CDRs 1, 2, and / or 3. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. In some embodiments, an antibody agent may be or comprise a polyclonal antibody preparation. In some embodiments, an antibody agent may be or comprise a monoclonal antibody preparation. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a particular organism, such as a camel, human, mouse, primate, rabbit, rat; in many embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a human. In some embodiments, an antibody agent may include one or more sequence elements that would be recognized by one skilled in the art as a humanized sequence, a primatized sequence, a chimeric sequence, etc. In some embodiments, an antibody agent may be a canonical antibody (e.g., may comprise two heavy chains and two light chains). In some embodiments, an antibody agent may be in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide- Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®;Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., poly-ethylene glycol, etc.)).

[0245] Antigen-. Those skilled in the art, reading the present specification, will appreciate that the term “antigen” refers to a molecule that is recognized by the immune system, e.g., in particular embodiments, the adaptive immune system, such that it elicits an antigen-specific immune response. In some embodiments, an antigen-specific immune response may be or comprise generation of antibodies and / or antigen-specific T cells. In some embodiments, an antigen is a peptide or polypeptide that comprises at least one epitope against which an immune response can be generated. In one embodiment, an antigen is presented by cells of the immune system such as antigen presenting cells like dendritic cells or macrophages. In one embodiments, an antigen or a processed product thereof such as a T-cell antigen is bound by a T- or B-cell receptor, or by an immunoglobulin molecule such as an antibody. Accordingly, an antigen or a processed product thereof may react specifically with antibodies or T lymphocytes (T cells). In one embodiment, an antigen is a parasitic antigen. In accordance with the present disclosure, in some embodiments, an antigen may be delivered by RNA molecules as described herein. In some embodiments, a peptide or polypeptide antigen can be 2-100 amino acids, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length. In some embodiments, a peptide or polypeptide antigen can be greater than 50 amino acids. In some embodiments, a peptide or polypeptide antigen can be greater than 100 amino acids. In some embodiments, an antigen is recognized by an immune effector cell. In some embodiments, an antigen, if recognized by an immune effector cell, is able to induce in the presence of appropriate co- stimulatory signals, stimulation, priming and / or expansion of the immune effector cell carrying an antigen receptor recognizing the antigen. In the context of the embodiments of the present disclosure, in some embodiments, an antigen can be presented or present on the surface of a cell, e.g., an antigen presenting cell. In one embodiment, an antigen is presented by a diseased cell such as a virus-infected cell. In one embodiment, an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC. In one embodiment, binding of a TCR when expressed by T cells and / or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and / or expansion of said T cells. In one embodiment, binding of a TCR when expressed by T cells and / or present on T cells to an antigen presented on diseased cells results in cytolysis and / or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g. perforins and granzymes.

[0246] Associated. Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite,microbe, etc) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0247] Binding-. Those skilled in the art, reading the present specification, will appreciate that the term “binding” typically refers to a non-covalent association between or among entities or moieties. In some embodiments, binding data are expressed in terms of “IC50”. As is understood in the art, IC50 is the concentration of an assessed agent in a binding assay at which 50% inhibition of binding of reference agent known to bind the relevant binding partner is observed. In some embodiments, assays are run under conditions in which (e.g., limiting binding target and reference concentrations), IC50 values approximate KD values. Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO 94 / 20127 and WO 94 / 03205, and other publications such Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol. 31:813 (1994). Alternatively, binding can be expressed relative to binding by a reference standard peptide. For example, can be based on its IC50, relative to the IC50 of a reference standard peptide. Binding can also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147: 189 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), cell free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol.Chem. 268: 15425 (1993)); high flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al.,Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992)).

[0248] Cap. As used herein, the term “cap” refers to a structure comprising or essentially consisting of a nucleoside-5 '-triphosphate that is typically joined to a 5'-end of an uncapped RNA (e.g., an uncapped RNA having a 5'- diphosphate). In some embodiments, a cap is or comprises a guanine nucleotide. In some embodiments, a cap is or comprises a naturally- occurring RNA 5’ cap, including, e.g., but not limited to a 7- methylguanosine cap, which has a structure designated as “m7G.” In some embodiments, a cap is or comprises a synthetic cap analog that resembles an RNA cap structure and possesses the ability to stabilize RNA if attached thereto, including, e.g., but not limited to anti -reverse cap analogs (ARC As) known in the art). Those skilled in the art will appreciate that methods for joining a cap to a 5’ end of an RNA are known in the art. For example, in some embodiments, a capped RNA may be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (including, e.g., but not limited to vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system). Alternatively, a capped RNA can be obtained by in vitro transcription (IVT) of a single-stranded DNA template in the presence of a dinucleotide or trinucleotide cap analog.

[0249] Cell-mediated, immunity. “Cell-mediated immunity,” “cellular immunity,” “cellular immune response,” or similar terms are meant to include a cellular response directed to cells characterized by expression of an antigen, in particular characterized by presentation of an antigen with class I or class II MHC. A cellular response relates to immune effector cells, in particular to T cells or T lymphocytes which act as either “helpers” or “killers.” The helper T cells (also termed CD4+T cells or CD4 T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8+T cells, CD8 T cells, or CTLs) kill diseased cells such as virus -infected cells, preventing the production of more diseased cells.

[0250] Co-administration. As used herein, the term “co-administration” refers to use of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent. The combined use of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agentmay be performed concurrently or separately (e.g., sequentially in any order). In some embodiments, a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent may be combined in one pharmaceutically- acceptable carrier, or they may be placed in separate carriers and delivered to a target cell or administered to a subject at different times. Each of these situations is contemplated as falling within the meaning of “co-administration” or “combination,” provided that a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent are delivered or administered sufficiently close in time that there is at least some temporal overlap in biological effect(s) generated by each on a target cell or a subject being treated.

[0251] Codon-optimized.'. As used herein, the term “codon-optimized” refers to alteration of codons in a coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, in some embodiments coding regions are codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein. In some embodiments, codon-optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of “rare codons.” In some embodiments, codon-optimization may include increasing guanosine / cytosine (G / C) content of a coding region of RNA described herein as compared to the G / C content of the corresponding coding sequence of a wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence.

[0252] Conserved Epitope: As used herein, a “conserved epitope” refers to an epitope that is retained in a variant polypeptide relative to a reference polypeptide. An epitope can be determined and / or inferred using methods that are well known in the art, including, e.g., antibody binding studies, B cell binding studies, structural analysis, and infection rates, among others.

[0253] Combination therapy. As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior toadministration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.

[0254] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0255] Corresponding to : As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example,need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHLLS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term “corresponding to” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and / or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.

[0256] Derived. '. In the context of an amino acid sequence (peptide or polypeptide)“derived from” a designated amino acid sequence (peptide or polypeptide), refers to a structural analogue of a designated amino acid sequence. In some embodiments, an amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.

[0257] Designed: As used herein, the term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and / or (iii) that is distinct from natural substances and other known agents.

[0258] Dosing regimen -. Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (z.e., is a therapeutic dosing regimen).

[0259] Encode. As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule (e.g., an mRNA) encodes a polypeptide if transcription and translation of mRNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of an RNA molecule encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such a target antigen. In some embodiments, a coding region of an RNA molecule encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.

[0260] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature aremanipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.

[0261] Epitope. As used herein, the term “epitope” refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. For example, an epitope may be recognized by a T cell, a B cell, or an antibody. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized). Accordingly, in some embodiments, an epitope of an antigen may include a continuous or discontinuous fragment of the antigen. In some embodiments, an epitope is or comprises a T cell epitope. In some embodiments, an epitope may have a length of about 5 to about 30 amino acids, or about 10 to about 25 amino acids, or about 5 to about 15 amino acids, or about 5 to 12 amino acids, or about 6 to about 9 amino acids.

[0262] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the generation of a gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0263] Five prime untranslated region . As used herein, the terms “five prime untranslated region” or “5' UTR” refer to a sequence of an mRNA molecule between a transcription start site and a start codon of a coding region of an RNA. In some embodiments, “5’ UTR” refers to a sequence of an mRNA molecule that begins at a transcription start site andends one nucleotide (nt) before a start codon (usually AUG) of a coding region of an RNA molecule, e.g., in its natural context.

[0264] Fragment. The term “fragment” as used herein in the context of a nucleic acid sequence (e.g. RNA sequence) or an amino acid sequence may typically be a fragment of a reference sequence. In some embodiments, a reference sequence is a full-length sequence of e.g. a nucleic acid sequence or an amino acid sequence. Accordingly, a fragment, typically, refers to a sequence that is identical to a corresponding stretch within a reference sequence. In some embodiments, a fragment comprises a continuous stretch of nucleotides or amino acid residues that corresponds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total length of a reference sequence from which the fragment is derived. In some embodiments, the term “fragment", with reference to an amino acid sequence (peptide or polypeptide), relates to a part of an amino acid sequence, e.g., a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. In some embodiments, a fragment of an amino acid sequence comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.

[0265] Homology: As used herein, the term “homology” or “homolog” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.

[0266] Humoral immunity: As used herein, the term “humoral immunity” or “humoral immune response” refers to antibody production and the accessory processes that accompany it, including: Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. It also refers to the effector functions of antibodies, which include pathogen neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

[0267] Identity: As used herein, the term “identity” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotidesequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0268] Immunologically equivalent: The term “immunologically equivalent” means that an immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect. In the context of the present disclosure, in some embodiments, the term “immunologically equivalent” is used with respect to the immunological effects or properties of antigens or antigen variants used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction having a specificity of reacting with the reference amino acid sequence.

[0269] In one embodiment, an antigen receptor is an antibody or B cell receptor which binds to an epitope of an antigen. In one embodiment, an antibody or B cell receptor binds to native epitopes of an antigen.

[0270] As used herein, “immune escaping” refers to a variant or strain of an infectious agent that can fully or partially evade an immune response (e.g., a B cell immune response).

[0271] As used here, “immune escape potential” refers to a likelihood of a given variant being able to evade previously developed immune responses. A skilled artisan is aware of various methods for assessing the immune escape potential of a given infectious agent. For example, in some embodiments, immune escape potential can be determined experimentally based on infection rates in a relevant population (e.g., infection rates in subjects previously infected and / or vaccinated with a previous variant of the infectious agent). In some embodiments, an immune escape potential can be determined using one or more in vitro assay(s) (e.g., neutralization assays as described herein). In some embodiments, an immune escape potential can be predicted using the sequence of the variant (e.g., predicted by in silico analysis, location of epitopes relative to previously determined neutralization epitopes, etc.).

[0272] Increased, Induced, or Reduced: As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a providedpharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) may be “increased” relative to that obtained with a comparable reference pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine). Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be “increased” relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term “reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term “increased” or “induced” refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.

[0273] Ionizable The term “ionizable” refers to a compound or group or atom that is charged at a certain pH. In the context of an ionizable amino lipid, such a lipid or a function group or atom thereof bears a positive charge at a certain pH. In some embodiments, an ionizable amino lipid is positively charged at an acidic pH. In some embodiments, an ionizable amino lipid is predominately neutral at physiological pH values, e.g., in some embodiments about 7.0-7.4, but becomes positively charged at lower pH values. In some embodiments, an ionizable amino lipid may have a pKa within a range of about 5 to about 7.

[0274] Isolated: The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated”, but the same nucleic acid or peptide partially or completely separated from the coexisting materialsof its natural state is “isolated”. An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0275] Lipid. As used herein, the terms “lipid” and “lipid-like material” are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.

[0276] Modified: As used herein, the term “modified”, in the context of an amino acid or nucleotide sequence, refers to a change relative to a reference sequence. In some embodiments, a modified amino acid or nucleotide sequence comprises a deletion (e.g., a deletion of a single residue, a short stretch of residues (e.g., 1 to 10 residues), a particular region (e.g., a particular region of a polypeptide, or a nucleotide sequence encoding said region), or a particular domain (e.g., a particular domain of a polypeptide or a nucleotide sequence encoding said domain). In some embodiments, a modified amino acid or nucleotide sequence comprises an insertion (e.g., an insertion of a single residue, a short stretch of residues (e.g., 1 to 10 residues), a particular region (e.g., a particular region of a polypeptide, or a nucleotide sequence encoding said region), or a particular domain (e.g., a particular domain of a polypeptide or a nucleotide sequence encoding said domain). In some embodiments a modified amino acid or nucleotide sequence comprises a substitution.

[0277] RNA lipid nanoparticle . As used herein, the term “RNA lipid nanoparticle” refers to a nanoparticle comprising at least one lipid and RNA molecule(s). In some embodiments, an RNA lipid nanoparticle comprises at least one ionizable amino lipid. In some embodiments, an RNA lipid nanoparticle comprises at least one ionizable amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size (e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, RNA lipid nanoparticles can have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanoparticles is determined by measuring the particle diameter. In some embodiments, RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.

[0278] Lipidoid: As used herein, a “lipidoid” refers to a lipid-like molecule. In some embodiments, a lipoid is an amphiphilic molecule with one or more lipid-like physical properties. In the context of the present disclosure, the term lipid is considered to encompass lipidoids.

[0279] Nanoparticle : As used herein, the term “nanoparticle” refers to a particle having an average size suitable for parenteral administration. In some embodiments, a nanoparticle has a longest dimension (e.g., a diameter) of less than 1,000 nanometers (nm). In some embodiments, a nanoparticle may be characterized by a longest dimension (e.g., a diameter) of less than 300 nm. In some embodiments, a nanoparticle may be characterized by a longest dimension (e.g., a diameter) of less than 100 nm. In many embodiments, a nanoparticle may be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 1 pm and about 500 nm, or between about 1 nm and 1,000 nm. In many embodiments, a population of nanoparticles is characterized by an average size (e.g., longest dimension) that is below about 1,000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm and often above about 1 nm. In many embodiments, a nanoparticle may be substantially spherical so that its longest dimension may be its diameter. In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health.

[0280] Naturally occurring: The term “naturally occurring” as used herein refers to an entity that can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

[0281] Neutralization: As used herein, the term “neutralization” refers to an event in which binding agents such as antibodies bind to a biological active site of a virus such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term “neutralization” refers to an event in which binding agents eliminate or significantly reduce ability of infecting cells.

[0282] As used herein, a “neutralization epitope” or “neutralization sensitive epitope” refers to an epitope that can be bound by a neutralizing antibody. Neutralization epitopes can be determined using methods that are well known in the art, including, e.g., neutralization assays and antibody binding studies, among other techniques.

[0283] Nucleic acid particle . A “nucleic acid particle” can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may comprise at least one cationic or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof and nucleic acid. In some embodiments, a nucleic acid particle is a lipid nanoparticle. In some embodiments, a nucleic acid particle is a lipoplex particle.

[0284] Nucleic acid / Polynucleotide. As used herein, the term “nucleic acid” refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded fragments. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxy thymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 - methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5 -fluorouridine, C5 -iodouridine, C5-propynyl-uridine, C5 - propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residuecomprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.

[0285] Nucleotide: As used herein, the term “nucleotide” refers to its art-recognized meaning. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0286] Patient: As used herein, the term “patient” refers to any organism who is suffering or at risk of a disease or disorder or condition. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disease or disorder or condition. In some embodiments, a patient has been diagnosed with one or more diseases or disorders or conditions. In some embodiments, a disease or disorder or condition that is amenable to provided technologies is or includes a HSV infection. In some embodiments, a patient is receiving or has received certain therapy to diagnose and / or to treat a disease, disorder, or condition. In some embodiments, a patient is a patient suffering from or susceptible to a HSV infection.

[0287] PEG-conjugated lipid. The term “PEG-conjugated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion.

[0288] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for parenteral administration, for example, by subcutaneous, intramuscular, or intravenous injection as, for example, a sterile solution or suspension formulation.

[0289] Pharmaceutically effective amount: The term “pharmaceutically effective amount” or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, a desired reaction in some embodiments relates to inhibition of the course of the disease. In some embodiments, such inhibition may comprise slowing down the progress of a disease and / or interrupting or reversing the progress of the disease. In some embodiments, a desired reaction in a treatment of a disease may be or comprise delay or prevention of the onset of a disease or a condition. An effective amount of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) described herein will depend, for example, on a disease or condition to be treated, the severity of such a disease or condition, individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0290] Poly(A) sequence: As used herein, the term “poly(A) sequence” or “poly-A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3 '-end of an RNA molecule. Poly(A) sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of theRNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0291] Polypeptide: As used herein, the term “polypeptide” refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class).For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.

[0292] Prevent: As used herein, the term “prevent” or “prevention” when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0293] Receptor Binding Domain: As used herein, a “receptor binding domain” (RBD), when used in the context of a generic infectious agent, refers to a region of an infectious agent polypeptide that plays a role in binding a host cell receptor, where the RBD is the region that binds the host cell receptor. In the context of a particular infectious agent, RBD can sometimes refer to a specific region of a protein (e.g., in the context of SARS-CoV-2, RBD refers to a particular region of the S protein).

[0294] Recombinant: The term “recombinant” in the context of the present disclosure means “made through genetic engineering”. In some embodiments, a “recombinant” entity such as a recombinant nucleic acid in the context of the present disclosure is not naturally occurring.

[0295] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneouslywith the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0296] In some embodiments, a reference antigen is an antigen that a subject has previously encountered or has a high likelihood of having previously encountered. For example, in some embodiments, a reference antigen is an antigen delivered by a vaccine that was previously administered to a subject. In some embodiments, a reference antigen is present in a strain or variant of an infectious agent that a subject was previously infected with and / or that was prevalent at a time and region in which the subject was previously infected. In some embodiments, a reference antigen is an antigen of the first strain or variant of an infectious agent that a subject encountered. In some embodiments, a reference antigen is an antigen of a strain or variant of an infectious agent that first became prevalent. In some embodiments, a reference antigen is an antigen delivered by one of the first vaccines that became widely available against a given infectious agent (e.g., for SARS-CoV-2, one of the first commercially approved vaccines delivering a Wuhan Spike protein). In some embodiments, a reference antigen is a Wuhan Spike protein, or an immunogenic portion thereof. In some embodiments, a reference antigen is a Spike protein of an Omicron variant (e.g., a BA.4 / 5 Omicron variant), or an immunogenic portion thereof.

[0297] Ribonucleic acid (RNA): As used herein, the term “RNA” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded fragments. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments where an RNA is a mRNA. In some embodiments where an RNA is a mRNA, a RNA typically comprises at its 3’ end a poly(A) region. In some embodiments where an RNA is a mRNA, an RNA typically comprises at its 5’ end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In someembodiments, a RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0298] Ribonucleotide: As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0299] Risk: As will be understood from context, “risk” of a disease, disorder, and / or condition refers to a likelihood that a particular individual will develop the disease, disorder, and / or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and / or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, risk may reflect one or more genetic attributes, e.g., which may predispose an individual toward development (or not) of a particular disease, disorder and / or condition. In some embodiments, risk may reflect one or more epigenetic events or attributes and / or one or more lifestyle or environmental events or attributes.

[0300] RNA lipoplex particle. As used herein, the term “RNA lipoplex particle” refers to a complex comprising liposomes, in particular cationic liposomes, and RNA molecules.Without wishing to bound by a particular theory, electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. In some embodiments, positively charged liposomes may comprise a cationic lipid, such as in some embodiments DOTMA, and additional lipids, such as in some embodiments DOPE. In one embodiment, a RNA lipoplex particle is a nanoparticle.

[0301] Selective or specific: The term “selective” or “specific”, when used herein in reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities, states, or cells. For example, in some embodiments, an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of a target -binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding moiety.

[0302] Stable: As used herein, the term “stable” in the context of the present disclosure refers to a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as a whole and / or components thereof meeting or exceeding pre-determined acceptance criteria. For example, in some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) exhibits no unacceptable levels of microbial growth, and substantially no or no breakdown or degradation of the active biological molecule component(s). In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to the integrity of RNA molecules being maintained at least above 90% or more. In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to at least 90% or more (including, e.g., at least 95%, at least 96%, at least 97%, or more) of RNA molecules being maintained to be encapsulated within lipid nanoparticles. In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to a formulation that remains capable of eliciting a desired immunologic response when administered to a subject. In some embodiments, a pharmaceuticalcomposition (e.g., immunogenic composition, e.g., vaccine) remains stable for a specified period of time under certain conditions.

[0303] Subject. As used herein, the term “subject” refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0304] Suffering from. An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0305] Susceptible to . An individual who is “susceptible to” a disease, disorder, and / or condition is one who has a higher risk of developing the disease, disorder, and / or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder,and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0306] Synthetic: As used herein, the term “synthetic” refers to an entity that is artificial, or that is made with human intervention, or that results from synthesis rather than naturally occurring. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized, e.g., in some embodiments by solid-phase synthesis. In some embodiments, the term “synthetic” refers to an entity that is made outside of biological cells. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., an RNA) that is produced by in vitro transcription using a template.

[0307] Therapy: The term “therapy” refers to an administration or delivery of an agent or intervention that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect (e.g., has been demonstrated to be statistically likely to have such effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0308] Three prime untranslated region : As used herein, the terms “three prime untranslated region” or “3' UTR” refer to a sequence of an mRNA molecule that begins following a stop codon of a coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence, e.g., in its natural context.

[0309] Threshold level (e.g., acceptance criteria) : As used herein, the term “threshold level” refers to a level that are used as a reference to attain information on and / or classify the results of a measurement, for example, the results of a measurement attained in an assay. Forexample, in some embodiments, a threshold level means a value measured in an assay that defines the dividing line between two subsets of a population (e.g. a batch that satisfy quality control criteria vs. a batch that does not satisfy quality control criteria). Thus, a value that is equal to or higher than the threshold level defines one subset of the population, and a value that is lower than the threshold level defines the other subset of the population. A threshold level can be determined based on one or more control samples or across a population of control samples. A threshold level can be determined prior to, concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold level can be a range of values.

[0310] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and / or condition.

[0311] Vaccination. As used herein, the term “vaccination” refers to the administration of a composition intended to generate an immune response, for example to a disease-associated (e.g., disease-causing) agent. In some embodiments, vaccination can be administered before, during, and / or after exposure to a disease-associated agent, and in certain embodiments, before, during, and / or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccine composition. In some embodiments, vaccination generates an immune response to an infectious agent.

[0312] Vaccine: As used herein, the term “vaccine” refers to a composition that induces an immune response upon administration to a subject. In some embodiments, an induced immune response provides protective immunity.

[0313] Variant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., inthe presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer thanabout 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature.

[0314] Vector, as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In some embodiments, known techniques may be used, for example, for generation or manipulation of recombinant DNA, for oligonucleotide synthesis, and for tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated herein by reference for any purpose.

[0315] All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.Detailed Description

[0316] In some embodiments, the present provides technologies (e.g., compositions, pharmaceutical compositions, immunogenic compositions, vaccines, and methods) that can be used to induce an immune response against an infectious agent. In some embodiments, technologies provided in the present disclosure can be used to mitigate immune imprinting effects and / or induce a stronger de novo immune response (e.g., as compared to other vaccination approaches).

[0317] Infectious agents have evolved various means of evading or subverting host defenses. One way in which an infectious agent can evade immune surveillance is by altering its antigens (e.g., its epitopes); this is particularly important for extracellular pathogens, against which a principal defense is the production of antibody against their surface proteins and / or glyco proteins.

[0318] Among other things, the present disclosure provides technologies that are useful for increasing the breadth of immune response. In some embodiments, such an immune response is or comprises a B cell immune response. In some embodiments, a B cell immune response is or comprises an antibody response (e.g., neutralizing antibody response) to arisen epitopes in variant polypeptides. In some embodiments, variant polypeptides are from various infectious agents.Natural Evolution of Infectious Agents

[0319] Infectious agents remain a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new strains e.g., viral strains, or bacterial strains, etc. emerge continuously because of the plasticity of their genome allowing them to adapt to changing conditions. Infectious agents can hereby be associates with circulating diseases. One notable feature of RNA viruses is their high mutation rate. Unlike DNA viruses which utilize the host replication machinery to detect and repair base- pairing errors during replication, RNA viruses use RNA-dependent RNA polymerases that lack proofreading ability and, therefore, are intrinsically error prone. This may necessitate reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations.

[0320] Infectious agents, such as viruses, display a wide diversity of sizes and shapes. A complete virus particle, known as a virion, comprises a nucleic acid surrounded by a protective coat of protein called a capsid and sometimes an outer envelope that comprises proteins, such as surface proteins, and phospholipid membranes derived from the host cell. Viruses may also contain additional proteins, such as enzymes, within the capsid or attached to the viral genome. Viruses can undergo genetic change by several mechanisms. These include a process called antigenic drift where individual bases in the DNA or RNA mutate to other bases. Most of these point mutations are "silent" — they do not change the protein that the gene encodes — but others can confer evolutionary advantages such as resistance to antiviral drugs. Antigenic shift occurs when there is a major change in the genome of the virus. This can be a result of recombination or reassortment. When this happens pandemics might result. In particular, human immunodeficiency virus (HIV) evades the immune system by constantly changing the amino acid sequence of the proteins on the surface of the virion. This is known as “escape mutation” as the viral epitopes escape recognition by the host immune response.

[0321] Infectious agent surface proteins and / or surface glycoproteins (e.g., antigens) can be immunodominant antigens that are targeted for antibody-mediated neutralization by the humoral immune response by the host. These surface proteins and / or surface glycoproteins (e.g., antigens) present numerous surfaces known as epitopes which are recognized by antibodies that are generated by the host immune system to specifically bind to these virus epitopes via the antibody’s functional ‘paratope’ domain in an epitope-paratope interaction (EPI). EPIs are key aspects of the dynamic interplay between the virus and the host immune response to neutralize the virus. Subsequently, the immune system retains a ‘memory’ of the antigen(s), along with the ability to produce the particular antibodies that target it, in the form of memory B and T cells.

[0322] Host antibody responses upon infectious agent infection vary widely depending on the infectious agent and the host’ s exposure history to the infectious agent, homologous infectious agent, and vaccines. Hosts that have been previously infected or vaccinated typically possess neutralizing antibodies (nAbs) against vulnerable epitopes (e.g., virus epitopes) which protect the host from infection upon infectious agent exposure, with the nAb titer often correlating with the degree of protection against future infections. However, for certain infectious agents pre-existing antibodies resulting from infections of different sub-types mayrecognize but not effectively neutralize the infectious agent, which may result in paradoxically worse disease in a mechanism known as antibody-dependent enhancement.

[0323] In some embodiments, poorly-neutralizing antibodies are undesirable as they may not protect a host from future exposures and thus lead to reinfection, though non- neutralizing antibodies can still play key roles in protection via Fc function. This neutralization ‘escape’ dynamic occurs, for example, in the case of influenza A strains and SARS-CoV-2 variants featuring mutations in vulnerable epitopes resulting in reinfection of hosts whose antibodies developed during prior infection or vaccination no longer effectively recognize the mutated epitopes. Antibody escape may be more or less pronounced depending on the host’s exposure history, with certain viruses tending to leave an imprint on the host antibody response based on the host’s first exposure to the virus in a mechanism known as original antigenic sin / seniority, which can occur divergently for antibodies (Abs) generated via vaccination versus infection as in the case of SARS-CoV-2 mRNA vaccines.

[0324] In this way, viruses experience continued pressure to evolve mutations in vulnerable epitopes toward acquiring the ability to escape existing antibodies and re-infect hosts. Likewise, hosts continually evolve new (in response to reinfection or additional vaccination) or matured (resulting from accumulation of somatic mutations within memory B cells) antibodies to neutralize viruses bearing mutated or homologous epitopes, wherein these responses are modulated by antigenic exposure history.Immune escape prone variants

[0325] Infectious agents have developed various means of evading or subverting host defenses.Antigenic variation

[0326] One way in which an infectious agent can evade immune surveillance is by altering its antigens (e.g., its epitopes); this is particularly important for extracellular pathogens, against which the principal defense is the production of antibody against their surface proteins and / or glyco proteins.Exemplary Infectious Agents

[0327] In some embodiments, an infectious agent is a virus, a bacteria, or a eukaryotic cell (e.g., a plasmodium).

[0328] In some embodiments, an infectious agent is a respiratory virus. In some embodiments, an infectious agent is an RNA virus. In some embodiments, an infectious agent is a coronavirus (e.g., MERS, SARS, or SARS-CoV-2). In some embodiments, an infectious agent is HIV. In some embodiments, an infectious agent is HSV (e.g., HSV-1 or HSV-2). In some embodiments, an infectious agent is RSV. In some embodiments, an infectious agent is a norovirus. In some embodiments, an infectious agent is an influenza virus. In some embodiments, an infectious agent is P. falciparum. In some embodiments, an antigen described herein is an antigen from a virus in the genus Orthopoxvirus. There are 12 species in this genus. Diseases associated with this genus include, but are not limited to smallpox, cowpox, horsepox, camelpox, and monkeypox.

[0329] In some embodiments, an infectious agent is a bacterium. In some embodiments, the bacterium is Mycobacterium. In some embodiments, the bacterium is selected from Haemophilus influenzae, Chlamydophila pneumoniae, Mycoplasma pneumonia, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, and Streptococcus pneumonia. In some embodiments, the bacterium is Streptococcus pneumonia.

[0330] In some embodiments, an infectious agent is an RNA virus. Compositions provided herein may provide a particular advantage in providing an immune response against RNA viruses, which have a relatively high mutation rate (high relative to other infectious agents).

[0331] In some embodiments, an infectious agent comprises a large number of strains, variants, or lineages. In some embodiments, an infectious agent has a relatively high mutation rate (e.g., relative to other infectious agents).

[0332] In some embodiments, an infectious agent is prone to immune escape.

[0333] In some embodiments, an infectious agent is one for which seasonal, variant- adapted booster shots are regularly provided.

[0334] In some embodiments, an infection agent is associated with a circulating infectious disease (e.g., for which variants can be expected to arise).

[0335] Exemplary viral infectious diseases include, but are not limited to coronavirus, ebolavirus, influenza viruses, norovirus, rotavirus, respiratory syncytial virus, alphaherpesvirus, etc.

[0336] In some embodiments, an antigen described herein is or comprises a B cell antigen. In some embodiments, such a B cell antigen comprises one or more antibody epitopes. In some embodiments, such epitopes are antibody binding epitopes. In some embodiments, such epitopes are antibody neutralizing epitopes.Exemplary Antigens

[0337] In some embodiments, an antigen described herein is or comprises an antigen of an infectious agent. In some embodiments, an infection agent is associated with a circulating infectious disease (e.g., for which variants can be expected to arise). In some embodiments, such circulating infectious disease is a bacterial infectious disease. In some embodiments, such circulating infectious disease is a parasitic infectious disease. An exemplary parasitic infectious disease is malaria. In some embodiments, such circulating infectious disease is a viral infectious disease. In some embodiments, a viral infectious disease is associated with an RNA virus. Exemplary viral infectious diseases include, but are not limited to coronavirus, ebolavirus, influenza viruses, norovirus, rotavirus, respiratory syncytial virus, alphaherpesvirus, etc.

[0338] In some embodiments, an antigen (e.g., SARS-CoV-2) described herein is or comprises a B cell antigen. In some embodiments, such a B cell antigen comprises one or more antibody epitopes. In some embodiments, such epitopes are antibody binding epitopes. In some embodiments, such epitopes are antibody neutralizing epitopes.

[0339] In some embodiments, an antigen described herein is or comprises a T cell antigen. In some embodiments, such a T cell antigen comprises one or more CD4 T cell and / or one or more CD8 T cell epitopes.

[0340] In some embodiments, an antigen described herein includes one or more variant sequences relative to a relevant reference antigen. For example, in some embodiments, a protease cleavage site is removed or blocked; alternatively or additionally, in some embodiments, aterminally truncated antigen is utilized, and / or one or more mutations associated with a viral variant is present in the antigen.

[0341] In some embodiments, utilized sequences may comprise one or more mutations associated with a viral variant (e.g., SARS-CoV-2) (e.g., a variant that prevalent and / or that is predicted to be highly immune escaping). In some embodiments, utilized sequences comprise one or more mutations associated with a variant of concern (e.g., a variant of concern identified by WHO). In some embodiments, utilized sequences comprise one or more mutations associated with a viral variant that has been determined to be or has been predicted to be highly immune escaping (e.g., highly immune escaping relative to an immune response developed in subjects administered a previously approved vaccine and / or a previously prevalent viral variant).

[0342] In some embodiments, an antigen described herein is or comprises a surface protein or a surface glycoprotein of an infectious agent (e.g., SARS-CoV-2) . In some embodiments, an antigen described herein is a surface protein or a surface glycoprotein of an infectious agent strain or variant (e.g., SARS-CoV-2) that was previously and / or is currently prevalent. In some embodiments, an antigen described herein is or comprises a surface protein or surface glycoprotein of an infectious agent (e.g., SARS-CoV-2) that has been previously delivered in a vaccine (e.g., a commercially available vaccine, an RNA vaccine, or a protein- based vaccine).

[0343] In some embodiments, an infectious agent antigen is solvent exposed on the surface of the infectious agent. In some embodiments, an infectious agent antigen is a glyocoprotein. In some embodiments, an infectious agent antigen is involved in host cell recognition. In some embodiments, an infectious agent antigen is involved in host cell entry. In some embodiments, an infectious agent antigen comprises one or more B cell epitopes (e.g., one or more neutralization epitopes).

[0344] In some embodiments, an antigen described herein is or comprises a full-length polypeptide antigen of an infectious agent. In some embodiments, an antigen described herein is or comprises an immunogenic fragment, portion, or domain of a polypeptide antigen of an infectious agent.

[0345] In some embodiments, a composition delivers a hypervariable domain. As used herein, a “hypervariable domain” refers to a domain or region having a high frequency ofmutation. In some embodiments, a hypervariable domain comprises a high number of mutations relative to other polypeptide encoded by the infectious agent. In some embodiments, a hypervariable domain comprises a higher frequency of mutations relative to other regions in the antigen (e.g., higher by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or more). In some embodiments, a hypervariable domain comprises a higher number or density of neutralization-sensitive epitopes (e.g., as compared to other antigens, other antigens encoded by an infectious agent, and / or other regions of the antigen). In some embodiments, a hypervariable domain corresponds to a region in an infectious agent antigen that has an increased frequency of mutation in variants or strains of an infectious agent that have an increased immune escape potential. In some embodiments, a hypervariable domain is a region or domain within a polypeptide (e.g., a viral polypeptide) that binds a host cell receptor and helps mediate cell entry.

[0346] In some embodiments, an antigen described herein is an antigen from a coronavirus. In some embodiments, an antigen described herein is from an alphacoronavirus. In some embodiments, an antigen described herein is from a betacoronavirus. In some embodiments, an antigen described herein is from a gammacoronavirus. In some embodiments, an antigen described herein is from a deltacoronavirus. Exemplary antigens from coronavirus include, but are not limited to spike (S) protein or immunogenic fragments or portions thereof (including, e.g., but not limited to receptor binding domain (RBD), N-terminal domain (NTD)), as well as membrane (M) protein, envelope (E) protein, nucleocapsid protein, or combinations thereof. In some embodiments, an exemplary antigen described herein is a SARS-CoV-2 S protein or an immunogenic fragment or portion thereof (including, e.g., but not limited to RBD or NTD). For example, in some embodiments, such a SARS-CoV-2 S protein or an immunogenic fragment or portion thereof (including, e.g., but not limited to RBD or NTD) is from a Wuhan strain or an Omicron BA.4 / 5 strain. In some embodiments, such a SARS-CoV-2 S protein or an immunogenic fragment or portion thereof (including, e.g., but not limited to RBD or NTD) is from a XBB strain (e.g., XBB1, XBB1.5 or sublineages thereof).

[0347] In some embodiments, an antigen described herein is an antigen from an influenza virus. In some embodiments, an antigen described herein is from influenza A virus, including, e.g., but not limited to A(H1N1), A(H3N2), etc. In some embodiments, an antigen described herein is from influenza B virus, including, e.g., B(Victoria), B(Yamagata), etc. Insome embodiments, an antigen described herein is from influenza C virus. In some embodiments, an antigen described herein is from influenza D virus. Exemplary antigens from influenza viruses include, but are not limited to hemagglutinin (HA), neuraminidase (NA), or immunogenic portions or fragments thereof, or combinations thereof.

[0348] In some embodiments, an antigen described herein is an antigen from a respiratory syncytial virus (RSV), e.g., as described herein.

[0349] In some embodiments, an antigen described herein is an antigen from a norovirus. Noroviruses are members of the Caliciviridae family of small, non-enveloped, positive-stranded RNA viruses. The Norovirus genus includes both human and animal (e.g., murine and canine) noroviruses. Exemplary antigens from noroviruses include, but are not limited to Viral Protein 1 (VP1), Viral Protein 2 (VP2), S domain, P domain, Pl, P2, non- structural proteins, N-terminal proteins (NS 1-2, p48), NTPase (NS3), P22(NS4), VPg (NS5), Protease (NS6), Polymerase (NS7), or immunogenic portions or fragments thereof, or combinations thereof. In some embodiments, a norovirus antigen that is useful in accordance with the present disclosure is a norovirus antigen described in the International Patent Application No. PCT / US22 / 46799, the relevant content of which is incorporated herein by reference for the purposes described herein.

[0350] In some embodiments, an antigen described herein is an antigen from malarial polypeptide (e.g., as described herein).

[0351] In some embodiments, an antigen described herein is an antigen from a virus in the genus Orthopoxvirus. There are 12 species in this genus. Diseases associated with this genus include, but are not limited to smallpox, cowpox, horsepox, camelpox, and monkeypox.

[0352] In some embodiments, an antigen described herein is an antigen from Herpes simplex virus (e.g., HSV-1 and HSV-2), for example, as described herein.

[0353] In some embodiments, an antigen described herein is useful as a reference antigen of an infectious agent.

[0354] In some embodiments, an antigen described herein is or comprises a variant polypeptide of a reference antigen of an infectious agent, or an immunogenic portion thereof.

[0355] In some embodiments, an antigen described herein is or comprises a full-length polypeptide antigen of an infectious agent. In some embodiments, an antigen described herein is or comprises an immunogenic fragment, portion, or domain of a polypeptide antigen of an infectious agent.

[0356] In some embodiments, an antigen that is useful in accordance with the present disclosure is an antigen described in a U.S. Provisional Application entitled “Immunogenic Compositions” and filed February 24, 2023, the entire content of which is incorporated herein by reference for the purposes described herein. In some embodiments, an antigen that is useful in accordance with the present disclosure is an antigen described in a U.S. Provisional Application entitled “SARS-CoV-2-specific Immunogenic Compositions” and filed February 24, 2023, the entire content of which is incorporated herein by reference for the purposes described herein.

[0357] In some embodiments, an antigen described herein is an engineered antigen. For example, in some embodiments, an engineered antigen is designed to promote tailored immune responses. In some embodiments, an engineered antigen is designed using systems and methods as described in US Provisional Application No. 63 / 448215, the entire content of which is incorporated herein by reference for the purposes described herein.Coronavirus Overview

[0358] Coronaviruses are enveloped, positive-sense, single-stranded RNA ((+) ssRNA) viruses. They have the largest genomes (26-32 kb) among known RNA viruses and are phylogenetically divided into four genera (a, 0, y, and 5), with betacoronaviruses further subdivided into four lineages (A, B, C, and D). Coronaviruses infect a wide range of avian and mammalian species, including humans. Some human coronaviruses generally cause mild respiratory diseases, although severity can be greater in infants, the elderly, and the immunocompromised. Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), belonging to betacoronavirus lineages C and B, respectively, are highly pathogenic. Both viruses emerged into the human population from animal reservoirs within the last 15 years and caused outbreaks with high case-fatality rates. The outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that causes atypical pneumonia (coronavirus disease 2019; CO VID- 19) has raged in China since mid- December 2019, and has developed to be a public health emergency of international concern.SARS-CoV-2 (MN908947.3) belongs to betacoronavirus lineage B. It has at least 70% sequence similarity to SARS-CoV.

[0359] In general, coronaviruses have four structural proteins, namely, envelope (E), membrane (M), nucleocapsid (N), and spike (S). The E and M proteins have important functions in the viral assembly, and the N protein is necessary for viral RNA synthesis. The S glycoprotein is responsible for virus binding and entry into target cells. WT S protein is synthesized as a single-chain inactive precursor that is cleaved by furin-like host proteases in the producing cell into two noncovalently associated subunits, SI and S2. SI contains a receptor-binding domain (RBD), which recognizes host-cell receptors. S2 contains a fusion peptide, two heptad repeats, and a transmembrane domain, all of which play a role in mediating fusion of viral and host-cell membranes by undergoing a large conformational rearrangement. S 1 and S2 trimerize to form a large prefusion spike complex.

[0360] In some embodiments, an antigen described herein is an antigen from a coronavirus. In some embodiments, an antigen described herein is from an alphacoronavirus. In some embodiments, an antigen described herein is from a betacoronavirus. In some embodiments, an antigen described herein is from a gammacoronavirus. In some embodiments, an antigen described herein is from a deltacoronavirus.

[0361] SARS-CoV-2 Spike (S) protein can be proteolytically cleaved into SI (685 aa) and S2 (588 aa) subunits. SI of SARS-CoV-2 comprises a receptor-binding domain (RBD), which mediates virus entry into host cells through the host angiotensin-converting enzyme 2 (ACE2) receptor.

[0362] The presentation of COVID-19 is generally with cough and fever, with chest radiography showing ground-glass opacities or patchy shadowing. However, many patients present without fever or radiographic changes, and infections may be asymptomatic which is relevant to controlling transmission. For symptomatic subjects, progression of disease may lead to acute respiratory distress syndrome requiring ventilation and subsequent multi-organ failure and death. Common symptoms in hospitalized patients (in order of highest to lowest frequency) include fever, dry cough, shortness of breath, fatigue, myalgias, nausea / vomiting or diarrhoea, headache, weakness, and rhinorrhoea. Anosmia (loss of smell) or ageusia (loss of taste) may be the sole presenting symptom in approximately 3% of individuals who have COVID-19.

[0363] All ages may present with the disease, but notably case fatality rates (CFR) are elevated in persons >60 years of age. Comorbidities are also associated with increased CFR, including cardiovascular disease, diabetes, hypertension, and chronic respiratory disease.Healthcare workers are overrepresented among CO VID- 19 patients due to occupational exposure to infected patients.

[0364] In most situations, a molecular test is used to detect SARS-CoV-2 and confirm infection. The reverse transcription polymerase chain reaction (RT-PCR) test methods targeting SARS-CoV-2 viral RNA is one method for diagnosing suspected cases of CO VID-19. Samples to be tested are collected from the nose and / or throat with a swab.SARS-CoV-2 Variants

[0365] Since the initial discovery of SARS-CoV-2, a number of variants have arisen around the world. The emergence of these novel circulating variants of SARS-CoV-2 has raised significant concerns about geographic and temporal efficacy of vaccine interventions. The emergence of Omicron (B.1.1.529) variants, which comprise a number of mutations in the S protein, has been of particular concern.

[0366] In some embodiments, the present disclosure refers to a SARS-CoV-2 variant that is prevalent and / or rapidly spreading in a relevant jurisdiction. In some embodiments, such variants may be identified based on publicly available data (e.g., data provided in the GISAID Initiative database: https: / / www.gisaid.org, and / or data provided by the World Health Organization WHO (e.g., as provided at https: / / www.who.int / activities / tracking-SARS-CoV-2- variants). In some embodiments, such a variant refers to a variant disclosed herein.

[0367] The Omicron BA.l variant was first reported to WHO on 24 November 24, 2021, and was detected in South Africa. Omicron and its sublineages have had a major impact on the epidemiological landscape of the COVID-19 pandemic since their initial emergence (WHO Technical Advisory Group on SARS-CoV-2 Virus Evolution (TAG-VE): Classification of Omicron (B.1.1.259): SARS-CoV-2 Variant of Concern (2021); WHO Headquarters (HQ), WHO Health Emergencies Programme, Enhancing Response to Omicron SARS-CoV-2 variant: Technical brief and priority actions for Member States (2022)). Significant alterations in the spike (S) glycoprotein of the first Omicron variant BA.l resulted in the loss of many neutralizing antibody epitopes (M. Hoffmann et al., “The Omicron variant is highly resistant against antibodymediated neutralization: Implications for control of the COVID- 19 pandemic”, Cell 185, 447- 456.el 1 (2022)) and rendered BA.l capable of partially escaping previously established SARS- CoV-2 wild-type strain (Wuhan-Hu- l)-based immunity (V. Servellita, et al., “Neutralizing immunity in vaccine breakthrough infections from the SARS-CoV-2 Omicron and Delta variants”, Cell 185, 1539-1548.e5 (2022); Y. Cao et al., “Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies”, Nature 602, 657-663 (2022)).

[0368] As a result, breakthrough infection of vaccinated individuals with Omicron is more common than with previous Variants of Concern (VOCs). While Omicron BA.l was displaced by the BA.2 variant in many countries around the globe, other variants such as BA.1.1 and BA.3 temporarily and / or locally gained momentum but did not become globally dominant (S. Xia et al., “Origin, virological features, immune evasion and intervention of SARS-CoV-2 Omicron sublineages. Signal Transduct. Target. Ther. 7, 241 (2022); H. Gruell et al., “SARS- CoV-2 Omicron sublineages exhibit distinct antibody escape patterns, Cell Host Microbe 7, 241 (2022).). Omicron BA.2.12.1 subsequently displaced BA.2 to become dominant in the United States, whereas BA.4 and BA.5 displaced BA.2 in Europe, parts of Africa, and Asia / Pacific (H. Gruell et al., “SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns,” Cell Host Microbe 7, 241 (2022); European Centre for Disease Prevention and Control, WeeklyCO VID-19 country overview -Country overview report: Week 31 2022 (2022); J. Hadfield et al., “Nextstrain: Real-time tracking of pathogen evolution,” Bioinformatics 34, 4121 4123 (2018)). Currently, Omicron BA.5 is dominant globally, including in the United States (Centers for Disease Control and Prevention. CO VID Data Tracker. Atlanta, GA: US Department of Health and Human Services, CDC; 2022, August 12. https: / / covid.cdc.gov / coviddata-tracker (2022)).

[0369] Omicron has acquired numerous alterations (amino acid exchanges, insertions, or deletions) in the S glycoprotein, among which some are shared between all Omicron VOCs while others are specific to one or more Omicron sublineages. Antigenically, BA.2.12.1 exhibits high similarity with BA.2 but not BA.l, whereas BA.4 and BA.5 differ considerably from their ancestor BA.2 and even more so from BA.l, in line with their genealogy (A. Z. Mykytyn et al., “Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.l and BA.2 are antigenically distinct,” Sci. Immunol. 7, eabq4450 (2022).). Major differences of BA.l from the remaining Omicron VOCs include A143-145, L212I, or ins214EPE in the S glycoprotein N-terminal domain and G446S or G496S in the receptor binding domain (RBD). Amino acid changesT376A, D405N, and R408S in the RBD are in turn common to BA.2 and its descendants but not found in BA.l. In addition, some alterations are specific for individual BA.2-descendant VOCs, including L452Q for BA.2.12.1 or L452R and F486V for BA.4 and BAA (BA.4 and BAA encode for the same S sequence). Most of these shared and VOC-specific alterations were shown to play an important role in immune escape from monoclonal antibodies and polyclonal sera raised against the wild-type S glycoprotein. In particular, the BA.4 / BA.5-specific alterations are strongly implicated in immune escape of these VOCs (P. Wang et al., “Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature 593, 130-135 (2021); Q. Wang et al., “Antibody evasion by SARS-CoV-2 Omicron subvariants BA.2.12.1, BA.4, & BAA. Nature 608, 603-608 (2022)).

[0370] Among other things, described herein are certain SARS-CoV-2 antigens for use in inducing an immunogenic response. In some embodiments, a SARS-CoV-2 antigen comprise immunogenic portions of a full-length SARS-CoV-2 polypeptide (e.g., an SI domain of a SARS- COV-2 S protein and / or an RBD of a SARS-CoV-2 S protein). In some embodiments, such antigens are delivered as protein antigens to induce an immunogenic response. In some embodiments, such antigens are delivered using RNA (e.g., modRNA encoding an SI domain and / or RBD of a SARS-CoV-2 S protein and formulated in LNP particles) to induce an immunogenic response.Exemplary Coronavirus Antigens

[0371] As used herein, a full length SARS-CoV-2 S protein comprising a “Wild-Type” or “Wuhan” sequence has a sequence corresponding to that of the first detected SARS-CoV-2 strain, consisting of 1273 amino acids and having an amino acid sequence according to SEQ IDNO: 1:MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTK RFDNPVLPFNDGVYFASTEKSNI IRGWI FGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLGVYYHKNNKSWME SEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNI DGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPI GINITRFQTLLALHRSYLTPGDS SSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTV EKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSAS FSTFKCYGVS PT KLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLK PFERDI STEP YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCV NFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEI LDITPCSFGGVSVITPGTNTSNQVAVLYQDVN CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNS YECDI PIGAGICASYQTQTNSPRRARSVASQSI I AYTMSLGAENSVAYSNNSIAI PTNFTI SVTTEI LPVSMTKTSVDCTMYI CGDSTECSNLLLQYGSFCTQLNRALTGI AVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRS FIEDLLFNKVTLADAGFIKQYGDCLGDIAAR DLI CAQKFNGLTVLPPLLTDEMI AQYTSAL LAGTIT SGWT EGA GAALQI PFAMQMAYRFNGIGVTQNVLYENQKLI A NQFNSAIGKIQDSLSSTASALGKLQDWNQNAQALNTLVKQLS SNFGAI SSVLNDI LSRLDKVEAEVQI DRLITGRL QSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTA PAI CHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQI ITTDNTFVSGNCDWIGIVNNTVYDPLQPELDS FKEELDKY FKNHTS PDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLI DLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTI MLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT ( SEQ ID NO : 1 )

[0372] Unless otherwise indicated, position numberings in a SARS-CoV-2 S protein given herein are in relation to the amino acid sequence of SEQ ID NO: 1. One of skill in the art reading the present disclosure will understand and be able to determine corresponding positions in a SARS-CoV-2 S protein variant sequence from locations of positions provided relative to the amino acid sequence of SEQ ID NO: 1 (i.e., a person of skill in the art provided positions relative to SEQ ID NO: 1, or another variant, will be able to determine corresponding positions in the S protein sequence of another SARS-CoV-2 variant or a fragment thereof).

[0373] In specific embodiments, a spike (S) protein described herein can be modified in such a way that the prototypical prefusion conformation is stabilized. Certain mutations that stabilize a prefusion confirmation are known in the art, e.g., as disclosed in WO 2021243122 A2 and Hsieh, Ching-Lin, et al. ("Structure-based design of prefusion-stabilized SARS-CoV-2 spikes," Science 369.6510 (2020): 1501-1505), the contents of each which are incorporated by reference herein in their entirety. In some embodiments, a SARS-CoV-2 S protein may be stabilized by introducing one or more proline mutations. In some embodiments, a SARS-CoV-2S protein comprises a proline substitution at positions corresponding to residues 986 and / or 987of SEQ ID NO: 1. In some embodiments, a SARS-CoV-2 S protein comprises a proline substitution at one or more positions corresponding to residues 817, 892, 899, and 942 of SEQID NO: 1. In some embodiments, a SARS-CoV-2 S protein comprises a proline substitution at positions corresponding to each of residues 817, 892, 899, and 942 of SEQ ID NO: 1. In some embodiments, a SARS-CoV-2 S protein comprises a proline substitution at positions corresponding to each of residues 817, 892, 899, 942, 986, and 987 of SEQ ID NO: 1.

[0374] In some embodiments, stabilization of the prototypical prefusion conformation of a SARS-CoV-2 S protein may be obtained by introducing two consecutive proline substitutions at residues 986 and 987. Specifically, spike (S) protein stabilized protein variants are obtained in a way that the amino acid residue at position 986 is exchanged to proline and the amino acid residue at position 987 is also exchanged to proline. In one embodiment, a SARS-CoV-2 S protein variant wherein the prototypical prefusion conformation is stabilized comprises the amino acid sequence shown in SEQ ID NO: 2:MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTK RFDNPVLPFNDGVYFASTEKSNI IRGWI FGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLGVYYHKNNKSWME SEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNI DGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPI GINITRFQTLLALHRSYLTPGDS SSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTV EKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPT KLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLK PFERDI STEP YQAGSTPCNGVEGFNCYFPLQS YGFQPTNGVGYQPYRVWLS FELLHAPATVCGPKKSTNLVKNKCV NFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEI EDIT PCS FGGVSVITPGTNTSNQVAVLYQDVN CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNS YECDI PIGAGICASYQTQTNSPRRARSVASQSI I AYTMSLGAENSVAYSNNSIAI PTNFTI SVTTEI LPVSMTKTSVDCTMYICGDSTECSNLLLQYGS FCTQLNRALTGI AVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRS FIEDLLFNKVTLADAGFIKQYGDCLGDIAAR DLI CAQKFNGLTVLPPLLTDEMI AQYTSAL LAGTIT SGWT EGA GAALQI PFAMQMAYRFNGIGVTQNVLYENQKLI A NQFNSAIGKIQDSLSSTASALGKLQDWNQNAQALNTLVKQLS SNFGAI SSVLNDI LSRLDPPEAEVQI DRLITGRL QSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTA PAI CHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQI ITTDNTFVSGNCDWIGIVNNTVYDPLQPELDS FKEELDKY FKNHTS PDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLI DLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTI MLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT( SEQ I D NO : 2 )

[0375] Those skilled in the art are aware of various SARS-COV-2 Spike variants, and / or resources that document them. For example, the following strains, their SARS-CoV-2 S protein amino acid sequences and, in particular, modifications thereof compared to wildtype SARS-CoV-2 S protein amino acid sequence, e.g., as compared to SEQ ID NO: 1, are useful herein.B.1.1.7 ("Variant of Concern 202012 / 01" (VOC-202012 / 01)

[0376] B.1.1.7 (“alpha variant”) is a SARS-CoV-2 variant that was first detected in October 2020 in the United Kingdom from a sample taken the previous month, and quickly began to spread by mid-December. It is correlated with a significant increase in the rate of COVID-19 infection; this increase is thought to be at least partly due to a change of N501Y inside the spike glycoprotein's receptor-binding domain, which is needed for binding to ACE2 in human cells. B.1.1.7 is defined by 23 mutations: 13 non-synonymous mutations, 4 deletions, and 6 synonymous mutations (z.e., there are 17 mutations that change proteins and six that do not). Spike protein changes in B.1.1.7 include deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.B.1.351 (501.V2)

[0377] B.1.351 lineage ( “Beta variant”), colloquially known as South African COVID-19 variant, has increased transmissibility relative to the original Wuhan strain. The B.1.351 variant is defined by multiple spike protein changes including: L18F, D80A, D215G, deletion 242-244, R246I, K417N, E484K, N501Y, D614G and A701V. There are three mutations of particular interest in the spike region of the B.1.351 genome: K417N, E484K, N501Y.B.1.1.298 (Cluster 5)

[0378] B .1.1.298 was discovered in North Jutland, Denmark, and is believed to have been spread from minks to humans via mink farms. Several different mutations in the spike protein of the virus have been confirmed. The specific mutations include deletion 69-70, Y453F, D614G, I692V, Ml 2291, and optionally S1147L.P.l (B.1.1.248)

[0379] Lineage B .1.1.248 (the “gamma variant”), known as the Brazil(ian) variant, is one of the variants of SARS-CoV-2 which has been named P.l lineage. P.l has a number of S- protein modifications (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, VI 176F) and is similar in certain key RBD positions (K417, E484, N501) to variant B.1.351 from South Africa.B.1.427 / B.1.429 (CAL.20C)

[0380] Lineage B.1.427 / B.1.429 (the “epsilon variant”), also known as CAL.20C, is defined by the following modifications in the S-protein: SI 31, W152C, L452R, and D614G, ofwhich the L452R modification is of particular concern. CDC has listed B.1.427 / B.1.429 as a "variant of concern".B.1.525

[0381] B.1.525 ( “eta variant”) carries the same E484K modification as found in the P.l, and B.1.351 variants, and also carries the same AH69 / AV70 deletion as found in B.1.1.7, and B.1.1.298. It also carries the modifications D614G, Q677H and F888L.B.1.526

[0382] B.1.526 ( “iota variant”) was detected as an emerging lineage of viral isolates in the New York region that shares mutations with previously reported variants. The most common sets of spike mutations in this lineage are L5F, T95I, D253G, E484K, D614G, and A701V.B.l.1.529

[0383] B .1.529 (“Omicron variant”) was first detected in South Africa in November2021. Omicron multiplies around 70 times faster than Delta variants, and quickly became the dominant strain of SARS-CoV-2 worldwide. Since its initial detection, a number of Omicron sublineages have arisen. Listed below are the current Omicron variants of concern, along with certain characteristic mutations associated with the S protein of each. The S protein of B A.4 and BA.5 have the same set of characteristic mutations, which is why the below table has a single row for “BA.4 or BA.5”, and why the present disclosure refers to a “BA.4 / 5” S protein in some embodiments. Similarly, the S proteins of the BA.4.6 and BF.7 Omicron variants have the same set of characteristic mutations, which is why the below table has a single row for “BA.4.6 or BF.7”).Table 1: Omicron Variants of Concern and Characteristic mutations

[0384] In some embodiments, SARS-CoV-2 S proteins described herein comprise one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) characteristic of a certain Omicron variant (e.g., one or more mutations of an Omicron variant listed in Table 1, e.g., each of the mutations associated with a given XBB variant in the above Table 1).Immunogenic Portions of Coronavirus S Protein

[0385] As noted elsewhere in the present disclosure, in some embodiments, compositions described herein deliver an immunogenic portion of a full length coronavirus S protein (e.g., SARS-CoV-2 S protein).

[0386] In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS- CoV-2) S protein lacks certain features that are in the full length polypeptide (e.g., features that have been shown or predicted to interfere with induction of a naive immune response). For example, in some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein lacks regions that have (i) a low number or density of B cell neutralization epitopes and / or (ii) a high number or density of B cell epitopes not associated with neutralization. For example, in some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein lacks a full S2 domain. In some embodiments, a coronavirus (e.g., SARS-CoV-2) S protein lacking a full S2 domain lacks regions of S2 that have (i) a low number or density of B cell epitopes associated with neutralization or (ii) a high number of B cell epitopes not associated with neutralization, but retains other portions of S2. In some embodiments, an immunogenicportion of a coronavirus (e.g., SARS-CoV-2) S protein lacks the entire S2 domain. In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein lacks a full S2 domain, but comprises certain sequences that can improve immunogenicity and / or stability of an immunogenic portion (e.g., in some embodiments, an immunogenic portion lacks a full S2 domain but retains a TM sequence).

[0387] A person of skill in the art reading the present disclosure will be able to identify B cell epitopes in a coronavirus (e.g., SARS-CoV-2) S protein and determine which epitopes are or are not associated with neutralization. For example, a person of skill in the art will be aware of numerous studies that have identified such regions using antibody binding studies (e.g., studies characterizing antibodies produced in subjects infected with or vaccinated against SARS- CoV-2).

[0388] In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS- CoV-2) S protein comprises certain regions that have been determined to have a high number or density of neutralization epitopes and optionally a high mutation rate. In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein comprises an N-terminal domain (NTD) of the S protein. In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) protein comprises a receptor binding domain (RBD) of the S protein. In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein comprises an S 1 domain of the S protein.

[0389] In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS- CoV-2) S protein comprises an RBD and an NTD and omits other features of the SI domain.

[0390] Coronavirus (e.g., SARS-CoV-2) S proteins are well characterized, and a person of skill in the art will be able to determine which portions of an S protein sequence correspond to immunogenic portions discussed herein (e.g., which portions of an S protein sequence correspond to the NTD, the RBD, the SI, and the S2 domains). In some embodiments, an RBD of a coronavirus (e.g., SARS-CoV-2) S protein comprises residues 327 to 528 of SEQ ID NO: 1 or a corresponding region.

[0391] In some embodiments, an RBD of a coronavirus (e.g., SARS-CoV-2) S protein comprises the amino acid sequence: VRFPNITNECPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKP SGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVG HQPYRVVVLSFELLHAPATVCGPK (SEQ ID NO: 3), or a corresponding region.

[0392] In some embodiments, an RBD of a coronavirus (e.g., SARS-CoV-2) S protein comprises the amino acid sequence:VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTK LNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKV GGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG YQPYRVVVLSFELLHAPATVCGPK (SEQ ID NO: 4), or a correspond.

[0393] In some embodiments, an SI domain of a coronavirus (e.g., SARS-CoV-2) S protein comprises amino acids 1 to 678 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant. In some embodiments, an SI domain of a SARS-CoV-2 S protein comprises amino acids 1 to 683 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant. In some embodiments, an SI domain of a SARS-CoV-2 S protein comprises amino acids 1 to 685 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant.

[0394] In some embodiments, an SI domain of a SARS-CoV-2 S protein comprises the amino acid sequence:MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVT WFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNA TNVVIKVCEFQFCNDPFLDVYQKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGK EGNFKNLREFVFKNIDGYFKIYS KHTPINLERDLPQGFS ALEPL VDLPIGINITRFQTLLAL HRSYLTPVDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL KSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVAD YSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYK LPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGV AGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF NFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGT NTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSY ECDIPIGAGICASYQTQT (SEQ ID NO: 5)

[0395] In some embodiments, an SI domain of a SARS-CoV-2 S protein comprises the amino acid sequence:MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMD LEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGST PCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKN KCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVS VITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEH VNNSYECDIPIGAGICASYQTQT (SEQ ID NO: 6).

[0396] In some embodiments, an S2 domain of a SARS-CoV-2 S protein comprises amino acids 679 to 1273 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS- CoV-2 variant. In some embodiments, an SI domain of a SARS-CoV-2 S protein comprises amino acids 684 to 1273 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS- CoV-2 variant. In some embodiments, an SI domain of a SARS-CoV-2 S protein comprises amino acids 686 to 1273 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS- CoV-2 variant.

[0397] In some embodiments, compositions described herein deliver an immunogenic portion of an S protein of a SARS-CoV-2 variant. In some embodiments, the variant is a variant of concern (e.g., a variant that has been predicted to and / or has been shown to spread rapidly in a relevant jurisdiction, e.g., as identified by certain public health agencies, e.g., the Center for Disease Control and Prevention (CDC), Public Health England and the COVID-19 Genomics UK Consortium for the UK, the Canadian CO VID Genomics Network (CanCOGeN), and / or the World Health Organization (WHO)). In some embodiments, a variant has been predicted to have a highly likelihood of becoming a variant of concern (e.g., using sequence-based algorithms that predict the ability of a variant to escape previously developed immune responses and / or measure the “fitness” of a given variant, such as described, e.g., in WO2022 / 235847 andWO2022 / 235853, the contents of each of which are incorporated by reference herein in their entirety).

[0398] In some embodiments, an RBD comprises mutations associated with a variant described herein. A person of skill in the art will be able to identify which portions of a given variant correspond to immunogenic portions described herein.

[0399] In some embodiments, a polypeptide comprises two or more SARS-CoV-2 subdomains (e.g., two or more SI domains or RBDs). In some embodiments, a polypeptide comprises two or more receptor binding domains linked in tandem, e.g., as described in Dai, Lianpan, et al. "A universal design of betacoronavirus vaccines against CO VID- 19, MERS, and SARS," Cell 182.3 (2020): 722-733, and Han, Yuxuan, et al. "mRNA vaccines expressing homo-prototype / Omicron and hetero-chimeric RBD-dimers against SARS-CoV-2," Cell Research 32.11 (2022): 1022-1025, the contents of each of which are incorporated by reference herein in their entirety. In some embodiments, the two or more subdomains are from the same SARS-CoV-2 variant (e.g., a variant described herein). In some embodiments, at least two of the two or more subdomains are from different SARS-CoV-2 variants (e.g., from different variants of concern, different Omicron variants, an Omicron variant and a non-Omicron variant, or a Wuhan strain and an Omicron variant).Malaria Overview

[0400] Malaria is a mosquito-borne infectious disease caused by single-celled eukaryotic Plasmodium parasites that are transmitted by the bite of Anopheles spp. mosquitoes (Phillips, M., et al. Malaria. Nat Rev Dis Primers 3, 17050 (2017), which is incorporated herein by reference in its entirety). Mosquitoes that transmit malaria must have been infected through a previous blood meal taken from an infected subject (e.g., a human). When a mosquito bites an infected subject a small amount of blood is taken in containing Malaria parasites. The infected mosquito can then subsequently bite a non-infected subject, infecting the subject.

[0401] Malaria remains one of the most serious infectious diseases, causing approximately 200 million clinical cases and 500,000-600,000 deaths annually. Although significant effort has been invested in developing therapeutic treatments for malaria, many malaria parasites have developed resistance to available therapeutics. According to MalariaEradication Research Agenda Initiative, malaria eradication will only be achievable through effective vaccination.

[0402] In 2015, the European Medicines Agency gave a positive review to a malaria vaccine candidate known as “RTS,S”, a milestone in malaria vaccine development. In 2019, the World Health Organization launched pilot programs that provide RTS,S to children at least 5 months of age in parts of three sub-Saharan African countries. RTS,S / AS01 is an adjuvanted protein subunit vaccine that consists of a portion of the major repeat region and the C-terminus of CSP from Plasmodium falciparum fused to the Hepatitis B surface antigen (HBsAg). The vaccine is a mix of this PfCSP-HBsAg compound with HBsAg that forms virus-like particles (RTS,S / AS01; Mosquirix™). RTS,S is administered according to a regimen that requires four doses: an initial 3-dose schedule given at least 1 month apart, and a 4th dose 15-18 months after dose 3 (see, for example, Vandoolaeghe & Schuerman Expert Rev Vaccines. 15: 1481, 2016; PATH_MVI_RTSS_Fact Sheet_042019, each of which is incorporated herein by reference in its entirety). Reports indicate that RTS,S protects approximately 30% to 50% of children from clinical disease over 18 months. RTS,S has been reported to induce protective antibody and CD4+ T-cell responses, but only negligible CD8+ T cell responses (see, for example, Moris et al. Hum Vaccin Immunother 14:17, 2018, which is incorporated herein by reference in its entirety). Phase III studies of RTS, S delivered as a three-dose series with a booster after 1 yr (year) showed moderate vaccine efficacy in children aged 5 to 17 months preventing 36% of clinical malaria cases over the full study period with a median follow-up of 4 yrs, with a range of 20% in high to 66% in low transmission settings. Furthermore, published literature suggests that protection wanes over time including reports of potential negative efficacy after 5 yrs in children with high malaria exposure (Olotu et al. 2016, N. Engl. J. Med. 374:2519-29, which is incorporated herein by reference in its entirety). Thus, an effective malaria vaccine remains an unmet medical need of critical importance for global health.A. Lifecycle

[0403] During a blood meal, infected mosquitos inject, along with their anticoagulating saliva, sporozoites known as the liver stage of Plasmodium spp. Sporozoites journey through the skin to the lymphatics and into hepatocytes of the liver. This journey happens very quickly; it can be complete within only a few minutes (Sinnis et al., Parasitol Int. 2007 Sep; 56(3): 171-8, which is herein incorporated by reference in its entirety). This is a time known to be a bottlekneck of Malaria infection most favorable for therapeutic intervention, as only a small number (thought to be a few hundred at maximum) of sporozoites are injected by the mosquito, with only fraction of that number establishing infection in the liver and developing into mature live-stage parasites (Flores-Garcia et al., mBio. 2018 Nov 20; 9(6):e02194-18, which is herein incorporated by reference in its entirety). Thus, a subject whose immune system is primed to clear sporozoites before they enter hepatocytes can efficiently clear an infection.

[0404] One particular challenge associated with clearing a malarial infection during this bottle neck is that the most abundant and immunogenic protein on the sporozoite surface, the circumsporozoite protein (CSP), is only exposed to the immune system in small quantities and for short duration of time due to the variably low inoculum from the mosquito and the kinetics of hepatocyte infection after inoculation. After liver infection is established, the parasite differentiates into a stage which no longer expresses CSP and instead has a different mosaic of surface antigens. Furthermore, due to the density and close proximity of neighboring CSPs on the surface of the parasite coupled with the bi-valency of antibodies, binding of antibodies to CSP can produce a phenomenon referred to as CSP precipitation reaction, whereby antibodies can crosslink neighboring CSP and cause them to precipitate and shed from the parasite surface, leaving a trail of precipitated antibody bound CSP that the parasite can replace through its normal CSP translocation process (Livingstone et al., Sci Rep 11, 5318 (2021); Steward et al., J Protozool. 1991 Jul-Aug; 38(4):411-21, which are herein incorporated by reference in their entirety).

[0405] When moving from an inoculation site in the skin to the liver, sporozoites traverse host cells (Mota et al., Science 2001 Jan 5;291(5501): 141-4, which is herein incorporated by reference in its entirety). Sporozoites traverse different types of host cells at the dermis, including fibroblasts and phagocytes (Amino et al., Cell Host Microbe. 2008 Feb 14;3(2): 88-96, which is herein incorporated by reference in its entirety), and the liver sinusoidal barrier, containing liver endothelial cellsand Kupffer cells (Frevert et al., PLoS Biol 3(6): el92. 2005, which is herein incorporated by reference in its entirety) and sinusoidal endothelial cells (Tavares et al., J Exp Med 2013 May 6;210(5):905- 15, which is herein incorporated by reference in its entirety), in order to gain access to hepatocytes. Sporozoites preferentially traverse cells with low-sulfated heparin sulfate proteoglycans (HSPGs) but preferentially invade cells withhigh-sulfated HSPGs (Coppi et al., Cell Host & Microbe 2, 316-327, November 2007, which is herein incorporated by reference in its entirety).

[0406] Cell traversal was first observed as non-phagocytic entry of P. berghei sporozoites into macrophages followed by “escape” from these cells (Vanderberg et al., J. Euk. Microbiol. 37:528-536, 1990, which is herein incorporated by reference in its entirety). The biochemical, biophysical, and stepwise processes of traversal are still being explored. However, it has been suggested by electron microscopy that host cell rupture occurs upon entry and exit from the host cell (Mota et al., 2001; Tavares et al., 2013, which is herein incorporated by reference in its entirety). It has also been shown that P. yoelii sporozoites can enter hepatocytes via a transient vacuole and that host membrane rupture occurs upon cell exit rather than cell entry (Risco-Castillo et al., Cell Host Microbe 2015 Nov 11; 18(5):593-603, which is herein incorporated by reference in its entirety).

[0407] Sporozoites also traverse hepatocytes before establishing a productive hepatocyte infection (Mota et al., 2001, which is herein incorporated by reference in its entirety). Several possibilities emerged as to why this occurs. The first hypothesis suggested that migration through hepatocytes primes parasites for invasion by activating apical exocytosis (Mota et al., Nat Med 2002 Nov; 8(11): 1318-22, which is herein incorporated by reference in its entirety). The second theory suggested that traversal releases hepatocyte growth factor (HGF), making neighboring hepatocytes more susceptible to infection (Carrolo et al., Nat Med. 2003 Nov;9(l l): 1363-9, which is herein incorporated by reference in its entirety). Lastly, other studies suggest that it takes some time for sporozoites to switch off the machinery for traversal and activate invasion machinery (Amino et al., 2008, Coppi et al., 2007, which are herein incorporated by reference in their entirety), and that traversal primarily functions to penetrate cell barriers and avoid phagocytosis en route to the liver (Amino et al., 2008, Coppi et al., 2007, Tavares et al., 2013, which are herein incorporated by reference in their entirety).

[0408] Although it has been shown that sporozoites traverse human cells (Behet et al., Malar J 2014 Apr 5; 13: 136; Cha et al., J Exp Med 2015 Aug 24; 212(9): 1391-403; Dumoulin et al., PLoS One 2015 Jun 12;10(6):e0129623; van Schaijk et al., PLoS ONE, 3 (10). e3549 2008, which are herein incorporated by reference in their entirety), the molecular basis for the traversal process is largely unstudied. Antibodies against circumsporozoite protein (CSP) impair traversal(Dumoulin et al., 2015, which is herein incorporated by reference in its entirety), but this is likely due to inhibition of motility rather than a direct effect (Cha et al., J Exp Med 2016 Sep 19; 213(10):2099-l 12, which is herein incorporated by reference in its entirety). Furthermore, antibodies induced by chloroquine prophylaxis with sporozoites interfere with cell traversal, and these may also target CSP (Behet et al., 2014). Recently it was shown that glyceraldehyde 3- phosphate dehydrogenase (GAPDH) on the parasite surface interacts with CD68 on Kupffer cells during traversal (Cha et al., 2015, Cha et al., 2016, which are herein incorporated by reference in their entirety).

[0409] In rodent malaria parasites such as P. berghei, two sporozoite microneme proteins have been identified that appear to be essential for cell traversal (sporozoite microneme protein essential for cell traversal [SPECT1; Ishino et al., PLoS Biol., 2 (2004), pp. 77-84] and SPECT2 [Ishino et al., Cell. Microbiol., 7 (2005), pp. 199-208], also called perforin-like protein 1 [PLP1] [Kaiser et al., Mol. Biochem. Parasitol., 133 (2004), pp. 15-26], which are herein incorporated by reference in their entirety). Even though genetic disruption of SPECT1 or SPECT2 rendered sporozoites unable to traverse murine cells, they still invaded hepatocytes in vitro (Ishino et al., 2004, Ishino et al., 2005, which are herein incorporated by reference in their entirety). When injected into rodents, sporozoites lacking SPECT1 or SPECT2 were impaired for liver infection, but a small number of sporozoites could still establish liver infection that resulted in subsequent patency. However, depletion of Kupffer cells allowed mutants to establish liver infection at levels comparable with wild-type parasites (Ishino et al., 2004, Ishino et al., 2005, which are herein incorporated by reference in their entirety). This data suggests that traversal by rodent-infecting sporozoites is important for navigating through the sinusoidal layer, but not for hepatocyte invasion, malarial exoerythrocytic forms development, or growth within erythrocytes (Ishino et al., 2004, Ishino et al., 2005, which are herein incorporated by reference in their entirety).

[0410] The ortholog of SPECT2 in P. yoelii, PLP1, has been shown to play a role in cell traversal. Although this protein is not required for hepatocyte entry, it plays a role in egress from transient vacuoles during traversal (Risco-Castillo et al., 2015, which are herein incorporated by reference in their entirety). Thus, sporozoites that infect rodents can traverse host cells by generating a vacuole at the entry step and use a perforin-like protein (e.g., SPECT2 / PLP1) to escape from this compartment and / or a host cell, during cell exit.

[0411] Once sporozoites have invaded liver cells, they differentiate into merozoites, a replicative form of the parasite capable of lysing hepatocytes after multiple rounds of replication. Within a few days, a few hundred sporozoites can become hundreds of thousands of merozoites. When infected liver cells rupture, they release the merozoites into the bloodstream, where they invade red blood cells and begin the asexual reproductive stage, which is the symptomatic stage of the disease. Within a small number of days, millions of merozoites can be present in blood.

[0412] Malaria symptoms typically develop 4-8 days after initial red blood cell invasion. Replication cycle of merozoites within the red blood cells continues for 36-72 hours, until hemolysis, releasing the merozoites for another round of red blood cell infection. Thus, in synchronous infections (infections that originate from a single infectious bite), fever occurs every 36-72 hours, when infected red blood cells lyse and release endotoxins en masse.

[0413] Plasmodium spp. parasites gain entry into red blood cells through specific ligand-receptor interactions mediated by proteins on the surface of the parasite that interact with receptors on the host erythrocyte (mature red blood cell) or reticulocyte (immature red blood cell), whereas P. falciparum can invade and replicate in erythrocytes and reticulocytes, P. vivax and other species predominantly invade reticulocytes, which are less abundant than erythrocytes. Most of the erythrocyte -binding proteins or reticulocyte -binding proteins that have been associated with invasion are redundant or are expressed as a family of variant forms; however, for P. falciparum, two essential red blood cell receptors (basigin and complement decay-accelerating factor (also known as CD55)) have been identified.

[0414] Plasmodium vivax and Plasmodium ovale can also enter a dormant state in the liver, the hypnozoite.

[0415] Merozoites released from red blood cells can invade other red blood cells and continue to replicate, or in some cases, they differentiate into male or female gametocytes. Gametocytes concentrate in skin capillaries and are then taken up by the mosquito vector in another blood meal. In the gut of the mosquito, each male gametocyte produces eight microgametes after three rounds of mitosis; the female gametocyte matures into a macrogamete. Male microgametes are motile forms with flagellae and seek the female macrogamete. The male and female gametocytes fuse, forming a diploid zygote, which elongates into an ookinete; this motile form secretes a chitinase in order to enter the peritrophic membrane and traverse themidgut epithelium to the basal lateral side of the midgut, establishing itself in the basal lamina as an oocyst Oocysts mature over 14-15 days, undergoing cycles of replication to form sporozoites that are ultimately liberated into the hemocoel, an environment rich in sugars and subtrates beneficial to the parasite’s survival. Thousands of sporozoites can form from a single oocyst and become randomly distributed throughout the hemocoel. These sporozoites are motile and rapidly destroy the hemolymph, with only approximately 20% successfully invading the salivary gland. Following invasion of the salivary gland, sporozoites are re -programmed via an unknown mechanism to prepare for liver invasion. Evidence of this reprogramming has been demonstrated by the inability of midgut sporzoites (directly from oocysts) to invade hepatocytes, and also by the fact that sporzoites which have successfully invaded a salivary gland are unable to do re- invade another salivary gland if presented one. Salivary gland sporozoites alter mosquito behavior and salivary gland function, as less saliva is produced resulting in an increase in mosquito probing behavior, increasing the chances of transmission to a human host via a mosquito bite.

[0416] Some drugs that prevent Plasmodium spp. invasion or proliferation in the liver have prophylactic activity, drugs that block the red blood cell stage are required for the treatment of the symptomatic phase of the disease, and compounds that inhibit the formation of gametocytes or their development in the mosquito (including drugs that kill mosquitoes feeding on blood) are transmission-blocking agents (Phillips, et al. Malaria. Nat Rev Dis Primers 3, 17050 (2017), which is incorporated herein by reference in its entirety).B. Genome

[0417] Since completion of the first sequence of P. falciparum 3D7 genome in 2002, genomic research on malaria parasites has rapidly advanced. Except for a short diploid phase after fertilization in the mosquito midgut, Plasmodium parasites are haploid throughout their life cycle. The genomes of different species range from 20 to 35 megabases, contain 14 chromosomes, a circular plastid genome of approximately 35 kilobases, and multiple copies of a 6 kilobase mitochondrial DNA. Comparison of genomes from different species showed that homologous genes are often found in synthetic blocks arranged in different orders among different chromosomes.

[0418] The adenine-thymine (AT) content of Plasmodium spp. can also be very different, e.g., ~80% AT in P. falciparum, P. reichenowi, and P. gallinaceum; ~75% AT in rodent malaria parasites; and ~60% AT in P. vivax, P. knowlesi, and P. cynomolgi. AT content is often higher in introns and intergenic noncoding regions than in protein-coding exons, with an average of 80.6% AT for the whole P. falciparum genome versus 86.5% for noncoding sequences. The high AT content of P. falciparum reflects large numbers of low-complexity regions, simple sequence repeats, and microsatellites, as well as a highly skewed codon usage bias. Polymorphisms of AT-rich repeats provide abundant markers for linkage mapping of drug resistance genes and for tracing the evolution and structure of parasite populations.

[0419] Malaria parasite genomes carry multigene families that serve important roles in parasite interactions with their hosts, including, for example, antigenic variation, signaling, protein trafficking, and adhesion. Among the gene families, genes encoding P. falciparum erythrocyte membrane protein 1 (PfEMPl) have been studied most extensively. Each individual P. falciparum parasite carries a unique set of 50 to 150 copies of the var gene in its genome, where switches of gene expression can produce antigenic variation. PfEMPl plays an important role in the pathogenesis of clinical developments such as in cerebral and placental malaria, in which it mediates the cytoadherence of infected red blood cells (iRBCs; infected erythrocytes) in the deep tissues. Different PfEMPl molecules bind to various host molecules, including a2- macroglobulin, CD36, chondroitin sulfate A (CSA), complement Iq, CR1, E-selectins and P- selectins, endothelial protein C receptor (EPCR), heparan sulfate, ICAM1, IgM, IgG, PECAM1, thrombospondin (TSP), and VCAM1. Such binding leads to activation of various host inflammatory responses. Hemoglobinopathies, including the hemoglobin C and hemoglobin S trait conditions, interfere with PfEMPl display in knob structures of the iRBCs. This poor display of PfEMPl on the host cell surface offers protection against malaria by reducing the cytoadherence and activation of inflammatory processes that promote the development of severe disease.

[0420] Members of the large Plasmodium interspersed repeat (pir) multigene family are named differently by parasite species, for example, yir in P. yoelii, bir in P. berghei, vir in P. vivax. Several P. falciparum gene families (stevor, rif, and PfMC-2TM) are classified with pir by their similar gene structures, which characteristically include a short first exon, a long second exon, and a third exon encoding a transmembrane domain. In a recent study, the pir genes fromP. chabaudi (cir) were shown to be expressed in different cellular locations, within and on the surface of iRBCs, and in merozoites. Malaria parasites devote large portions of their genomes to gene families that ensure evasion of host immune defenses and protection of molecular processes essential to infection. These families emphasize the importance of research on their roles in parasite-host interactions and virulence, despite the difficulties inherent to their investigation.

[0421] An additional, exemplary polymorphic gene family comprises a group of 14 genes encoding proteins with six cysteines (6-Cys). These proteins often localize on the parasite surface interacting with host proteins and are expressed at different parasite developmental stages. 6-Cys proteins also demonstrate diverse functions and have been shown to play roles in, for example, parasite fertilization, mating interactions, evasion of immune responses, and invasion of hepatocytes. The proteins expressed in asexual stages are generally polymorphic and / or under selection, suggesting that they could be targets of the host immune response; however, their functions in parasite development remain largely unknown.

[0422] Plasmodium genomes can be highly polymorphic. Early studies demonstrated polymorphisms involving tens to hundreds of kilobases and that the chromosome structure in P. falciparum is largely conserved in central regions but extensively polymorphic is both length and sequence near the telomeres. Much of the subtelomeric variation was explained by recombination within blocks of repetitive sequences and families of genes.

[0423] The frequency of simple sequence repeats (microsatellites) in P. falciparum is estimated to be approximately one polymorphic microsatellite per kb DNA. Without wishing to be bound by any one theory, this high rate may reflect the AT-rich nature of the genome.Microsatellites seem to be less frequent in other Plasmodium species that have genomes with lower AT contents. In addition to the highly polymorphic and repetitive structure of Plasmodium genomes, there are also large numbers of Single Nucleotide Polymorphisms (SNPs) and Copy Number Variations (CNVs) (Su et al., Plasmodium Genomics and Genetics: New Insights into Malaria Pathogenesis, Drug Resistance, Epidemiology, and Evolution. Clin Microbiol Rev. 2019 Jul 31;32(4), which is incorporated herein by reference in its entirety).C. Malarial Proteins

[0424] Plasmodium parasites are known to express various proteins at different stages of their lifecycles. Exemplary malarial proteins are described below, and exemplary amino acid sequences are provided in Table 2.

[0425] Circumsporozoite protein (CSP) is a multifunctional protein that is involved in Plasmodium life cycle, as it is required for the formation of sporozoites in the mosquito midgut, the release of sporozoites from the oocyst, invasion of salivary glands, attachment of sporozoites to hepatocytes in the liver, and sporozoite invasion of hepatocytes (see, e.g., Zhao et al. (2016) PLoS ONE 11(8): e0161607). CSP is present in all Plasmodium species, and although variation exists in the amino acid sequence across species, the overall domain structure of a central repeat region and nonrepeat flanking regions is well conserved (see, e.g., Zhao et al. (2016) PLoS ONE 11(8): e0161607; Wahl et al. (2022) J. Exp. Med. 219: e20201313, which are herein incorporated by reference in their entirety). CSP sequences are known (see, e.g., UniProt accession numbers A0A2L1CF52, AOA2L,1CF88, C6FGZ3, C6FH2,7 C6FHG7, M1V060, M1V0A3, M1V0B0, M1V0C4, M1V0E0, M1V9I4, M1VFN9, M1VKZ2, P02893, Q5EIJ9, Q5EIK2, Q5EIK8, Q5EIL3, Q5EIL5, Q5EIL8, Q5R2L2, Q7K740, Q8I9G5, Q8I9J3, Q8I9J4), and Table 1 includes exemplary sequences for CSP P. falciparum isolates from Asia, South America and Africa.

[0426] Table 1A: Exemplary Sequences for CSP P. falciparum isolates from Asia, South America and Africa

[0427] Exemplary CSP amino acid sequence is provided in Table 2A.

[0428] RH5 is found in Plasmodium falciparum (P . falciparum) and not found in the other species of Plasmodium that infect humans. RH5 orthologues are also found in other species belonging to the Lavarenia subgenus, which includes parasites that infect chimpanzees and gorillas, indicating a unique role in P. falciparum invasion of human erythrocytes. See, e.g., Ragotte, et al. Trends Parasitol. 36(6) 2020, which is incorporated herein by reference in its entirety. RH5 is expressed during the mature schizont stages and can complex with Cysteine -rich Protective Antigen (CyRPA) and RH5 -interacting Protein (Ripr) to form an elongated protein trimer on the merozoite surface that binds to erythrocyte surface protein basigin. See, e.g., Ragotte Trends Parasitol 2020 Jun;36(6):545-559, which is herein incorporated by reference in its entirety).

[0429] In humans, RH5 binding to basigin plays an essential role in invasion, acting downstream of membrane deformation. Binding of RH5 to basigin is required for the induction of a spike in calcium within the erythrocyte, which is blocked when merozoites attempt to invade in the presence of anti-RH5, anti-Ripr, or anti-basigin antibodies or soluble basigin. See, e.g., Ragotte (2020).

[0430] RH5 is a 63 kDa protein expressed during the mature schizont stage. It is processed and cleaved to a 45 kDa form which is shed by the parasite. The structure of PfRH5 reveals a kite-like architecture formed from the coming together of two three -helical bundles. See, e.g., Ragotte (2020).

[0431] RH5 sequences are known (see, e.g., UniProt accession numbers A0A159SK44, A0A159SK99, A0A159SKS8, A0A159SKW8, A0A159SL23, A0A159SL78, A0A159SL96, A0A159SLM7, A0A159SMC8, A0A159SMR9, A0A161FQT0, A0A1B1UZE2, A0A1B1UZE4, A0A1B1UZE5, A0A346RCI1, A0A346RCJ0, A0A346RCJ2, A0A346RCJ3, A0A346RCJ4, A0A346RCK4, A0A346RCK5, A0A346RCK6, A0A346RCK9, B2L3N7, Q8IFM5), and exemplary RH5 amino acid sequence is provided in Table 2A.

[0432] Pl 13 is a glycosylphosphatidylinositol (GPI)-linked protein that interacts directly with the N terminus of unprocessed RH5, providing a mechanism by which the RH5 invasion complex is tethered to the merozoite surface. See, e.g., Ragotte (2020). Pl 13 orthologues are found in all Plasmodium species sequenced thus far, suggestive of a common and conserved function(s) (Bullen et al. (2022) Molecular Microbiology 117:1245-1262, which is herein incorporated by reference in its entirety). Despite this, in rodent model of malaria, P. berghei, pl 13 knockout parasites were viable indicating the protein was not essential for asexual blood stage growth and invasion. The knockout parasites do, however, display defects in natural sporozoite transmission, leading to delayed patency in infected mice (Offeddu et al. (2014) Mol. Biochem. Parasitology 193: 101-109, which is herein incorporated by reference in its entirety).

[0433] Plasmodium Pl 13 sequences are known (see, e.g., Uniprot accession number Q8ILP3). Exemplary Pl 13 amino acid sequence is provided in Table 2A.

[0434] Cysteine-Rich Protective Antigen (CyRPA) is a 43 kDa protein with a predicted N-terminal secretion signal. CyRPA is part of a multi-protein complex, including RH5 and Ripr, important for triggering Ca2+release and establishment of tight junctions. PfCyRPA is highly conserved, with only a single SNP above 5% prevalence, is essential for invasion (as conditional knockdown causes the loss of invasion activity), and has poor sero-reactivity from natural exposure (See, e.g., Ragotte (2020)).

[0435] Plasmodium CyRPA sequences are known (see, e.g., Uniprot accession number A0A2S1Q7P0, A0A2S1Q7P5, A0A2S1Q7Q4, Q8IFM8). Exemplary CyRPA amino acid sequence is provided in Table 2A.

[0436] RH5 -interacting Protein (Ripr) is an approximately 120 kDa protein and localized to micronemes during the schizont stage of the P. falciparum life cycle. The full-length 120 kDa protein is processed into two fragments of similar size, an N-terminal fragment(including EGF domains 1 and 2) and a C-terminal fragment (including EGF domains 3-10). Ripr colocalizes with RH5 and CyRPA during parasite invasion at the junction between merozoites and erythrocyte. Parasites with conditional knockouts of PfRipr induce membrane deformation, but cannot complete invasion (See, e.g., Ragotte (2020)).

[0437] Plasmodium Ripr sequences are known (see, e.g., UniProt accession numbers A0A193PDI9, A0A193PDK3, A0A193PDK8, A0A193PDL3, A0A193PDL9, A0A193PDP4, A0A193PDQ8, A0A193PE01, A0A193PE05, A0A193PE07, 097302, A0A193PE17). Exemplary Ripr amino acid sequence is provided in Table 2A.

[0438] El 40 is found in every Plasmodium species for which genomic sequence is available, and is well conserved, with amino acid identity ranging from 34-92% among species. See, e.g., Smith , et al. PLoS one 15.5 (2020): e0232234; http: / / doi: 10.1371 / journal.pone.023223; and U.S. Patent Publication No. US 2019 / 0117752; which are incorporated herein by reference in their entirety. E140 is also highly conserved (95-99%) in P. falciparum strains isolated from different locations around the world, and exhibits a low mutation frequency. E140 is expressed at different life stages of malaria parasites (specifically, E140 has been detected in sporozoites, liver, and blood stage parasites).

[0439] Protein structure algorithms predict that the El 40 protein has five transmembrane domains, presumable spanning a parasite or host-derived membrane. El 40 displays distinct patterns of protein expression in mature sporozoites, late liver, and late schizont stages. It traffics to the anterior and posterior ends of the sporozoite, the parasitophorous vacuole space of the late liver stage and around developing merozoites in the late schizont stage. It is also known to be expressed in mature salivary gland sporozoites as well as oocyst-derived sporozoites and oocysts.

[0440] E140 sequences are known (see, e.g., UniProt accession numbers A0A650D649,A0A650D653, A0A650D672, A0A650D687, A0A650D690, A0A650D694, A0A650D6A3, A0A650D6B8, A0A650D6L3, A0A650D6L7, Q8I299), and exemplary E140 amino acid sequence is provided in Table 2A.

[0441] CelTOS is required for sporozoite traversal through Kupfer cells during the liver invasion process. CelTOS forms a pore from within the cell, allowing for sporozoite egress into the liver. Antibody epitopes have been characterized from immunized mice and infected humanpopulations (Pf and Pv). In mouse studies, immunization with CelTOS has been shown to provide protection and against challenge. Vaccination with CelTOS may generate antibodies that can bind the extracellular domain of the pore-forming complex, blocking complete formation of the pore and preventing sporozoite traversal into the liver. See, e.g., Jimah et al., Elife 2016 Dec 1; 5:e20621. doi: 10.7554 / eLife.20621, which is incorporated herein by reference in its entirety.

[0442] Plasmodium CelTOS sequences are known (see, e.g., Uniprot accession number M1ETJ8, Q53UB7, A0A2R4QLA5, A0A2R4QLI0, A0A2R4QLI5, A0A2R4QLJ1, A0A2R4QLJ4, M1ETJ8, Q53UB8, Q8I5P1). Exemplary CelTOS amino acid sequence is provided in Table 2A.

[0443] SPECT1 and SPECT2 (the latter also sometimes referred to as perforin-like protein 1 (PLP1)) are essential Plasmodium proteins that may play a role in cell traversal. See Yang et al., Cell Rep. 2017 Mar 28; 18(13):3105-3116. doi: 10.1016 / j.celrep.2017.03.017, which is incorporated herein by reference in its entirety. Targeted disruption of P . falciparum SPECT1 or SPECT2 has been shown to reduce infectivity of sporozoites in liver-stage development in humanized mice. However, mechanisms of cell traversal of these two proteins are yet to be defined in P. falciparum. See Y ang et al.

[0444] SPECT1 and SPECT2 are considered attractive pre-erythrocytic immune targets due to the key role they are thought to play in the crossing of the malaria parasite across the dermis and the liver sinusoidal wall, prior to invasion of hepatocytes. Recombinant P. falciparum SPECT2 has been shown to cause lysis of red blood cells in a Ca2+-dependent manner, as has the MACPF / CDC domain of PfSPECT2. PfSPECT2 has also been implicated in the Ca2+- dependent egress of P. falciparum merozoites from red blood cells.

[0445] Plasmodium SPECT1 and SPECT2 sequences are known (see, e.g., UniProt accession numbers Q8IDR4 and Q9U0J9), and exemplary amino acid sequence is provided in Table 2A.

[0446] Exported protein 1 (EXP1) is a single pass transmembrane protein with an N- terminal signal peptide expressed during intraerythrocytic stage and liver stage (see, e.g., Spielmann et al., Int J Med Microbiol. 2012 Oct; 302(4-5): 179-86, which is herein incorporated by reference in its entirety). EXP1 was shown to initially localize to dense granules in merozoites and then be transported to parasitophorous vacuolar membrane (PVM) after invasion (see, e.g.,Iriko et al., Parasitol Int. 2018 Oct; 67(5):637-639, which is herein incorporated by reference in its entirety). Once localized to the PVM, EXP1 forms homo-oligomers with a N-terminus that is exposed to the parasitophorous vacuolar lumen and a C-terminus that is exposed to the red blood cell cytosol (see, e.g., Mesen-Rarmrez et al., PLoS Biol. 2019 Sep 30;17(9):e3000473, which is herein incorporated by reference in its entirety).

[0447] EXP1 has been demonstrated to possess glutathione S-transferase (GST) activity that may protect Plasmodium from oxidative damage (see, e.g., Mesen-Rarmrez et al., PLoS Biol 17(9) 2019 Sep 30; 17(9):e3000473, which is herein incorporated by reference in its entirety). Recently, it was demonstrated that EXP1 is important for Plasmodium survival by maintaining correct localization of EXP2, a nutrient-permeable channel in the PVM (see, e.g., Mesen- Rarmrez et al., 2020).

[0448] P. falciparum EXP1 polypeptide sequences are known (see, e.g., UniProt accession number Q8IIF0, W7JTD3, Q25840, Q548U2, Q5VKK2, Q5VKK5, Q5WRH8, Q6V9G4, Q6V9G6, Q6V9G9, Q6V9H1, Q6V9H2, Q9U590, P04923, P04926). Exemplary EXP1 amino acid sequence is provided in Table 2A.

[0449] Upregulated in infective sporozoites gene 3 (UIS3) is a membrane -bound protein localized to sporozoite parasitophorous vacuolar membrane (PVM) in infected hepatocytes.UIS3 was shown to interact with liver fatty acid-binding protein (L-FABP) and be involved in fatty acid and / or lipid import during phases of Plasmodium growth (see, e.g., Sharma et al., J Biol Chem. 2008 Aug 29; 283(35): 24077-24088; Mikolajczak et al., Int J Parasitol. 2007 Apr;37(5):483-9, which are herein incorporated by reference in their entirety).

[0450] After sporozoite invasion of host liver cells, there is synthesis of vitalPlasmodium structural features (e.g., parasitophorous vacuolar membrane). During hepatocytic stages, the Plasmodium relies on host fatty acids for rapid synthesis of its membranes (see, e.g., Sharma et al., J Biol Chem. 2008 Aug 29; 283(35): 24077-24088, which is herein incorporated by reference in its entirety). UIS3 insertion in the PVM provides Plasmodium a method to import essential fatty acids and / or lipids during rapid sporozoites growth phases (see, e.g., Sharma et al., 2008).

[0451] Immunization with UIS 3 -deficient Plasmodium berghei sporozoites protected against malaria in rodent malaria model (see, e.g., Mueller et al., Nature. 2005 Jan 13;433(7022): 164-7, which is herein incorporated by reference in its entirety). UIS 3 -deficient Plasmodium berghei can start the transformation process in the liver; however, they show severe defects during transformation into trophozoites (see, e.g., Mueller et al., 2005). UIS 3 -deficient Plasmodium berghei are also unable to develop into mature liver schizonts and therefore abort malaria infection within the liver itself (see, e.g., Mueller et al., 2005). Further, it was previously demonstrated that UIS3 derived from Plasmodium berghei and UIS3 derived from Plasmodium falciparum exhibited a low (i.e. 34%) amino acid sequence identity (see, e.g., Mueller et al., 2005).

[0452] Plasmodium UIS3 sequences are known (see, e.g., UniProt accession number A0A509ARS3, A0A1C6YLP3, Q8IEU1, A0A384KLI1, A0A1G4H423, A0A077YB01, Q9NFU4). Exemplary UIS3 amino acid sequence is provided in Table 2A.

[0453] Upregulated in infective sporozoites gene 4 (UIS4) contains a single transmembrane domain and localizes to secretory organelles of sporozoites and to the parasitophorous vacuole membrane (PVM) of liver stages. UIS4 is not expressed in blood stages or early sporozoites that are produced in oocysts (see, e.g., Mackellar et al., Eukaryot Cell. 2010 May; 9(5): 784-794, which is herein incorporated by reference in its entirety).

[0454] Deletion of UIS4 gene is associated with arrest of early liver stage development (see, e.g., Vaughan and Kappe, Cold Spring Harb Perspect Med. 2017 Jun 1; 7(6):a025486, which is herein incorporated by reference in its entirety). Recently, UIS4 was demonstrated to be involved in Plasmodium berghei survival by eluding host actin structures deployed as part of host cytosolic defense (see, e.g., Bana et al., iScience. 2022 Apr 22;25(5): 104281. doi: 10.1016 / j.isci.2022.104281. eCollection 2022 May 20, which is herein incorporated by reference in its entirety). P. falciparum has an ortholog to UIS4 named ETRAMP10.3 which is not able serve as a functional compliment to P. yoelii UIS4, indicating it likely serves a different function in P. falciparum ’s life cycle (see Mackellar et al., Eukaryot. Cell 9:784-94 (2010), which is herein incorporated by reference in its entirety).

[0455] Plasmodium UIS4 sequences are known (see, e.g., UniProt accession number Q8IJM9). Exemplary UIS4 amino acid sequence is provided in Table 2A.

[0456] Liver specific protein 1 (LISP-1) is expressed during Plasmodium development in hepatocytes and localized to the parasitophorous vacuolar membrane (PVM) (see, e.g., Ishinoet al., Cell Microbiol. 2009 Sep; 11(9): 1329-1339). LISP-1 was shown to be expressed at high levels during late liver stages development and to be involved in PVM breakdown and subsequent merozoite release (see, e.g., Ishino et al., Cell Microbiol. 2009 Sep; 11(9): 1329- 1339, which is herein incorporated by reference in its entirety).

[0457] Intracellular Plasmodium deficient in LISP- 1 develop into hepatic merozoites and display normal infectivity to erythrocytes (see, e.g., Ishino et al., Cell Microbiol. 2009 Sep;11(9): 1329-1339, which is herein incorporated by reference in its entirety). However, LISP1- deficient liver-stage Plasmodium do not rupture PVM and remain trapped inside hepatocytes (see, e.g., Ishino et al., 2009).

[0458] Plasmodium LISP-1 sequences are known (see, e.g., UniProt accession number A0A2I0C2X6, Q8ILR5). Exemplary LISP-1 amino acid sequence is provided in Table 2.

[0459] Liver specific protein 2 (LISP-2) contains a modified 6-cys domain and is expressed during Plasmodium development in hepatocytes (see, e.g., Orito et al., Mol Microbiol. 2013 Jan; 87(l):66-79, which is herein incorporated by reference in its entirety). LISP-2 was shown to be expressed by liver stages Plasmodium, exported to hepatocytes, and be distributed throughout the host cell, including the nucleus (see, e.g., Orito et al., 2013).

[0460] Intracellular Plasmodium deficient in LISP2 do not mature effectively during merozoites development (see, e.g., Orito et al., 2013).

[0461] Plasmodium LISP-2 sequences are known (see, e.g., UniProt accession number A0A2I0BZR4, Q8I1X6, Q9U0D4). Exemplary LISP-2 amino acid sequence is provided in Table 2A.

[0462] Thrombospondin-related adhesion protein (TRAP) contains an N-terminal domain that is commonly referred to as von Willebrand factor A domain, although it is most similar to an integrin I domain because it contains a metal ion-dependent adhesion site (MIDAS) with a bound Mg2+ion that is required for sporozoite motility in vitro and infection in vivo (see, e.g., Lu et al., PLoS One. 2020; 15(1): e0216260, which is herein incorporated by reference in its entirety). The I domain is inserted in an extensible P-ribbon and followed by a thrombospondin repeat (TSR) domain, a proline -rich segment at the C-terminus, a single-pass transmembrane domain, and a cytoplasmic domain (see, e.g., Lu et al., 2020). Sequence analysis of the proline-rich segment revealed the presence of SH3 -domain binding PxxP motifs in Plasmodium TRAPs (Akhouri et al., Malar J. 2008 Apr 22; 7:63. doi: 10.1186 / 1475-2875-7-63, which is herein incorporated by reference in its entirety).

[0463] TRAP is stored in the micronemes and becomes surface exposed at the sporozoite anterior tip when parasite comes in contact with host cells (Akhouri et al., Malar J. 2008 Apr 22;7:63. doi: 10.1186 / 1475-2875-7-63, which is herein incorporated by reference in its entirety). TRAP also plays an important role in liver cell invasion of sporozoites by helping sporozoites in gliding motility and in recognition of host receptors on the mosquito salivary gland and hepatocytes (Akhouri et al., Malar J. 2008 Apr 22;7:63. doi: 10.1186 / 1475-2875-7-63, which is herein incorporated by reference in its entirety).

[0464] Plasmodium TRAP sequences are known (see, e.g., UniProt accession numbers A0A5Q2EXK8, A0A5Q2EZD7, A0A5Q2F1F6, A0A5Q2F2B8, A0A5Q2F2H6, A0A5Q2F4G9, 076110, P16893, Q01507, Q26020, Q76NM2, W8VNB6), and exemplary TRAP amino acid sequence is provided in Table 2A.

[0465] Liver-stage-associated protein (LS AP- 1 ) has been shown to be found mainly at the periphery of the intracellular hepatic parasite throughout its development, but not in blood stage parasites and possibly in minor quantities in salivary gland sporozoites (see, e.g., Siau et al., PLoS Pathog. 2008 Aug 8;4(8):el000121, which is herein incorporated by reference in its entirety). LSAP-1 is among the most abundant transcripts in the salivary gland transcriptome but has not been detected in proteomic surveys of sporozoites. Rather, expression has only been detected only in liver stages (see, e.g., Siau et al., 2008).

[0466] Plasmodium LSAP-1 sequences are known (see, e.g., UniProt accession number Q8I632, W7JR53). Exemplary LSAP-1 amino acid sequence is provided in Table 2A.

[0467] Like LS AP- 1 , LS AP-2 is also among the most abundant transcripts in the salivary gland transcriptome but has not been detected in proteomic surveys of sporozoites. LSAP-2 has shown some efficacy as a vaccine when combined with other antigens. See, e.g., Halbroth et al., Infect Immun. 2020 Jan 22; 88(2):e00573-19. doi: 10.1128 / IAI.00573-19. Print 2020 Jan 22, which is incorporated herein by reference in its entirety.

[0468] Plasmodium LSAP-2 sequences are known (see, e.g., UniProt accession number Q8I632, W7JR53). Exemplary LSAP-2 amino acid sequence is provided in Table 2.

[0469] Liver-Stage Antigen 1 (LSA-1) is expressed after Plasmodium have invaded hepatocytes and antigen accumulates in the parasitophorous vacuole (see, e.g., Tucker, K. et al., 2016, 'Pre-Erythrocytic Vaccine Candidates in Malaria', in A. J. Rodriguez-Morales (ed.), Current Topics in Malaria, IntechOpen, London. 10.5772 / 65592, which is herein incorporated by reference in its entirety). The function of LSA-1 remains currently not known (see, e.g., Tucker, K. et al., 2016).

[0470] LS A- 1 is a 230 kDa preerythrocytic stage protein containing a large central region consisting of over eighty 17 amino acid residue repeat units flanked by highly conserved C- and N-terminal regions (Richie, T.L. and Parekh, L.K. (2009) Malaria. In Vaccines for Biodefense and Emerging and Neglected Diseases (Barrett, A.D.T. and Stanberry L.R., eds), pp. 1309-1364, Elsevier, which is herein incorporated by reference in its entirety). LSA1 is expressed only by liver stage Plasmodium and not by sporozoites (Richie, T.L. and Parekh, L.K. (2009) Malaria, which is herein incorporated by reference in its entirety). In Vaccines for Biodefense and Emerging and Neglected Diseases (Barrett, A.D.T. and Stanberry L.R., eds, pp. 1309-1364, Elsevier, which is herein incorporated by reference in its entirety). The repeat region results in significant variation of the protein between strains of Plasmodium falciparum (see, e.g., Tucker, K. et al., 2016).

[0471] Plasmodium LSA-1 sequences are known (see, e.g., UniProt accession number Q25886, Q25887, Q25893, Q26028, Q9GTX5, 096125). Exemplary LSA-1 amino acid sequence is provided in Table 2A.

[0472] Liver stage antigen 3 (LSA-3) is a 200-kDa protein that is composed of three nonrepeating regions (NR- A, NR-B, and NR-C) flanking two short repeat regions and one long repeat region (see, e.g., Tucker, K. et al., 2016). The nonrepeat regions are well conserved across geographically diverse strains of Plasmodium falciparum (see, e.g., Tucker, K. et al., 2016). The most significant variation is in the repeating regions due to organization and number of repeating subunits rather than composition of the repeating regions (see, e.g., Tucker, K. et al., 2016).

[0473] Recently, in vitro data has shown that antibodies against LSA-3 (in particular, the C-terminal portion of LSA-3) may provide some protection (see, e.g., Morita et al, Sci Rep. 2017Apr 5; 7:46086. doi: 10.1038 / srep46086, which is herein incorporated by reference in its entirety).

[0474] Plasmodium LSA-3 sequences are known (see, e.g., UniProt accession number C7DU21, C7DU22, C7DU23, C7DU24, C7DU25, C7DU26, C7DU27, C7DU28, C7DU29, C7DU32, C7DU33, C7DU34, C7DU36, C7DU37, C7DU38, C7DU39, C7DU40, Q8I042, Q8I0A5, Q8I0D0, Q8IFR1, Q8IFR2, Q8IFR3, Q8IFR4, Q8IFR5, Q8IFR6, Q8IFR7, Q8IFR8, Q8IFR9, Q8IFS0, Q8IFS1, Q8IFS2, Q8IFS3, Q8IFS4, Q8IFS5, Q8IFS6, Q8IFS7, Q8IFS8, Q8IFS9, Q8IFT0, Q8IFT1, Q8IFT2, Q8IFT3, Q8IFT4, Q9U0N9, Q9U0P0, A0A2I0BVD6, A0PFM9, 096275). Exemplary LSA-3 amino acid sequence is provided in Table 2A.

[0475] Glutamic acid-rich protein (GARP) is a 80kDA protein which derives its name from its glutamic rich amino acid sequence which comprises 24% of all its residues. GARP is predominantly expressed in ring stages and trophozoites and has been shown to be a non- essential gene in cell culture but highly immunogenic in animal models (Hon et al., Trends Parasitol. 2020 Aug; 36(8):653-655, which is herein incorporated by reference in its entirety). Although GARP is non-essential in cell culture, its localization to the periphery of infected erythrocytes may indicate a role in the sequestration of infected erythrocytes. GARP’s involvement in sequestration has been proposed to occur by way of binding with a chloride / bicarbonate anion exchanger (Lau et al., PLoS Pathog. 10, el004135. 2014, which is herein incorporated by reference in its entirety). Antibodies against GARP have been proposed to serve as signatures of protection against severe malaria and have shown efficacy in experimental trials in monkeys. See, e.g., Hon et al, Trends in Paras 2020 Aug; 36(8):653-655. doi: 10.1016 / j.pt.2020.05.012 and Laue et al, Pios Path. 2014 10, el004135, which are herein incorporated by reference in their entirety. GARP sequences are known (see, e.g., UniProt accession number, Q9GTW3, Q9U0N1), and exemplary GARP amino acid sequence is provided in Table 2A.

[0476] Parasite-infected erythrocyte specific protein 2 (PIESP2) (see, e.g., UniProt accession number Q8I488) is a highly immunogenic protein first expressed in the trophozoite stage and believed to be important for the clinical progression of cerebral malaria. Although this protein is predominantly found within erythrocytes, it has been shown to be present on the surface of erythrocytes, allowing them to adhere to endothelial cells in the vasculature of thebrain. Antibodies against PIESP2 have been shown to prevent vascular adherence of plasmodium and could prove valuable in preventing the preventing inflammatory response in the brain and impairment of the blood-brain barrier during cerebral malaria progression (see, e.g., Liu et al, Int J Biol Macromol. 2021 Apr 30;177:535-547. doi: 10.1016, (j.ijbiomac.2021.02.145, which is herein incorporated by reference in its entirety). PIESP2 sequences are known (see, e.g., UniProt accession number Q8I488), and exemplary PIESP2 amino acid sequence is provided in Table 2A.

[0477] Shizont egress antigen- 1 (SEA1) is a large 244 kDA protein lacking transmembrane domains or known targeting signals. The function of SEA1 is not known; however, it has been shown to be effective in rodent vaccine studies and has even been proposed as a target of protective antibodies found in children. SEA1 received its name after it was reported that antibodies agasint this protein inhibited egress of plasmodium merizoites. SEA1 localizes closely to centromers during nuclear division, implicating its role in the essential process of replication. To date, various studies have proposed a role for SEA1 in egress, but also in mitotic division of nuclei during replication, (see, e.g., Perrin et al, mBio. 2021 Mar 9;12(2):e03377-20. doi: 10.1128 / mBio.03377-20, which is herein incorporated by reference in its entirety). SEA1 sequences are known (see, e.g., UniProt accession number A0A143ZXM2), and exemplary SEA1 amino acid sequence is provided in Table 2A.D. Embodiments of Malarial Sequences

[0478] An exemplary full length CSP polypeptide amino sequence from Plasmidum falciparum isolate 3D7 is presented in Table 2A as SEQ ID NO:A, and includes the following: a secretory signal (amino acids 1-18); an N-terminal domain (amino acids 19-104); a junction region (amino acids 93-104), a central domain (amino acids 105-272); and a C-terminal domain (amino acids 273-397). In exemplary SEQ ID NO: A, the N-terminal domain includes an N- terminal region (amino acids 19-80); an N-terminal end region (amino acids 81-92); and a junction region (amino acids 93-104). In exemplary SEQ ID NO:A, the junction region includes an R1 region (amino acids 93-97) and amino acids ADGNPDP (SEQ ID NO: B) at positions 98- 104. In exemplary SEQ ID NO:A, the central domain includes a minor repeat region (amino acids 105-128) and a major repeat region (amino acids 129-272). In exemplary SEQ ID NO:1, the minor repeat region includes three repeats of the amino acid sequence NANPNVDP (SEQ ID NO:C). In exemplary SEQ ID NO: 1, the major repeat region includes 35 repeats of the aminoacid sequence NANP (SEQ ID NO: D), wherein 35 repeats of the amino acid sequence NANP are separated into two contiguous stretches, and wherein one stretch includes 17 repeats of the amino acid sequence NANP and one includes 18 repeats of the amino acid sequence NANP which flank an amino acid sequence of NVDP (SEQ ID NO: E). The major repeat region includes the amino acid sequences NPNANP (SEQ ID NOT) and NANPNA (SEQ ID NO:G). In exemplary SEQ ID NO:A, the C-terminal domain includes a C-terminal region (amino acids 273-375) and a transmembrane domain (amino acids 376-397). In exemplary SEQ ID NO:A, the C-terminal region includes a Th2R region (amino acids 314-327) and a Th3R region (amino acids 352-363).

[0479] Table 2A: Exemplary amino acid sequencesInfluenza Overview

[0480] Influenza illness is caused by influenza viruses, of which there are four types: A, B, C, and D. Types A and B are responsible for the seasonal epidemics that occur every winter in the United States (also known as flu season). Type A viruses are the only type to date that have caused a pandemic (i.e., a global epidemic). Type C viruses generally cause mild illness and are not thought to cause human epidemics, while type D viruses primarily affect cattle, and are not known to infect or cause illness in humans.

[0481] Influenza A viruses are divided into subtypes based on two surface proteins: hemagglutinin (HA) and neuraminidase (NA). 18 HA subtypes and 11 different NA subtypes are known to exist, and more than 130 influenza A subtype combinations have been observed, although many more subtype combinations are possible, given the virus’s propensity for “reassortment” (i.e., the process in which influenza viruses swap gene segments, which can occur when two viruses infect a host at the same time). Subtypes H1N1 and H3N2 are the type A viruses that are currently common in humans. Subtypes can be further broken down into“clades” and “sub-clades” (also known as “groups” and “sub-groups”, respectively), which are organized based on HA gene sequences.

[0482] Clades and sub-clades may be genetically distinct from one another while not being antigenically distinct. For example, it may be possible for two viruses to have distinct HA gene sequences, and thus be genetically distinct, and yet still be bound and neutralized by a given antibody, and thus not antigenically distinct.

[0483] Currently circulating influenza A (H1N1) viruses are related to the 2009 H1N1 virus that emerged in the spring of 2009 and caused the flu pandemic of that year. These viruses, also called A(HlNl)pdm09 viruses or “2009 HINT’, have continued to circulate seasonally since first being discovered, and have undergone several changes both genetically and antigenically.

[0484] Influenza A (H3N2) viruses also comprise many separate, genetically different clades in recent years that contine to circulate.

[0485] Influenza B viruses are classified by lineage rather than subtype. Two lineages of influenza B viruses exist: B / Yamagata and B / Victoria, each of which can be further divided into clades and sub-clades. Influenza B viruses generally change more slowly than influenza A viruses, both genetically and antigenetically. In recent years, both B / Yamagata and B / Victoria have been in co-circulation, although the proportion from each lineage can vary depending on location and season.

[0486] Influenza virus names usually indicate type (A, B, C, D), host of origin (although for humans, the host of origin is usually not indicated), geographical origin, strain number, and year of collection. For influenza A viruses, HA and NA descriptions are provided in parenthesis.Seasonal flu vaccines are typically formulated to provide protection against multiple influenza viruses that are known to cause epidemics. In recent years, vaccines have been formulated as tetravalent vaccines, to provide antigens against H1N1, H3N2, B / Victoria, and B / Yamagata viruses. In some embodiments, an influenza vaccine can protect both against the viruses that the vaccine comprises or delivers antigens from, and antigenically similar viruses.Norovirus Overview

[0487] Noroviruses are members of the Caliciviridae family of small, non-enveloped, positive-stranded RNA viruses. The Norovirus genus includes both human and animal (e.g., murine and canine) noroviruses.

[0488] Noroviruses typically have a 24-48 hour incubation period between infection and development of symptoms. Symptoms typically persist for 12-72 hours, but reports have indicated that viral shedding can continue long after symptoms have resolved. It is believed that viral shedding can continue for several days or even 1-2 weeks after symptoms have resolved; immunocompromised individuals may continue shedding virus even longer, up to several (e.g., 3, 4, 5, 6, 7, 8 or more) months after infection..

[0489] Noroviruses are highly infectious; it has been reported that doses as low as 20 viral particles may be sufficient to establish infection. Exposure is typically via inhalation or ingestion (e.g., commonly by oral exposure, such as by ingestion of contaminated food). Norovirus virions withstand acidic pH and can survive passage through the stomach.

[0490] Given the above-noted low infection dose and long shedding periods, combined with the high levels of shedded virus ( 108- 1010copies of RNA per g) often detected in feces, norovirus infections can spread rapidly within communities.

[0491] Norovirus infection can be asymptomatic, particularly in children (see, for example, Robilotti et al., Clin. Microbiol. Rev., 28: 134, 2015 and references cited therein).Symptomatic infection typically results in acute gastroenteritis, characterized by symptoms such as vomiting and diarrhea, and / or nausea and severe abdominal cramps. Other reported associated conditions include encephalopathy, intravascular coagulation, necrotizing enterocolitis in premature infants, postinfectious irritable bowel syndrome, and benign infantile seizures. Young children, the elderly, and immunocompromised individuals (e.g., transplant patients or other subjects receiving immunosuppressive medication or therapy) are among those most susceptible to development of serious disease.

[0492] Although it has been reported that 20-30% of cases of norovirus infection in humans can be asymptomatic or “mild” (e.g., resolving within a few days) (Qi et al. Am J Infect Control. 43:833, 2015, doi: 10.1016 / j.ajic.2015.04.182; Marshall et al., J Med Virol. 69:568, 2003, doi: 10.1002 / jmv.10346), norovirus infection remains a significant risk. Mortality may be as high as 3%, and norovirus infections are believed to be responsible for up to 20% ofemergency room visits and hospitalizations, even in middle- to high- income countries (Lopman et al., PLoS Med. 13:el001999, 2016, doi: 10.1371 / journal.pmed.l001999). Dehydration associated with norovirus infection can be particularly problematic, particularly in the elderly and / or the very young. Furthermore, in some instances (e.g., in immunocompromised subjects including, for example, transplant patients, patients receiving chemotherapy or immunosuppressive therapy, subjects infected with HIV, etc.) (Cardemil et al. Infect Dis Clin North Am. 31:839, 2017, doi: 10.1016 / j .idc.2017.07.012), norovirus infection can become chronic, with serious consequences (Bok et al., Oncol Nurs Forum. 40:434, 2013, doi: 10.1188 / 13. ONF.434-436; Kaufman et al. Antiviral Res. 105C:80, 2014, doi: 10.1016 / j.antiviral.2014.02.012; Trivedi et al. Am J Infect Control. 41:654, 2013, doi:10.1016 / j.ajic.2012.08.002). Without wishing to be bound by any particular theory, the present disclosure proposes that a robust T cell immunization, e.g., as may be achieved as described herein (e.g., via administration or delivery of one or more T cell epitopes as described here, for example via string constructs), may be particularly useful or effective to protect against chronic infection, e.g., by facilitating removal of infected cells.

[0493] No commercial vaccines or specific antivirals are currently available to treat or prevent human norovirus infections. Standard of care remains supportive therapy, particularly to address dehydration and / or electrolyte abnormalities. Some reports have suggested that administration of nitazoxanide may be helpful, for example, to reduce the duration of illness (see, for example, Rossignol et al. Aliment Pharmacol Ther 24:1423, 2006, doi.org / 10. I l l 1 / j.1365-2036.2006.03128.x.). Enteric administration of human immunoglobulin has also been reported to assist in resolution of diarrhea associated with norovirus infection (see, for example, Chagla et al, J Clin Virol 55:306, 2013, doi.org / 10.1016 / j.jcv.2013.06.009).

[0494] Furthermore no small animal models have been described that mimic human disease; only recently has an in vivo model (in zebrafish larvae) been shown to support norovirus replication (e.g., of GII.3 and GII.4 variants; see Van Dycke et al., PLoS Pathog. 15:el008009, 2019, doi: 10.1371 / journal.ppat.1008009). Some in vitro replication models have been described; specifically, some strains (e.g., Gii.4-Sydney) have been shown to replicate in human B cells (see, for example, Lindesmith et al., J Infect Dis. 216: 1227, 2017, doi:10.1093 / infdis / jix385); and some (e.g., some GII.3 and some GII.4 strains) have been shown to replicate in human intestinal enteroid monolayer cultures (see, for example, Ettayebi et al.,Science. 353:1387, 2016, doi: 10.1126 / science.aaf5211). Also, a monoclonal antibody (NV8812; see White et al. J Virol. 70:6589-97. doi: 10.1128 / JVI.70.10.6589, 1996) to the viral VP1 protein that has been reported to bind to the C-terminal region at residues 300-384, has been reported to block binding of virus-like particles (VLPs) comprising the norovirus VP1 protein to relevant human and animal cells.

[0495] An effective norovirus vaccine remains an unmet medical need of critical importance for global health.Lifecycle

[0496] Infection by a norovirus begins when the viral capsid binds to a host cell surface receptor; histo-blood group antigens (HBGA) have been described as potential receptors or co- receptors. HBGAs are polymorphic glucans found on the surfaces of red blood cells and of certain epithelial cells (de Graaf et al. Nat Rev Microbiol. 14:421, 2016, doi: 10.1038 / nrmicro.2016.48; Mallagaray et al. Nat Commun. 10: 1320, 2019, doi: 10.1038 / s41467- 019-09251-5, each of which is incorporated herein by reference in its entirety). It has been reported that individuals who do not express fucosyltransferase 2 (Fut2), which generates HBGAs, are not susceptible to norovirus infection (de Graaf et al. Nat Rev Microbiol. 14:421, 2016, doi: 10.1038 / nrmicro.2016.48, which is incorporated herein by reference in its entirety). Moreover, studies have shown that noroviruses recognize a determined group of HBGAs (Huang et al. J Virol. (2005) 79:6714, 2005, doi: 10.1128 / JVI.79.11.6714-6722.2005, which is incorporated herein by reference in its entirety); at least four different binding patterns of human noroviruses have been described based on ABO blood type, Lewis blood group, and fut2 status (secretor / nonsecretor) (Huang et al. J Infect Dis. 188: 19, 2003, doi: 10.1086 / 375742, which is incorporated herein by reference in its entirety). Generally, noroviruses that have HBGA type A / B binding patterns recognize the A and / or B and H antigens, but not the Lewis antigens; and noroviruses that have Lewis binding patterns bind only to Lewis antigens and / or the H antigen (Huang et al. J Virol. 79:6714, 2005, doi: 10.1128 / JVI.79.11.6714-6722.2005, which is incorporated herein by reference in its entirety).

[0497] After binding, virus becomes internalized, uncoated, and disassembled; host factors are recruited to replicate and translate the genome {reviewed in de Graaf et al., Nat Rev Microbiol. 14:421, 2016, which is incorporated herein by reference in its entirety).

[0498] The genomes of noroviruses that infect humans comprise a linear, positive-sense RNA strand about 7.3-8.3 kb long (often about 7.5-7.7 kb). The 5’ end of the norovirus genome is covalently linked to one of the nonstructural proteins (the VPg protein) it encodes; the 3’ end is polyadenylated.

[0499] Upon internalization, the viral genome is released from the VPg protein, which then recruits host translation initiation factors (e.g., eIF3) and initiates assembly of the translation complex.

[0500] As described in more detail below, translation produces three proteins: structural VP1 and VP2 proteins, and a polyprotein that is autocleaved to produce six (6) non-structural viral proteins, via a cascade that first generates three protein precursors, each of which becomes cleaved into two viral proteins.

[0501] Replication proceeds by transcribing the (+-strand) genome to generate (- strand) RNAs that become templates for synthesis of new (+-strand) genomic and subgenomic RNAs. These subgenomic RNAs contain the ORFs for VP1 and VP2, and are translated to produce these proteins. Replicated genomic RNAs are assembled into new virions that are released from the infected host cells.Genome

[0502] The norovirus genome includes short untranslated regions (UTRs) at either end; these contain evolutionarily conserved structures that are thought to participate in replication, translation, and / or pathogenesis.

[0503] The norovirus genome includes three open reading frames (ORFs 1, 2, and 3) that together encode eight viral proteins (reviewed in, Robilotti et al., Clin Microbiol Rev. 28:134, 2015, which is incorporated herein by reference in its entirety). ORF-2 and ORF-3 encode the structural components of the virion, viral protein 1 (VP1) and VP2, respectively. ORF-1 encodes the above-mentioned polyprotein that is proteolytically processed into the six nonstructuralproteins of the virus: p48 (NA1 / NS2), NTPase (NS3), p22(NS4), VPg (N5), Pro (NS6), and Pol (NS7; RdRp), these last two being the norovirus protease and RNA-dependent RNA polymerase, respectively. See, review of norovirus proteins in Compillay- Veliz et al. Front Immunol 11:961, 2020, which is incorporated herein by reference in its entirety)

[0504] VP1 is the primary structural protein of the capsid; 90 dimers of VP1 assemble into the icosahedral (T = 3) capsid, with only a few copies of VP2 included. VP1 includes a shell (S) domain and a protruding (P) domain, with Pl and P2 components (see, for example, Prasad et al., Science 286:287, 1999, doi: 10.1126 / science.286.5438.287, which is incorporated herein by reference in its entirety). The S domain makes up the core of the capsid, from which the P domain protrudes. The S domain is involved in binding VP2, thereby associating it with the capsid. The P domain, and particularly, P2, mediates binding to host HBGA molecules (see, e.g., Campillay- Veliz el al., Front. Immunol. 11:961, 2020, doi: 10.3389 / fimmu.2020.00961, which is incorporated herein by reference in its entirety). The P domain also mediates interactions between VP1 proteins and therefore impacts size and stability of viral capsids.

[0505] The S domain is located in the N-terminal portion of the VP1 protein, for example extending from about residue 225 to the end, according to canonical numbering systems. The Pl domain is typically considered to begin at residue 226 according to canonical numbering systems, and to be interrupted by the P2 domain, so that Pl includes residues 226-278 and 406-52, and P2 includes residues 278-406 according to canonical numbering systems.

[0506] The P2 subdomain is the most variable region of the VP1 protein, and is believed to be surface exposed on the viral capsid. P2 variants have been reported to be associated with particular epidemic outbreaks (see, for example, 22). The Pro protein is responsible for cleaving the polyprotein generated by translation of ORF1, first into p48 / NTPase, p22 / VPg and Pro / Pol precursor proteins, and ultimately into the six individual proteins.

[0507] The Pol, VPg, NTPase and p48 proteins have all been reported to play roles in viral replication. NTPase has been reported to have helicase, NTP hydrolase, and chaperone activities; p48 has been reported to increase Pol activity, and also disassembly of the trans-Golgi network, resulting in interference with host cell signaling pathways involved in immune response.

[0508] P22 has also been reported to contribute to trans-Golgi disassembly (36), and also to facilitate virion release from cells.

[0509] At least ten (10) different genogroups (GI-GX) of noroviruses have been defined {see, for example, Chhabra et al. J Gen Virol. 100: 1393 406, 2019, doi: 10.1099 / jgv.0.001318; see also, Campillay- Veliz et al., Front. Immunol, 11:961, 2020, doi: 10.3389 / fimmu.2020.00961, each of which is incorporated herein by reference in its entirety) based on similarity of highly- conserved regions of either the Pol (NS7; RdRp) protein or (ii) VP1 {e.g., the amino acidic regions of VP1, such as are found in the S domain); three of these genogroups (specifically, GI, GII, and GIV) infect and cause acute gastroenteritis in humans. Norovirus genogroups have been further subdivided into genotypes, which in turn include strains and variants {e.g., that arise by mutation). Recombination between or among variants also gives rise to new strains {see, for example, Cannon J Virol. 83:5363, 2009, doi: 10.1128 / JVI.02518-08; see also Vinje J Infect Dis. 176:1374, 1997, doi: 10.1086 / 517325, each of which is incorporated herein by reference in its entirety). The GII.4 genotype is the most prevalent worldwide; its Sydney and New Orleans variants are particularly prevalent {see, Glass et al N Engl J Med. 361: 1776, 2009. doi: 10.1056 / NEJMra0804575; Vinje et al. J Infect Dis. 176: 1374, 1997, doi: 10.1086 / 517325; Tamminen et al Viruses. 11:91, 2019 doi: 10.3390 / vl 1020091, each of which is incorporated herein by reference in its entirety) and a GII.P16-GII.4 Sydney recombinant strain was responsible for a 2015 pandemic {see, Lindesmith et al. J Infect Dis. 217: 1145, 2017, doi: 10.1093 / infdis / jix651, which is incorporated herein by reference in its entirety). Other highly infectious genotypes include GII.17 {see, for example, Lindesmith et al. J Infect Dis. 217:1145, 2017, doi: 10.1093 / infdis / jix651; see also, Lindesmith et al. J Infect Dis. 216:1227, 2017, doi: 10.1093 / infdis / jix385, each of which is incorporated herein by reference in its entirety).

[0510] To give an example of norovirus genogrouping, a system has been described in which viruses whose VP1 protein sequences differ by less than 14.3% are classified in the same strain; those whose VP1 protein sequences differ by 14.3-43.8% are classified in the same genotype, and those whose VP1 protein sequences differ between 45-61.4% are classified in the same genogroup {see Zheng et al. Virology 346:312, 2006, doi: 10.1016 / j.virol.2005.11.015, which is incorporated herein by reference in its entirety).

[0511] It has been reported that individuals infected with norovirus of one genogroup do not typically develop immunity to other genogroups (see, e.g., Esposito and Principi, Front. Immunol. 11: 1383, 2020; see also, Atmar et al., Curr. Opin. Infect. Dis. 31: 422 (2018); and Brown et al. J. Clin. Virol. 96:44 (2017), each of which is incorporated herein by reference in its entirety) even when they may develop immunity to other strains or variants within the genogroup with which they were infected. In some embodiments, as described herein, provided technologies administer or deliver (e.g., by administration of an encoding RNA) polypeptides that, together, are or comprise epitopes from multiple genotypes (e.g., GI and GII) and / or multiple clades.Norovirus Antigens

[0512] In some embodiments, the present disclosure provides certain norovirus antigen constructs particularly useful in effective vaccination.

[0513] Antigens utilized in accordance with the present disclosure are or include norovirus components (e.g., proteins or fragments or epitopes thereof, including epitopes that may comprise non-amino acid, e.g., carbohydrate moieties), which components induce immune responses when administered to humans (or other animals such as rodents and non-human primates susceptible to norovirus infection).Norovirus Protein Sequences

[0514] In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., norovirus vaccine) comprises or delivers (e.g., causes expression of in a recipient organism, for example by administration of a nucleic acid construct, such as an RNA construct as described herein, that encodes it) an antigen that is or comprises one or more epitopes (e.g., one or more B-cell and / or one or more T-cell epitopes) of a norovirus protein. In some embodiments, a pharmaceutical composition described herein induces a relevant immune response effective against norovirus (e.g., by targeting a norovirus protein).

[0515] In some embodiments a provided pharmaceutical composition (e.g., immunogenic composition, e.g., norovirus vaccine) comprises or delivers an antigen that is orcomprises a full-length norovirus protein. In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., norovirus vaccine) comprises or delivers an antigen that is or comprises a portion of a norovirus protein that is less than a full-length norovirus protein. In some embodiments, a provide...

Claims

CLAIMS1. A method of inducing an immune response in a subject who was previously exposed to a first SARS-CoV-2 Spike (S) protein, the method comprising a step of delivering a polypeptide comprising a fragment of a second SARS-CoV-2 S protein to the subject, wherein the fragment of the second SARS-CoV-2 S protein comprises or consists a Receptor Binding Domain (RBD) or an SI domain of the second SARS-CoV-2 S protein, and wherein the fragment of the second SARS-CoV-2 S protein comprises one or more mutations of one or more SARS-CoV-2 variants.

2. The method of claim 1, wherein the first SARS-CoV-2 S protein is from a strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction.

3. The method of claim 1 or 2, wherein the subject was previously exposed to the first SARS-CoV-2 S protein by:(a) administration of one or more doses of one or more vaccines that deliver the first SARS-CoV-2 S protein,(b) previous infection by a SARS-CoV-2 virus comprising the first SARS-CoV-2 S protein, and / or(c) presence in a jurisdiction where a SARS-CoV-2 strain or variant comprising the first SARS-CoV-2 S protein was prevalent.

4. The method of claim 1 or 2, wherein the fragment of the second SARS-CoV-2 S protein does not comprise one or more regions of a SARS-CoV-2 S protein that are infrequently mutated in SARS-CoV-2 variants.

5. The method of any one of claims 1-4, wherein the fragment of the second SARS-CoV- 2 S protein does not comprise an S2 domain.

6. The method of any one of claims 1-5, wherein the fragment of the second SARS-CoV- 2 S protein does not comprise an N-terminal domain (NTD).

7. The method of any one of claims 1-6, wherein the fragment of the second SARS-CoV- 2 S protein comprises or consists of the RBD.

8. The method of any one of claims 1-5, wherein the fragment of the second SARS-CoV- 2 S protein comprises or consists of the SI domain.

9. The method of any one of the preceding claims, wherein the fragment of the second SARS-CoV-2 S protein comprises one or mutations associated with a SARS-CoV-2 variant that is prevalent, predicted to be prevalent, predicted to continue to be prevalent, and / or predicted to increase in prevalence in a relevant jurisdiction.

10. The method of any one of the preceding claims, wherein the fragment of the second SARS-CoV-2 S protein comprises one or more mutations associated with a SARS-CoV-2 variant that has a high immune escape potential.

11. The method of claim 10, wherein the SARS-CoV-2 variant has been determined to have a high immune escape potential using an in vitro assay (e.g., a viral neutralization assay), in silico analysis (e.g., sequence analysis and / or molecular dynamic simulations), in vivo studies (e.g., mouse or rat studies), and / or based on an infection rate and / or growth rate in a human population.

12. The method of claim 10 or 11, wherein the SARS-CoV-2 variant is an Omicron variant.

13. The method of claim 12, wherein the Omicron variant is an XBB variant (e.g., an XBB.l or XBB.1.5 variant), a BQ.l variant, a BA.2.86 variant, or a JN variant.

14. The method of claim 13, wherein the one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N,N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, or N969K, or a combination thereof, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

15. The method of any one of claims 1-14, wherein the fragment of the second SARS- CoV-2 S protein comprises or consists of an RBD of an XBB.1.5 SARS-CoV-2 variant, and wherein the RBD comprises one or more of the following mutations relative to SEQ ID NO: 1: G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, or Y505H, or any combination thereof.

16. The method of any one of claims 1-15, wherein the fragment of the second SARS- CoV-2 S protein comprises or consists of an SI domain, and wherein the one or more mutations associated with an XBB.1.5 variant are selected from: T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, and P681H, or any combination thereof, wherein the positions of the one or more mutations are shown relative to SEQ ID NO: 1.

17. The method of any one of claims 1-16, wherein the polypeptide comprising the fragment of the second SARS-CoV-2 S protein is delivered by administering an RNA that comprises a nucleotide sequence encoding the fragment of the second SARS-CoV-2 protein.

18. The method of any one of claims 1-16, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 3.

19. The method of any one of claims 1-18, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 5.

20. The method of any one of the preceding claims, wherein the polypeptide comprises a secretion signal.

21. The method of claim 20, wherein the secretion signal is a homologous secretion signal.

22. The method of claim 20, wherein the secretion signal is a heterologous secretion signal.

23. The method of any one of claims 20-22, wherein the secretion signal is present at or near the N-terminus of the polypeptide.

24. The method of claim 20, wherein the secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gDl secretion signal, a gBl secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal.

25. The method of claim 24, wherein the SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 15.

26. The method of claim 24, wherein the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9.

27. The method of claim 24, wherein the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 16.

28. The method of claim 24, wherein the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 8.

29. The method of claim 24, wherein the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 13.

30. The method of claim 24, wherein the gDl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 12.

31. The method of claim 24, wherein the gBl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 37.

32. The method of claim 24, wherein the gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 35.

33. The method of claim 24, wherein the gI2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 11.

34. The method of claim 24, wherein the gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 38.

35. The method of claim 24, wherein the EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39.

36. The method of claim 24, wherein the HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.

37. The method of any one of the preceding claims, wherein the polypeptide further a multimerization domain.

38. The method of claim 37, wherein the multimerization domain in the C-terminal region (e.g., at the C-terminus).

39. The method of claim 37 or 38, wherein the multimerization domain is a fibritin domain.

40. The method of claim 39, wherein the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 95.

41. The method of claim 39, wherein the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 96.

42. The method of any one of the preceding claims, wherein the polypeptide comprises a transmembrane (TM) domain.

43. The method of claim 42, wherein the TM domain is a homologous TM domain.

44. The method of claim 42, wherein the TM domain is a heterologous TM domain.

45. The method of any one of claims 42-44, wherein the TM domain is present in the C- terminal portion of the SARS-CoV-2 S protein variant or immunogenic portion thereof (e.g., at the C-terminus).

46. The method of claim 45, wherein the polypeptide comprises a multimerization domain and a TM domain at or near the C-terminus.

47. The method of claim 46, wherein the TM domain is C-terminal to the multimerization domain.

48. The method of claim 47, wherein:(a) the multimerization domain is directly adjacent to the fragment of the second SARS- CoV-2 protein or connected to the fragment of the second SARS-CoV-2 protein via a flexible linker, and / or(b) the TM domain is directly adjacent to the multimerization domain or connected to the multimerization domain via a flexible linker.

49. The method of any one of claims 42-48, wherein the TM domain is a SARS-CoV-2 S protein TM domain or an influenza TM domain.

50. The method of claim 49, wherein the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 89.

51. The method of claim 49, wherein the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 90.

52. The method of any one of claims 17-51, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 120.

53. The method of any one of claims 17-52, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 130.

54. The method of any one of claims 17-52, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 135.

55. The method of any one of claims 17-52, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 145.

56. The method of any one of claims 17-52, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 150.

57. The method of any one of claims 17-56, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has been codon-optimized for expression in mammalian subjects.

58. The method of any one of claims 17-57, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has been codon-optimized for expression in human subjects.

59. The method of any one of claims 17-58, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has an enriched G / C content relative to wild- type sequence.

60. The method of claim 59, wherein G / C content has been increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%.

61. The method of any one of claims 17-60, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a heterologous 3’ UTR or 5’UTR.

62. The method of claim 61, wherein the heterologous 5' UTR comprises or consists of a modified human alpha-globin 5 '-UTR.

63. The method of claim 61 or 62, wherein the heterologous 3’ UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

64. The method of any one of claims 17-63, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a poly(A) sequence.

65. The method of claim 64, wherein the poly(A) sequence has a length of about 100-150 nucleotides.

66. The method of claim 64 or 65, wherein the poly (A) sequence is a disrupted poly(A) sequence.

67. The method of any one of claims 17-66, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a 5' cap.

68. The method of any one of claims 17-67, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 122 or 124.

69. The method of any one of claims 17-67, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 131 or 133.

70. The method of any one of claims 17-67, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 136 or 138.

71. The method of any one of claims 17-67, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 146 or 148.

72. The method of any one of claims 17-67, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 151 or 153.

73. The method of any one of claims 17-72, wherein the RNA is unmodified RNA.

74. The method of any one of claims 17-73, wherein the RNA comprises one or more modified nucleotides.

75. The method of claim 74, wherein the modified nucleotide is pseudouridine (e.g., Nl- methyl-pseudouridine).

76. The method of claim 74 or 75, wherein the RNA comprises a modified nucleotide in place of each uridine.

77. The method of any one of claims 17-76, wherein the RNA is an self-amplifying RNA or trans-amplifying RNA.

78. The method of any one of claims 17-77, wherein the RNA is fully or partially encapsulated within lipid nanoparticles (LNP), polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

79. The method of claim 78, wherein the RNA is fully or partially encapsulated within LNP.

80. The method of claim 79, wherein the LNP comprise a cationically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.

81. The method of any one of claims 1-80, wherein the first SARS-CoV-2 S protein is from a strain or variant that the subject was first exposed to and / or that was first prevalent in a population of subjects.

82. The method of any one of claims 1-81, wherein the first SARS-CoV-2 S protein is a Wuhan SARS-CoV-2 S protein or an Omicron BA.4 / 5 SARS-CoV-2 S protein.

83. The method of any one of claims 1-82, where the first SARS-CoV-2 S protein is from a strain or variant that the subject has previously been vaccinated against or is delivered by one or more vaccines that a significant proportion of the population (e.g., at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least abut 45%, at least about 50%, at least about 55%, or at least about 60%) has previously been administered.

84. The method of claim 83, wherein the vaccine previously administered to the subject or a significant proportion of the population was a first generation vaccine.

85. The method of claim 83, wherein the first SARS-CoV-2 S protein is from a SARS- CoV-2 strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction.

86. The method of claim 83, wherein the first SARS-CoV-2 S protein is from a variant that first became prevalent in a relevant jurisdiction.

87. The method of any one of claims 1-86, wherein the immune response comprises a B cell immune response.

88. The method of claim 87, wherein the immune response comprises a naive B cell immune response.

89. The method of any one of claims 1-88, wherein:(a) the immune response comprises a reduced memory B cell immune response as compared to an immune response induced by administering the full length sequence of the second SARS-CoV-2 S protein,(b) the immune response comprises an increased naive B cell immune response as compared to an immune response induced by administering the full length sequence of the second SARS-CoV-2 S protein, and / or(c) the ratio of the naive B cell immune response to the memory B cell immune response is increased.

90. The method of claim 89, wherein:(a) the memory B cell immune response is reduced by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein;(b) the memory B cell immune response is increased by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein; and / or(c) the ratio of the naive immune response to the memory B cell immune response is increased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein.

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