Optimized vaccine compositions and methods for making the same

Abstract

To provide optimized vaccine compositions, and to provide methods for making the same.SOLUTION: Described herein are compositions of and methods of making vaccines which can provide broad serological reactivity, an inverse dose response, and a swarm effect. Provided herein is a vaccine comprising a set of antigens that are representative of at least 60% of fifth order clades of a microbe. Provided herein is a vaccine comprising a set of antigens of a microbe, the smallest pairwise edit distance between two of the antigens being at least 10% of the average size of the antigens, and the largest pairwise edit distance between two of the antigens being no more than 98% of the average size of the antigens.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 808,760, filed on February 21, 2019, and U.S. Provisional Patent Application No. 62 / 817,902, filed on March 13, 2019, the entire contents of each of which are incorporated herein by reference in their entirety.

Background Art

[0002] Pathogens such as infectious bacteria, parasites, fungi, viruses, and cancer have evolved various strategies to avoid detection and neutralization by the host immune response. Such strategies can often weaken and complicate successful vaccine development against these pathogens. Rapid mutations can complicate vaccine design. Through vaccination, a host can generate protective antibodies before infection. This can be achieved by exposing the host's immune system to one or more antigens of one or more pathogens. When this is done against a vaccine that mutates relatively slowly, the vaccine can be successful because a multi - year time frame can be obtained to match and provide protection with a single vaccine.

[0003] Vaccination against rapidly mutating pathogens may provide only limited protection, for example, because the immune system's response to the vaccine may become outdated. In some cases, the antigen changes over time, so the vaccine can become outdated. This can result in the need for new vaccines regularly or semi - regularly, such as annually. In some cases, the protection provided by such vaccines can vary, and some vaccines may provide inadequate protection for some period or years. This can be due to an incorrect prediction of the virus strain likely to circulate in the next season. In some cases, such as with the common cold, such rapid mutations can prevent the effectiveness of the vaccine.

Summary of the Invention

Means for Solving the Problem

[0004] The present disclosure provides a vaccine comprising a set of antigens representing at least 60% of the fifth clade of microorganisms.

[0005] The present disclosure also provides a vaccine comprising a set of antigens of a microorganism, wherein the minimum pairwise edit distance between two antigens is at least 10% of the average size of the antigens, and the maximum pairwise edit distance between two antigens is 98% or less of the average size of the antigens.

[0006] The present disclosure also provides a vaccine comprising a set of antigens representing at least 60% of all operational taxonomic units (OTUs) of the microorganism.

[0007] In some embodiments, an antigen represents a clade if its sequence is up to 40% of the average size of the antigens of the represented clade that differ from other members of the represented clade. In some embodiments, an antigen represents a clade if the edit distance is up to 5% of the size of another strain of the represented clade. In some embodiments, an antigen represents a clade if its sequence is up to 20% of the average size of the antigens of the represented clade that differ from at least 95%, 96%, 97%, 98%, or 99% of all strains of the represented clade. In some embodiments, an antigen represents a clade if its sequence is up to 10% of the average size of the antigens of the represented clade that differ from at least 95%, 96%, 97%, 98%, or 99% of all strains of the represented clade. In some embodiments, an antigen represents a clade if the edit distance is up to 5% of the size of 95%, 96%, 97%, 98%, or 99% of all strains of the represented clade. In some embodiments, an antigen represents a clade if its sequence is at an edit distance of up to 25 from all strains of the represented clade. In some embodiments, an antigen represents a clade if its sequence is at an edit distance of up to 100 from all strains of the represented clade. In some embodiments, an antigen represents a clade if its sequence is present in the clade.

[0008] Also provided in the present disclosure are vaccine compositions comprising a set of antigens, wherein the antigens are derived from a library of microbial variants and a) two antigens of the set having the maximum edit distance have an edit distance S; b) two antigens of the library having the maximum edit distance have an edit distance L; and c) S is at least 60% of L.

[0009] In addition, the present disclosure also provides a vaccine composition comprising at least four influenza virus hemagglutinin antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to another influenza virus hemagglutinin antigen polypeptide, and each polypeptide contains an antigen that is at least 90% identical among the four influenza virus hemagglutinin antigen polypeptides.

[0010] In addition, the present disclosure also provides a vaccine composition comprising at least four influenza virus neuraminidase antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to another influenza virus neuraminidase antigen polypeptide, and each polypeptide contains an antigen that is at least 90% identical among the four influenza virus neuraminidase antigen polypeptides.

[0011] In addition, the present disclosure also provides a vaccine composition comprising at least four HIV gp160 antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to another HIV gp160 antigen polypeptide, and each polypeptide contains an antigen that is at least 90% identical among the four HIV gp160 antigen polypeptides.

[0012] In addition, the present disclosure also provides a vaccine composition comprising at least four HIV gp120 antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to another HIV gp120 antigen polypeptide, and each polypeptide contains an antigen that is at least 90% identical among the four HIV gp120 antigen polypeptides.

[0013] In addition, the present disclosure also provides at least four HIVs each represented by a sequence that is at least 20% identical and at most 95% identical to another HIV gp41 antigen polypeptide A vaccine composition comprising a gp41 antigen polypeptide is also provided, wherein each polypeptide comprises an antigen that is at least 90% identical among four HIV gp41 antigen polypeptides.

[0014] Also provided herein is a vaccine composition comprising a set of antigens that activate an immune response in a subject against at least six strains identified in Table 1.

[0015] In some embodiments, the immune response is detectable using head-specific antibodies in a hemagglutination inhibition assay. In some embodiments, when tested using a hemagglutination inhibition assay, the immune response is at least 2-fold greater when using the antigen than when using H1N1 + H3N2 + HAB. In some embodiments, when tested using a hemagglutination inhibition assay, the immune response is at least 10-fold greater when using the antigen than when using H1N1 + H3N2 + HAB. In some embodiments, when tested using a hemagglutination inhibition assay, the immune response is at least 100-fold greater when using the antigen than when using H1N1 + H3N2 + HAB.

[0016] In some embodiments, the microorganism is a bacterium. In some embodiments, the microorganism is a virus. In some embodiments, the virus is influenza. In some embodiments, the influenza is type A. In some embodiments, the influenza is type B. In some embodiments, type A influenza is H1N1, H1N2, H3N1, H3N2, or H2N3. In some embodiments, type A influenza is H1N1. In some embodiments, type A influenza is H3N2. In some embodiments, the virus is human immunodeficiency virus (HIV). In some embodiments, the HIV is HIV-1. In some embodiments, the HIV-1 is HIV-1 of subtype A, subtype B, or subtype C.

[0017] In some embodiments, the antigen is a broadly neutralizing antigen of surface-exposed residues adjacent in three-dimensional space. In some embodiments, the broadly neutralizing antigen is at the base of hemagglutinin. In some embodiments, the broadly neutralizing antigen is at the head of hemagglutinin. In some embodiments, the broadly neutralizing antigen is in neuraminidase. In some embodiments, the broadly neutralizing antigen is in gp160. In some embodiments, the broadly neutralizing antigen is in gp120. In some embodiments, the broadly neutralizing antigen is in gp41.

[0018] In some embodiments, the antigen is a broadly conserved fragment of hemagglutinin. In some embodiments, the antigen is a broadly conserved fragment of the head of hemagglutinin. In some embodiments, the antigen is a broadly conserved fragment of the base of hemagglutinin. In some embodiments, the antigen is a broadly conserved fragment of neuraminidase. In some embodiments, the antigen is a broadly conserved fragment of gp160.

[0019] In some embodiments, the vaccine comprises at least one antigen selected from SEQ ID NOs: 1 to 87. In some embodiments, the vaccine comprises at least one antigen that is at least 60% identical to any one of SEQ ID NOs: 1 to 87. In some embodiments, the vaccine comprises at least one antigen that is at least 70% identical to any one of SEQ ID NOs: 1 to 87. In some embodiments, the vaccine comprises at least one antigen that is at least 80% identical to any one of SEQ ID NOs: 1 to 87. In some embodiments, the vaccine comprises at least one antigen that is at least 90% identical to any one of SEQ ID NOs: 1 to 87. In some embodiments, the vaccine comprises at least one antigen selected from SEQ ID NOs: 88 to 127. In some embodiments, the vaccine comprises at least one antigen that is at least 60% identical to any one of SEQ ID NOs: 88 to 127. In some embodiments, the vaccine comprises at least one antigen that is at least 70% identical to any one of SEQ ID NOs: 88 to 127. In some embodiments, the vaccine comprises at least one antigen that is at least 80% identical to any one of SEQ ID NOs: 88 to 127. In some embodiments, the vaccine comprises at least one antigen that is at least 90% identical to any one of SEQ ID NOs: 88 to 127. In some embodiments, the vaccine comprises at least one antigen selected from SEQ ID NOs: 128 to 171. In some embodiments, the vaccine comprises at least one antigen that is at least 60% identical to any one of SEQ ID NOs: 128 to 171. In some embodiments, the vaccine comprises at least one antigen that is at least 70% identical to any one of SEQ ID NOs: 128 to 171. In some embodiments, the vaccine comprises at least one antigen that is at least 80% identical to any one of SEQ ID NOs: 128 to 171. In some embodiments, the vaccine comprises at least one antigen that is at least 90% identical to any one of SEQ ID NOs: 128 to 171. In some embodiments, the vaccine comprises at least one antigen selected from SEQ ID NOs: 172 to 267. In some embodiments, the vaccine comprises at least one antigen that is at least 60% identical to any one of SEQ ID NOs: 172 to 267.In some embodiments, the vaccine comprises at least one antigen that is at least 70% identical to any one of SEQ ID NOs: 172 to 267. In some embodiments, the vaccine comprises at least one antigen that is at least 80% identical to any one of SEQ ID NOs: 172 to 267. In some embodiments, the vaccine comprises at least one antigen that is at least 90% identical to any one of SEQ ID NOs: 172 to 267.

[0020] In some embodiments, the set of antigens comprises at least 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 different antigens. In some embodiments, the set of antigens comprises at least 30 antigens. In some embodiments, the set of antigens comprises at least 50 antigens. In some embodiments, the antigen has an average edit distance from each of the other antigens that is at least 5% of the average size of the antigens within the clade.

[0021] In some embodiments, the minimum pairwise edit distance between two or more antigens within the set is 1 or less, and the maximum pairwise edit distance is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100. In some embodiments, the minimum pairwise edit distance between two or more antigens within the set is 5% or less of the size of the antigen, and the maximum pairwise edit distance is at least 75% of the size of the antigen.

[0022] In some embodiments, a clade is a clade of a phylogenetic tree that is a neighbor joining clustering tree or a most parsimonious tree. In some embodiments, a primary clade is a clade that is not wholly or partially included within another higher-level clade. In some embodiments, each Xth clade is phylogenetically under X - 1 branching nodes in the phylogenetic tree of the microorganism. In some embodiments, the vaccine comprises an antigen representing at least 60% of each of the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, or 15th clades of the microorganism. In some embodiments, the clade being represented is a clade Y that is included in clade Y - 1. In some embodiments, a representative clade is a node-based clade. In some embodiments, a representative clade is a stem-based clade. In some embodiments, a representative clade is an apomorphy-based clade. In some embodiments, the average number of branches between each pair of antigens in the set is at least 1, 3, 5, or 10. In some embodiments, the minimum number of branches between any two antigens in the set is 1 or less, and the maximum number of branches between any two antigens in the set is at least 5, 10, or 15.

[0023] In some embodiments, each OTU comprises sequences that are at least 95% homologous to each other. In some embodiments, each OTU is composed of at least 3 different sequences.

[0024] In some embodiments, each antigen in the set shares at least 90%, 95, or 99% sequence identity with at least one other antigen in the set. In some embodiments, each antigen in the set shares at least 90%, 95, or 99% sequence identity with at least one other antigen in the set over at least 100 amino acids in length. In some embodiments, each antigen in the set differs in sequence identity by at least 5% from each of the other antigens in the set. In some embodiments, each antigen in the set differs in sequence identity by 75% or less from each of the other antigens in the set.

[0025] In some embodiments, each antigen is a peptide comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 85, 100, 110, or 120 amino acids. In some embodiments, the antigen is selected from Table 2, Table 3, Table 4, Table 5, or fragments or homologs thereof. In some embodiments, the vaccine comprises two or more antigens from Table 2, Table 3, Table 4, Table 5, or fragments or homologs thereof. In some embodiments, the vaccine comprises three or more antigens from Table 2, Table 3, Table 4, Table 5, or fragments or homologs thereof. In some embodiments, the vaccine comprises five or more antigens from Table 2, Table 3, Table 4, Table 5, or fragments or homologs thereof. In some embodiments, the vaccine comprises ten or more antigens from Table 2, Table 3, Table 4, Table 5, or fragments or homologs thereof. In some embodiments, the fragment comprises at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, or 120 amino acids. In some embodiments, the fragment comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, or 120 amino acids. In some embodiments, the homolog comprises a sequence having at least 90% sequence identity with the antigen of Table 1.

[0026] In some embodiments, the library comprises at least 1×10 4 、1×10 5 、1×10 6 different variants of the microorganism. In some embodiments, the library comprises at least 90% of all known sequences of the microorganism.

[0027] Also provided in the present disclosure is a pharmaceutical composition comprising the vaccine composition recited in any of the preceding claims and a pharmaceutically acceptable diluent, adjuvant, excipient, or any combination thereof.

[0028] In some embodiments, the vaccine is in the form of an aerosol formulation. In some embodiments, the vaccine is in the form of an injectable formulation.

[0029] In some embodiments, each antigen is at a concentration that alone does not provide a significant prophylactic immune response against a broad neutralizing antigen of interest, but collectively, the plurality of antigens have a combined concentration that provides an immune response against the broad neutralizing antigen of interest. In some embodiments, the subject is a bird. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a pig.

[0030] Also provided in the present disclosure are virus-like particles (VLPs) comprising the vaccine composition recited in any of the preceding claims.

[0031] Also provided in the present disclosure are recombinant expression vectors comprising: a) a set of antigens representing at least 60% of each of the primary and secondary clades of a microorganism; b) a set of antigens having a minimum pairwise edit distance between two antigens of 25 or less and a maximum pairwise edit distance between two antigens of at least 300; c) a set of antigens representing at least 60% of all operational taxonomic units (OTUs) of a microorganism; or d) a nucleic acid molecule encoding a set of antigens representing at least 60% of all operational taxonomic units (OTUs) of a microorganism.

[0032] Also provided in the present disclosure are recombinant expression vectors comprising: a) a set of antigens representing at least 60% of each of the primary and secondary clades of a microorganism; b) a set of antigens wherein the minimum pairwise edit distance between two antigens is 5% or less of the size of the antigen and the maximum pairwise edit distance between two antigens is at least 75% of the size of the antigen; c) a set of antigens representing at least 60% of all operational taxonomic units (OTUs) of a microorganism; or d) a nucleic acid molecule encoding a set of antigens representing at least 60% of all operational taxonomic units (OTUs) of a microorganism.

[0033] Also provided herein are methods for treating an infection or reducing the likelihood of infection in a subject, comprising administering to the subject a vaccine composition recited in any of the preceding claims.

[0034] Also provided herein are methods for treating or reducing the likelihood of influenza infection in a subject, comprising administering to the subject a vaccine composition that provides immunostimulation effective against seasonal influenza for at least 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0035] In some methods, the subject is a human. In some methods, the subject is a domesticated animal.

[0036] Also provided herein are methods for making a vaccine composition, comprising selecting: (a) a set of antigens that represent at least 60% of each of the primary and secondary clades of a microorganism; (b) a set of antigens wherein the minimum pairwise edit distance between two antigens is 5% or less of the size of the antigens and the maximum pairwise edit distance is at least 75% of the size of the antigens; (c) a set of antigens that represent at least 60% of all of the operational taxonomic units (OTUs) of a microorganism; or (d) a set of antigens that represent at least 60% of all of the operational taxonomic units (OTUs) of a microorganism.

[0037] In some embodiments, the method further comprises obtaining a plurality of antigen sequences from a library of microbial strains; and aligning the plurality of antigen sequences to create a phylogenetic tree of the antigens. Incorporation by reference

[0038] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0039] The novel features of the present invention are described in detail in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description which illustrates exemplary embodiments that utilize the principles of the present invention, and to the accompanying drawings.

Brief Description of the Drawings

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[0054] Rapidly evolving pathogens can pose fundamental challenges to vaccine design because mutations in such pathogens can render previous vaccine responses obsolete. For example, in the annual redesign of influenza vaccines, it is possible to attempt to predict the evolved influenza variants that are likely to circulate generally in the following year. Such attempts can often fail. Conserved epitopes on identified influenza coat proteins may be missed by antibodies induced by most vaccine recipients.

[0055] Provided herein are immunogenic compositions (e.g., vaccines) that can induce a response against one or more pathogens. In some examples, the vaccines provided herein incorporate an antigen that can induce an immune response capable of obtaining broadly neutralizing antibodies. The present disclosure also provides methods of using and manufacturing the vaccines provided herein.

[0056] Broadly neutralizing antibodies can provide "universal" or longer-term vaccines. Such broadly neutralizing antibodies can be directed against broadly conserved residues, e.g., residues of an antigen. Broadly conserved antigens can be conserved across multiple strains of a microorganism. A microorganism can contain broadly conserved antigens even if it is a rapidly mutating pathogen. Antibodies that can identify and / or bind these conserved antigens or sets of antigens can provide protection against many strains of a given virus.

[0057] Non-conserved residues can be present more abundantly (e.g., far more abundantly) than widely conserved residues and thus can be immunodominant, so the present disclosure provides an approach that can shift the abundance distribution of residues of an antigen to favor responses to more conserved antigens. By incorporating into a vaccine widely conserved antigens that represent various variations (e.g., variants, different strains, or different clades) of a microorganism, immunity against many or substantially all variations of the microorganism can be conferred on a subject. I. Microorganisms

[0058] The vaccine compositions described herein can elicit an immune response against a microorganism or against a strain or subset of strains of a microorganism. In some examples, such a vaccine can provide broad immunity (immunity not limited to a particular strain) against multiple strains of a microorganism. Such a vaccine can include a set of antigens that can include some or many different antigens from some or many different strains of a microorganism.

[0059] A microorganism can be a pathogen against which a subject can be vaccinated. In some examples, a microorganism can be pathogenic or can cause a disease, condition, or death. A microorganism can be a bacterium, virus, fungus, parasite, or a derivative thereof, such as a nucleic acid, protein, toxin, or peptide secreted by or isolated from a microorganism.

[0060] A bacterium can be a unicellular prokaryotic microorganism. A bacterium can be Gram-positive or Gram-negative. A bacterium can be infectious.

[0061] Fungi can be organisms that produce spores. Fungi present in the skin, lungs, blood, or another organ, tissue, or biological fluid can potentially cause disease in a subject. In some examples, the fungi can be of the genus Aspergillus, Blastomyces, Candida, Coccidioides, or Cryptococcus (e.g., C. neoformans and C. gattii). Other suitable fungi are also within the scope of the present disclosure.

[0062] Parasites can be protozoa, helminths, ectoparasites, or any other suitable parasite. In some examples, the parasite can be a Plasmodium parasite. In some examples, the parasite can be a Plasmodium parasite or a Trypanosoma parasite. In the case of a Plasmodium parasite, the vaccine can be against Plasmodium malariae. In the case of a Trypanosoma parasite, the vaccine can be against Trypanosoma cruzi, Trypanosoma brucei, or Leishmania parasites.

[0063] Protozoa can be single-celled organisms that can live and / or multiply within the blood or tissues of a subject. Examples of protozoa can include, but are not limited to, sarcodines (e.g., Entamoeba), mastigophores (e.g., Giardia and Leishmania), ciliates (e.g., Balantidium), and sporozoans (e.g., Plasmodium, Cryptosporidium, etc.).

[0064] Helminths can be multicellular organisms. Helminths can be divided into three groups: flatworms, acanthocephalans, and nematodes. Examples of helminths can include, but are not limited to, parasitic flatworms, flukes, tapeworms, acanthocephalans, roundworms, and pinworms. Helminths can live in the gastrointestinal tract, blood, lymphatic system, or other tissues. Ectoparasites can be organisms that can inhabit the surface of a subject and, in some cases, can attach to or lurk on the skin of the subject. Examples of ectoparasites can include ticks, lice, fleas, and mites.

[0065] In some examples, the virus can be a retrovirus, flavivirus, filovirus, coronavirus, or paramyxovirus. In the case of a retrovirus, the vaccine can be against the human immunodeficiency (HIV) virus. In the case of a flavivirus, the vaccine can be against dengue virus, Zika virus, or West Nile virus. In the case of a filovirus, the vaccine can be against Ebola virus, Marburg virus, or Ravn virus. In the case of a coronavirus, the vaccine can be against Middle East respiratory syndrome (MERS) virus, severe acute respiratory syndrome (SARS) virus, or a novel coronavirus (e.g., 2019-nCoV). In the case of a paramyxovirus, the vaccine can be against respiratory syncytial virus (RSV) or Nipah virus.

[0066] The vaccine can be against any type of influenza virus, such as any one or more of the influenza viruses described herein, although not limited thereto. The influenza virus can be influenza A, B, C, or D. Influenza A viruses can be divided into subtypes based on two proteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA). Different influenza strains can have different subtypes of hemagglutinin, neuraminidase, or both. The predicted combination of influenza A subtypes can be at least 198. Influenza B viruses can be further classified into lineages, which can include, for example, B / Yamagata and B / Victoria.

[0067] If the virus is an influenza A virus, it may be any subtype including, but not limited to, H1N1, H1N2, H1N3, H2N2, H3N2, H3N8, H4N2, H4N4, H4N6, H4N8, H5N1, H5N2, H5N3, H5N8, H6N1, H6N4, H6N5, H6N6, H6N8, H7N1, H7N2, H7N3, H7N7, H7N8, H7N9, H8N4, H9N2, H9N5, H9N8, H10N3, H10N4, H10N7, H10N8, H10N9, H11N2, H11N6, H11N9, H12N1, H12N3, H12N5, H13N6, H13N8, H14N5, H15N2, H15N8, H16N3, H17N, H18N11, or variants thereof. If the virus is an influenza B virus, it can be any strain of the influenza B virus or variants thereof. A non-exhaustive list of influenza viruses is included in Table 1.

[0068] The influenza virus can be a strain described in Table 1. Some vaccines can include a set of antigens capable of activating an immune response in a subject against at least 80% of the strains in Table 1. In some examples, the vaccine can include a set of antigens capable of activating an immune response in a subject against at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the strains in Table 1.

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Table 1-5

[0069] The vaccine can be directed against any species of HIV virus, such as HIV-1 and HIV-2. HIV-1 can have group M (main), group O (outlier), or group N (non-M, non-O). HIV-1 group M can have subtypes A, B, C, D, F, G, H, J, or another subtype. HIV-2 can have subtype A or subtype B. In some examples, the vaccine against HIV can be a vaccine against either HIV-1 or HIV-2. In some examples, the vaccine against HIV can be a vaccine against both HIV-1 and HIV-2.

[0070] The vaccine can be directed against variants of HIV or, in some cases, simian immunodeficiency virus (SIV). In some examples, the vaccine against HIV can also be a vaccine against SIV. In some examples, the vaccine against HIV can also provide protection against variants of SIV.

[0071] The HIV virus can be the strains described in FIGS. 26 - 34. Some vaccines can include a set of antigens that can activate an immune response in a subject against at least 80% of the strains in FIGS. 26 - 34. In some examples, the vaccine can include a set of antigens that can activate an immune response in a subject against at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the strains in FIGS. 26 - 34.

[0072] Microorganisms can have variants. Variants of microorganisms can include microorganisms with variations in their genome, microorganisms with variations in their post - translational modifications, microorganisms with variations in their epigenome, microorganisms with variations in their protein expression, microorganisms with variations in their RNA expression, microorganisms with variations in their antigen content, or combinations thereof. In some examples, microorganisms with variations in their antigen content can be of particular interest.

[0073] Microorganisms can have multiple variants that can be represented using a phylogenetic tree. A phylogenetic tree can be a model or tool that can be used to investigate relationships. In some examples, a phylogenetic tree can be a graphical representation of the phylogenetic or evolutionary history in the sense of a minimally - connected graph. A phylogenetic tree can include several clades, where a clade can be a grouping of nested branches. A clade can be a grouping that includes a common ancestor and all of its descendants (both living and extinct). An example of a clade of a phylogenetic tree is shown in FIG. 1. An example of a clade of a phylogenetic tree is shown in FIG. 1. An example of a grouping of a phylogenetic tree without a clade is also shown in FIG. 1. A vaccine can include antigens that represent a clade of microorganisms.

[0074] Groups of microorganisms can be divided into clades, or groups on a phylogenetic tree. In some examples, a phylogenetic tree can be an organization of microorganisms that shows how closely related individual microorganisms are to each other.

[0075] Clades can be placed based on the similarity of one or more sequences of antigens of the microorganisms. Clades and subclades on a phylogenetic tree have similar genetic changes such as nucleotide or amino acid changes and can be shown as groups having a single common ancestor (e.g., a common node or branch).

[0076] Genetically distinct clades may have different antigenicity. Clades that differ in antigenicity can refer to clades whose members can have a difference in their ability to affect the immunity of a subject.

[0077] A clade can be a clade of a phylogenetic tree that can be a neighbor-joining clustering tree or a most parsimonious tree. In some examples, a phylogenetic tree can be modeled by a Bayesian model, a maximum likelihood model, a weighted pair group method with arithmetic mean (WPGMA), or an unweighted pair group method with arithmetic mean (UPGMA). The phylogenetic trees provided herein can also be modeled by other suitable statistical models. A neighbor-joining clustering tree can be created using an agglomerative clustering method. A most parsimonious tree can be created to minimize the total number of character state changes.

[0078] A clade can include nodes and branches. A node can be a point or vertex of a phylogenetic tree and can represent a division of one lineage of microorganisms to form two or more lineages (e.g., an internal node within a phylogenetic tree) or the current lineage (e.g., a terminal node). A branch can be a line on a phylogenetic tree and can be used to represent a lineage, whether ancestral or terminal.

[0079] ​A clade may be a node-based clade, a branch-based clade, or an apomorphy-based clade. A node-based clade can be a clade that starts from a specific node of a phylogenetic tree and can include that node, the branches and nodes descending from that node. An example of a node-based clade is shown in the upper panel of Figure 2. A branch-based clade can be a clade that starts from a specific node of a phylogenetic tree and can include that branch, the branches and nodes descending from that branch. An example of a branch-based clade is shown in the central panel of Figure 2. In some examples, the clade may be an apomorphy-based clade. An apomorphy-based clade can be a clade that is derived from an ancestor in which a specific character state, e.g., a feature of an array or structure, occurred. An example of an apomorphy-based clade is shown in the lower panel of Figure 2. The horizontal line of the apomorphy-based clade shown in Figure 2 indicates where the specific character state occurred.

[0080] In some examples, a clade can be a clade of a phylogenetic tree that can be a rooted tree, an unrooted tree, or a bifurcating tree. A phylogenetic tree can be a rooted tree if any unique node can correspond to the most recent common ancestor of all entities on the phylogenetic tree. A phylogenetic tree can be an unrooted tree if it can be created with few assumptions about ancestors. A phylogenetic tree can be a bifurcating tree if each node has exactly two descendants.

[0081] A clade can be described by its degree. The degree of a clade can explain how many clades it contains. A clade of degree X can enclose a clade of degree X+1. For example, a clade of degree 1 can include clades of degree 2, 3, 4, etc., and a clade of degree 2 can include clades of degree 3, 4, 5, etc. A diagram of a first-degree clade, a second-degree clade, and a third-degree clade within the same phylogenetic tree is shown in Figure 3. Here, the first-degree clade includes the second-degree clade, and the second-degree clade includes the third-degree clade.

[0082] Similarly, a clade of degree Y can be included in a clade of degree Y-1. For example, a clade of degree 5 can be included in a clade of degree 4, a clade of degree 6 can be included in a clade of degree 5, and a clade of degree 7 can be included in a clade of degree 6.

[0083] If the clade is a clade of order X, the clade of order X may be phylogenetically below the branching node of X-1 in the phylogenetic tree of the microorganism. For example, a clade of degree 5 can be phylogenetically below 4 branching nodes, a clade of degree 6 can be phylogenetically below 5 branching nodes, and a clade of degree 7 can be phylogenetically below 6 branching nodes.

[0084] The clade of the microorganism can be of degree 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or any other suitable degree. If the clade is a first-degree clade, the whole or part of that clade cannot be included within another higher-level clade.

[0085] For example, influenza subtypes (e.g., influenza A) can be classified into various genetic clades and subclades. Influenza clades can be used to subdivide influenza viruses based on the similarity of the genetic sequences (e.g., hemagglutinin sequences) of the influenza viruses.

[0086] As another example, HIV subtypes (e.g., HIV-1 and / or HIV-2) can be classified into various genetic clades and subclades. HIV clades can be used to subdivide HIV viruses based on the similarity of the genetic sequences (e.g., the env gene encoding the gp160 protein) of the HIV viruses. II. Antigen

[0087] The vaccine composition described in this specification can contain an antigen. The antigen in the vaccine composition can be an antigen of a microorganism or a strain of microorganism capable of inducing an immune response by the vaccine composition.

[0088] An antigen can be a molecule that can be recognized by an immune system including antibodies, B cells, and / or T cells. For example, an antigen can be a foreign substance capable of inducing an immune response. In some examples, the induced immune response can include the production of antibodies. In some examples, antibodies can recognize, attach to, or bind to an antigen.

[0089] An antigen can be an immunogen, such as any antigenic determinant on any primary immunogen to which an antibody binds via an antigen-binding site. The determinant or antigenic determinant on an antigen usually consists of chemically active surface groupings of molecules such as amino acid or sugar side chains and usually has specific three-dimensional structural characteristics as well as specific charge characteristics.

[0090] An antigen can be a neoantigen, self-antigen, endogenous antigen, exogenous antigen, viral antigen, bacterial antigen, fungal antigen, parasitic antigen, toxin, or tumor antigen. Some vaccines can contain an antigen from one source, and some vaccines can contain antigens from 2, 3, 4, or more sources.

[0091] The vaccine can contain an antigen of the strain in Table 1. Some vaccines can contain one or more homologs of one or more antigens of the strain in Table 1. In some examples, such homologs can contain at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the antigen in Table 1.

[0092] The antigen can be an influenza antigen. In some examples, the influenza antigen can be hemagglutinin or neuraminidase. In some examples, the influenza antigen can be a fragment, derivative, or modified form of hemagglutinin or neuraminidase. In certain cases, the vaccine can include a combination of hemagglutinin and neuraminidase, or a fragment, derivative, or modified form thereof. In various examples, the antigen can be a hemagglutinin-neuraminidase complex protein, or a fragment, derivative, or modified form thereof.

[0093] Hemagglutinin can be a homotrimeric glycoprotein that can be found on the surface of influenza virus. Hemagglutinin can be a class 1 fusion protein and can have multifunctional activities as an attachment factor and / or a membrane fusion protein. In some examples, hemagglutinin can play a role in binding the influenza virus to sialic acid on the surface of target cells or host cells. After this binding, the influenza virus can translocate internally. In some examples, hemagglutinin can play a role in the fusion of the viral envelope of the influenza virus and the late endosome membrane in a low pH (e.g., 5.0 - 5.5) environment.

[0094] Hemagglutinin can be from an influenza A or B virus. Hemagglutinin can have a structure including a head and a base and can include three identical monomers. The monomer can include an intact HA0 single polypeptide chain having HA1 and HA2 regions linked by two disulfide bridges. The HA2 region can have an α - helical coiled - coil structure and can be located above the HA1 region. The HA1 region can be a small globular domain that can include a mixture of α / β structures.

[0095] The vaccine composition can include the head of hemagglutinin, the base of hemagglutinin, or a combination or fragment thereof. In some examples, the vaccine composition can include a broadly conserved fragment of hemagglutinin, a broadly conserved fragment of the head of hemagglutinin, a broadly conserved fragment of the base of hemagglutinin, or a combination thereof. A broadly conserved sequence can be an amino acid sequence or a nucleic acid sequence that can be identical or similar across microbial species or strains.

[0096] The vaccine composition can include the HA0 region, the HA1 region, the HA2 region, or a combination or fragment thereof. The hemagglutinin antigen can be of the subtype of hemagglutinin. For example, the hemagglutinin of the hemagglutinin antigen of influenza A virus can be of 18 or more subtypes. The subtypes of hemagglutinin can be H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, or H18.

[0097] Some vaccine compositions can comprise at least 5, at least 10, at least 20, or at least 30 influenza virus hemagglutinin antigen polypeptides. Such hemagglutinin antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 95% identical to other polypeptides in the vaccine, and each polypeptide comprises a target segment that is at least 90% identical within the influenza virus hemagglutinin antigen polypeptides. In some examples, such hemagglutinin antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 95% identical to other polypeptides in the vaccine. In certain cases, such hemagglutinin antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 10% identical and at most 95% identical to other polypeptides in the vaccine. In various examples, such hemagglutinin antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 98% identical to other polypeptides in the vaccine. In some examples, such hemagglutinin antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 10% identical and at most 98% identical to other polypeptides in the vaccine.

[0098] Neuraminidase can be a protein found on the surface of influenza virus. Neuraminidase can enable the release of influenza virus from host cells. Neuraminidase can be a member of glycoside hydrolase family 34. Neuraminidase can be a protein shaped like a mushroom. Neuraminidase can comprise a head portion that can include four substantially spherical subunits in the same plane and a hydrophobic region. In some examples, the hydrophobic region can be embedded within the viral membrane. Neuraminidase can comprise a single polypeptide chain. The neuraminidase polypeptide can be a single chain of six conserved polar amino acids, followed by variable amino acids.

[0099] The vaccine can include spherical subunits of neuraminidase, hydrophobic regions of neuraminidase, or combinations thereof. In some examples, the vaccine composition can include broadly conserved fragments of neuraminidase, broadly conserved fragments of spherical subunits of neuraminidase, broadly conserved fragments of hydrophobic regions of neuraminidase, or combinations thereof.

[0100] In some examples, the neuraminidase antigen can be of a subtype of neuraminidase. The neuraminidase antigen of an influenza virus can be of 11 or more subtypes. The subtypes of neuraminidase can be N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, or N11. In some examples, the neuraminidase antigen can be of a virus other than an influenza virus. In some examples, the neuraminidase can be of a bacterium. For example, the neuraminidase can be of Bacteroides fragilis or Pseudomonas aeruginosa or another bacterium.

[0101] Some vaccine compositions can comprise at least 5, at least 10, at least 20, or at least 30 influenza virus neuraminidase antigen polypeptides. Such neuraminidase antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 95% identical to other polypeptides in the vaccine, and each polypeptide comprises a target segment that is at least 90% identical within the influenza virus neuraminidase antigen polypeptide. In some examples, such neuraminidase antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 95% identical to other polypeptides in the vaccine. In certain cases, such neuraminidase antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 10% identical and at most 95% identical to other polypeptides in the vaccine. In various examples, such neuraminidase antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 98% identical to other polypeptides in the vaccine. In some examples, such neuraminidase antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 10% identical and at most 98% identical to other polypeptides in the vaccine.

[0102] The antigen can be an HIV antigen. In some examples, the HIV antigen can be the glycoprotein gp160. In some examples, the HIV antigen can be a fragment, derivative, or modified form of gp160. In certain cases, the vaccine can comprise a combination of gp160, or a fragment, derivative, or modified form thereof.

[0103] gp160 can be a protein encoded by the env gene of the HIV virus, can form a homotrimer, and can be found on the surface of the HIV virus. gp160 can be cleaved into gp120 and gp41 by proteases such as furin in a subject. gp120 and gp41 can be transported to the cell membrane of the host cell of the subject, where gp41 can anchor gp120 to the membrane of the infected cell. In some examples, gp120 can be an antigen. In some examples, gp41 can be an antigen.

[0104] gp160 can be of HIV-1 or HIV-2. gp160 can include three subunits that can have an extracellular glycoprotein portion attached to a membrane-binding portion. The interface between GP160 subunits can be polar.

[0105] The vaccine composition can include a glycoprotein segment of gp160, a membrane-binding portion of gp160, a broadly conserved fragment of gp160, a broadly conserved fragment of a glycoprotein segment of gp160, a broadly conserved fragment of a membrane-binding portion of gp160, or a combination thereof.

[0106] Some vaccine compositions can contain at least 5, at least 10, at least 20, or at least 30 HIV gp160 antigen polypeptides. Such gp160 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 95% identical to other polypeptides in the vaccine, and each polypeptide contains a target segment that is at least 90% identical within the gp160 antigen polypeptides. In certain cases, such gp160 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 95% identical to other polypeptides in the vaccine. In various examples, such gp160 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 10% identical and at most 95% identical to other polypeptides in the vaccine. In some examples, such gp160 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 98% identical to other polypeptides in the vaccine. In certain cases, such gp160 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 10% identical and at most 98% identical to other polypeptides in the vaccine.

[0107] gp120 can be of HIV-1 or HIV-2. gp120 can contain three subunits that can have an extracellular glycoprotein portion attached to a membrane-bound portion. The interface between GP120 subunits can be polar.

[0108] The vaccine composition can include a glycoprotein segment of gp120, a membrane-bound portion of gp120, a broadly conserved fragment of gp120, a broadly conserved fragment of the glycoprotein segment of gp120, a broadly conserved fragment of the membrane-bound portion of gp120, or a combination thereof.

[0109] Some vaccine compositions can include at least 5, at least 10, at least 20, or at least 30 HIV gp120 antigen polypeptides. Such gp120 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 95% identical to other polypeptides in the vaccine, and each polypeptide includes a target segment that is at least 90% identical within the gp120 antigen polypeptides. In certain cases, such gp120 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 95% identical to other polypeptides in the vaccine. In various examples, such gp120 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 10% identical and at most 95% identical to other polypeptides in the vaccine. In some examples, such gp120 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 98% identical to other polypeptides in the vaccine. In certain cases, such gp120 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 10% identical and at most 98% identical to other polypeptides in the vaccine.

[0110] gp41 can be from HIV-1 or HIV-2. gp41 can include three subunits that can have an extracellular glycoprotein portion attached to a membrane-binding portion. The interface between GP41 subunits can be polar.

[0111] The vaccine composition can include a glycoprotein segment of gp41, a membrane-binding portion of gp41, a broadly conserved fragment of gp41, a broadly conserved fragment of the glycoprotein segment of gp41, a broadly conserved fragment of the membrane-binding portion of gp41, or a combination thereof.

[0112] Some vaccine compositions can include at least 5, at least 10, at least 20, or at least 30 HIV gp41 antigen polypeptides. Such gp41 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 95% identical to other polypeptides in the vaccine, and each polypeptide includes a target segment that is at least 90% identical among the gp41 antigen polypeptides. In certain cases, such gp41 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 95% identical to other polypeptides in the vaccine. In various examples, such gp41 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 10% identical and at most 95% identical to other polypeptides in the vaccine. In some examples, such gp41 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 20% identical and at most 98% identical to other polypeptides in the vaccine. In certain cases, such gp41 antigen polypeptides can be represented by sequences (e.g., amino acid sequences) that are at least 10% identical and at most 98% identical to other polypeptides in the vaccine.

[0113] The antigen can be broadly neutralizing. A broadly neutralizing antigen can be recognized by broadly neutralizing antibodies. Broadly neutralizing antibodies can be antibodies that can affect multiple strains of the virus. Some broadly neutralizing antigens can be broadly neutralizing antigens of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the strains.

[0114] Broadly neutralizing antigens can include surface-exposed residues that are adjacent in three-dimensional space. That is, broadly neutralizing antigens can be neutralized based on their surface properties. In some examples, broadly neutralizing antigens can include neutralizing residues in non-surface regions of the antigen, such as pockets, active sites, or the interior of the antigen.

[0115] Antigens such as broad neutralizing antigens can be antigens of influenza virus. Such broad neutralizing antigens can be located in hemagglutinin. Such broad neutralizing antigens can be present at the base of hemagglutinin, the head of hemagglutinin, or a part thereof. In some examples, the broad neutralizing antigen can be similar to the base of hemagglutinin, the head of hemagglutinin, or a part thereof. In some examples, such a portion can be broadly neutralizing. For example, the broad neutralizing antigen can be at least about 60% similar, at least about 70% similar, at least about 80% similar, at least about 85% similar, or at least about 90% similar to the head of hemagglutinin or the base of hemagglutinin. In some examples, a part of the head of hemagglutinin or a part of the base of hemagglutinin can be at least about 60% similar, at least about 70% similar, at least about 80% similar, at least about 85% similar, or at least about 90% similar. In some examples, a broadly conserved fragment of hemagglutinin, a broadly conserved fragment of the head of hemagglutinin, or a broadly conserved fragment of the base of hemagglutinin can be at least about 60% similar, at least about 70% similar, at least about 80% similar, at least about 85% similar, or at least about 90% similar.

[0116] In some examples, the broad neutralizing antigen can be located in neuraminidase or a part thereof. In some examples, the broad neutralizing antigen can be similar to neuraminidase. For example, the broad neutralizing antigen can be at least about 60% similar, at least about 70% similar, at least about 80% similar, at least about 85% similar, or at least about 90% similar to neuraminidase or a part thereof. In some examples, such a portion can be broadly neutralizing. In some examples, the broad neutralizing antigen can be at least about 60% similar, at least about 70% similar, at least about 80% similar, at least about 85% similar, or at least about 90% similar to a part of neuraminidase. In some examples, the broad neutralizing antigen can be at least about 60% similar, at least about 70% similar, at least about 80% similar, at least about 85% similar, or at least about 90% similar to a broadly conserved fragment of neuraminidase.

[0117] Broadly neutralizing antigens can be those of the HIV virus. In some examples, the broadly neutralizing antigen can be located in gp160 or a portion thereof. In some examples, the broadly neutralizing antigen can be similar to gp160. For example, the broadly neutralizing antigen can be at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, or at least about 90% similar to gp160 or a portion thereof. In certain cases, such portions can be broadly neutralizing. In various examples, the broadly neutralizing antigen can be at least about 60%, at least about 70%, at least about 80%, at least about 85%, or at least about 90% similar to a portion of gp160. In some examples, the broadly neutralizing antigen can be at least about 60%, at least about 70%, at least about 80%, at least about 85%, or at least about 90% similar to a broadly conserved fragment of gp160.

[0118] Broadly neutralizing antigens can be those of the HIV virus. In some examples, the broadly neutralizing antigen can be located in gp120 or a portion thereof. In some examples, the broadly neutralizing antigen can be similar to gp120. For example, the broadly neutralizing antigen can be at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, or at least about 90% similar to gp120 or a portion thereof. In certain cases, such portions can be broadly neutralizing. In various examples, the broadly neutralizing antigen can be at least about 30%, at least 40%, at least 50%, at least 60%, at least about 70%, at least about 80%, at least about 85%, or at least about 90% similar to a portion of gp120. In some examples, such portions can be broadly conserved fragments.

[0119] Broadly neutralizing antigens can be those of the HIV virus. In some examples, the broadly neutralizing antigen can be located in gp41 or a portion thereof. In some examples, the broadly neutralizing antigen can be similar to gp41. For example, the broadly neutralizing antigen can be at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, or at least about 90% similar to gp41 or a portion thereof. In certain cases, such portions can be broadly neutralizing. In various examples, the broadly neutralizing antigen can be at least about 60%, at least about 70%, at least about 80%, at least about 85%, or at least about 90% similar to a portion of gp41. In some examples, such portions can be broadly conserved fragments.

[0120] In some examples, a vaccine can include antigens that correspond to multiple microorganisms. Some vaccines can include antigens against 2, 3, 4, 5, 6, or more microorganisms. The antigens can each be present in the vaccine at a lower dose than currently available vaccines. By a diverse combination of antigens, it is possible to generate B cells and / or T cells that broadly recognize many strains of the microorganism, even strains not included in the vaccine. In some examples, this is sometimes referred to as the herd effect. The herd effect can reduce the dose of each antigen as well as the total antigen dose required to induce an immune response. For example, a vaccine can include antigens that represent a given number of clades of a clade of microorganisms. By including representatives of diverse clades, it may be possible to make the vaccine effective against evolutionarily distinct strains. As another example, a vaccine can include antigens that represent a particular amount of operational taxonomic units (OTUs) of the microorganism. OTUs can be used to classify groups of closely related entities such as antigens.

[0121] An OTU can be a group of microorganisms. Microorganisms within a given OTU can be closely related. An overview of OTUs can be found in Sokal et al. (Principles of Numerical Taxonomy. W.H. Freeman and Co., San Francisco and London (1963)) and Schloss et al. (Applied and Environmental Microbiology, Oct. 2006, p. 6773-6779, Vol. 72, No. 10).

[0122] OTUs generally may refer to clusters of antigens grouped by sequence similarity and may serve as practical surrogates for "species" at different taxonomic levels. By administering a vaccine targeting antigens that represent the clades of diverse OTUs, immunity can be conferred to the entire set of genetically diverse antigens.

[0123] In some examples, an OTU can be based on a specific nucleic acid or amino acid sequence of a microorganism. For example, an OTU can be based on the sequence of 16S rRNA or the sequence of the gene encoding 16S rRNA. Such an OTU can be called a 16S OUT. In various examples, an OTU can be based on the sequence of 18S rRNA or the sequence of the gene encoding 18S rRNA.

[0124] An OTU can be a group of microorganisms with a threshold similarity. For example, an OUT can be a group of microorganisms having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity. In some examples, different microorganisms can have a similarity higher than the threshold. Such microorganisms can be present in a merged OTU that can include multiple species. In a particular case, a single species of microorganism can have paralogs with a similarity lower than the threshold. Such paralogs can be split into two or more OTUs.

[0125] Methods for assigning OTUs can include rarefaction curves. Such methods can evaluate species richness and alpha and beta diversity estimators, and it is implicitly assumed that the OTUs are biological observations with negligible error and / or that the number of observations is well correlated with the total number of monophyletic groups.

[0126] In some examples, for instance, due to artifacts such as sequencing errors and / or chimeras, clusters can be spurious. In certain cases, an OTU can be based on the genetic distance between the sequences of the microorganisms within the OTU. In various examples, an OTU can provide an estimate of the richness and / or diversity of a microbial population. In some examples, the microorganisms belonging to a given OTU can have structural similarities. In certain cases, the microorganisms belonging to different OTUs can have different structures.

[0127] Microbial sequences can be assigned to OTUs using software or tools. Examples of such tools include, but are not limited to, the Distance-Based OTU and Richness (DOTUR) tool, the Shared OTU and Similarity (SONS) tool, the LIBSHUFF / ∫-LIBSHUFF tool, the TreeClimber tool, the UniFrac tool, or the Analysis of Molecular Variance (AMOVA) tool.

[0128] Based on an input containing information on multiple microbial sequences, the tool can assign microbial sequences to OTUs. Examples of such inputs can include a distance matrix, a phylogenetic tree, and / or a distance matrix. The DOTUR tool can assign sequences to OTUs by using the farthest neighbor algorithm, the average neighbor algorithm, or the nearest neighbor algorithm for one, several, or each distance level. The DOTUR tool can assign OTUs based on an input of a distance matrix. The SONS tool can calculate an incidence curve to estimate the proportion and abundance of OTUs shared among communities. The SONS algorithm can assign OTUs based on an OTU designation input.

[0129] The LIBSHUFF / ∫-LIBSHUFF tool can use the Cramer-von Mises statistic to test whether the structures of two communities are the same, different, or subsets of each other. The LIBSHUFF / ∫-LIBSHUFF tool can assign OTUs based on an input of a distance matrix. The TreeClimber tool can implement a parsimony-based test to determine whether the community structures of two or more communities are significantly different. The TreeClimber tool can assign OTUs based on an input of a phylogenetic tree.

[0130] The UniFrac tool can compare the phylogenetic distances between pairs of communities to describe the similarity of their structures. The UniFrac tool can assign OTUs based on an input of a phylogenetic tree. The AMOVA tool can use an analysis of variance formulation to determine whether the genetic diversities of two or more community structures are significantly different. The AMOVA tool can assign OTUs based on an input of a distance matrix.

[0131] OTUs generally refer to clusters of antigens grouped by sequence similarity and can sometimes serve as practical proxies for "species" at different taxonomic levels. By including representative clades of diverse OTUs, a set of genetically diverse antigens can be included. Each OTU can contain similar microbial sequences. Each OTU can contain microbial sequences that are at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to each other. Each OTU can contain microbial sequences that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to each other.

[0132] In further examples, a set of antigens derived from a library of variants may be included such that the set of antigens broadly represents the library of variants. In some examples, a set of antigens derived from a library of variants may be included such that the set of antigens broadly represents at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% of the library of variants. In some examples, such a set of antigens can further represent one or more variants not included in the library of variants.

[0133] The antigen may be a single peptide or protein of interest, or a complex of 2, 3, 4, 5, 6, or more peptides and / or proteins. In some examples, the antigen may refer to an antigen domain, or a region or section of a larger protein or protein complex. In some examples, the antigen may refer to 2 or more antigen domains, or 2 or more regions or sections of a larger protein or protein complex. In some examples, the antigen may be a whole protein, a fragment of a whole protein, a functional fragment of a whole protein, a peptide, a multimeric polypeptide, a polypeptide sequence, or a combination thereof. In some examples, the antigen may be modified (e.g., artificially modified). Such modifications can stabilize or improve antigenicity. Examples of modifications include changes to the polypeptide sequence (e.g., truncation, extension, or mutation). In some examples, the antigen may comprise 2 or more whole proteins, fragments of whole proteins, functional fragments of whole proteins, peptides, or multimeric polypeptides. Sometimes, the antigen may comprise a lipid, nucleic acid, polysaccharide, or a combination thereof.

[0134] The antigen can be a native (i.e., whole or entire) antigen or a functional portion of the antigen. The antigen can be a peptide functional portion of the antigen. As used herein, "native" generally refers to the full-length antigen as it exists in nature. A native antigen adopts the natural protein folding as seen in nature and can present both primary sequence epitopes as well as three-dimensional conformational epitopes. This can be in stark contrast to the delivery of only a small part or only peptides of the antigen. By delivering a native antigen to a cell, a broad immune response can be induced rather than an immune response to only a single or selected few epitopes. In some examples, at the time of vaccine or library development, one, several, or all of the epitopes on the antigen may be unknown.

[0135] Alternatively, depending on the size of the initial antigen, the antigen can be divided into parts. Generally, when the whole antigen is a multimeric polypeptide, the whole protein can be divided into subunits and / or domains, and the individual subunits or domains of the antigen can be associated with a polymer according to the methods disclosed herein. In some examples, the part of the antigen can be a functional fragment or portion.

[0136] The vaccine can include homologs of the antigen (e.g., hemagglutinin homolog, neuraminidase homolog, gp160 homolog, gp120 homolog, or gp41 homolog). A homolog can be a molecule having a common ancestor with the antigen. In some examples, a homolog of the antigen can be the antigen.

[0137] In some examples, the antigen can be modified. Modifications can include truncation, tagging, amino acid addition, glycosylation, methylation, ubiquitination, insertion, deletion, mutation, or other modifications. For example, the HIV env-glycoprotein complex (trimer) is usually artificially mutated, such as by disulfide bonds, to keep the antigen stable and / or prevent or delay the dissociation of the protein derived from HIV env. This can include, but is not limited to, artificial cleavage sites, removal of entire functional regions (e.g., cleavage of transmembrane regions).

[0138] In some examples, the antigen can be present at a lower concentration than currently available vaccines. In some examples, the antigen can be present at a concentration that is insufficient to induce an immune response when the antigen is administered alone. In some examples, each antigen in the vaccine can be present at a concentration that is insufficient to induce an immune response when that antigen is administered alone. In such examples, it can be the total antigen concentration, rather than the concentration of an individual antigen, that is the determinant of whether an immune response is induced. When the total antigen concentration is the determinant, the required concentration of an individual antigen can be a function of the total concentration or a function of the number of antigens.

[0139] In some examples, one or more antigens can provide immunity to a subject against a wide variety of variants of a microorganism. For example, the antigen can provide immunity against variants of a microorganism that are variants based on year of origin, species of origin, specific mutations, or any other suitable variant basis.

[0140] Two antigens within a set of antigens can have an edit distance (e.g., pairwise edit distance). The edit distance can describe the difference between the two antigens. The edit distance can be the Levenshtein distance. The Levenshtein distance between a first antigen and a second antigen can be the minimum number of edits required to change the first antigen into the second antigen. In calculating the edit distance, the permitted edits can include insertions, deletions, and / or substitutions. If the first antigen and the second antigen are identical, the Levenshtein distance can be zero. If the first antigen and the second antigen are the same size, the Levenshtein distance can be at most the size of the first antigen. If the sizes of the first antigen and the second antigen are different, the Levenshtein distance can be at least the difference in size between the first antigen and the second antigen.

[0141] Another way to calculate the edit distance can be the longest common subsequence method, in which only insertions and deletions are permitted edits in the calculation. Yet another way to calculate the edit distance can be the Hamming distance, in which only antigens of the same size can be used.

[0142] The minimum edit distance between two or more antigens can be determined. In some examples, the minimum edit distance can be the minimum edit distance between any two of the two or more antigens. The minimum edit distance can be a measure of the similarity of the two or more most similar antigens within the set.

[0143] The maximum edit distance between two or more antigens can be determined. In some examples, the maximum edit distance can be the maximum edit distance between any two antigens of two or more antigens. The maximum edit distance can be a measure of the similarity of the two or more most different antigens within the set.

[0144] Both the minimum pairwise edit distance and the maximum pairwise edit distance between two or more antigens within the set can be measured or described. The combination of both edit distances can describe the most similar and the least similar antigens within the set and, in some examples, can serve as a measure of the boundaries of the various antigens within the set.

[0145] The edit distance can be measured as a percentage of the size of the antigen. As used herein, the size of the antigen can be the average size of the antigen, the maximum size of the antigen, the minimum size of the antigen, or another measure of the size of the antigen. For example, for two antigens each of size 100 amino acids and an edit difference of 5, the pairwise edit distance can be expressed as 5%.

[0146] A vaccine can include a set of antigens of a microorganism where the minimum pairwise edit distance between two antigens is less than a particular threshold. A vaccine can include a set of antigens where the maximum pairwise edit distance between two antigens is at least a different particular threshold. In some examples, requiring a minimum and maximum difference between the included antigens can help ensure that immunity is conferred across a variety of antigens with minimal bias.

[0147] A library or vaccine can be described by the difference between the maximum edit distance and the minimum edit distance. Two antigens of a set with the maximum edit distance can have an edit distance called "S". Two antigens of a library with the maximum edit distance can have an edit distance called "L". The difference between "S" and "L" can indicate the range of differences in the antigens present in the vaccine. The greater the difference, the greater the range of antigen differences, or the greater the diversity. In some examples, "S" can be at least 60%, 70%, 80%, or 90% of "L". In other words, the antigens with the most different edit distances in the vaccine composition can fall within the top 10%, 20%, 30%, or 40% of the antigens with different edit distances in the library.

[0148] The average number of nodes between antigens can be calculated for a given vaccine. In some examples, the average number of nodes between each antigen within a set of antigens in the vaccine can be at least 1, at least 3, at least 5, at least 10, at least 15, or at least 20. In some examples, the average number of nodes between each antigen can be a function of the number of antigens in the set. Sometimes, when the number of antigens in the set is large, the average number of nodes between each antigen can be low.

[0149] The antigens in a vaccine can be compared using the number of nodes that separate them on a phylogenetic tree. For example, closely related antigens can be separated by fewer nodes than antigens that are not as closely related. When describing the number of nodes that separate two antigens, antigens separated by fewer nodes are more likely to have a more closely related structure or sequence, while antigens separated by more nodes are more likely to have a less closely related or less similar structure or sequence. For example, in some examples, two antigens in a vaccine can be separated by at least 1, at least 3, at least 5, at least 10, at least 15, or at least 20 nodes.

[0150] The minimum number of nodes between any two antigens can be 1 for some vaccines. The maximum number of nodes between any two antigens can be at least 5, at least 10, at least 15, or at least 20. For some vaccines, the minimum number of nodes between any two antigens can be 1 and the maximum number of nodes between any two antigens can be at least 5. For some vaccines, the minimum number of nodes between any two antigens can be 1 and the maximum number of nodes between any two antigens can be at least 10. For some vaccines, the minimum number of nodes between any two antigens can be 1 and the maximum number of nodes between any two antigens can be at least 15. For some vaccines, the minimum number of nodes between any two antigens can be 1 and the maximum number of nodes between any two antigens can be at least 20. For some vaccines, the minimum number of nodes between any two antigens can be less than 5 and the maximum number of nodes between any two antigens can be at least 5. For some vaccines, the minimum number of nodes between any two antigens can be less than 5 and the maximum number of nodes between any two antigens can be at least 10. For some vaccines, the minimum number of nodes between any two antigens can be less than 5 and the maximum number of nodes between any two antigens can be at least 15. For some vaccines, the minimum number of nodes between any two antigens can be less than 5 and the maximum number of nodes between any two antigens can be at least 20. For some vaccines, the minimum number of nodes between any two antigens can be less than 10 and the maximum number of nodes between any two antigens can be at least 10. For some vaccines, the minimum number of nodes between any two antigens can be less than 10 and the maximum number of nodes between any two antigens can be at least 15. For some vaccines, the minimum number of nodes between any two antigens can be less than 10 and the maximum number of nodes between any two antigens can be at least 20.

[0151] The average number of branches between antigens can be calculated for a given vaccine. In some examples, the average number of branches between each antigen within a set of antigens in a vaccine can be at least 1, at least 3, at least 5, at least 10, at least 15, or at least 20. In some examples, the average number of branches between each antigen can be a function of the number of antigens in the set. Sometimes, when the number of antigens in the set is large, the average number of branches between each antigen can be low.

[0152] The antigens in a vaccine can be compared using the number of branches that separate them on a phylogenetic tree. For example, closely related antigens can be separated by fewer branches than antigens that are not as closely related. When describing the number of branches that separate two antigens, antigens separated by fewer branches are more likely to have a more closely related structure or sequence, while antigens separated by more branches are less likely to have a closely related structure or sequence. For example, in some examples, two antigens in a vaccine can be separated by at least 1, at least 3, at least 5, at least 10, at least 15, or at least 20 branches.

[0153] The minimum number of branches between any two antigens can be 1 in some vaccines. The maximum number of branches between any two antigens can be at least 5, at least 10, at least 15, or at least 20. In some vaccines, the minimum number of branches between any two antigens can be 1, and the maximum number of branches between any two antigens can be at least 5. In some vaccines, the minimum number of branches between any two antigens can be 1, and the maximum number of branches between any two antigens can be at least 10. In some vaccines, the minimum number of branches between any two antigens can be 1, and the maximum number of branches between any two antigens can be at least 15. In some vaccines, the minimum number of branches between any two antigens can be 1, and the maximum number of branches between any two antigens can be at least 20. In some vaccines, the minimum number of branches between any two antigens can be less than 5, and the maximum number of branches between any two antigens can be at least 5. In some vaccines, the minimum number of branches between any two antigens can be less than 5, and the maximum number of branches between any two antigens can be at least 10. In some vaccines, the minimum number of branches between any two antigens can be less than 5, and the maximum number of branches between any two antigens can be at least 15. In some vaccines, the minimum number of branches between any two antigens can be less than 5, and the maximum number of branches between any two antigens can be at least 20. In some vaccines, the minimum number of branches between any two antigens can be less than 10, and the maximum number of branches between any two antigens can be at least 10. In some vaccines, the minimum number of branches between any two antigens can be less than 10, and the maximum number of branches between any two antigens can be at least 15. In some vaccines, the minimum number of branches between any two antigens can be less than 10, and the maximum number of branches between any two antigens can be at least 20.

[0154] Vaccines can be described by their sequence identity. As used herein, the term "identical" or "identity" percent in the context of two or more nucleic acid or polypeptide sequences generally refers to two or more sequences or subsequences that are the same or have a particular percentage of amino acid residues or nucleotides that are the same.

[0155] Alternatively, an indication that two nucleic acid sequences or polypeptides are identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, for example, if two peptides differ only by conservative substitutions, the polypeptides are generally identical to the second polypeptide. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described herein.

[0156] Alternatively, an indication that two nucleic acid sequences or polypeptides are identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, for example, if two peptides differ only by conservative substitutions, the polypeptides are generally identical to the second polypeptide. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described herein.

[0157] Alternatively, an indication that two nucleic acid sequences or polypeptides are identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, for example, if two peptides differ only by conservative substitutions, the polypeptides are generally identical to the second polypeptide. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described herein. In some examples, each antigen in a set can share at least 90%, at least 95%, or at least 99% sequence identity with at least one antigen in the set. In some examples, sequence identity can be a measure of how similar or different the sequences of the antigens in a vaccine are.

[0158] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein and generally refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been naturally modified or modified by any other manipulation or modification, such as, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation to a labeling component. Also included in this definition are polypeptides containing, for example, one or more analogs of amino acids (including, for example, non-natural amino acids), as well as other suitable modifications. Since the polypeptides of the present disclosure are antibody-based, it is understood that the polypeptides can exist as single chains or related chains.

[0159] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Polynucleotides may include modified nucleotides such as methylated nucleotides and their analogs. Where present, modifications to the nucleotide structure may be imparted before or after construction of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution with one or more naturally occurring nucleotide analogs, internucleotide modifications such as those by uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) and charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those by intercalators (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those containing modified linkages (e.g., α-anomeric nucleic acids, etc.), as well as the unmodified forms of one or more polynucleotides.

[0160] Furthermore, any of the hydroxyl groups normally present in the sugar can be replaced with, for example, a phosphonic acid group, a phosphate group, protected with a standard protecting group, or activated to create an additional bond to an additional nucleotide, or can be attached to a solid support. The OH at the 5' and 3' ends can be phosphorylated or substituted with an amine or a moiety of an organic capping group of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized with standard protecting groups. The polynucleotide can include ribose or deoxyribose sugars in analogous forms including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomer sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments where the phosphate is replaced with P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR’, CO, or CH2 (“formacetal”), where each R or R’ is independently H or a substituted or unsubstituted alkyl (1-20C) optionally containing an ether (--O--) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all of the bonds of the polynucleotide need to be the same. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0161] "Identity", "sameness", or "similarity" can refer to sequence similarity between two peptides or two nucleic acid molecules. Identity, similarity, and sameness can each be determined by comparing the positions of each sequence that can be aligned for the purposes of comparison. When equivalent positions of the compared sequences are occupied by the same base or amino acid, the molecules are identical at that position, and when equivalent sites are occupied by the same or similar amino acid residues (e.g., residues having similar steric and / or electronic properties), the molecules can be referred to as homologous (similar) at that position. The expression as a percentage of identity / similarity or sameness refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. "Unrelated" or "non-homologous" sequences can share less than 40% identity, or less than 25% identity, with the sequences of the present disclosure. When comparing two sequences, identity and homology / similarity are also decreased if there are residues (amino acids or nucleic acids) missing or extra residues present.

[0162] The term "identity" describes a mathematically based comparison of sequence similarity used to identify genes or proteins having similar functions or motifs. The sequence may be used, for example, as a "query sequence" to perform a search against public databases to identify other family members, related sequences or homologs. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. A BLAST nucleotide search can be performed using the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. BLAST amino acids can be performed using the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the protein molecules of the present disclosure. To obtain a gap alignment for comparison purposes, gapped BLAST as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402 can be utilized. When using the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and BLAST) can be used (see www.ncbi.nlm.nih.gov).

[0163] As used herein, "identity" can mean the percentage of identical nucleotides or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity is not limited to, but includes, Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). It can be easily calculated by known methods including the methods described in these references. The methods for determining identity are designed to give the maximum match between the sequences being tested. Further, the methods for determining identity are embodied in publicly available computer programs. Computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990) and Altschul et al., Nuc. Acids Res. 25:3389-3402 (1997)). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)). Identity may be determined using the well-known Smith Waterman algorithm.

[0164] The desirable range of sequence identity is from about 80% to about 100%, and integer values in between. In general, this disclosure encompasses sequences having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any sequence described herein.

[0165] The letter "X" used in the amino acid sequences herein is intended to indicate that any of the 20 standard amino acids may be positioned at this location, unless otherwise specified. III. Vaccine

[0166] A vaccine can be a composition capable of stimulating the immune system. This can include stimulation of antibody production and can provide immunity against one or more microorganisms. A vaccine can be prepared from a microorganism that can be a disease-causing agent, its product, or a synthetic substitute thereof. Examples of microorganisms are described above.

[0167] A vaccine can be prepared as a pharmaceutical composition. A pharmaceutical composition can be a formulation of a vaccine containing an antigen in a form that can be administered to a subject. In some examples, the pharmaceutical composition can be ready for use. The pharmaceutical composition can be optimized with respect to ease of use, minimization of adverse reactions, ease of storage, storage temperature range, ease of dosing, ease of manufacture, or any other suitable factor.

[0168] The pharmaceutical composition can comprise the vaccine composition described herein and a pharmaceutically acceptable diluent, adjuvant, additive, or any combination thereof. In some examples, the pharmaceutical composition can comprise a diluent. The diluent can be a diluting agent. The diluent can help to lower the viscosity or density of the vaccine, for example, to improve its fluidity when passing through a needle. In some examples, the diluent can be pre-mixed with the vaccine. In some examples, the diluent can be provided separately from the vaccine and mixed prior to administration.

[0169] In some examples, the pharmaceutical composition can comprise an adjuvant. The adjuvant can bring about an improvement in immunogenicity. The adjuvant can bring about a slow release of the antigen (for example, the adjuvant can be a liposome) or can itself be immunogenic and thus be an adjuvant that functions synergistically with the antigen. For example, the adjuvant can be a known adjuvant or other substance that promotes the uptake of nucleic acids, recruits immune system cells to the site of administration, or boosts the immunostimulation of responsive lymphocyte cell lines. Adjuvants include, but are not limited to, immunomodulatory molecules (for example, cytokines), oil-based and water-based emulsions, aluminum hydroxide, glucan, dextran sulfate, iron oxide, sodium alginate, Bacto-Adjuvant, synthetic polymers such as polyamino acids and amino acid copolymers, saponins, paraffin oil, PS-GAMP, and muramyl dipeptide.

[0170] In some examples, the adjuvant can be an immunomodulatory molecule. For example, the immunomodulatory molecule can be a recombinant protein cytokine, chemokine, or immunostimulant, or a nucleic acid encoding a cytokine, chemokine, or immunostimulant designed to enhance the immune response.

[0171] The pharmaceutical composition described herein can be packaged in virus-like particles (VLPs). In some examples, the virus-like particles can comprise the vaccine.

[0172] In certain cases, the pharmaceutical composition can include additives. In various examples, the additives can be inert substances that can serve as a vehicle or medium for the vaccine. The additives can include binders, coating agents, coloring agents, disintegrants, flavors, flow promoters, lubricants, preservatives, adsorbents, sweeteners, vehicles, or any combination thereof.

[0173] The vaccine composition or pharmaceutical composition herein can be formulated into one of several formulations. In some examples, the vaccine can be in the form of an aerosol formulation, an injectable formulation, an oral formulation, or any other suitable formulation.

[0174] The vaccine composition provided herein can be formulated as a unit dose. The unit dose can be a dose of the vaccine appropriate for one subject. In some embodiments, the vaccine can be packaged as a single unit dose. In some other embodiments, the vaccine can be packaged as multiple unit doses.

[0175] The immune response that occurs in response to the vaccine can depend on the total amount of antigen rather than the amount of each antigen present. In some examples, each of the antigens can be at a concentration that alone does not result in a significant immune response against a broadly neutralizing antigen of the subject. Such an immune response can include mobilization and / or activation in the case of immune cells such as B cells or T cells. In some examples, such an immune response can be a prophylactic immune response. In some examples, the antigen can have a combined concentration that provides an immune response against a broadly neutralizing antigen of the subject. IV. Method of Use

[0176] Provided herein are methods for treating or reducing adverse events resulting from infection and / or exposure to a microorganism or toxin. Also provided herein are methods for suppressing or reducing the likelihood of a disease or condition resulting from a microorganism or toxin in a subject. The method can include administering to the subject a vaccine composition described herein. In some examples, the methods described herein can confer immunity to a disease or condition caused by a microorganism in the subject.

[0177] The terms "treating" or "treatment" are used interchangeably herein. These terms generally refer, herein, to an approach for obtaining a beneficial or desirable result, which result includes, but is not limited to, a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit can mean eradication or amelioration of the underlying disease being treated. Also, a therapeutic benefit can be achieved by eradication or amelioration of one or more physiological conditions associated with the underlying disease such that improvement is observed in the subject even though the subject still suffers from the underlying disease. Prophylactic effects include delaying, preventing, or eliminating the occurrence of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, delaying, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or a subject reporting one or more physiological symptoms of a disease, may be treated even if the disease has not been diagnosed.

[0178] The subject to whom a vaccine is administered herein can be a mammal, a bird, or any other suitable subject. In some examples, the subject can be a human. In certain cases, the subject can be a pig, or another domesticated animal, such as a cow, a goat, a sheep, a horse, a chicken, a duck, a turkey, a parrot, a monkey, a hamster, a guinea pig, a rat, a mouse, a dog, or a cat.

[0179] Such subjects can be administered the vaccine orally, as an aerosol, by injection, or in combinations thereof. In some examples, multiple doses of the vaccine may be administered. In some examples, a subject can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of the vaccine. Booster administrations can be performed on the same day, or at intervals of at least 1 day, at least 1 week, at least 1 month, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, or at least 10 years. In some examples, booster administrations can be performed as a booster immunization and can be the same dose, a higher dose, or a lower dose than the previous dose. A booster immunization can be a booster immunization after a primary stimulation.

[0180] Individual doses of the vaccine for which booster administrations are performed can contain the same antigen or different antigens. If the individual doses contain different antigens, they can differ by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens. In some examples, if the individual doses contain different antigens, the difference can be to maximize exposure to as many different antigens as possible. In some examples, if the individual doses contain different antigens, the additional dose can be an "update" dose that can confer immunity against newly evolved strains, adapted strains, or resistant strains.

[0181] In certain embodiments, the vaccine to be injected can be formulated as a subcutaneous injection. In some examples, the vaccine to be injected can be formulated for intramuscular, intraperitoneal, intravenous, intradermal, or any other suitable type of injection. The vaccine can be administered to healthy subjects, non-healthy subjects, or subjects of unknown health status. In some examples, the vaccine can be more effective when the subject is healthy. The vaccine can contain a recombinant expression vector. In some examples, the vaccine can be produced by a recombinant expression vector. In various examples, the vaccine can be stored as a recombinant expression vector.

[0182] In some examples, the recombinant expression vector can include a set of antigens representing at least 60% of each of the primary and secondary clades of a microorganism; a set of antigens where the minimum pairwise edit distance between two antigens is 25 or less and the maximum pairwise edit distance between two antigens is at least 300; a set of antigens representing at least 60% of the OTUs of a microorganism; or a nucleic acid molecule encoding a set of antigens representing at least 60% of the OTUs of a microorganism.

[0183] In certain cases, the recombinant expression vector can include a nucleic acid molecule encoding a recombinant expression vector that includes a set of antigens representing at least 60% of each of the primary and secondary clades of a microorganism; a set of antigens where the minimum pairwise edit distance between two antigens is 5% or less of the size of the antigen and the maximum pairwise edit distance between two antigens is at least 75% of the size of the antigen; a set of antigens representing at least 60% of all of the OTUs of a microorganism; or a nucleic acid molecule encoding a set of antigens representing at least 60% of all of the OTUs of a microorganism.

[0184] In various examples, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens representing at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of each of the primary clades of a microorganism. In some examples, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens representing at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of each of the secondary clades of a microorganism. In certain cases, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens representing at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of each of the tertiary clades of a microorganism.

[0185] In some examples, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the minimum pairwise edit distance between two antigens is 1% or less, 5% or less, 10% or less, 15% or less, or 20% or less of the size of the antigen. In some examples, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the maximum pairwise edit distance is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the size of the antigen. In some examples, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the minimum pairwise edit distance between two antigens is 1% or less and the maximum pairwise edit distance is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the size of the antigen. In certain cases, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the minimum pairwise edit distance between two antigens is 5% or less and the maximum pairwise edit distance is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the size of the antigen. In various examples, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the minimum pairwise edit distance between two antigens is 10% or less and the maximum pairwise edit distance is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the size of the antigen. In some examples, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the minimum pairwise edit distance between two antigens is 15% or less and the maximum pairwise edit distance is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the size of the antigen. In certain cases, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the minimum pairwise edit distance between two antigens is 20% or less and the maximum pairwise edit distance is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the size of the antigen.

[0186] In some examples, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the minimum pairwise edit distance between two antigens is at least 1%, at least 5%, at least 10%, at least 15%, or at least 20% of the size of the antigen. In certain cases, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the maximum pairwise edit distance is 60% or less, 70% or less, 80% or less, 90% or less, 95% or less, or 98% or less of the size of the antigen. In various examples, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the minimum pairwise edit distance between two antigens is at least 1% and the maximum pairwise edit distance is 60% or less, 70% or less, 80% or less, 90% or less, 95% or less, or 98% or less of the size of the antigen. In certain cases, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the minimum pairwise edit distance between two antigens is at least 5% and the maximum pairwise edit distance is 60% or less, 70% or less, 80% or less, 90% or less, 95% or less, or 98% or less of the size of the antigen. In various examples, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the minimum pairwise edit distance between two antigens is at least 10% and the maximum pairwise edit distance is 60% or less, 70% or less, 80% or less, 90% or less, 95% or less, or 98% or less of the size of the antigen. In some examples, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the minimum pairwise edit distance between two antigens is at least 15% and the maximum pairwise edit distance is 60% or less, 70% or less, 80% or less, 90% or less, 95% or less, or 98% or less of the size of the antigen. In certain cases, the recombinant expression vector can include a nucleic acid molecule encoding a set of antigens where the minimum pairwise edit distance between two antigens is at least 20% and the maximum pairwise edit distance is 60% or less, 70% or less, 80% or less, 90% or less, 95% or less, or 98% or less of the size of the antigen.

[0187] A method for preparing a vaccine composition can include selecting a set of antigens that represent at least 60% of the primary and secondary clades of a microorganism, where the minimum pairwise edit distance between two antigens is 5% or less of the size of the antigen, and the maximum pairwise edit distance is at least 75% of the size of the antigen, and represents at least 60% of all OTUs of the microorganism, or represents at least 90% of all OTUs of the microorganism.

[0188] In some examples, a method for preparing a vaccine composition can include selecting a set of antigens that represent at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of each of the primary clades of a microorganism. In certain cases, a method for preparing a vaccine composition can include selecting a set of antigens that represent at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of each of the secondary clades of a microorganism. In various examples, a method for preparing a vaccine composition can include selecting a set of antigens that represent at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of each of the tertiary clades of a microorganism.

[0189] In some examples, a method for preparing a vaccine composition can include selecting a set of antigens where the minimum pairwise edit distance between two antigens is 1% or less, 5% or less, 10% or less, 15% or less, or 20% or less of the size of the antigen. In certain cases, a method for preparing a vaccine composition can include selecting a set of antigens where the maximum pairwise edit distance is at least 60%, at least 70%, at least 80%, or at least 90% of the size of the antigen. In various examples, a method for preparing a vaccine composition can include selecting a set of antigens where the minimum pairwise edit distance between two antigens is 1% or less and the maximum pairwise edit distance is at least 60%, at least 70%, at least 80%, or at least 90% of the size of the antigen. In some examples, a method for preparing a vaccine composition can include selecting a set of antigens where the minimum pairwise edit distance between two antigens is 5% or less and the maximum pairwise edit distance is at least 60%, at least 70%, at least 80%, or at least 90% of the size of the antigen. In certain cases, a method for preparing a vaccine composition can include selecting a set of antigens where the minimum pairwise edit distance between two antigens is 10% or less and the maximum pairwise edit distance is at least 60%, at least 70%, at least 80%, or at least 90% of the size of the antigen. In various examples, a method for preparing a vaccine composition can include selecting a set of antigens where the minimum pairwise edit distance between two antigens is 15% or less and the maximum pairwise edit distance is at least 60%, at least 70%, at least 80%, or at least 90% of the size of the antigen. In some examples, a method for preparing a vaccine composition can include selecting a set of antigens where the minimum pairwise edit distance between two antigens is 20% or less and the maximum pairwise edit distance is at least 60%, at least 70%, at least 80%, or at least 90% of the size of the antigen.

Example

[0190] V. Example For example, a vaccine has been developed that can be different from (and / or provide advantages compared to) typical commercially available vaccines in at least one of several methods, including: (1) containing a large number (e.g., 6 or more) of antigens, such as 30 antigens; (2) having relatively small amounts of individual antigens, such as 0.05 μg of each antigen, enabling a relatively low total antigen content; and (3) the breadth of the immunogenic response induced by the vaccine against a shared site, e.g., each of the 30 antigens is 0.05 μg, but the shared site is effective at a high dose 30 times that, or 1.5 μg.

[0191] In the following examples, influenza vaccine compositions were administered to pigs. Several influenza vaccine compositions were used. The vehicle contained squalene and phosphate buffered saline for all compositions. The vehicle control composition contained squalene and phosphate buffered saline. A bivalent control influenza vaccine designed to mimic a commercially available influenza vaccine contained either two influenza antigen variants: H1N1_2007 (A / Brisbane / 2007) (5 μg) and H3N2_1997 (A / Sydney / 5 / 1997) or H3N2_2007 (A / Brisbane / 2007) (5 μg). The first single antigen influenza vaccine composition contained 50 ng of the first hemagglutinin variant (H1N1_2007; A / Brisbane / 2007). The second single antigen influenza vaccine composition contained 50 ng of the second hemagglutinin variant (H3N2_2007; A / Brisbane / 2007). The 30 antigen influenza vaccine composition contained 30 influenza antigens (30 hemagglutinin variants) representative of influenza antigens from 1918 to 2015 selected as described in Example 4. The 27 antigen influenza vaccine composition contained 27 influenza antigens (27 hemagglutinin variants) representative of influenza antigens from 1918 to 2008. The 30 antigen influenza vaccine composition was also employed in a general vaccine composition test. The 27 antigen influenza vaccine was employed to test the protective ability of the vaccine against "future" strains. This can perhaps be explained as the ability of the vaccine to provide protection against antigen variants that do not yet exist but may exist in the future.

[0192] The following examples are representative of embodiments of the compositions and methods described herein and are in no way meant to be limiting. Example 1: Quantification of the number of innate antibodies induced after vaccination

[0193] An adult can contain a repertoire of approximately 100 million unique B cell receptors (BCRs). After influenza vaccination, approximately 1,000 unique BCRs can be induced to initiate affinity maturation. Serological analysis has shown that approximately 10% of these can be efficiently converted into productive plasma cells. This can result in influenza vaccination that can provide serological protection with approximately 100 unique antibodies. This concept is shown in Figure 4. Example 2: Estimation of the number of unique B cell epitopes on hemagglutinin

[0194] Human antibodies can contact approximately 20 - 30 residues on the antigen surface they recognize. In some examples, approximately 12 - 16 such residues can form an epitope that can be called a "potentially important epitope". In some examples, such an epitope can be important for maintaining antigen recognition by the antibody.

[0195] Considering that approximately 100 unique antibodies are induced by vaccination from about 1,000 B cell lineages, the following questions arose: (1) How many unique epitopes are present on the surface of the influenza hemagglutinin protein (HA)? (2) If those epitopes are conserved, what is the probability that individual antibodies will cross - react widely? (3) For the approximately 100 unique antibodies as a whole, what are the probabilities and proportions that the vaccine response of the subject includes broad - spectrum protective antibodies?

[0196] To generate a database of unique potentially important epitopes on the surface of the influenza hemagglutinin protein (HA), Zdock was used with a representative HA structure (PDB Deep computational protein - protein docking of 559 crystallized human antibody structures was performed against ID:3FKU). This resulted in a set of 3,718,346 unique contact residue sets, each containing 25 + / - 12 HA - contacting residues. Next, repeated random subsampling of 14 contact residues from the unique contact residue sets on HA that could potentially form important epitopes was performed, resulting in a database of 263,022,674 unique 14 - residue potential important epitopes that can define the binding determinants of a single antibody. Although not exhaustive, this database provides a basis for modeling the epitope conservation probability of human antibodies that bind to HA. This concept is shown in Figure 4. Example 3: Quantification of Amino Acid Conservation of All Hemagglutinin Epitopes

[0197] The conservation of 263 million potential important epitopes was analyzed across a panel of 82 HA protein sequences from strains spanning 1918 to 2018. This analysis identified that the potentially important epitopes that are universally conserved across all HAs are less than 1 in 1 million. As shown in Figure 5, it was observed that potentially important epitopes are conserved at a rate of approximately 1 in 300,000 and are 90% conserved across all strains, while the rate of being universally conserved within a given HA antigen group was observed to be between 1% and 3%. Potentially important epitopes that were 90% conserved in the H1 or H3 antigen groups were not very common (6.9% and 5.8% in H1 and H3, respectively). This may suggest that in some cases, epitopes that are not widely conserved can become immunodominant because there are overwhelmingly more of them than widely conserved epitopes.

[0198] This model suggests that truly universally conserved epitope-binding antibodies are so rare that they rarely occur in most immune systems, and that while a broad range of antibodies that recognize epitopes conserved within a strain may arise, they are a minority within the response and are unlikely to be present at a high enough concentration to mediate sufficient protection the following year. For example, if there are 100 responding antibodies, most subjects are predicted not to induce antibodies against widely conserved epitopes. In the case of subjects given 100 responding antibodies, for those subjects who can or may be able to induce antibodies against widely conserved epitopes, in most cases, those antibodies can account for approximately 1% of the serum response. In such cases, seasonal antigen drift may be sufficient to render the remaining 99% of the subject's antibody titer obsolete or nearly obsolete. Combined with the natural decline in titer over time, such subjects may ultimately remain unprotected.

[0199] This model was consistent with the observation that seasonal variation of approximately 10% - 15% of amino acid positions is sufficient to disrupt most of the epitopes against hemagglutinin, which could lead to the need for continued vaccination by current methods.

[0200] Furthermore, immunizing only the base of hemagglutinin may provide some benefit, but seasonal variation within the base may continue to render most of the epitopes obsolete. Variation of immunogens containing consensus hemagglutinin variants may provide limited benefits. Even with enhanced adjuvants, the total number of responding antibodies can be increased, and the probability distribution of conservation of basic important epitopes may be altered, so the issue of breadth may not be resolved.

[0201] The conserved epitopes are present within the base and head of hemagglutinin, but their ratios may be too low to elicit the dominant ratio of immune response by most subjects, including most humans or most pigs, and there is a low likelihood that this can be resolved by optimizing the sequence of the immunogen or enhancing the adjuvant. Example 4: Generation of Influenza Vaccine Compositions

[0202] Currently, commercially available vaccines generally deliver about 10 - 50 μg of total antigen. Such commercially available vaccines often contain only two antigens (bivalent vaccines), three antigens (trivalent vaccines), or four antigens (quadrivalent vaccines).

[0203] To identify an optimally dispersed population of hemagglutinin variants, a method was developed that receives as input a large database of homologous variants of the target antigen and outputs an optimally dispersed representative subset generated probabilistically based on the percent amino acid identity. Herein, the input was a large database of homologous variants of hemagglutinin antigens against influenza, and the output was a subset of antigens representative of the input group of antigens described herein. In this example, two separate vaccine compositions were designed for administration to a subject.

[0204] This method was repeatedly applied 1000 probabilistic iterations to 8600 hemagglutinin variants over the period from 1918 to 2015, and a set of 30 representative influenza antigen variants was output. These variants include 30 antigen vaccine compositions and are detailed in Table 2. The antigens were purchased from Sino Biological, Inc. (Beijing, China). [Table 2]

[0205] In some embodiments, 27 antigen vaccine compositions designed to represent antigen variants over the period from 1918 to 2008 were used. Most of the antigens of these 27 antigen vaccine compositions are also included in 30 antigen vaccine compositions. This vaccine composition can be used to determine the effectiveness of such compositions having antigens representing epitopes of microorganisms against "future antigens" and includes the antigens in Table 3. The antigens were purchased from Sino Biological, Inc. (Beijing, China).

Table 3

[0206] In some embodiments, a vaccine composition containing 73 influenza hemagglutinin antigens from human, porcine, and unknown hosts from 1918 to 2018 was used. The antigens in this composition are listed in Table 4. The antigens were purchased from Sino Biological, Inc. (Beijing, China). The amino acid sequences of these antigens are SEQ ID NOs: 1-87 as described in Table 6.

Table 4-1

Table 4-2

Table 4-3

[0207] In some embodiments, a vaccine composition containing 28 influenza neuraminidase antigens from human, porcine, and unknown hosts from 1930 to 2019 was used. The antigens in this composition are listed in Table 5. The antigens were purchased from Sino Biological, Inc. (Beijing, China). The amino acid sequences of these antigens are SEQ ID NOs: 88-127 as described in Table 7.

Table 5-1

Table 5-2

[0208] Additional amino acid sequences of antigens that can be included in the vaccine are provided as SEQ ID NOs: 128 to 171 as described in Table 8. Example 5: Hemagglutination inhibition assay

[0209] The hemagglutination inhibition assay can be used to measure the immune response conferred by the vaccine. The sample protocol for the hemagglutination inhibition assay can start with the preparation of a serial dilution of the virus and can include the preparation of at least one negative control or positive control sample, or both a negative control sample and a positive control sample. For example, antibodies that may be against the virus in a serum sample can be incubated with the virus. Next, red blood cells can be added to each sample and incubated to observe hemagglutination. In some examples, the absence of a button can be considered a positive reaction. In some examples, the titer can be calculated and reported. Example 6: Influenza neutralization assay

[0210] The sample protocol for the influenza neutralization assay can begin with the preparation of serial dilutions of an antibody or a solution containing an antibody, such as serum. A sample method for generating such serial dilutions is described below. The antibody diluent can be prepared using a virus diluent. The virus diluent can be added to the wells of a microtiter plate, such as a 96-well plate. In some examples, 50 μL of the virus diluent can be added to each well. Next, 50 μL of the antibody or the solution containing the antibody can be added to the first well. In some examples, this can be a stock solution of 1 mg / mL. In some examples, this can be serum. In some examples, this can be diluted serum. A two-fold serial dilution can be achieved by transferring 50 μL from the first well to each successive well to achieve dilutions in the range of 1:1 to 1:128 (antibody solution:diluent). Additionally, negative control wells can be prepared that contain the virus diluent but neither the antibody nor the solution containing the antibody. The last 50 μL can be discarded. In some examples, this can be done, for example, in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 replicates.

[0211] The plate can be covered and placed in an incubator. In some examples, the conditions can be about 37 °C, about 5% CO2. In some examples, the conditions can be exactly 37 °C, about 5% CO2. In some examples, the conditions can be about 37 °C, exactly 5% CO2. In some examples, the conditions can be exactly 37 °C, exactly 5% CO2. During the preparation of the diluted virus, the plate can be kept in the incubator. In some examples, it is necessary to maintain the pH to avoid harmful pH effects on the virus when adding the virus.

[0212] The virus can be diluted to a working dilution of 100 TCID 50 / 50 μL with the virus diluent. TCID 50 can be the 50% tissue culture infectious dose and 100 TCID 50 can be equal to 100 times the value of TCID 50 .

[0213] 50 μL of the diluted virus can be added to wells containing the antibody and control wells without the antibody. In some examples, additional control wells having the antibody or a solution containing the antibody but without the virus can be prepared. In some examples, a row of wells can be reserved for reverse titration of the virus. The plate can be gently mixed, and a virus diluent can be added to all wells to equalize each well. Reverse titration can be performed using standard protocols.

[0214] Cover the plate and it can be placed in the incubator again for 1 hour as described above. After 1 hour, 100 μL of diluted MDCK cells (which should be at 70 - 95% confluence and low passage <30) can be added to each well of the microtiter plate. Each well can contain 1.5×10 4 cells.

[0215] Cover the plate and it can be placed in the incubator again for 18 - 20 hours as described above. After incubation, the liquid can be removed from the wells, and for example, the wells can be washed using 200 μL of phosphate - buffered saline (PBS). 100 μL of cold 80% acetone can be added to each well, and the plate can be incubated at room temperature for 10 - 12 minutes for fixation. Next, the acetone can be removed from the wells, and the plate can be dried, for example, by air drying for about 10 minutes or until dry.

[0216] Thereafter, an influenza enzyme-linked immunosorbent assay (ELISA) can be performed using a standard ELISA protocol to measure the amount of neutralized virus. In some examples, an influenza neutralization assay can be performed using the serum of a subject inoculated with a vaccine composition developed according to the present disclosure. In some examples, such an influenza neutralization assay can be performed using porcine serum, human serum, or the serum of any mammal. In some examples, the serum can contain antibodies that are head-specific antibodies, base-specific antibodies, broadly neutralizing antibodies, universal antibodies, 90% universal antibodies, broadly strain-specific antibodies, and / or 90% strain-specific antibodies.

[0217] In an influenza neutralization assay, control samples obtained from subjects vaccinated with a control vaccine can be included. In such cases, the control vaccine can be a monovalent vaccine, a bivalent vaccine, a trivalent vaccine, or a quadrivalent vaccine. If the control vaccine is a trivalent vaccine, it can include antigens from the H1N1 strain, the H3N2 strain, and the HAB strain. In some examples, the minimum neutralization dilution can be at least two-fold greater by using a vaccine developed rather than using a control vaccine. In some examples, when tested using a hemagglutinin inhibition assay or influenza neutralization, the minimum neutralization dilution can be at least two-fold greater by using a vaccine developed rather than using a control vaccine that includes H1N1, H3N2, and HAB antigens. In some examples, when tested using a hemagglutinin inhibition assay or influenza neutralization, the minimum neutralization dilution can be at least two-fold greater by using a vaccine developed rather than using a control quadrivalent vaccine. In some examples, the minimum neutralization dilution can be at least ten-fold greater by using a vaccine developed rather than using a control vaccine. In some examples, when tested using a hemagglutinin inhibition assay or influenza neutralization, the minimum neutralization dilution can be at least ten-fold greater by using a vaccine developed rather than using a control vaccine that includes H1N1, H3N2, and HAB antigens. In some examples, when tested using a hemagglutinin inhibition assay or influenza neutralization, the minimum neutralization dilution can be at least ten-fold greater by using a vaccine developed rather than using a control quadrivalent vaccine. In some examples, the minimum neutralization dilution can be at least one hundred-fold greater by using a vaccine developed rather than using a control vaccine. In some examples, when tested using a hemagglutinin inhibition assay or influenza neutralization, the minimum neutralization dilution can be at least one hundred-fold greater by using a vaccine developed rather than using a control vaccine that includes H1N1, H3N2, and HAB antigens.In some examples, when tested using a hemagglutinin inhibition assay or influenza neutralization, the minimum neutralization dilution can be at least 100-fold greater when using the developed vaccine than when using a control quadrivalent vaccine. In certain cases, the minimum neutralization dilution can be at least 1,000-fold greater when using the developed vaccine than when using a control vaccine. In various examples, when tested using a hemagglutinin inhibition assay or influenza neutralization, the minimum neutralization dilution can be at least 1,000-fold greater when using the developed vaccine than when using a control vaccine that includes H1N1, H3N2, and HAB antigens. In some examples, when tested using a hemagglutinin inhibition assay or influenza neutralization, the minimum neutralization dilution can be at least 1,000-fold greater when using the developed vaccine than when using a control quadrivalent vaccine. In certain cases, the minimum neutralization dilution can be at least 10,000-fold greater when using the developed vaccine than when using a control vaccine. In various examples, when tested using a hemagglutinin inhibition assay or influenza neutralization, the minimum neutralization dilution can be at least 10,000-fold greater when using the developed vaccine than when using a control vaccine that includes H1N1, H3N2, and HAB antigens. In some examples, when tested using a hemagglutinin inhibition assay or influenza neutralization, the minimum neutralization dilution can be at least 10,000-fold greater when using the developed vaccine than when using a control quadrivalent vaccine. In certain cases, the improvement in the minimum neutralization dilution can be with respect to one antigen, multiple antigens, or all of the antigens tested. In various examples, the improvement in the minimum neutralization dilution can be with respect to one antigen, multiple antigens, or all of the antigens tested.

[0218] In some examples, the developed vaccine can neutralize more strains than the control vaccine. For example, the developed vaccine can neutralize at least one more strain than the control vaccine. In certain cases, the developed vaccine can neutralize at least 2, at least 5, at least 10, at least 25, at least 50, or at least 100 more strains than the control vaccine. In various examples, the developed vaccine can neutralize at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, or at least 10,000-fold more strains than the control vaccine. Example 7: ELISA Assay

[0219] ELISA can be used to detect antibodies present in serum that are directed against antigens such as influenza antigens. In some embodiments, multiple different antigens (e.g., hemagglutinin variants from various strains) are investigated. Briefly, hemagglutinin antigen variants can be immobilized in the wells of a 96-well plate. In some examples, a dilution series can be prepared. A negative control well without antigen can be prepared.

[0220] A solution containing serum suspected of containing hemagglutinin can be incubated in wells containing hemagglutinin antigen variants or control wells. After the incubation period, the solution containing serum can be washed away. In some examples, if one or more antibodies that can react with hemagglutinin are present in the serum, the antibodies will remain bound to the plate.

[0221] A secondary antibody produced against the antibodies of the animal from which the serum is derived can be applied to the wells. After the incubation period, the secondary antibody can be washed away. If there is an interaction between the hemagglutinin and the antibody in the serum, the secondary antibody can label the hemagglutinin-antibody complex.

[0222] The secondary antibody can be labeled by any acceptable means, and signals can be detected in the wells where the secondary antibody is present. In some examples, the signal may correspond to the amount of secondary antibody present. In some examples, the signal may correspond to the amount of serum-derived antibody present. In some examples, the signal may correspond to the amount of hemagglutinin-antibody interaction in the well. Example 8: Expansion of Average Epitope Width by Association of Conservation and Concentration

[0223] We tested whether it was possible to design a vaccination strategy that shifts the probability distribution of epitope conservation in vivo to enrich vaccine-induced antibodies against conserved epitopes.

[0224] Without being bound by any one particular theory, the hypothesis was that B cells that can recognize widely conserved epitopes and can affinity mature against them could be selectively rewarded by associating the conservation of these epitopes with their concentration in the vaccine formulation (known as Conservation-Concentration Coupling or C3). A mixture of 30 diverse variants of HA was administered individually at doses insufficient to induce an effective strain-specific immune response, effectively removing a dominant population of single-variant epitopes from the distribution. Thus, this preferentially selects B cells that recognize common epitopes across the components, and broadly reactive B cells will receive a dose up to 30-fold higher than strain-specific B cells.

[0225] Figure 6 schematically shows how C3 functions. B cells carrying receptors targeting conserved epitopes receive the stimuli necessary to differentiate into plasma cells that can produce antibodies, i.e., antigens with a relatively high load. Other B cells with receptors capable of recognizing strain-specific epitopes can receive antigens with a relatively low load, which may be below the threshold required to initiate an immune response. Example 9: Epitope focusing by study design - association of conservation and concentration (C3)

[0226] Three in vivo experiments were designed using domesticated outbred pigs (Sus scrofa), a model organism, and a veterinary target organism for influenza A infection and vaccination. In the first in vivo study, 35 pigs were divided into five cohorts, with seven pigs in each cohort. Each vaccine (C3) formulation contained a diverse set of 30 HA variants (H1, H2, H3, H5, and H7 spanning 1918 - 2014). The pig cohorts were inoculated with either a bivalent formulation (BIV, 2 HA antigens H1N1 2007 and H3N2 1997, 5,000 ng / antigen), C3 - 500 (30 HA antigens, 500 ng / antigen), C3 - 100 (30 HA antigens, 100 ng / antigen), C3 - 50 (30 HA antigens, 50 ng / antigen), or a vehicle control (PBS + squalene). The animals were inoculated three times at three - week intervals, and sera were collected 3 - 4 weeks after each vaccination (days 29, 50, and 71). RNA was isolated from PBMC collected 7 days after the third vaccination (day 50).

[0227] In the second and third in vivo studies, representatives of all HAs from 2008 onwards were removed to create a "pre - 2009" C3 formulation for examining serological responses to de facto future virus strains and HAs, including pandemic H1N1 2009 through H3N2 2018. Seasonal bivalent controls for 2008 were also formulated using H1N1 2007 and H3N2 2007.

[0228] In the second in vivo study, 20 pigs were separated into five cohorts, with four pigs per cohort. Each cohort was inoculated with a formulation containing either two HA antigens at 5,000 ng / antigen (BIV), 27 HA antigens at 50 ng / antigen (C3-50), 50 ng of H1N1 2007 (single low dose H1), 50 ng of H3N2 2007 (single low dose H3), or PBS + squalene (vehicle control). The single HA cohorts administered at the low dose (50 ng) were included to thresh (verify below the stochiometric limit where the given antigen does not induce an immune response). The animals were each inoculated six times at 21-day intervals. Additionally, three cohorts (BIV, C3-50, and vehicle control) received a seventh inoculation nine weeks after the sixth inoculation. In the case of C3-50, the seventh inoculation included a high-dose boost (27 HA, 500 ng / antigen). Serum was collected immediately prior to each inoculation on the same day. Additional serum was collected four weeks after the sixth inoculation (day 135) and four weeks after the seventh inoculation (day 195). RNA was isolated from peripheral blood mononuclear cells collected seven days after the sixth inoculation (day 112). In the third in vivo study, 25 pigs were separated into five cohorts, with five pigs per cohort. Each cohort was inoculated with a formulation containing either 5,000 ng / antigen + squalene (BIV), C3-50 + squalene, C3-50 + alum, C3-50 + TLR, or vehicle control (PBS + squalene + alum + MPLA + imiquimod). The animals received three inoculations at 21-day intervals, and serum was collected immediately prior to each inoculation on the same day. Additional serum was collected four weeks after the third inoculation (day 70). All three C3-50 cohorts were administered 28 HA antigens at 50 ng / antigen for the first two inoculations and 500 ng / antigen for the third inoculation.

[0229] Example 10: Herd effect

[0230] To support the theory of the association between concentration and storage, L thresh threshIt was determined in vivo. First, by administering either a single antigen of H1N1 2007 or H3N2 2007 at a dose of 50 ng, it was confirmed that the HA at a dose of 50 ng was below the threshold for immune activation. A total of six doses were administered to pigs at 21-day intervals (corresponding to the second study of the previous example). In both cases, as shown in Figure 7, the serum response was not detected by ELISA for either antigen 28 days after the sixth inoculation.

[0231] However, when the two antigens were combined with 25 other HAs from 1918 to 2008 and each was similarly administered at 50 ng (C3-50), a serum response to both antigens was detected. Furthermore, inoculation with C3-50 resulted in a serum response against a panel of "future" post-2008 strains not included in the formulation, including other HAs of C3 and H1N1, H3N2, H5N1, and H7N9 from 2009 to 2013. Thus, even when individual components may be below the threshold dose, a low-dose ensemble may be able to induce an immune response.

[0232] In this case, if 27 antigens are individually in a pool below the physiological dose, the shared epitope can be at a dose up to 27 times higher, and thus there is a possibility of successful targeting and obtaining a preferentially broad response that may cross heterologous strains.

[0233] Figure 7 shows the ELISA signals (%max OD ) of individual porcine sera against 10 HA antigens (H1, H3, H5, and H7 from 1934 to 2013) across the entire immunized cohort of C3-50 (27HA, 50 ng / HA), single low-dose (50 ng) H1N1 450nm 2007 antigen, single low-dose (50 ng) H3N2 2007 antigen, and vehicle control (PBS + squalene) (n = 4 each). The box-and-whisker plot shows the median and IQR. The whiskers show 1.5 times the IQR of the lower and upper quartiles. The dots represent outliers. The antigens shown within the dashed frame represent the HAs present in the 1918 - 2008 C3 vaccine formulation. Example 11: The response by association of conservation and concentration is mainly subtype-specific and requires a minimum total dose of the subtype antigen.

[0234] Analysis of the elicited binding response to C3 was separated by HA subtype. The C3-50 formulation, the first study described in Example 9, contained 10 H1, 7 H3, 5 H5, 3 H7, and 2 H2 individually at 50 ng each. Serum responses were correlated with the total subtype dose, with H1 and H3 showing the strongest responses, H5 showing a weaker response, and H2 and H7 appearing to have an insufficient response to C3-50. These results support the prediction that the response to one subtype does not necessarily enhance the response to other subtypes. Thus, in some cases, a broad response within a subtype may be far more common than a complete universal antibody. The data suggest a potential minimum clade dose of 7-10 members (i.e., 350-500 ng of the most conserved epitopes) for optimal effect.

[0235] Data illustrating this experiment are shown in FIG. 8. The ELISA signals (% max OD 450nm ) of pigs in the C3-50 cohort (Study #1) are shown separated into the various HA antigen classes present in the C3-50 formulation (H1, H2, H3, H5, and H7). The x-axis shows the number of HA per class. Serum was collected 28 days after the third vaccination (day 71). Pearson's correlation coefficient for the number of HA antigens against the ELISA signal is shown as R 2 with the corresponding p-value. Box and whisker plots show the median and IQR. Whiskers show 1.5 times the IQR of the lower and upper quartiles. P-values are calculated using the Kruskal-Wallis rank sum test with Dunn's correction for multiple comparisons. Selected P-values are reported as adjusted, * P<0.05, ** P<0.01, *** P<0.001, and **** P<0.0001. Example 12: Association of broad reactivity and inverse dose response with concentration

[0236] In some examples, decreasing the dose of individual components can increase the pressure targeting only the conserved epitopes, and thus may broaden the breadth of serological responses. To evaluate the relationship between the dose of C3 and the response, three dose cohorts of C3 were tested together with the BIV group and the vehicle control group. Serum responses to 36 antigen panels obtained from each of 7 pigs per cohort from the first study described in Example 9 showed that the lowest dose of C3 (50 ng / HA) provided the greatest serum response against most antigens spanning H1N1, H1N3, H1N3, H2N2, H3N2, H5N1, H6N2, and H17N10 from 1918 to 2014. This inverse dose response is consistent with the prediction of the model and is a unique feature of this vaccination strategy. As shown in Panel B of FIG. 9, all three C3 formulations had improved median serum responses compared to the bivalent formulation measured by ELISA. Furthermore, as shown in Panel A of FIG. 9, the maximum median serum response was obtained at the minimum dose of C3, and an inverse dose response was observed that resulted in the largest fold change in serum titers over time from 7 to 28 days after the third vaccination, as shown in Panel B of FIG. 9. This is consistent with the prediction of the model that the association of conservation and concentration results in a shift in breadth by diluting competing epitopes.

[0237] Panel A of Figure 9 shows the mean ELISA signal (% max OD450nm) per pig across all test strains per pig (n = 36, spanning H1, H2, H3, H5, H7, and H17 from 1918 - 2014) (Study #1). Serum was collected 28 days after the third vaccination (day 71). Panel B of Figure 9 shows the fold change in the mean ELISA signal (% max OD450nm) per pig from C) between 7 and 28 days after the third vaccination. Box - and - whisker plots show the median and IQR. Whiskers show 1.5 times the IQR of the lower and upper quartiles. P - values were calculated using the Kruskal - Wallis rank - sum test with Dunn's correction for multiple comparisons. The selected P - values are reported as adjusted, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as shown.

[0238] When analyzing the responses per cohort and per animal, as shown in the heatmap of Figure 10, more animals were observed to respond more broadly as a function of the inverse dose - response, with the lowest dose (C3 - 50 ng / antigen) showing the largest breadth.

[0239] Figure 10 shows a heatmap of the ELISA signal (% max OD450nm) from individual pig sera of five immune cohorts and broadly neutralizing human antibodies (C05, F10, and CR9114) that bind 36 HAs (H1, H2, H3, H5, H6, H7, and H17 spanning 1918 - 2014). The immune cohorts were three different C3 formulations of 30 HAs at 500 ng / HA, 100 ng / HA, and 50 ng / HA, the BIV cohorts (H1N1 2007 and H3N2 1997, 5,000 ng each), and the vehicle control cohort (PBS + squalene). Serum was collected 28 days after the third inoculation (day 71, Study #1). The shading of the heatmap was adjusted using quantile breaks to account for the distribution of the ELISA data. The coverage rate of positive strains was determined as the percentage value above the mean of the vehicle control for each strain + 3×SD.

[0240] In summary, the data shows that the width of the response is related to the concentration of the conserved epitope, and that the C3 formulation provided the broadest average strain bound per porcine serum at a minimum dose of 50 ng / antigen in most pigs. Example 13: Association of conservation with concentration induces uniform B cell expansion

[0241] Next, the IgM and IgG B cell receptor (BCR) repertoires of porcine peripheral blood mononuclear cells (PBMCs) were analyzed for the pigs described in the second study (described in Example 9) 7 days after the sixth inoculation. In contrast to the high variability in clonal expansion observed among pigs receiving either a bivalent or vehicle immunization schedule, significantly more uniform BCR repertoire clonotype expansion was observed in pigs administered the C3-50 formulation. Thus, C3 appeared to generate a more uniform and thus potentially more predictable immune response.

[0242] Data demonstrating these observations are shown in Figure 11 as the peripheral BCR repertoire occupied by the top clonotypes separated by isotype. The black line shows the average repertoire occupied as a percentage (%) of the total sequences of the top n clonotypes across all pigs for each immunization group. The grey area shows the 95% confidence interval. The immunization cohort was from the second in vivo study described in Example 9; C3 formulations formulated with 27 HAs, 50 ng / HA (n = 4), bivalent cohort (H1N1 2007 and H3N2 2007, 5,000 ng each, n = 3) and vehicle control (PBS + squalene, n = 4). Lymphocytes were harvested 7 days after the sixth inoculation (day 112). Isotypes were determined by C-region specific priming during library preparation. Clonotypes were defined as sequences with the same CDRH3 amino acid sequence. Example 14: Association of conservation with concentration induces broadly neutralizing antibodies

[0243] To isolate monoclonal porcine anti-HA antibodies produced in C3-vaccinated animals, a library of phage-displayed porcine antibodies was generated from the C3 animals of the first in vivo study described in Example 9. From these libraries, porcine anti-HA antibodies, including bnAb 9C5, were isolated from pigs administered low doses of C3 (50 ng / antigen).

[0244] Data representing the results of this experiment are shown in FIG. 12. Sequence alignment of the broadly neutralizing porcine monoclonal antibody 9C5 isolated by phage display. The heavy and light chain nucleotide sequences were aligned using IMGT V Quest against the porcine (Sus scrofa) germline genes. Mutations compared to the detected germline genes are underlined and CDRH3 is marked in bold. CDRH3 was reported by IMGT at seven amino acid positions 111.1 - 111.3 and 112.4 - 112.1 added in the IMGT numbering scheme. The phage library was constructed from B cell pools for each immune cohort of lymphocytes 7 days after the third vaccination (day 50, study #1) and panned four times against H3N2 2007 and H1N1 2007. Monoclonal 9C5 was derived from one pig (C3 - 50 cohort, 30HA, 50 ng / HA).

[0245] The data show that the C3 antigen preparation can induce broadly neutralizing antibodies and that the performance is improved compared to known human bnAbs. This indicates that the approach provided herein effectively overcomes the stochastic hurdles of these very rare epitopes and that antibodies targeting them can broadly neutralize a range of influenza strains even if they were not part of the original antigen preparation.

[0246] Furthermore, experiments were conducted to determine the minimum antibody concentration (μg / mL) sufficient for in vitro neutralization of a panel of five influenza A strains (H1N1 / California / 2009, H1N1 / Michigan / 2015, H1N1 / Michigan / 2017, H3N2 / Brisbane / 2007, and H3N2 / Victoria / 2011). The results are shown in Figure 13. Porcine monoclonal 9C5 isolated from one pig (C3-50, in vivo study #1) using a phage display library was tested as an scFv-huFc fusion chimera construct in 2Ml HEK293 supernatant and quantified with a Protein A standard curve on an OctetQK at 32 μg / Ml. It is shown in comparison to control bnAbs CR9114, F10, and C05 tested for neutralization capacity as purified human IgG1. Example 15: Neutralization of "future" influenza strains

[0247] Here, it was determined whether the C3 formulation induced neutralizing antibodies by a micro-neutralization assay with sera from pigs inoculated with C3. For sera from the second in vivo study described in Example 9, similar neutralizing capacities were observed from sera of the C3-50 vaccinated cohort and sera of pigs vaccinated with a bivalent control of strains (H1N1 and H3N2 in 2007) that were part of the bivalent control cohort. Furthermore, for each of the future strains tested, C3-50 pigs were able to neutralize 5 / 5 (H1N1 2009, H3N2 2009, H3N2 2011, H3N2 2014, and H1N1 2015), while the bivalent control group showed neutralizing titers for only 2 / 5 (H3N2 2009 and 2011).

[0248] For the sera from the third in vivo study described in Example 9, pigs in the bivalent control group neutralized 2 / 6 strains tested with one “future” virus, H3N2 2009, which is not part of the formulation. Alum and squalene in the C3-50 cohort showed improved neutralization and neutralized 4 / 6 and 5 / 6 of the strains tested. Neutralizing three and four future viruses is not part of the C3-50 formulation. Pigs administered C3-50 + TLR agonist as an adjuvant were unable to neutralize “future” strains and neutralized only H3N2 2007. Overall, the data show that the C3 formulation administered at 50 ng generates sera with broad neutralizing capacity at levels similar to the bivalent formulation while also neutralizing a broad range of “future” viruses that are not part of the antigen formulation. Thus, the conservation and concentration association approach can focus the immune response on highly conserved epitopes on the HA coat protein. Furthermore, these epitopes can induce a neutralizing serum response at levels similar to those of a normal bivalent formulation and can further cover virus strains that emerge nearly 10 years after the evolution of the virus.

[0249] Data showing these results are presented in Figure 14. Panel A of Figure 14 provides the ELISA signals (% max OD450nm) of porcine sera from an initial in vivo study against 30 HA antigens (H1, H2, H3, H5, H6, H7, and H17 spanning 1918 - 2014) in immunized cohorts of C3 - 50 (30HA, 50 ng / HA), BIV cohort (H1N1 2007 and H3N2 1997, 5,000 ng each) and vehicle control (PBS + squalene) (n = 7 pigs each). Sera were collected 28 days after the third vaccination (day 71). Bars represent the mean of all pigs in the cohort and error bars indicate the positive standard deviation. Arrows indicate HAs not present in the C3 vaccine formulation (in vivo study #1). Asterisks indicate statistically significant differences between C3 - 50 and bivalent formulations with adjusted p - values less than or equal to 0.05. P - values were determined using the Wilcoxon rank - sum test for each lineage, using the alternative hypothesis that the BIV signal is less than the C3 - 50 signal. The Benjamini Hochberg correction was applied to adjust for multiple testing.

[0250] Panel B of Figure 14 shows the breadth of neutralization as a scatter plot of the mean number of neutralizing strains per pig per cohort and the number of positive tests per cohort. Sera were collected 28 days after the seventh vaccination (day 195, study #2). Viruses tested included H1N1 / Puerto Rico / 1934, H1N1 / Brisbane / 2007, H1N1 / California / 2009, H1N1 / Michigan / 2015, H3N2 / Brisbane / 2007, H3N2 / Perth / 2009, H3N2 / Victoria / 2011, and H3N2 / Hong Kong / 2014. Sera were not pooled. Cohorts were vehicle control (PBS + squalene), seasonal BIV (H1N1 2007 and H3N2 2007, 5,000 ng each), single low - dose H1 antigen (H1N1 2007, 50 ng), single low - dose H3 antigen (H3N2 2007, 50 ng), and C3 - 50 (27 HAs, 50 ng / HA), in vivo study #2. Error bars indicate the standard deviation.

[0251] Panel C of Figure 14 shows the neutralization potency of Study #2 reported as the reciprocal neutralizing dilution per strain across the entire cohort using a bar graph. The bars represent the mean of all pigs (n = 4) in the cohort, and the error bars represent the standard deviation. The tested "future" strains that are not part of the 2008 C3 formulation are indicated by arrows. Serum was collected 28 days (day 195) after the 7th vaccination.

[0252] Panel D of Figure 14 shows the neutralization potency of Study #3 reported as the reciprocal neutralizing dilution per strain across the entire cohort using a bar graph. The bars represent the mean of all pigs (n = 5) in the cohort, and the error bars represent the standard deviation. The points represent the individual values per pig. The tested "future" strains that are not part of the 2008 C3 formulation are indicated by arrows. Serum was collected 28 days (day 70) after the 3rd vaccination. The cohort consisted of a vehicle control (PBS + squalene + TLR-agonist + alum), seasonal BIV (H1N1 2007 and H3N2 2007, 5,000 ng each + squalene), and three C3-50 (28 types of HA, 50 ng / HA) cohorts each containing squalene, TLR-agonist, and alum adjuvant. Example 16: Selection of the 32 most diverse HIV gp160 sequences for each of HIV clades A, B, and C.

[0253] The HIV sequences were downloaded from www.hiv.lanl.gov / and separated into clades. The sequences were filtered by length, excluding sequences shorter than 800 amino acids and sequences from clades A, B, or C of (HIV-1 group M) selected for further processing.

[0254] The sequences were aligned using a custom-made HMM profile for the HIV gp160 protein with hmmalign (HMMER v.3.0, hmmer.org / ). Next, a pairwise Hamming distance matrix was generated using a custom-made python (v.3.7.2) script. Subsequently, the sequences were clustered based on the Hamming distance matrix using UPGMA clustering (UPGMA: unweighted pair group method with arithmetic mean) implemented in the hclust function of the R (v.3.6.1) base statistics package. The clustered sequences were split into 32 subclusters using the cutree function of the R (v 3.6.1) base statistics package.

[0255] The medoids function of the GDA tool (v.1.4) was used to extract the representative sequences of each cluster as their medoids. The output of this analysis was 96 amino acid sequences, 32 for each of HIV clades A, B, and C. These sequences are listed in Table 9. They are sequence numbers 172 - 267. VI. Compositions

[0256] The composition can be a vaccine or a library of antigens from which the vaccine antigen is selected. The library of antigens can contain multiple antigens. Such a composition can contain a minimum number of variants of the microorganism. In some examples, such a library can contain at least 1×10 1 , at least 1×10 2 , at least 1×10 3 , at least 1×10 4 , at least 1×10 5 , at least 1×10 6 , at least 1×10 7 , at least 1×10 8 , at least 1×10 9 or at least 1×10 10 different variants of the microorganism.

[0257] The antigen library can include a subset of all known sequences of the microorganism. A library containing more sequences can be considered more complete and, in some embodiments, may be considered more desirable. In some examples, the library can include at least 80%, 85%, 90%, or 95% of all known sequences of the microorganism.

[0258] The antigen in the vaccine can contain a specified number of amino acids. In some examples, the antigen in the vaccine can contain 10 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 35 or fewer, 40 or fewer, 45 or fewer, 50 or fewer, 55 or fewer, 60 or fewer, 65 or fewer, 70 or fewer, 75 or fewer, 80 or fewer, 85 or fewer, 90 or fewer, 95 or fewer, 100 or fewer, 110 or fewer, or 120 or fewer amino acids. In some examples, the antigen in the vaccine can contain at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, or at least 120 amino acids.In some examples, the antigen in the vaccine can comprise from 10 to 120 amino acids, from 10 to 100 amino acids, from 10 to 90 amino acids, from 10 to 80 amino acids, from 10 to 70 amino acids, from 10 to 60 amino acids, from 10 to 50 amino acids, from 10 to 40 amino acids, from 10 to 30 amino acids, from 20 to 120 amino acids, from 20 to 110 amino acids, from 20 to 100 amino acids, from 20 to 90 amino acids, from 20 to 80 amino acids, from 20 to 70 amino acids, from 20 to 60 amino acids, from 20 to 50 amino acids, from 20 to 40 amino acids, from 20 to 30 amino acids, from 30 to 120 amino acids, from 30 to 110 amino acids, from 30 to 100 amino acids, from 30 to 90 amino acids, from 30 to 80 amino acids, from 30 to 70 amino acids, from 30 to 60 amino acids, from 30 to 50 amino acids, from 30 to 40 amino acids, from 40 to 120 amino acids, from 40 to 110 amino acids, from 40 to 100 amino acids, from 40 to 90 amino acids, from 40 to 80 amino acids, from 40 to 70 amino acids, from 40 to 60 amino acids, from 40 to 50 amino acids, from 50 to 120 amino acids, from 60 to 110 amino acids, from 50 to 100 amino acids, from 50 to 90 amino acids, from 50 to 80 amino acids, from 50 to 70 amino acids, from 50 to 60 amino acids, from 60 to 120 amino acids, from 60 to 110 amino acids, from 60 to 100 amino acids, from 60 to 90 amino acids, from 60 to 80 amino acids, from 60 to 70 amino acids, from 70 to 120 amino acids, from 70 to 110 amino acids, from 70 to 100 amino acids, from 70 to 90 amino acids, from 70 to 80 amino acids, from 80 to 120 amino acids, from 80 to 110 amino acids, from 80 to 100 amino acids, from 80 to 90 amino acids, from 90 to 120 amino acids, from 90 to 110 amino acids, from 90 to 100 amino acids, from 100 to 120 amino acids, from 100 to 110 amino acids, or from 110 to 120 amino acids.

[0259] In some examples, the antigen in the vaccine can contain at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids. In some examples, the antigen in the vaccine can contain 200 or fewer, 300 or fewer, 400 or fewer, 500 or fewer, 600 or fewer, 700 or fewer, or 800 or fewer amino acids. In some examples, the antigen in the vaccine contains 10 - 1000 amino acids, 10 - 900 amino acids, 10 - 800 amino acids, 10 - 700 amino acids, 10 - 600 amino acids, 10 - 500 amino acids, 10 - 400 amino acids, 10 - 300 amino acids, 10 - 200 amino acids, 100 - 1000 amino acids, 100 - 800 amino acids, 100 - 700 amino acids, 100 - 600 amino acids, 100 - 500 amino acids, 100 - 400 amino acids, 100 - 300 amino acids, 100 - 200 amino acids, 200 - 1000 amino acids, 200 - 900 amino acids, 200 - 800 amino acids, 200 - 700 amino acids, 200 - 600 amino acids, 200 - 500 amino acids, 200 - 400 amino acids, 200 - 300 amino acids, 300 - 1000 amino acids, 300 - 900 amino acids, 300 - 800 amino acids, 300 - 700 amino acids, 300 - 600 amino acids, 300 - 500 amino acids, 300 - 400 amino acids, 400 - 1000 amino acids, 400 - 900 amino acids, 400 - 800 amino acids, 400 - 700 amino acids, 400 - 600 amino acids, 400 - 500 amino acids, 500 - 1000 amino acids, 500 - 900 amino acids, 500 - 800 amino acids, 500 - 700 amino acids, 500 - 600 amino acids, 600 - 1000 amino acids, 600 - 900 amino acids, 600 - 800 amino acids, 600 - 700 amino acids, 700 - 1000 amino acids, 700 - 900 amino acids, 700 - 800 amino acids, 800 - 1000 amino acids, 800 - 900 amino acids, or 900 - 1000 amino acids.

[0260] The vaccine can contain a specified number of antigens. The antigens included in the vaccine can be selected based on the total number of available antigens, similarity to pathogenic strains, similarity to other included antigens, or differences from other included antigens. The vaccines herein can include a set of antigens that can contain at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 different antigens. In selected examples, the vaccine can contain at least 30 antigens. In some examples, the vaccine can include a set of antigens that can contain 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 15 or fewer, 20 or fewer, 30 or fewer, 40 or fewer, 50 or fewer, 60 or fewer, 70 or fewer, 80 or fewer, 90 or fewer, 100 or fewer, 200 or fewer, 300 or fewer, 400 or fewer, 500 or fewer, 600 or fewer, 700 or fewer, 800 or fewer, 900 or fewer, or 1000 or fewer different antigens. In selected examples, the vaccine can contain 30 or fewer antigens. In some examples, the vaccine can contain from 5 to 1000, from 5 to 900, from 5 to 800, from 5 to 700, from 5 to 600, from 5 to 500, from 5 to 400, from 5 to 300, from 5 to 200, from 5 to 100, from 5 to 90, from 5 to 80, from 5 to 70, from 5 to 60, from 5 to 50, from 5 to 40, from 5 to 30, from 5 to 20, from 5 to 10, from 10 to 1000, from 10 to 900, from 10 to 800, from 10 to 700, from 10 to 600, from 10 to 500, from 10 to 400, from 10 to 300, from 10 to 200, from 10 to 100, from 10 to 90, from 10 to 80, from 10 to 70, from 10 to 60, from 10 to 50, from 10 to 40, from 10 to 30, from 10 to 20, from 20 to 1000, from 20 to 900, from 20 to 800, from 20 to 700, from 20 to 600, from 20 to 500, from 20 to 400, from 20 to 300, from 20 to 200,It can contain 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, 30 to 1000, 30 to 900, 30 to 800, 30 to 700, 30 to 600, 30 to 500, 30 to 400, 30 to 300, 30 to 200, 30 to 100, 30 to 90, 30 to 80, 30 to 70, 30 to 60, 30 to 50, 30 to 40, 40 to 1000, 40 to 900, 40 to 800, 40 to 700, 40 to 600, 40 to 500, 40 to 400, 40 to 300, 40 to 200, 40 to 100, 40 to 90, 40 to 80, 40 to 70, 40 to 60, 40 to 50, 50 to 1000, 50 to 900, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 50 to 90, 50 to 80, 50 to 70, 50 to 60, 100 to 1000, 100 to 900, 100 to 800, 100 to 700, 100 to 600, 100 to 500, 100 to 400, 100 to 300, 100 to 200, 200 to 1000, 200 to 900, 200 to 800, 200 to 700, 200 to 600, 200 to 500, 200 to 400, 200 to 300, 300 to 1000, 300 to 900, 300 to 800, 300 to 700, 300 to 600, 300 to 500, 300 to 400, 400 to 1000, 400 to 900, 400 to 800, 400 to 700, 400 to 600, 400 to 500, 500 to 1000, 500 to 900, 500 to 800, 500 to 700, 500 to 600, 600 to 1000, 600 to 900, 600 to 800, 600 to 700, 700 to 1000, 700 to 900, 700 to 800, 800 to 1000, 800 to 900, or 800 to 1000 different antigens.,

[0261] In some examples, the intact antigen can be split into functional fragments or portions of the whole antigen, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95 or at least 100 portions (e.g., pieces or fragments) (including all), and the individual functional fragments of the antigen can be associated with a polymer according to the methods disclosed herein. In some examples, the antigen may be split into at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 30, at most 35, at most 40, at most 45, at most 50, at most 55, at most 60, at most 65, at most 70, at most 75, at most 80, at most 85, at most 90, at most 95, or at most 100 portions (e.g., pieces or fragments) (including all).

[0262] In some examples, a broadly conserved array can be conserved at least 40% across a series of strains of a microorganism (e.g., at least 10% of the strains of the microorganism, at least 25% of the strains, at least 50% of the strains, at least 75% of the strains, at least 90% of the strains, at least 95% of the strains, or all of the strains). In some examples, a broadly conserved array may be an array with limited variation between species or strains of a microorganism. An array conserved across multiple microorganisms can be the same or substantially the same across the multiple microorganisms. For example, an array conserved 40%, 50%, 60%, 70%, 80%, 90%, or 100% can have 40%, 50%, 60%, 70%, 80%, 90%, or 100% structural or sequence identity across a series of microorganisms.

[0263] In certain cases, a broadly conserved array can be conserved at least 50% across a series of strains of a microorganism (e.g., at least 10% of the strains of the microorganism, at least 25% of the strains, at least 50% of the strains, at least 75% of the strains, at least 90% of the strains, at least 95% of the strains, or all of the strains). In various examples, a broadly conserved array can be conserved at least 60% across a series of strains of a microorganism (e.g., at least 10% of the strains of the microorganism, at least 25% of the strains, at least 50% of the strains, at least 75% of the strains, at least 90% of the strains, at least 95% of the strains, or all of the strains). In some examples, a broadly conserved array can be conserved at least 70% across a series of strains of a microorganism (e.g., at least 10% of the strains of the microorganism, at least 25% of the strains, at least 50% of the strains, at least 75% of the strains, at least 90% of the strains, at least 95% of the strains, or all of the strains). In certain cases, a broadly conserved array can be conserved at least 80% across a series of strains of a microorganism (e.g., at least 10% of the strains of the microorganism, at least 25% of the strains, at least 50% of the strains, at least 75% of the strains, at least 90% of the strains, at least 95% of the strains, or all of the strains). In various examples, a broadly conserved array can be conserved at least 90% across a series of strains of a microorganism (e.g., at least 10% of the strains of the microorganism, at least 25% of the strains, at least 50% of the strains, at least 75% of the strains, at least 90% of the strains, at least 95% of the strains, or all of the strains).

[0264] One antigen represents a certain clade if its sequence is up to 10% of the average size of the antigens of the represented clade and is different from other members of the represented clade. In some examples, one antigen represents a certain clade if its sequence is up to 15%, up to 20%, up to 25%, or up to 30% of the average size of the antigens of the represented clade and is different from other members of the represented clade.

[0265] An antigen can represent a represented clade if its sequence is 75% or less of the average size of the antigens of the represented clade and is different from the other members of the represented clade. In some clades, an antigen can represent the clade if its sequence differs by 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less, or 95% or less compared to the other members of the represented clade.

[0266] An antigen can represent a clade if its sequence differs from a subset of the strains of the represented clade by up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acids. In some examples, an antigen can represent a clade if its sequence differs from at least 50% of the strains of the represented clade by up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acids. In some examples, an antigen can represent a clade if its sequence differs from at least 60% of the strains of the represented clade by up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acids. In some examples, an antigen can represent a clade if its sequence differs from at least 70% of the strains of the represented clade by up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acids. In some examples, an antigen can represent a clade if its sequence differs from at least 80% of the strains of the represented clade by up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acids.In some examples, an antigen may represent a clade if its sequence differs from that of the strains of the represented clade by at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acids, up to at least 90%. In some examples, an antigen may represent a clade if its sequence differs from that of all strains of the represented clade by at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acids.

[0267] An antigen may represent a clade if its edit distance is up to 5% of the average size of the antigens of the represented clade. In some examples, an antigen may represent a clade if its edit distance is up to 10%, 15%, or 20% of the average size of the antigens of the represented clade. An antigen may represent a clade if its sequence differs from that of all strains of the represented clade by at most 10% of the average size of the antigens of the represented clade and is at least 95%, 96%, 97%, 98%, or 99% different. In some examples, an antigen may represent a clade if its sequence differs from that of all strains of the represented clade by at most 15% of the average size of the antigens of the represented clade and is at least 95%, 96%, 97%, 98%, or 99% different. In some examples, an antigen may represent a clade if its sequence differs from that of all strains of the represented clade by at most 20% of the average size of the antigens of the represented clade and is at least 95%, 96%, 97%, 98%, or 99% different.

[0268] An antigen can represent a clade if its edit distance is 75% or less of the average size of the antigens of the represented clade. In some examples, an antigen can represent a clade if its edit distance is 70% or less, 80% or less, 90% or less, 95% or less, or 99% or less of the average size of the antigens of the represented clade.

[0269] An antigen can represent a clade if it is 90% or less of the average size of the antigens of the represented clade and its sequence differs from at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all strains of the represented clade. In some examples, an antigen can represent a clade if it is 80% or less of the average size of the antigens of the represented clade and its sequence differs from at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all strains of the represented clade. In some examples, an antigen can represent a clade if it is 70% or less of the average size of the antigens of the represented clade and its sequence differs from at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all strains of the represented clade.

[0270] An antigen can represent a clade if its edit distance is up to 5% of the average size of at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all strains of the represented clade. In some examples, an antigen can represent a clade if its edit distance is up to 10% of the average size of at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all strains of the represented clade. In some examples, an antigen can represent a clade if its edit distance is up to 15% of the average size of at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all strains of the represented clade.

[0271] An antigen can represent a clade if its edit distance is less than or equal to 90% of the average size of all strains in the represented clade at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some examples, an antigen can represent a clade if its edit distance is less than or equal to 80% of the average size of all strains in the represented clade at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some examples, an antigen can represent a clade if its edit distance is less than or equal to 70% of the average size of all strains in the represented clade at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0272] Some vaccines can include an antigen with an average edit distance from each of the other antigens that is at least 5% of the average size of the antigens within the clade. Some vaccines can include an antigen with an average edit distance from at least one of the other antigens that is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25% of the average size of the antigens within the clade. In such vaccines, some vaccines can include an antigen with an average edit distance from each of the other antigens that is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25% of the average size of the antigens within the clade. In such vaccines, the actual edit distances may vary or be similar. In some examples, there will be no antigen with an actual edit distance that is exactly at least 5% of the average size of the antigens within the clade.

[0273] In some examples, the minimum pairwise edit distance between two or more antigens in the set may be 1 or less. In some examples, the minimum pairwise edit distance between two or more antigens in the set may be 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 20 or less, 30 or less, 40 or less, or 50 or less. In some examples, the minimum pairwise edit distance between two or more antigens in the set may be at least 1. In some examples, the minimum pairwise edit distance between two or more antigens in the set may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, or at least 50.

[0274] In some examples, the minimum pairwise edit distance may be about 1% or less, about 2% or less, about 3% or less, about 4% or less, about 5% or less, about 6% or less, about 7% or less, about 8% or less, about 9% or less, about 10% or less, about 15% or less, about 20%, or about 25% or less of the size of the antigen. In some examples, the minimum pairwise edit distance may be at least about 5% of the size of the antigen. In some examples, the minimum pairwise edit distance may be at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, or at least about 25% of the size of the antigen.

[0275] In some examples, the maximum pairwise edit distance can be at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 700, or at least 800. In some examples, the maximum pairwise edit distance can be 5 or less, 10 or less, 20 or less, 30 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, 100 or less, 150 or less, 200 or less, 250 or less, 300 or less, 350 or less, 400 or less, 450 or less, 500 or less, 550 or less, 600 or less, 700 or less, or 800 or less.

[0276] In some examples, the maximum pairwise edit distance can be about 75% or less of the size of the antigen. In some examples, the maximum pairwise edit distance can be about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, about 75% or less, about 80% or less, about 85% or less, about 90% or less, or about 95% or less of the size of the antigen. In some examples, the maximum pairwise edit distance can be at least about 75% of the size of the antigen. In some examples, the maximum pairwise edit distance can be at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the size of the antigen.

[0277] In some examples, the minimum pairwise edit distance between two or more antigens in the set can be 1 or less, and the maximum pairwise edit distance can be at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be at least 1, and the maximum pairwise edit distance can be at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be at least 1, and the maximum pairwise edit distance can be 5 or less, 10 or less, 20 or less, 30 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, 100 or less, 200 or less, 300 or less, 400 or less, 500 or less, 600 or less, 700 or less, or 800 or less. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be 1 or less, and the maximum pairwise edit distance can be 5 or less, 10 or less, 20 or less, 30 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, 100 or less, 200 or less, 300 or less, 400 or less, 500 or less, 600 or less, 700 or less, or 800 or less.

[0278] In some examples, the minimum pairwise edit distance between two or more antigens in a set can be about 1% or less, and the maximum pairwise edit distance can be at least about 75%. In some examples, the minimum pairwise edit distance between two or more antigens in a set can be at least about 1%, and the maximum pairwise edit distance can be at least about 75%. In some examples, the minimum pairwise edit distance between two or more antigens in a set can be at least about 1%, and the maximum pairwise edit distance can be 75% or less. In some examples, the minimum pairwise edit distance between two or more antigens in a set can be about 1% or less, and the maximum pairwise edit distance can be about 75% or less.

[0279] In some examples, the minimum pairwise edit distance between two or more antigens in a set can be about 5% or less, and the maximum pairwise edit distance can be at least about 75%. In some examples, the minimum pairwise edit distance between two or more antigens in a set can be at least about 5%, and the maximum pairwise edit distance can be at least about 75%. In some examples, the minimum pairwise edit distance between two or more antigens in a set can be at least about 5%, and the maximum pairwise edit distance can be 75% or less. In some examples, the minimum pairwise edit distance between two or more antigens in a set can be about 5% or less, and the maximum pairwise edit distance can be about 75% or less.

[0280] In some examples, the minimum pairwise edit distance between two or more antigens in the set can be about 20% or less, and the maximum pairwise edit distance can be at least about 75%. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be at least about 20%, and the maximum pairwise edit distance can be at least about 75%. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be at least about 20%, and the maximum pairwise edit distance can be 75% or less. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be about 20% or less, and the maximum pairwise edit distance can be about 75% or less.

[0281] In some examples, the minimum pairwise edit distance between two or more antigens in the set can be about 1% or less, and the maximum pairwise edit distance can be at least about 90%. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be at least about 1%, and the maximum pairwise edit distance can be at least about 90%. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be at least about 1%, and the maximum pairwise edit distance can be 90% or less. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be about 1% or less, and the maximum pairwise edit distance can be about 90% or less.

[0282] In some examples, the minimum pairwise edit distance between two or more antigens in the set can be about 5% or less, and the maximum pairwise edit distance can be at least about 90%. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be at least about 5%, and the maximum pairwise edit distance can be at least about 90%. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be at least about 5%, and the maximum pairwise edit distance can be 90% or less. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be about 5% or less, and the maximum pairwise edit distance can be about 90% or less.

[0283] In some examples, the minimum pairwise edit distance between two or more antigens in the set can be about 20% or less, and the maximum pairwise edit distance can be at least about 90%. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be at least about 20%, and the maximum pairwise edit distance can be at least about 90%. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be at least about 20%, and the maximum pairwise edit distance can be 90% or less. In some examples, the minimum pairwise edit distance between two or more antigens in the set can be about 20% or less, and the maximum pairwise edit distance can be about 90% or less.

[0284] The vaccine can include a set of antigens where the minimum pairwise edit distance between two antigens can be 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 11% or less, 12% or less, 13% or less, 14% or less, 15% or less, 16% or less, 17% or less, 18% or less, 19% or less, or 10% or less (inclusive). The vaccine can include a set of antigens where the minimum pairwise edit distance between two antigens can be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 10% (inclusive). The vaccine can include a set of antigens where the maximum pairwise edit distance between two antigens can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% (inclusive). The vaccine can include a set of antigens where the maximum pairwise edit distance between two antigens can be 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less, or 95% or less (inclusive).

[0285] In some examples, each antigen in the set can share at least 90%, at least 95%, or at least 99% sequence identity with at least one antigen in the set over a length of at least 100 amino acids. In some examples, each antigen in the set can share at least 90%, at least 95%, or at least 99% sequence identity with at least one antigen in the set over a length of at least 300 amino acids. In some examples, each antigen in the set can share at least 90%, at least 95%, or at least 99% sequence identity with at least one antigen in the set over a length of at least 500 amino acids.

[0286] The vaccine can include an antigen within the set that has at least 1%, at least 5%, or at least 10% sequence identity different from other antigens within the set. In some examples, the vaccine can include an antigen within the set that has at least 1%, at least 5%, or at least 10% sequence identity different from all other antigens within the set.

[0287] The vaccine can include an antigen within the set that has 75% or less sequence identity different from at least one other antigen within the set. In some examples, the vaccine can include an antigen within the set that has 75% or less sequence identity different from all other antigens within the set.

[0288] The vaccine can include a set of antigens representing at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the fifth clade of the microorganism. In some examples, the vaccine can include a set of antigens representing at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the sixth clade of the microorganism. In further examples, the vaccine can include a set of antigens representing at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the seventh clade of the microorganism. In some examples, the vaccine can include a set of antigens representing at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the eighth clade of the microorganism. In some examples, the vaccine can include a set of antigens representing at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the ninth clade of the microorganism. In some examples, the vaccine can include a set of antigens representing at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the tenth clade of the microorganism.In some examples, the vaccine can include a set of antigens that represent at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the 11th clade of the microorganism. In some examples, the vaccine can include a set of antigens that represent at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the 12th clade of the microorganism. In some examples, the vaccine can include a set of antigens that represent at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the 13th clade of the microorganism. In some examples, the vaccine can include a set of antigens that represent at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the 14th clade of the microorganism. In some examples, the vaccine can include a set of antigens that represent at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the 15th clade of the microorganism. In some examples, the vaccine can include a set of antigens that represent at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the 20th clade of the microorganism.In some examples, the vaccine can include a set of antigens that represent at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the 25th clade of the microorganism. In some examples, the vaccine can include a set of antigens that represent at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the 30th clade of the microorganism.

[0289] The vaccine can include a set of antigens that represent at least 60% of the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, or other suitable clades of the microorganism. In some examples, the vaccine can include a set of antigens that represent at least 70% of the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, or other suitable clades of the microorganism. In further examples, the vaccine can include a set of antigens that represent at least 80% of the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, or other suitable clades of the microorganism. In still further examples, the vaccine can include a set of antigens that represent at least 90% of the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, or other suitable clades of the microorganism.

[0290] The minimum number of branches between any two antigens can be 1 in some vaccines. The maximum number of branches between any two antigens can be at least 5, at least 10, at least 15, or at least 20. In some vaccines, the minimum number of branches between any two antigens can be 1, and the maximum number of branches between any two antigens can be at least 5. In some vaccines, the minimum number of branches between any two antigens can be 1, and the maximum number of branches between any two antigens can be at least 10. In some vaccines, the minimum number of branches between any two antigens can be 1, and the maximum number of branches between any two antigens can be at least 15. In some vaccines, the minimum number of branches between any two antigens can be 1, and the maximum number of branches between any two antigens can be at least 20. In some vaccines, the minimum number of branches between any two antigens can be less than 5, and the maximum number of branches between any two antigens can be at least 5. In some vaccines, the minimum number of branches between any two antigens can be less than 5, and the maximum number of branches between any two antigens can be at least 10. In some vaccines, the minimum number of branches between any two antigens can be less than 5, and the maximum number of branches between any two antigens can be at least 15. In some vaccines, the minimum number of branches between any two antigens can be less than 5, and the maximum number of branches between any two antigens can be at least 20. In some vaccines, the minimum number of branches between any two antigens can be less than 10, and the maximum number of branches between any two antigens can be at least 10. In some vaccines, the minimum number of branches between any two antigens can be less than 10, and the maximum number of branches between any two antigens can be at least 15. In some vaccines, the minimum number of branches between any two antigens can be less than 10, and the maximum number of branches between any two antigens can be at least 20.

[0291] The vaccine, which is an influenza vaccine, can contain an antigen selected from Table 2, or a fragment or homolog thereof. When the vaccine contains a fragment or homolog of the antigen, the fragment or homolog can be at least a certain size. In some examples, the fragment or homolog can be at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids. In some examples, the fragment or homolog can be at most 10, at most 15, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, at most 50, at most 55, at most 60, at most 65, at most 70, at most 75, at most 80, at most 85, at most 90, at most 95, at most 100, at most 110, or at most 120 amino acids, at most 150 amino acids, at most 200 amino acids, at most 300 amino acids, at most 400 amino acids, at most 500 amino acids, at most 600 amino acids, at most 700 amino acids, at most 800 amino acids, at most 900 amino acids, or at most 1000 amino acids.

[0292] In some examples, the antigen in the vaccine can be selected from SEQ ID NOs: 1 to 87. In some examples, the antigen in the vaccine can be at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or at least about 95% identical to a sequence selected from SEQ ID NOs: 1 to 87.

[0293] In some examples, the antigen in the vaccine can be selected from SEQ ID NOs: 88 to 127. In some examples, the antigen in the vaccine is at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or at least about 95% identical to a sequence selected from SEQ ID NOs: 88 to 127.

[0294] In some examples, the antigen in the vaccine can be selected from SEQ ID NOs: 128 to 171. In some examples, the antigen in the vaccine is at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or at least about 95% identical to a sequence selected from SEQ ID NOs: 128 to 171.

[0295] In some examples, the antigen in the vaccine can be selected from SEQ ID NOs: 172 to 267. In some examples, the antigen in the vaccine is at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or at least about 95% identical to a sequence selected from SEQ ID NOs: 172 to 267. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 9-1

Table 9-2

Table 9-3

Table 9-4

Table 9-5

Table 9-6

Table 9-7

Table 9-8

Table 9-9

Table 9-10

Table 9-11

Table 9-12

Table 9-13

Table 9-14

Table 9-15

Table 9-16

Table 9-17

Table 9-18

Table 9-19

Table 9-20

Table 9-21

Table 9-22

Table 9-23

Table 9-24

Table 9-25

Table 9-26

Table 9-27

Table 9-28

Table 9-29

Table 9-30

Table 9-31

[0296] Notwithstanding the appended claims, the disclosure herein is also defined by the following clauses. 1. A vaccine comprising a set of antigens representing at least 60% of the fifth clade of a microorganism. 2. A vaccine comprising a set of antigens of a microorganism, wherein the minimum pairwise edit distance between two antigens is at least 10% of the average size of the antigens, and the maximum pairwise edit distance between two antigens is 98% or less of the average size of the antigens. 3. A vaccine comprising a set of antigens representing at least 60% of all operational taxonomic units (OTUs) of a microorganism. 4. The vaccine according to clause 1 or clause 3, wherein an antigen represents a clade if the sequence of the antigen is up to 40% of the average size of the antigens of the represented clade that are different from other members of the represented clade. 5. The vaccine according to clause 1 or clause 3, wherein an antigen represents a clade if the edit distance is up to 5% of the size of another strain of the represented clade. 6. The vaccine according to clause 1 or clause 3, wherein an antigen represents a clade if the sequence of the antigen is up to 20% of the average size of the antigens of the represented clade and is different from at least 95%, 96%, 97%, 98%, or 99% of all strains of the represented clade. 7. The vaccine according to clause 1 or clause 3, wherein an antigen represents a clade if the sequence of the antigen is up to 10% of the average size of the antigens of the represented clade and is different from at least 95%, 96%, 97%, 98%, or 99% of all strains of the represented clade. 8. The vaccine according to clause 1 or clause 3, wherein an antigen represents a clade if the edit distance is up to 5% of the size of at least 95%, 96%, 97%, 98%, or 99% of all strains of the represented clade. 9. If the antigen sequence is at an edit distance of up to 25 from all strains of the represented clade, the vaccine according to clause 1 or clause 3, wherein the antigen represents the clade. 10. If the antigen sequence is at an edit distance of up to 100 from all strains of the represented clade, the vaccine according to clause 1 or clause 3, wherein the antigen represents the clade. 11. If the antigen sequence is present within the clade, the vaccine according to clause 1 or clause 3, wherein the antigen represents the clade. 12. The antigen is derived from a library of microbial variants, a. Two antigens within the set having the maximum edit distance have an edit distance S; b. Two antigens within the library having the maximum edit distance have an edit distance L; c. S is at least 60% of L, A vaccine composition comprising a set of antigens. 13. A vaccine composition comprising at least four influenza virus hemagglutinin antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to another influenza virus hemagglutinin antigen polypeptide, wherein each polypeptide comprises an antigen that is at least 90% identical among the four influenza virus hemagglutinin antigen polypeptides. 14. A vaccine composition comprising at least four influenza virus neuraminidase antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to another influenza virus neuraminidase antigen polypeptide, wherein each polypeptide comprises an antigen that is at least 90% identical among the four influenza virus neuraminidase antigen polypeptides. 15. A vaccine composition comprising at least four HIV gp160 antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to another HIV gp160 antigen polypeptide, wherein each polypeptide comprises an antigen that is at least 90% identical among the four HIV gp160 antigen polypeptides. 16. A vaccine composition comprising at least four HIV gp120 antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to another HIV gp120 antigen polypeptide, wherein each polypeptide comprises an antigen that is at least 90% identical among the four HIV gp120 antigen polypeptides. 17. A vaccine composition comprising at least four HIV gp41 antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to another HIV gp41 antigen polypeptide, wherein each polypeptide comprises an antigen that is at least 90% identical among the four HIV gp41 antigen polypeptides. 18. A vaccine composition comprising a set of antigens that activate an immune response in a subject against at least six strains specified in Table 1. 19. The vaccine composition according to clause 13 or clause 18, wherein the immune response is detectable using head-specific antibodies in a hemagglutination inhibition assay. 20. The vaccine composition according to clause 19, wherein when tested using a hemagglutination inhibition assay, the immune response is at least 2-fold greater when using the antigen than when using H1N1 + H3N2 + HAB. 21. The vaccine composition according to clause 14, wherein when tested using a hemagglutination inhibition assay, the immune response is at least 10-fold greater when using the antigen than when using H1N1 + H3N2 + HAB. 22. The vaccine composition according to clause 19, wherein when tested using a hemagglutination inhibition assay, the immune response is at least 100-fold greater when using the antigen than when using H1N1 + H3N2 + HAB. 23. The vaccine according to clause 1, clause 2, clause 3, or clause 12, wherein the microorganism is a bacterium. 24. The vaccine according to clause 1, clause 2, clause 3, or clause 12, wherein the microorganism is a virus. 25. The vaccine composition according to clause 24, wherein the virus is influenza. 26. The vaccine composition according to clause 25, wherein the influenza is type A. 27. The vaccine composition according to clause 25, wherein the influenza is type B. 28. The vaccine according to clause 26, wherein the type A influenza is H1N1, H1N2, H3N1, H3N2, or H2N3. 29. The vaccine according to clause 28, wherein the type A influenza is H1N1. 30. The vaccine according to clause 28, wherein the type A influenza is H3N2. 31. The vaccine composition according to clause 24, wherein the virus is human immunodeficiency virus (HIV). 32. The vaccine composition according to clause 31, wherein the HIV is HIV-1. 33. The vaccine composition according to clause 32, wherein the HIV-1 is HIV-1 of subtype A, subtype B, or subtype C. 34. The vaccine according to clause 1, clause 2, clause 3, or clause 12, wherein the antigen is a broadly neutralizing antigen of surface-exposed residues adjacent in three-dimensional space. 35. The vaccine composition according to clause 34, wherein the broadly neutralizing antigen is at the base of hemagglutinin. 36. The vaccine composition according to clause 34, wherein the broadly neutralizing antigen is at the head of hemagglutinin. 37. The vaccine composition according to clause 34, wherein the broadly neutralizing antigen is at neuraminidase. 38. The vaccine composition according to clause 34, wherein the broadly neutralizing antigen is at gp160. 39. The vaccine composition according to clause 34, wherein the broadly neutralizing antigen is at gp120. 40. The vaccine composition according to clause 34, wherein the broadly neutralizing antigen is at gp41. 41. The vaccine according to clause 1, clause 2, clause 3, or clause 12, wherein the antigen is a widely conserved fragment of hemagglutinin. 42. The vaccine according to clause 41, wherein the antigen is a widely conserved fragment of the head of hemagglutinin. 43. The vaccine according to clause 41, wherein the antigen is a widely conserved fragment of the base of hemagglutinin. 44. The vaccine according to clause 1, clause 2, clause 3, or clause 12, wherein the antigen is a widely conserved fragment of neuraminidase. 45. The vaccine according to clause 1, clause 2, clause 3, or clause 12, wherein the antigen is a widely conserved fragment of gp160. 46. The vaccine according to clause 1, clause 2, clause 3, or clause 12, comprising at least one antigen selected from SEQ ID NOs: 1 to 87. 47. The vaccine according to clause 1, clause 2, clause 3, or clause 12, comprising at least one antigen that is at least 60% identical to any one of SEQ ID NOs: 1 to 87. 48. The vaccine according to clause 1, clause 2, clause 3, or clause 12, comprising at least one antigen that is at least 70% identical to any one of SEQ ID NOs: 1 to 87. 49. The vaccine according to clause 1, clause 2, clause 3, or clause 12, comprising at least one antigen that is at least 80% identical to any one of SEQ ID NOs: 1 to 87. 50. The vaccine according to clause 1, clause 2, clause 3, or clause 12, comprising at least one antigen that is at least 90% identical to any one of SEQ ID NOs: 1 to 87. 51. The vaccine according to clause 1, clause 2, clause 3, or clause 12, comprising at least one antigen selected from SEQ ID NOs: 88 to 127. 52. The vaccine according to clause 1, clause 2, clause 3, or clause 12, comprising at least one antigen that is at least 60% identical to any one of SEQ ID NOs: 88 to 127. 53. The vaccine according to clause 1, clause 2, clause 3, or clause 12, comprising at least one antigen that is at least 70% identical to any one of SEQ ID NOs: 88 to 127. The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 80% identical to any one of SEQ ID NOs: 88 to 127. 55. The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 90% identical to any one of SEQ ID NOs: 88 to 127. 56. The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen selected from SEQ ID NOs: 128 to 171. 57. The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 60% identical to any one of SEQ ID NOs: 128 to 171. 58. The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 70% identical to any one of SEQ ID NOs: 128 to 171. 59. The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 80% identical to any one of SEQ ID NOs: 128 to 171. 60. The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 90% identical to any one of SEQ ID NOs: 128 to 171. 61. The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen selected from SEQ ID NOs: 172 to 267. 62. The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 60% identical to any one of SEQ ID NOs: 172 to 267. 63. The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 70% identical to any one of SEQ ID NOs: 172 to 267. 64. The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 80% identical to any one of SEQ ID NOs: 172 to 267. The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 90% identical to any one of SEQ ID NOs: 172 to 267. 66. The vaccine according to item 1, item 2, item 3, or item 12, wherein the set of antigens comprises at least 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 different antigens. 67. The vaccine composition according to item 1, item 2, item 3, or item 12, wherein the set of antigens comprises at least 30 antigens. 68. The vaccine composition according to item 1, item 2, item 3, or item 12, wherein the set of antigens comprises at least 50 antigens. 69. The vaccine according to item 1, item 2, item 3, or item 12, wherein the antigen has an average edit distance from each of the other antigens that is at least 5% of the average size of the antigens within the clade. 70. The vaccine according to item 1, item 3, or item 12, wherein the minimum pairwise edit distance between two or more antigens within the set is 1 or less, and the maximum pairwise edit distance is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100. 71. The vaccine according to item 1, item 3, or item 4, wherein the minimum pairwise edit distance between two or more antigens within the set is 5% or less of the size of the antigen, and the maximum pairwise edit distance is at least 75% of the size of the antigen. 72. The vaccine according to item 1, wherein the clade is a clade of a phylogenetic tree that is a neighbor-joining clustering tree or a most parsimonious tree. 73. The vaccine according to item 1, wherein the primary clade is a clade that is not entirely or partially contained within another higher-level clade. 74. The vaccine according to item 1, wherein each X-level clade is phylogenetically below X-1 branching nodes in the phylogenetic tree of the microorganism. The vaccine according to claim 1, further comprising an antigen representing at least 60% of each of the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, or 15th clade of the microorganism. The vaccine according to claim 75, wherein the represented clade is clade Y included in clade Y-1. The vaccine according to claim 1, wherein the representative clade is a node-based clade. The vaccine according to claim 1, wherein the representative clade is a stem-based clade. The vaccine according to claim 1, wherein the representative clade is an apomorphy-based clade. The vaccine according to claim 1, wherein the average number of branches between each antigen in the set is at least 1, 3, 5, or 10. The vaccine according to claim 1, wherein the minimum number of branches between any two antigens in the set is 1 or less, and the maximum number of branches between any two antigens in the set is at least 5, 10, or 15. The vaccine according to claim 3, wherein each OTU comprises sequences that are at least 95% identical to each other. The vaccine according to claim 3, wherein each OTU is composed of at least 3 different sequences. The vaccine composition according to claim 1, claim 2, claim 3, or claim 12, wherein each antigen in the set shares at least 90%, 95, or 99% sequence identity with at least one other antigen in the set. The vaccine composition according to claim 1, claim 2, claim 3, claim 12, wherein each antigen in the set shares at least 90%, 95, or 99% sequence identity with at least one other antigen in the set over at least 100 amino acids in length. The vaccine composition according to claim 1, claim 2, claim 3, or claim 12, wherein each antigen in the set differs from each of the other antigens in the set by at least 5% sequence identity. The vaccine composition according to claim 1, claim 2, claim 3, claim 12, wherein each antigen in the set differs from each of the other antigens in the set by 75% or less sequence identity. 88. The microbial vaccine composition according to clause 1, clause 2, clause 3, or clause 12, wherein each antigen is a peptide comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 85, 100, 110, or 120 amino acids. 89. The vaccine composition according to clause 1, clause 2, or clause 3, wherein the antigen is selected from Table 2, Table 3, Table 4, Table 5, or a fragment or homolog thereof. 90. The vaccine composition according to clause 89, comprising two or more antigens from Table 2, Table 3, Table 4, Table 5, or a fragment or homolog thereof. 91. The vaccine composition according to clause 90, comprising three or more antigens from Table 2, Table 3, Table 4, Table 5, or a fragment or homolog thereof. 92. The vaccine composition according to clause 91, comprising five or more antigens from Table 2, Table 3, Table 4, Table 5, or a fragment or homolog thereof. 93. The vaccine composition according to clause 92, comprising ten or more antigens from Table 2, Table 3, Table 4, Table 5, or a fragment or homolog thereof. 94. The vaccine composition according to clause 89, wherein the fragment comprises at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, or 120 amino acids. 95. The vaccine composition according to clause 89, wherein the fragment comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, or 120 amino acids. 96. The vaccine composition according to clause 89, wherein the homolog comprises a sequence having at least 90% sequence identity with the antigen of Table 1. 97. The library comprises at least 1×10 4 、1×10 5 、1×10 6 different variants of microorganisms, and the vaccine composition according to clause 12. 98. The vaccine composition according to clause 12, wherein the library comprises at least 90% of all known sequences of the microorganism. 99. A pharmaceutical composition comprising the vaccine composition according to any of the preceding clauses and a pharmaceutically acceptable diluent, adjuvant, additive, or any combination thereof. 100. The vaccine composition according to clause 99, wherein the vaccine is in the form of an aerosol formulation. 101. The vaccine composition according to clause 99, wherein the vaccine is in the form of an injectable formulation. 102. Each antigen is at a concentration that does not alone provide a significant prophylactic immune response against the broad neutralizing antigen of the subject, but collectively, a plurality of antigens have a combined concentration that provides an immune response against the broad neutralizing antigen of the subject. The vaccine composition according to any of the preceding clauses. 103. The vaccine composition according to clause 102, wherein the subject is a bird. 104. The vaccine composition according to clause 102, wherein the subject is a mammal. 105. The vaccine composition according to clause 103, wherein the subject is a human. 106. The vaccine composition according to clause 103, wherein the subject is a pig. 107. A virus-like particle (VLP) comprising the vaccine composition according to any of the preceding clauses. 108. A recombinant expression vector comprising a nucleic acid molecule encoding: (a) A set of antigens representing at least 60% of each of the primary and secondary clades of the microorganism; (b) A set of antigens wherein the minimum pairwise edit distance between two antigens is 25 or less and the maximum pairwise edit distance between two antigens is at least 300; (c) A set of antigens representing at least 60% of all operational taxonomic units (OTUs) of the microorganism; or (d) A set of antigens representing at least 60% of all operational taxonomic units (OTUs) of the microorganism. 109. A recombinant expression vector comprising a nucleic acid molecule encoding: (a) A set of antigens representing at least 60% of each of the primary and secondary clades of a microorganism; (b) A set of antigens in which the minimum pairwise edit distance between two antigens is 5% or less of the size of the antigen and the maximum pairwise edit distance is at least 75% of the size of the antigen; (c) A set of antigens representing at least 60% of all operational taxonomic units (OTUs) of a microorganism; or (d) A set of antigens representing at least 60% of all operational taxonomic units (OTUs) of a microorganism. 110. A method for treating an infection or reducing the likelihood of infection in a subject, comprising administering the vaccine composition according to any of the preceding clauses to the subject. 111. A method for treating or reducing the likelihood of influenza infection in a subject, comprising administering a vaccine composition that provides effective immune activation against seasonal influenza to the subject for at least 3, 4, 5, 6, 7, 8, 9, or 10 years. 112. The method according to clause 110 or clause 111, wherein the subject is a human. 113. The method according to clause 110 or clause 111, wherein the subject is a domesticated animal. 114. A method for preparing a vaccine composition, comprising: (a) A set of antigens representing at least 60% of each of the primary and secondary clades of a microorganism; (b) A set of antigens in which the minimum pairwise edit distance between two antigens is 5% or less of the size of the antigen and the maximum pairwise edit distance is at least 75% of the size of the antigen; (c) A set of antigens representing at least 60% of all operational taxonomic units (OTUs) of a microorganism; or (d) A set of antigens representing at least 60% of all operational taxonomic units (OTUs) of a microorganism A method comprising. 115. The method of clause 114, Obtaining a plurality of antigen sequences from a library of microbial strains; and Aligning a plurality of antigen sequences to create a phylogenetic tree of the antigens A method further comprising.

[0297] As used herein, the term "about" generally refers to a range that is 2%, 5%, 10%, or 15% greater or less (±) than the recited numerical value within the context of a particular usage. For example, "about 10" includes the range from 8.5 to 11.5. As used herein, the terms "about" and "approximately" when used to modify a numerical value or numerical range indicate that deviations up to about 0.2%, about 0.5%, about 1%, about 2%, about 5%, about 7.5%, or about 10% (or any integer from about 1% to 10%) above or below the value or range remain within the scope of the intended meaning of the recited value or range.

[0298] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a method" includes one or more methods, and / or steps of the kind described herein, and / or those that will be apparent to those skilled in the art upon reading this disclosure.

[0299] Preferred embodiments of the invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby. In certain embodiments, for example, the following items are provided. (Item 1) A vaccine comprising a set of antigens representing at least 60% of the fifth clade of a microorganism. (Item 2) A vaccine comprising a set of antigens of a microorganism, wherein the minimum pairwise edit distance between two of the antigens is at least 10% of the average size of the antigens, and the maximum pairwise edit distance between two of the antigens is 98% or less of the average size of the antigens. (Item 3) A vaccine comprising a set of antigens representing at least 60% of all operational taxonomic units (OTUs) of the microorganism. (Item 4) An antigen of a represented clade, wherein the sequence of the antigen differs from other members of the represented clade and is up to 40% of the average size of the antigen, and the antigen represents the clade, the vaccine according to item 1 or item 3. (Item 5) If the edit distance is up to 5% of the size of another strain of the represented clade, the antigen represents the clade, the vaccine according to item 1 or item 3. (Item 6) An antigen of a represented clade, wherein the sequence of the antigen differs from at least 95%, 96%, 97%, 98%, or 99% of all strains of the represented clade and is up to 20% of the average size of the antigen, and the antigen represents the clade, the vaccine according to item 1 or item 3. (Item 7) An antigen of a represented clade, wherein the sequence of the antigen differs from at least 95%, 96%, 97%, 98%, or 99% of all strains of the represented clade and is up to 10% of the average size of the antigen, and the antigen represents the clade, the vaccine according to item 1 or item 3. (Item 8) If the edit distance is up to 5% of the size of 95%, 96%, 97%, 98%, or 99% of all strains of the represented clade, the antigen represents the clade, the vaccine according to item 1 or item 3. (Item 9) If the sequence of the antigen is at an edit distance of up to 25 from all strains of the represented clade, the antigen represents the clade, the vaccine according to item 1 or item 3. (Item 10) If the antigen sequence is at an edit distance of up to 100 from all strains of the represented clade, the vaccine according to item 1 or item 3, wherein the antigen represents the clade. (Item 11) If the antigen sequence is present within the clade, the vaccine according to item 1 or item 3, wherein the antigen represents the clade. (Item 12) A vaccine composition comprising a set of antigens, wherein the antigen is derived from a library of microbial variants, a. Two antigens of the set having the maximum edit distance have an edit distance S, b. Two antigens of the library having the maximum edit distance have an edit distance L, c. S is at least 60% of L, A vaccine composition comprising a set of antigens. (Item 13) A vaccine composition comprising at least four influenza virus hemagglutinin antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to other influenza virus hemagglutinin antigen polypeptides, each polypeptide comprising an antigen that is at least 90% identical among the four influenza virus hemagglutinin antigen polypeptides. (Item 14) A vaccine composition comprising at least four influenza virus neuraminidase antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to other influenza virus neuraminidase antigen polypeptides, each polypeptide comprising an antigen that is at least 90% identical among the four influenza virus neuraminidase antigen polypeptides. (Item 15) A vaccine composition comprising at least four HIV gp160 antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to other HIV gp160 antigen polypeptides, each polypeptide comprising an antigen that is at least 9 0% identical among the four HIV gp160 antigen polypeptides. (Item 16) A vaccine composition comprising at least four HIV gp120 antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to other HIV gp120 antigen polypeptides, each polypeptide comprising an antigen that is at least 90% identical among the four HIV gp120 antigen polypeptides. (Item 17) A vaccine composition comprising at least four HIV gp41 antigen polypeptides, each represented by a sequence that is at least 20% identical and at most 95% identical to other HIV gp41 antigen polypeptides, each polypeptide comprising an antigen that is at least 90% identical among the four HIV gp41 antigen polypeptides. (Item 18) A vaccine composition comprising a set of antigens that activate an immune response of a subject against at least six strains specified in Table 1. (Item 19) The vaccine composition according to item 13 or item 18, wherein the immune response is detectable using head-specific antibodies in a hemagglutination inhibition assay. (Item 20) The vaccine composition according to item 19, wherein when tested using the hemagglutination inhibition assay, the immune response is at least twice as large as when using the antigen compared to H1N1 + H3N2 + HAB. (Item 21) The vaccine composition according to item 14, wherein when tested using the hemagglutination inhibition assay, the immune response is at least ten times as large as when using the antigen compared to H1N1 + H3N2 + HAB. (Item 22) The vaccine composition according to item 19, wherein when tested using the hemagglutinin inhibition assay, the immune response is at least 100-fold greater than when using the antigen compared to H1N1 + H3N2 + HAB. (Item 23) The vaccine according to item 1, item 2, item 3, or item 12, wherein the microorganism is a bacterium. (Item 24) The vaccine according to item 1, item 2, item 3, or item 12, wherein the microorganism is a virus. (Item 25) The vaccine composition according to item 24, wherein the virus is influenza. (Item 26) The vaccine composition according to item 25, wherein the influenza is type A. (Item 27) The vaccine composition according to item 25, wherein the influenza is type B. (Item 28) The vaccine according to item 26, wherein the type A influenza is H1N1, H1N2, H3N1, H3N2, or H2N3. (Item 29) The vaccine according to item 28, wherein the type A influenza is H1N1. (Item 30) The vaccine according to item 28, wherein the type A influenza is H3N2. (Item 31) The vaccine composition according to item 24, wherein the virus is human immunodeficiency virus (HIV). (Item 32) The vaccine composition according to item 31, wherein the HIV is HIV-1. (Item 33) The vaccine composition according to item 32, wherein the HIV-1 is HIV-1 of subtype A, subtype B, or subtype C. (Item 34) The vaccine according to item 1, item 2, item 3, or item 12, wherein the antigen is a broadly neutralizing antigen of surface-exposed residues adjacent in three-dimensional space. (Item 35) The vaccine composition according to item 34, wherein the broadly neutralizing antigen is at the base of hemagglutinin. (Item 36) The vaccine composition according to item 34, wherein the broadly neutralizing antigen is at the head of hemagglutinin. (Item 37) The vaccine composition according to item 34, wherein the broadly neutralizing antigen is in neuraminidase. (Item 38) The vaccine composition according to item 34, wherein the broadly neutralizing antigen is in gp160. (Item 39) The vaccine composition according to item 34, wherein the broadly neutralizing antigen is in gp120. (Item 40) The vaccine composition according to item 34, wherein the broadly neutralizing antigen is in gp41. (Item 41) The vaccine according to item 1, item 2, item 3, or item 12, wherein the antigen is a broadly conserved fragment of hemagglutinin. (Item 42) The vaccine according to item 41, wherein the antigen is a broadly conserved fragment of the head of hemagglutinin. (Item 43) The vaccine according to item 41, wherein the antigen is a broadly conserved fragment of the base of hemagglutinin. (Item 44) The vaccine according to item 1, item 2, item 3, or item 12, wherein the antigen is a broadly conserved fragment of neuraminidase. (Item 45) The vaccine according to item 1, item 2, item 3, or item 12, wherein the antigen is a broadly conserved fragment of gp160. (Item 46) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen selected from SEQ ID NOs: 1 to 87. (Item 47) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 60% identical to any one of SEQ ID NOs: 1 to 87. (Item 48) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 70% identical to any one of SEQ ID NOs: 1 to 87. (Item 49) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 80% identical to any one of SEQ ID NOs: 1 to 87. (Item 50) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 90% identical to any one of SEQ ID NOs: 1 to 87. (Item 51) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen selected from SEQ ID NOs: 88 to 127. (Item 52) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 60% identical to any one of SEQ ID NOs: 88 to 127. (Item 53) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 70% identical to any one of SEQ ID NOs: 88 to 127. (Item 54) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 80% identical to any one of SEQ ID NOs: 88 to 127. (Item 55) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 90% identical to any one of SEQ ID NOs: 88 to 127. (Item 56) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen selected from SEQ ID NOs: 128 to 171. (Item 57) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 60% identical to any one of SEQ ID NOs: 128 to 171. (Item 58) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 70% identical to any one of SEQ ID NOs: 128 to 171. (Item 59) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 80% identical to any one of SEQ ID NOs: 128 to 171. (Item 60) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 90% identical to any one of SEQ ID NOs: 128 to 171. (Item 61) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen selected from SEQ ID NOs: 172 to 267. (Item 62) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 60% identical to any one of SEQ ID NOs: 172 to 267. (Item 63) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 70% identical to any one of SEQ ID NOs: 172 to 267. (Item 64) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 80% identical to any one of SEQ ID NOs: 172 to 267. (Item 65) The vaccine according to item 1, item 2, item 3, or item 12, comprising at least one antigen that is at least 90% identical to any one of SEQ ID NOs: 172 to 267. (Item 66) The vaccine according to item 1, item 2, item 3, or item 12, wherein the set of antigens comprises at least 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 different antigens. (Item 67) The vaccine composition according to item 1, item 2, item 3, or item 12, wherein the set of antigens comprises at least 30 antigens. (Item 68) The vaccine composition according to item 1, item 2, item 3, or item 12, wherein the set of antigens comprises at least 50 antigens. (Item 69) The vaccine according to item 1, item 2, item 3, item 12, wherein the antigen has an average edit distance from each of the other antigens that is at least 5% of the average size of the antigens within the clade. (Item 70) The vaccine according to item 1, item 3, or item 12, wherein the minimum pairwise edit distance between two or more of the antigens within the set is 1 or less, and the maximum pairwise edit distance is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100. (Item 71) The vaccine according to item 1, item 3, or item 4, wherein the minimum pairwise edit distance between two or more antigens within the set is 5% or less of the size of the antigen, and the maximum pairwise edit distance is at least 75% of the size of the antigen. (Item 72) The vaccine according to item 1, wherein the clade is a clade of a phylogenetic tree that is a neighbor-joining clustering tree or a most parsimonious tree. (Item 73) The vaccine according to item 1, wherein the primary clade is a clade that is not entirely or partially included within another higher-level clade. (Item 74) The vaccine according to item 1, wherein each X-th clade is phylogenetically below X-1 branching nodes in the phylogenetic tree of the microorganism. (Item 75) The vaccine according to item 1, further comprising an antigen representing at least 60% of each of the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, or 15th clades of the microorganism. (Item 76) The vaccine according to item 75, wherein the represented clade is clade Y included in clade Y-1. (Item 77) The vaccine according to item 1, wherein the representative clade is a node-based clade. (Item 78) The vaccine according to item 1, wherein the representative clade is a stem-based clade. (Item 79) The vaccine according to item 1, wherein the representative clade is an apomorphy-based clade. (Item 80) The vaccine according to item 1, wherein the average number of branches between each of the antigens in the set is at least 1, 3, 5, or 10. (Item 81) The vaccine according to item 1, wherein the minimum number of branches between any two antigens in the set is 1 or less, and the maximum number of branches between any two antigens in the set is at least 5, 10, or 15. (Item 82) The vaccine according to item 3, wherein each OTU comprises sequences that are at least 95% homologous to each other. (Item 83) The vaccine according to item 3, wherein each OTU is composed of at least 3 different sequences. (Item 84) The vaccine composition according to item 1, item 2, item 3, or item 12, wherein each of the antigens in the set shares at least 90%, 95, or 99% sequence identity with at least one other antigen in the set. (Item 85) The vaccine composition according to item 1, item 2, item 3, or item 12, wherein each of the antigens in the set shares at least 90%, 95%, or 99% sequence identity with at least one other antigen in the set over at least 100 amino acids in length. (Item 86) The vaccine composition according to item 1, item 2, item 3, or item 12, wherein each of the antigens in the set differs from each of the other antigens in the set by at least 5% sequence identity. (Item 87) The vaccine composition according to item 1, item 2, item 3, or item 12, wherein each of the antigens in the set differs from each of the other antigens in the set by 75% or less sequence identity. (Item 88) The microbial vaccine composition according to item 1, item 2, item 3, or item 12, wherein each antigen is a peptide comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 85, 100, 110, or 120 amino acids. (Item 89) The vaccine composition according to item 1, item 2, or item 3, wherein the antigen is selected from Table 2, Table 3, Table 4, Table 5, or a fragment or homolog thereof. (Item 90) The vaccine composition according to item 89, comprising two or more antigens from Table 2, Table 3, Table 4, Table 5, or fragments or homologs thereof. (Item 91) The vaccine composition according to item 90, comprising three or more antigens from Table 2, Table 3, Table 4, Table 5, or fragments or homologs thereof. (Item 92) The vaccine composition according to item 91, comprising five or more antigens from Table 2, Table 3, Table 4, Table 5, or fragments or homologs thereof. (Item 93) The vaccine composition according to item 92, comprising ten or more antigens from Table 2, Table 3, Table 4, Table 5, or fragments or homologs thereof. (Item 94) The vaccine composition according to item 89, wherein the fragment contains at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, or 120 amino acids. (Item 95) The vaccine composition according to item 89, wherein the fragment contains at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, or 120 amino acids. (Item 96) The vaccine composition according to item 89, wherein the homolog contains a sequence having at least 90% sequence identity with the antigen in Table 1. (Item 97) The vaccine composition according to item 12, wherein the library contains at least 1×10 4 , 1×10 5 , 1×10 6 different variants of the microorganism. (Item 98) The vaccine composition according to item 12, wherein the library contains at least 90% of all known sequences of the microorganism. (Item 99) A pharmaceutical composition comprising the vaccine composition according to any of the preceding items and a pharmaceutically acceptable diluent, adjuvant, additive, or any combination thereof. (Item 100) The vaccine composition according to item 99, wherein the vaccine is in the form of an aerosol formulation. (Item 101) The vaccine composition according to item 99, wherein the vaccine is in the form of an injectable formulation. (Item 102) For each of the antigens, the concentration is such that it does not provide a significant prophylactic immune response against the broad neutralizing antigen of the subject alone, but collectively, the plurality of antigens has a combined concentration that provides an immune response against the broad neutralizing antigen of the subject. The vaccine composition according to any of the preceding claims. (Item 103) The vaccine composition according to item 102, wherein the subject is a bird. (Item 104) The vaccine composition according to item 102, wherein the subject is a mammal. (Item 105) The vaccine composition according to item 103, wherein the subject is a human. (Item 106) The vaccine composition according to item 103, wherein the subject is a pig. (Item 107) Virus-like particles (VLPs) comprising the vaccine composition according to any of the preceding items. (Item 108) A recombinant expression vector comprising a nucleic acid molecule encoding the following: (a) A set of antigens representing at least 60% of each of the primary and secondary clades of a microorganism. (b) A set of antigens wherein the minimum pairwise edit distance between two of the antigens is 25 or less and the maximum pairwise edit distance between two of the antigens is at least 300. (c) A set of antigens representing at least 60% of all the operational taxonomic units (OTUs) of the microorganism, or (d) A set of antigens representing at least 60% of all the operational taxonomic units (OTUs) of the microorganism. (Item 109) A recombinant expression vector comprising a nucleic acid molecule encoding the following: (a) A set of antigens representing at least 60% of each of the primary and secondary clades of a microorganism. (b) A set of antigens wherein the minimum pairwise edit distance between two of the antigens is 5% or less of the size of the antigens and the maximum pairwise edit distance between two of the antigens is at least 75% of the size of the antigens. (c) A set of antigens representing at least 60% of all the operational taxonomic units (OTUs) of the microorganism, or (d) A set of antigens representing at least 60% of all the operational taxonomic units (OTUs) of the microorganism. (Item 110) A method for treating an infection or reducing the likelihood of infection in a subject, comprising administering to the subject a vaccine composition as described in any of the preceding items. (Item 111) A method for treating or reducing the likelihood of influenza infection in a subject, comprising administering to the subject a vaccine composition that provides immunostimulation effective against seasonal influenza for at least 3, 4, 5, 6, 7, 8, 9, or 10 years. (Item 112) The method according to item 110 or item 111, wherein the subject is a human. (Item 113) The method according to item 110 or item 111, wherein the subject is a domesticated animal. (Item 114) A method for preparing a vaccine composition, comprising (a) a set of antigens representing at least 60% of each of the primary and secondary clades of a microorganism, (b) a set of antigens wherein the minimum pairwise edit distance between two of said antigens is 5% or less of the size of said antigens and the maximum pairwise edit distance is at least 75% of the size of said antigens, (c) a set of antigens that is representative of at least 60% of all operational taxonomic units (OTUs) of said microorganism, or (d) a set of antigens that is representative of at least 60% of all operational taxonomic units (OTUs) of said microorganism The method comprising selecting. (Item 115) Obtaining a plurality of antigen sequences from a library of strains of said microorganism, and Aligning said plurality of antigen sequences to create a phylogenetic tree of said antigens The method according to item 114, further comprising.

Claims

[Claim 1] The invention described in the specification.

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