Nanoparticle based subunit vaccine for african swine fever virus

Abstract

Disclosed herein is a vaccine composition, wherein the vaccine is effective against African Swine Fever virus (ASFV), wherein the vaccine comprises at least one immunogenic peptide, wherein said immunogenic peptide comprises CD2v, EP153R, P72, p10, CP530R, E199L, or a functional variant or functional fragment thereof. These compositions can be present in the form of a vaccine for administration. The vaccine can be present in a kit, for example. The composition can be administered to a subject in need thereof in order to prevent, or lessen the severity of, ASFV infection in the subject.

Description

[0001] NANOPARTICLE BASED SUBUNIT VACCINE FOR AFRICAN

[0002] SWINE FEVER VIRUS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of, and priority to, U.S. Provisional Patent Application No. 63 / 581,095 filed September 7, 2023, the content of which is incorporated herein by reference in its entirety.

[0005] REFERNCE TO SEQUENCE LISTING

[0006] The sequence listing submitted on September 9, 2024, as an .XML file entitled “103361- 569W01_ST26.xml” created on September 9, 2024, and having a file size of 27,056 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0007] BACKGROUND

[0008] African swine fever virus (ASFV), the virus that causes African swine fever (ASF), is a highly contagious and deadly virus affecting both domestic and feral pig populations with mortality rates approaching one hundred percent within seven days of infection (Salguero et al. 2020). ASFV was first identified in domestic pigs in Kenya in the early 1900s, and since then, it has spread to the European Union, Russia, China, southeastern Asia, the Dominican Republic, Haiti, and, most recently, Nepal in 2022 resulting in huge economic impacts to the pork industry and trade economy (Sanchez-Cordon et al. 2018; Subedit et al. 2022).

[0009] Current control strategies of ASFV involve rapid detection and implementation of widespread infected animal depopulation procedures, as there is not a commercially available ASF vaccine. Although attempts to engineer ASFV vaccines have been made, the large genome and intricate immune evasion mechanisms of ASFV has rendered therapeutic development challenging. Because of the complex genetic makeup of ASFV, a subunit vaccine containing a single or a couple virus antigen(s) may not be sufficient to stimulate the immune system to prevent infection.

[0010] What is needed in the art are key ASFV immunogenic epitopes that enhance host immunogenicity, thereby lowering the risk of re-infection upon virus encounter. Further, what is needed is coupling the multiepitope protein vaccine with nanotechnology and delivering the vaccine in a manner that stimulates the mucosal immune system, further enhancing the efficacy of the ASFV vaccine. SUMMARY

[0011] Disclosed herein is a vaccine composition, wherein the vaccine is effective against African Swine Fever virus (ASFV), wherein the vaccine comprises at least one immunogenic peptide, wherein said immunogenic peptide comprises CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

[0012] Also disclosed herein is a method of eliciting or enhancing an immune response to ASFV in a subject, the method comprising administering to the subject a vaccine, wherein the vaccine comprises at least one immunogenic peptide, wherein said immunogenic peptide comprises CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

[0013] Further disclosed is a method of preventing or lessening the severity of symptoms associated with ASFV infection in a subject, the method comprising administering to the subject a vaccine, wherein the vaccine comprises at least one immunogenic peptide, wherein said immunogenic peptide comprises CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

[0014] DESCRIPTION OF DRAWINGS

[0015] Figs. 1A-1B show bacterial expression pRSET A vector with bacteriophage T7 promoter, N-terminal 6x His tag for purification, and Xpress tag (Fig. 1 A). Eukaryotic expression pSeqTag 2B vector with CMV promoter, IgK leader sequence for secretion, and C- terminal 6x His tag for purification (Fig. IB).

[0016] Figs. 2A-2B show bacterial expression of the multi epitope protein in BL21 (DE3) cells. Multiepitope protein was detected in the total (Fig. 2A, lane 2) and insoluble fractions (Fig. 2A, lane 4). Expression was confirmed using Western blot (Fig. 2B).

[0017] Figs. 3A-3C show eukaryotic expression of the multiepitope protein in ExpiCHO cells. Expression was detected in the supernatant and cell lysate fractions using a Western blot (Fig. 3 A). Multiepitope protein from the supernatant (Fig. 3B) and cell lysate (Fig. 3C) fractions was purified using His tag purification.

[0018] Fig- 4 shows the ASFV vaccine study experimental design in pigs. Details are described in Example 2.

[0019] Fig. 5 shows the timeline for the ASFV vaccine study. Details are described in Example Fig- 6 shows mean pig weights at day 42 post vaccination of the ASFV study from Example 2.

[0020] Fig- 7 shows a T cell proliferation assay from Example 2.

[0021] Figs. 8A-8B show results of an ELISA for serum collected from pigs on days 0, 22, and 42 from Example 2. Fig. 8 A shows M-CS ASF Protein + M-CS ADU SI 00. Fig. 8B shows AS Protein + M-CS ADU SI 00.

[0022] Fig. 9 shows FACS-T helper cell population (unstimulated vs. stimulated) from Example 2.

[0023] Fig. 10 shows FACS- Cytotoxic T cell population (unstimulated vs. stimulated) from Example 2.

[0024] DETAILED DESCRIPTION

[0025] Definitions

[0026] Disclosed herein are methods and compositions for treating or preventing ASFV in a subject that involve combining ASFV antigens with a nanoparticle.

[0027] An “immunogenic composition” is a composition of matter suitable for administration to a human or animal subject (e.g., in an experimental setting) that is capable of eliciting a specific immune response, e.g., against a pathogen, such as African Swine Fever Virus (ASFV). As such, an immunogenic composition includes one or more antigens (for example, whole purified virus or antigenic subunits, e.g., polypeptides, thereof) or antigenic epitopes. An immunogenic composition can also include one or more additional components capable of eliciting or enhancing an immune response, such as an excipient, carrier, and / or adjuvant. In certain instances, immunogenic compositions are administered to elicit an immune response that protects the subject against symptoms or conditions induced by a pathogen. In some cases, symptoms or disease caused by a pathogen is prevented (or treated, e.g., reduced or ameliorated) by inhibiting replication of the pathogen following exposure of the subject to the pathogen. In the context of this disclosure, the term immunogenic composition will be understood to encompass compositions that are intended for administration to a subject or population of subjects for the purpose of eliciting a protective or palliative immune response against the virus (that is, vaccine compositions or vaccines).

[0028] An “antigen” is a compound, composition, or substance that can stimulate the production of antibodies and / or a T cell response in an animal, including compositions that are injected, absorbed or otherwise introduced into an animal. The term “antigen” includes all related antigenic epitopes. The term “epitope” or “antigenic determinant” refers to a site on an antigen to which B and / or T cells respond. The “dominant antigenic epitopes” or “dominant epitope” are those epitopes to which a functionally significant host immune response, e.g., an antibody response or a T-cell response, is made. Thus, with respect to a protective immune response against a pathogen, the dominant antigenic epitopes are those antigenic moieties that when recognized by the host immune system result in protection from disease caused by the pathogen. The term “T-cell epitope” refers to an epitope that when bound to an appropriate MHC molecule is specifically bound by a T cell (via a T cell receptor). A “B-cell epitope” is an epitope that is specifically bound by an antibody (or B cell receptor molecule). An antigen can also affect the innate immune response.

[0029] An “immune response” is a response of different cell types of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen specific neutralizing antibodies. An immune response can also be a T cell response, such as a CD4+ T cell response and / or a CD8+ T cell response. In some cases, the response is specific for a particular antigen (that is, an “antigen-specific response”). An immune response can also include the innate response. If the antigen is derived from a pathogen, the antigen-specific response is a “pathogenspecific response.” A “protective immune response” is an immune response that inhibits a detrimental function or activity of a pathogen, reduces infection by a pathogen, or decreases symptoms (including death) that result from infection by the pathogen. A protective immune response can be measured, for example, by the inhibition of viral replication or plaque formation in a plaque reduction assay or virus-neutralization assay, or by measuring resistance to pathogen challenge in vivo.

[0030] The immunogenic compositions disclosed herein are suitable for preventing, ameliorating and / or treating disease caused by infection of the virus.

[0031] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0032] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0033] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

[0034] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0035] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0036] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces virus titer” means reducing the amount of virus in a given volume.

[0037] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0038] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, swine, bovine, avian, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Specifically disclosed here is a swine subject.

[0039] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0040] "Biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.

[0041] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. “Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0042] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative." For example, a negative control can be an untreated or mock treated control. A positive control, can be a control with a known positive response.

[0043] A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0044] "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0045] “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

[0046] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0047] “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

[0048] As used herein, the term “intranasal(ly)” refers to application of the compositions of the present invention to the surface of the skin and mucosal cells and tissues of the nasal passages, e.g., nasal mucosa, sinus cavity, nasal turbinates, or other tissues and cells which line the nasal passages.

[0049] As used herein, the terms "drug composition" and "drug", "vaccinal composition" and "vaccine" and "vaccine composition" and "drug-vaccine composition" and "drug-vaccine dual agent" and "therapeutic composition" and "therapeutic-immunologic composition" cover any composition that induces protection against a pathogen. In some embodiments, the protection may be due to an inhibition or prevention of infection by a pathogen. In other embodiments, the protection may be induced by an immune response against the antigen(s) of interest, or which efficaciously protects against the antigen; for instance, after administration or injection into the subject, elicits a protective immune response against the targeted antigen or immunogen or provides efficacious protection against the antigen or immunogen expressed from the inventive adenovirus vectors of the invention.

[0050] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0051] General Description

[0052] Disclosed herein is a vaccine composition, wherein the vaccine is effective against African Swine Fever virus (ASFV). The vaccine can comprise at least one immunogenic peptide (also referred to herein as an “antigen”) of ASFV, wherein said immunogenic peptide is selected from CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof. These genes are represented by SEQ ID NOS: 13-18, respectively. The vaccine can comprise any combination of antigens derived from CD2v, EP153R, P72, plO, CP530R, and E199L. For example, the vaccine can comprise one, two, three, four, five, or all six immunogenic peptides, in any combination. By way of specific example, the vaccine can comprise any one of CD2v, EP153R, P72, plO, CP530R, or E199L. Or the vaccine can comprise two antigens, such that the vaccine comprises CD2v and EP153R, or CD2v and P72, or CD2v and plO, or CD2v and CP530R, or CD2v and E199L, or EP153R and P72, or EP153R and plO, or EP153R and CP530R, or EP153R and E199L, or P72 and pl0, or P72 and CP530R, or P72 and E199L, or plO and CP530R, or plO and E199L, or CP530R and E199L. In another embodiment, the vaccine can comprise three antigens, four antigens, five antigens, or all six antigens selected from CD2v, EP153R, P72, plO, CP530R, or E199L.

[0053] The vaccine can further comprise other immunogenic peptides of ASFV. It can also include immunogenic peptides to other microorganisms. Examples include, but are not limited to, porcine circoviruses, porcine parvoviruses, and pseudorabies virus, as well as influenza A strains H1N1, H1N2 or H3N2 (known as “swine flu”). Classic swine flu antigens (pestivirus) can also be included. The vaccine can also comprise immunogenic peptides from bacteria, such as swine brucellosis, or other microorganisms.

[0054] The vaccine can comprise multiple immunogenic peptides which are linked together. These immunogenic peptides can be linked by spacers, or linkers. For example, two different linkers were used in the multiepitope vaccine antigen. Each linker consists of short amino acid sequences used between each epitope. The amino acid sequence of each is listed below, along with a short reason why it was chosen. It is noted that a variety of linkers can be used, and these are intended to be exemplary only:

[0055] 1. GPGPGP- Literature has shown that glycine-rich linkers have the capacity to improve sequence flexibility without affecting the function of proteins they attach to.

[0056] 2. AAY- This amino acid sequence is a cleavage site of proteasomes in mammalian cells, helps form natural epitopes and prevent the formation of ‘junctional epitopes’, which enhances epitope presentation and increases immunogenicity, according to previous studies.

[0057] In a specific example, the vaccine comprises SEQ ID NO: 1, or a functional fragment or variant of SEQ ID NO: 1, wherein the fragment or variant is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or any amount below, above, or in-between this value. Functional fragments and variants are described in more detail below.

[0058] SEQ ID NO: 1 is represented by

[0059] MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSTYQLVYSRNRAAYSPPPKPC

[0060] PPPKPCPPAAYSFLNLT KLYHHHSHYGPGPGNRKKSHYTDLLFICSGPGPGAGKQDITPIT DATYGPGPGSVDSPTITYAAYYQNGSRLTKAAYFLNHNLSKLGPGPGDLLEIINNLAAYL TICQNIAKAAYEGAVYTLVDPFGRPIGGSGGSVPTIVMVDAYKPTK

[0061] Antigens

[0062] As discussed above, the immunogenic peptides (also referred to herein as the antigens) used in the vaccine described herein are derived from ASFV. These antigens can be used alone or in combination as described above. In one embodiment, “native” or “natural” immunogenic peptides can be used, such that they are identical to, or derived from, a naturally occurring immunogenic peptide from ASFV. Of course, there exists many variations and strains of ASFV, so it is expected that even “naturally derived” epitopes can vary depending on the source. Also disclosed herein are variants or fragments of these naturally occurring antigens.

[0063] By “functional variant” or “functional fragment” is meant that while not the entire immunogenic peptide itself is used, the fragment or variant is capable of eliciting an immune response. This immunogenic response can be either in vivo or in vitro. The immune response elicited from the fragment or variant can be less than the response elicited by the full peptide, but still significant enough to be considered “immunogenic.” For example, the functional variant or fragment can elicit a response which is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the immune response elicited by the peptide itself, or any amount below, above, or in between these values.

[0064] By “functional fragment” is meant that the immunogenic peptide is less than the full length protein for any of CD2v, EP153R, P72, plO, CP530R, or E199L. For example, the fragment can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acids of the full length immunogenic peptide. Examples of functional fragments can be found in SEQ ID NOS: 2-12 (see Example 1).

[0065] A ’’functional variant” can comprise the full length immunogenic peptide, or can comprise a fragment of the immunogenic peptide, wherein the variant comprises one or more amino acid variations compared to the native peptide. As such, the antigen or antigenic fragment used in the compositions disclosed herein can be identical to a known antigen, or can be a derivative (variation) thereof. When a derivative is used, the derivative can comprise substitutions, deletions, or insertions in the amino acid sequence in comparison to the known, or native, sequence of an antigen. These variations can render the amino acid sequence of the derivative antigen different than the known antigen, yet still capable of eliciting an immune response in a subject. For example, the derivative can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 ,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,

[0066] 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,

[0067] 92, 93, 94, 95, 96, 97, 98, 99, 100, or more mutated amino acid residues compared to the original, known, or native sequence. These differences can be engineered, of can have occurred naturally through evolution of the virus into different variants.

[0068] The composition can be bivalent, or can be multivalent, meaning it can comprise two, three, four, five, or more antigens or fragments thereof. Multiple antigenic proteins or fragments thereof in the same composition can all come from ASF virus. These multiple antigenic proteins or fragments thereof can be from the same variant, or can be from different variants. Furthermore, the antigenic proteins or fragments thereof can be from different viruses. This is discussed in more detail above.

[0069] Adjuvants

[0070] Compositions of the invention can be administered in conjunction with other immunoregulatory agents, including adjuvants. As used herein, the term "adjuvant" refers to a compound or mixture that enhances an immune response. In particular, the compositions disclosed herein can include an adjuvant. The invention can also comprise combinations of aspects of one or more of the adjuvants identified herein. Some specific antigens contemplated herein are ADU-S100, Mycobacterium smegmatis whole cell lysate, CPD-ODN, or Poly (I:C).

[0071] Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis o Mycobacterium tuberculosis derived proteins. Certain adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Rahway, N.J.); AS-2 (GlaxoSmithKline, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.

[0072] The adjuvant composition can be a composition that induces an anti-inflammatory immune response (antibody or cell-mediated). Accordingly, high levels of anti-inflammatory cytokines (anti-inflammatory cytokines may include, but are not limited to, interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 10 (IL- 10), and transforming growth factor beta (TGFP).

[0073] Optionally, an anti-inflammatory response would be mediated by CD4+ T helper cells. Bacterial flagellin has been shown to have adjuvant activity (McSorley et al., J. Immunol. 169:3914-19, 2002). Also disclosed are polypeptide sequences that encode flagellin proteins that can be used in adjuvant compositions.

[0074] Optionally, the adjuvants used in conjunction with the disclosed compositions increase lipopolysaccharide (LPS) responsiveness. Illustrative adjuvants include but are not limited to, monophosphoryl lipid A (MPL), aminoalkyl glucosaminide 4-phosphates (AGPs), including, but not limited to RC-512, RC-522, RC-527, RC-529, RC-544, and RC-560 (Corixa, Hamilton, Mont.).

[0075] In addition, the adjuvant composition can be one that induces an immune response predominantly of the Thl type. High levels of Thl-type cytokines tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a subject will support an immune response that includes Thl - and Th2-type responses. Optionally, the level of Thl-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. The level of Th2-type cytokines can increase to a greater extent than the level of Thl-type cytokines.

[0076] Certain adjuvants for eliciting a predominantly Thl-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt adjuvants are available from Corixa Corporation (Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094, which are hereby incorporated by reference for their teaching of the same). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Thl response. Such oligonucleotides are well known and are described, for example, in WO 96 / 02555, WO 99 / 33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996. Another adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins. Other formulations can include more than one saponin in the adjuvant combinations, for example combinations of at least two of the following group comprising QS21, QS7, Quil A, P-escin, or digitonin.

[0077] Saponin formulations can also be combined with vaccine vehicles composed of other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc. The saponins can also be formulated in the presence of cholesterol to form particulate structures such as liposomes or immune-stimulating complexes (ISCOMs). Furthermore, the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM. The saponins can also be formulated with excipients such as CARBOPOLTM (Noveon, Cleveland, Ohio) to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose.

[0078] Optionally, the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and 3D-MPL. adjuvant, as described in WO 94 / 00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96 / 33739. Other formulations comprise an oil-in-water emulsion and tocopherol. Another adjuvant formulation employing QS21, 3D-MPL.RTM. adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95 / 17210.

[0079] Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 is disclosed in WO 00 / 09159. Optionally the formulation additionally comprises an oil in water emulsion and tocopherol.

[0080] Additional illustrative adjuvants for use in the disclosed compositions Montamide ISA 720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from GlaxoSmithKline, Philadelphia, Pa.), Detox (EnhanzynTM) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. patent application Ser. Nos. 08 / 853,826 and 09 / 074,720, the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99 / 52549A1.

[0081] Other specific adjuvants contemplated herein include CPG-ODN, ADU-S100, and Poly(I:C). ADU-S100 has increased stability and high affinity for mouse and human Stimulator of Interferon Genes (STING). A TLR-3 adjuvant poly(I:C) is a potent mucosal adjuvant for intranasal delivery of vaccines shown for killed influenza virus antigens in a vaccine trial in pigs (Renu 2020). A TLR-9 adjuvant CPG-ODN is known to induce antigen-specific slgA antibody response in mucosal surfaces in intranasal vaccinated pigs against two respiratory viruses (Dhakal 2019; Zhang 2007). The combination adjuvant targets multiple signaling pathways resulting in synergistic activation of immune cells and a balanced immune response. Nanoparticles

[0082] The compositions, immunogenic compositions and vaccines described herein can comprise one or more nanoparticles (NP). Examples of nanoparticles (used interchangeably with the term “nanocarrier”) include, but are not limited to, nanocarriers composed of mannose conjugated chitosan. Nanoparticle (NP)-based protein subunit delivery helps protect the vaccine antigens from premature degradation, increases its stability, and ensures targeted delivery of immunogen to antigen presenting cells (APC) (Pati 2018; Means 2003). The NP-forming a natural carbohydrate polymer chitosan is biocompatible and bioavailable, and its positively charged amino groups electrostatically interact with negatively charged sialic acid mucus and epithelial surfaces, becoming a mucoadhesive vaccine vehicle (Renu 2020). Surface labeling NP with mannose, a calcium-dependent (type C) mannose receptor of the lectin family binds readily to dendritic cells and macrophages, providing a pronounced adjuvant effect to the vaccine (Dhakal 2018; Han 2020).

[0083] Chitosan is a natural, biodegradable, biocompatible, and non-cytotoxic carbohydrate polymer with high charge density. It is low cost, non-immunogenic, and has intrinsic adjuvanticity and mucoadhesive properties, making the spectrum of its applicability much broader (Carroll, Jin et al. , Islam and Ferro 2016, Malik, Gupta et al. 2018, Islam, Dmour et al. 2019, Shim and Yoo 2020). Chitosan NP -based vaccine antigens are efficiently processed leading to activation of B and T cells via cross-linking (Foged, Brodin et al. 2005, Heit, Schmitz et al. 2007, Schliehe, Redaelli et al. 2011, Woodrow, Bennett et al. 2012). Chitosan has intrinsic adjuvant properties by activating cGAS-STING and NLRP3 inflammasome (Carroll, Jin et al. 2016, Turley, Moran et al. 2021), and activate maturation of DCs with expression of various costimulatory molecules and secretion of cytokines (Dhakal, Renu et al. 2018, Zhuo, Wu et al. 2022). Chitosan and its derivatives are used in therapeutics and tested safely in >100 clinical trials (Kantak and Bharate 2022).

[0084] Glycan mannose is a ligand for mannose-binding lectin receptor and mannose surface- coated NP is rapidly shuttled to the follicular dendritic cells that are concentrated in the germinal centers and triggers adaptive immune responses (Tokatlian, Read et al. 2019). It has been demonstrated that mannose (m)-Chitosan NP-entrapped inactivated influenza virus delivered intranasal induces cross-protective B and T cell responses, with robust slgA and IgG antibodies in the respiratory tract and systemically, resulting in reduced virus load and clinical disease in pigs, a natural host of influenza (Dhakal, Renu et al. 2018, Renu, Feliciano-Ruiz et al. 2021). The nanoparticle can be comprised of one or more polymers. In some embodiments, the one or more polymers is a water soluble, non-adhesive polymer. In some embodiments, polymer is polyethylene glycol (PEG) or polyethylene oxide (PEO). In some embodiments, the polymer is polyalkylene glycol or polyalkylene oxide. In some embodiments, the one or more polymers is a biodegradable polymer. In some embodiments, the one or more polymers is a biocompatible polymer that is a conjugate of a water soluble, non-adhesive polymer and a biodegradable polymer. In some embodiments, the biodegradable polymer is polylactic acid (PLA), poly(glycolic acid) (PGA), or poly(lactic acid / glycolic acid) (PLGA). In some embodiments, the nanocarrier is composed of PEG-PLGA polymers.

[0085] In some embodiments, the nanocarrier is formed by self-assembly. Self-assembly refers to the process of the formation of a nanocarrier using components that will orient themselves in a predictable manner forming nanocarriers predictably and reproducibly. In some embodiments, the nanocarriers are formed using amphiphillic biomaterials which orient themselves with respect to one another to form nanocarriers of predictable dimension, constituents, and placement of constituents. In some embodiments, the nanocarrier is a microparticle, nanoparticle, or picoparticle. In some embodiments, the microparticle, nanoparticle, or picoparticle is self-assembled.

[0086] In some embodiments, the nanocarrier has a positive zeta potential. In some embodiments, the nanocarrier has a net positive charge at neutral pH. In some embodiments, the nanocarrier comprises one or more amine moieties at its surface. In some embodiments, the amine moiety is a primary, secondary, tertiary, or quaternary amine. In some embodiments, the amine moiety is an aliphatic amine. In some embodiments, the nanocarrier comprises an amine- containing polymer. In some embodiments, the nanocarrier comprises an amine-containing lipid. In some embodiments, the nanocarrier comprises a protein or a peptide that is positively charged at neutral pH. In some embodiments, the nanocarrier is a latex particle. In some embodiments, the nanocarrier with the one or more amine moieties on its surface has a net positive charge at neutral pH.

[0087] Nanoparticles can aid the delivery of the antigens disclosed herein, and / or can also be immunogenic. Delivery can be to a particular site of interest, e.g. the mucosa. In some embodiments, the nanoparticle can create a timed release of antigens to enhance and / or extend the immune response. In some embodiments, the nanoparticle is associated with the antigens such that the composition can elicit an immune response. The association can be, for example, wherein the nanoparticle is entrapped or encapsulated with the ASFV antigens and / or other antigens. By entrapped is meant that there is a physical encasing the antigens in nanoparticles. In some embodiments, the antigens are entrapped within the nanoparticle by a water / oil / water emulsion method. In some embodiments, the nanoparticle is poly(lactide co- glycolide) (PLGA). Depending on the ratio of lactide to glycolide used for the polymerization, different forms of PLGA can be obtained and utilized. These forms are typically identified in regard to the monomers' ratio used (e.g. PLGA 75:25 identifies a copolymer whose composition is 75% lactic acid and 25% glycolic acid). Different ratios can be used in this invention, e.g. 90: 10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and numbers above and in between these ratios. Additional examples of suitable nanoparticles include calcium phosphate, lipids of various bacteria like E. coli, mycobactera, leptospira and mixtures thereof. In one example, the composition can be derived mixing about 180 mg of PLGA to about 5 mg of antigenic material (or about 36 mg PLGA to Img antigenic material). The entrapment (encapsulation) efficiency of antigens can vary. In one embodiment the nanoparticles were 50-55% entrapped / encapsulated, calculated based on amount of total antigens used in the entrapment. Entrapped antigens can be administered as mixtures of entrapped / encapsulated and unentrapped / unencapsulated antigens or the entrapped / encapsulated antigens can be further purified.

[0088] Additional Ingredients

[0089] Additional compounds suitable for use with the compositions of the invention include, but are not limited to, one or more solvents, such as an organic phosphate-based solvent, bulking agents, coloring agents, pharmaceutically acceptable excipients, a preservative, pH adjuster, buffer, chelating agent, etc. The additional compounds can be admixed into a previously formed composition comprising a nanoparticle, or the additional compounds can be added to the original mixture to be emulsified. In certain of these embodiments, one or more additional compounds are admixed into existing compositions immediately prior to its use.

[0090] Suitable preservatives for use with the compositions of the invention include, but are not limited to, cetylpyridinium chloride, benzalkonium chloride, benzyl alcohol, chlorhexidine, imidazolidinyl urea, phenol, potassium sorbate, benzoic acid, bronopol, chlorocresol, paraben esters, phenoxyethanol, sorbic acid, alpha-tocophernol, ascorbic acid, ascorbyl palmitate, butylated hydroxy anisole, butylated hydroxytoluene, sodium ascorbate, sodium metabisulphite, citric acid, edetic acid, semi -synthetic derivatives thereof, and combinations thereof. Other suitable preservatives include, but are not limited to, benzyl alcohol, chlorhexidine (bis (p- chlorophenyldiguanido) hexane), chlorphenesin (3-(-4-chloropheoxy)-propane-l,2-diol), Kathon CG (methyl and methylchloroisothiazolinone), parabens (methyl, ethyl, propyl, butyl hydrobenzoates), phenoxyethanol (2-phenoxyethanol), sorbic acid (potassium sorbate, sorbic acid), Phenonip (phenoxyethanol, methyl, ethyl, butyl, propyl parabens), Phenoroc (phenoxyethanol 0.73%, methyl paraben 0.2%, propyl paraben 0.07%), Liquipar Oil (isopropyl, isobutyl, butylparabens), Liquipar PE (70% phenoxyethanol, 30% liquipar oil), Nipaguard MPA (benzyl alcohol (70%), methyl & propyl parabens), Nipaguard MPS (propylene glycol, methyl & propyl parabens), Nipasept (methyl, ethyl and propyl parabens), Nipastat (methyl, butyl, ethyl and propyel parabens), Elestab 388 (phenoxyethanol in propylene glycol plus chlorphenesin and methylparaben), and Killitol (7.5% chlorphenesin and 7.5% methyl parabens).

[0091] The compositions disclosed herein can further comprise at least one pH adjuster. Suitable pH adjusters include, but are not limited to, diethyanolamine, lactic acid, monoethanolamine, triethylanolamine, sodium hydroxide, sodium phosphate, semi -synthetic derivatives thereof, and combinations thereof. In addition, the nanoemulsion vaccine can comprise a chelating agent. In one embodiment of the invention, the chelating agent is present in an amount of about 0.0005% to about 1%. Examples of chelating agents include, but are not limited to, ethylenediamine, ethylenediaminetetraacetic acid (EDTA), phytic acid, polyphosphoric acid, citric acid, gluconic acid, acetic acid, lactic acid, and dimercaprol, and a preferred chelating agent is ethylenediaminetetraacetic acid.

[0092] The compositions disclosed herein can further comprise a buffering agent, such as a pharmaceutically acceptable buffering agent. Examples of buffering agents are known to those of skill in the art. The vaccine can also comprise one or more emulsifying agents to aid in the formation of emulsions. Emulsifying agents include compounds that aggregate at the oil / water interface to form a kind of continuous membrane that prevents direct contact between two adjacent droplets. The compositions disclosed herein can be readily diluted with water or another aqueous phase to a desired concentration without impairing their desired properties.

[0093] Vaccine Administration

[0094] Specifically disclosed herein is a vaccine comprising the compositions disclosed herein. The vaccine can be given in a variety of manners, and a variety of doses and timing. For example, the vaccine can be given via oral administration (i.e., drops in the mouth), by intranasal route (through a drop or mist, for example), by subcutaneous route (as an injection into fatty tissue), intradermally, or by intramuscular injection (injection in muscle tissue). One of skill in the art can readily ascertain how, when, and how often to provide the vaccine to a subject in need. Illustrative embodiments follow.

[0095] In a specific embodiment, the vaccine disclosed herein can be given nasally. Intranasal administration includes administration via the nose, either with or without concomitant inhalation during administration. Such administration is typically through contact by the composition comprising the vaccine with the nasal mucosa, nasal turbinates or sinus cavity. Administration by inhalation comprises intranasal administration, or may include oral inhalation. Such administration may also include contact with the oral mucosa, bronchial mucosa, and other epithelia. The vaccine can be administered as a drop or a mist, for example.

[0096] Exemplary dosage forms for pharmaceutical administration are described herein. Examples include but are not limited to liquids, ointments, creams, emulsions, lotions, gels, bioadhesive gels, sprays, aerosols, pastes, foams, sunscreens, capsules, microcapsules, suspensions, pessary, powder, semi-solid dosage form, etc.

[0097] The pharmaceutical compositions may be formulated for immediate release, sustained release, controlled release, delayed release, or any combinations thereof, into the epidermis or dermis. In some embodiments, the formulations may comprise a penetration-enhancing agent. Suitable penetration-enhancing agents include, but are not limited to, alcohols such as ethanol, triglycerides and aloe compositions. The amount of the penetration-enhancing agent may comprise from about 0.5% to about 40% by weight of the formulation.

[0098] The vaccines of the invention can be applied and / or delivered utilizing electrophoretic delivery / electrophoresis. Further, the composition may be a transdermal delivery system such as a patch or administered by a pressurized or pneumatic device (i.e., “gene gun”). Such methods, which comprise applying an electrical current, are well known in the art.

[0099] The pharmaceutical compositions for administration may be applied in a single administration or in multiple administrations. For example, one dose can be placed in each nostril during vaccination. For example, bi-dose delivery can be used with the vaccines according to the invention. Bi-dose devices contain two sub-doses of a single vaccine dose, one sub-dose for administration to each nostril. Generally, the two sub-doses are present in a single chamber and the construction of the device allows the efficient delivery of a single sub-dose at a time. Alternatively, a mono-dose device may be used for administering the vaccines according to the invention.

[0100] The vaccine can be given in one, two, three, four, or more doses, so that the subject is given a first dose (which can be a bi-dose or mono-dose, as described above), and then a second dose is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 days, or 4 ,5, 6, 7, 8, 9, 10, 11, or 12 weeks, or 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more years apart. The subject can also receive a booster dose, which can be given at any time after the initial dose or doses. The booster dose can comprise the same, or different, antigens than the initial vaccination(s). For example, the vaccine can be updated with more recent variant antigens. In one specific example, dosing occurs from one to three times.

[0101] One of skill in the art can readily determine the proper dosage of the vaccine to be given to the subject. For example, the dosage can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg of protein per dose, or more, less, or any amount in between these values.

[0102] The invention is not necessarily limited to spray delivery of liquid formulations. Vaccines according to the invention may be administered in other forms e.g. as a powder.

[0103] Methods

[0104] Disclosed herein are methods of eliciting or enhancing an immune response to ASFV in a subject, the method comprising administering to the subject a vaccine, wherein the vaccine comprises at least one immunogenic peptide, wherein said immunogenic peptide comprises CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

[0105] The subject, or patient, being given the vaccine can be a mammal, such as a swine. The subject may have previously been vaccinated against ASFV, for example by intramuscular injection. Such vaccination does not preclude the patient from also receiving the vaccine disclosed herein. For example, the vaccine that was initially given can be against a different variant, or the same variant. In the case of a multivalent vaccine, the subject may have been exposed to one, or all, of the antigens in the vaccine disclosed herein, previous to the vaccine of the present invention being administered. For example, the subject may have previously been vaccinated, or may have natural immunity through exposure.

[0106] In one example, if the patient has antibodies to ASFV either through natural immunity or vaccination, the subject may be given a smaller dose, or fewer separate doses, than a subject who does not have natural immunity.

[0107] Also disclosed is a method of preventing or lessening the severity of symptoms or markers of disease associated with ASFV infection in a subject, the method comprising administering to the subject a vaccine, wherein the vaccine comprises an ASFV antigen associated with a nanoparticle, wherein the ASFV antigen comprises CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment

[0108] By “preventing” is meant reducing the chance that the subject will be infected with ASFV by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 ,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% compared with a subject who has not been vaccinated with the vaccine disclosed herein.

[0109] By “lessening severity of symptoms” is meant that the subject contracts ASFV, but has less severe symptoms as compared to what symptoms the subject would have had, had they not been vaccinated. For example, the subject can have a “mild” infection instead of a “moderate” infection.

[0110] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0111] EXAMPLES

[0112] Example 1: Engineering an African Swine Fever Virus Multiepitope Protein for Use in an ASF Nanoparticle-based Subunit Vaccine

[0113] Disclosed herein is a vaccine, wherein the vaccine is comprised of highly immunogenic epitopes of ASFV. These epitopes can be placed into a single, synthetic multiepitope protein. Using in-silico modeling and prediction tools, key ASFV immunogenic epitopes are identified that enhance host immunogenicity thereby lowering the risk of re-infection upon virus encounter. Furthermore, the entrapment of vaccine antigens in chitosan nanoparticles can readily traffic the epitopes to lymphoid tissues upon uptake by antigen presenting cells (APCs) and induce mucosal cross-protective B and T cell responses, as well as slgA, in pigs (Jia et al. 2018; Dhakal et al. 2018) / Therefore, coupling the multiepitope protein vaccine with nanotechnology and delivering the vaccine intranasally stimulated the mucosal immune system, further enhancing the efficacy of the ASFV vaccine.

[0114] TABLE 1: ASF Immunogenic Genes Providing Partial Protection

[0115] RESULTS

[0116] • Six previously investigated, partially protective ASF genes were chosen for further cross- protective epitope identification across the 24 genotypes of ASFV and analysis of SLA binding affinity (Table 1).

[0117] • Eleven epitopes (Table 2) were combined into a single multiepitope protein using flexible linkers, and the DNA sequence was cloned into an expression vector in bacterial (Fig. 1 A) or eukaryotic cells (Fig. IB).

[0118] • Multiepitope protein (predicted size ~24 kDa) was detected using SDS-PAGE (Fig. 2A) in inclusion bodies of lysed BL21 (DE3) E.coli cells. Multiepitope expression was confirmed via Western blot using an anti-Express primary antibody (Fig. 2B).

[0119] • Multi epitope protein (predicted size ~38 kDa) was detected via Western blot using an anti- Myc tag primary antibody in the culture supernatant and cell lysate of eukaryotic ExpiCHO cells (Fig. 3 A). However, protein purification revealed low protein yields in elution fractions (Fig. 3B, C).

[0120] TABLE 2: Epitopes Selected for Multiepitope Vaccine

[0121] CONCLUSIONS

[0122] ASF is a deadly and highly infectious disease of pigs, for which there is currently no vaccine available. Combining nanoparticle vaccine technology and intranasal delivery, this novel multi epitope vaccine should prove effective at eliciting a protective ASF-specific systemic and mucosal immune response against circulating and emerging ASFV strains.

[0123] Example 2: ASF Virus Vaccine Study

[0124] The ASFV vaccine study was carried out in 18 pigs which were 4 weeks old. There were 6 pigs per group, and they were placed in 2 rooms. IM was used for the vaccine delivery method. This setup is shown in Figure 4.

[0125] The following groups were studied:

[0126] • Mock: given PBS instead of vaccine

[0127] • M-CS ASF Protein + M-CS ADU S100: multiepitope protein entrapped in mannose chitosan nanoparticle plus mannose chitosan entrapped ADU SI 00 adjuvant

[0128] • ASF Protein + M-CS ADU SI 00: multi epitope protein (unentrapped) plus mannose chitosan entrapped ADU SI 00 adjuvant.

[0129] The experimental timeline is shown in Figure 5.

[0130] Results are shown in Figures 6-10.

[0131] In Figure 6, the mean pig weights at 42 days post vaccine are shown. Liveweight of each pig (in kg) was estimated using a measuring tape. Weight was estimated using the following formula:

[0132] Weight (lbs) = Girth measurement (inches)A2 x length (inches) / 400. Weight (kg) = weight (lbs) x 0.455 Using One-way ANOVA (p<0.05), no statistical difference was found between the weights of mock vaccinated and any experimentally vaccinated groups suggesting that our vaccine did not cause any adverse effects. Additionally, no injection site reactions were observed. Together, these results suggest that our vaccine formulation is safe and nontoxic within the limits of these clinical parameters.

[0133] Figure 7 shows a T cell proliferation assay. PBMCs were harvested from pigs day post vaccination (DPV) 22 and DPV 42. lxlOA6 PBMCs per animal were stimulated with 10 ug / mL of multiepitope protein for 4 hours. Stimulation index is determined using an MTS assay kit (abeam) and is interpreted as the average of stimulated vs. unstimulated absorbance read at 490 nm. Significance was analyzed using Two-way ANOVA with Tukey’s post-hoc test. Significance level set at a=0.05. Plotted here is mean +- SEM.

[0134] No statistical significance observed between groups. However, at DPV 42 (pink) there appears to be an increase in the stimulation index between stimulated mock and both stimulated vaccinate groups, suggesting that T cells harvested from vaccinated animals are proliferating in response to vaccine antigen stimulation.

[0135] Serum collected on DPVO, DPV22, and DPV42 was tested for the presence of antivaccine antigen (anti-multiepitope protein) antibodies by ELISA. Results are shown in Figure 8A-B. In M-CS ASF protein + M-CS AUD S100 vaccinates (A), positive antibody response was detected at DPV22 respective to ASF protein + M-CS ADU SI 00 (B). In both vaccinated groups, a significantly higher antibody response was detected at DPV42 compared to the mock and DPV22 titers. Significance was assessed using One-way ANVOA with comparisons between groups ( * = p< 0.05, ** = p< 0.01, *** = p< 0.001, **** = p< 0.0001).

[0136] Fluorescent Activated Cell Sorting (FACS) was carried out. 5 million PBMCs / well / animal were seeded in duplicate in 1 mL of RPMI medium in a 48 well plate. Cells were supplemented with 2 ng / ul of recombinant porcine IL-2 and stimulated with 10 ug / mL of ASF multiepitope protein (stimulated plate) for 48 hours. One plate was left unstimulated (IL-2 + RPMI only). After 42 hours, protein transport inhibitor Brefeldin A was added for the last 6 hours of incubation. After 48 hours, cell culture supernatants were harvested for cytokine ELISA.

[0137] 1 million cells (from the original 5 million) were transferred to a 96 well plate for intracellular cytokine labeling and surface staining. Appropriate isotype control antibody staining was included as negative control for the flow cytometry. Cells were fixed using 1% paraformaldehyde at 4 °C for 30 min and resuspended in FACS buffer. The results of the FACS assay on T helper cells for stimulated vs. unstimulated cells can be seen in Figure 9. No significant (p<0.05) differences were observed between the groups using Two-way ANOVA with multiple comparisons. However, there appears to be an increase in the mean number of T helper cells between stimulated mock and the vaccinated groups, suggesting that the vaccine induces a T helper cell response. Induction of T helper cells is important because once activated by antigen, T helper cells proliferate into memory T cells (long lasting memory to a specific antigen) and effector T helper cells (release cytokines, activates B cell to produce antibodies).

[0138] The results of the FACS assay on cytotoxic T cell population (unstimulated vs. stimulated) is shown in Figure 10. No significant (p<0.05) differences were observed between the groups using Two-way ANOVA with multiple comparisons. However, there appears to be an increase in the mean number of cytotoxic T cells between stimulated Mock and the M-CS ASF protein + M-CS ADU SI 00 vaccinated group, suggesting that the entrapped multiepitope protein vaccine induces a cytotoxic T cell response better than the unentrapped protein formulation. Induction of cytotoxic T cells is important because cytotoxic T cells are responsible for recognizing and killing infected cells.

[0139] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0140] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0141] SEQUENCES

[0142] SEQ ID NO: 1 ASFV strain is Georgia 2007 / 1 :

[0143] MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSTYQLVYSRNRAAYSPPPKP CPPPKPCPPAAYSFLNLT KLYHHHSHYGPGPGNRKKSHYTDLLFICSGPGPGAGKQDIT PITDATYGPGPGSVDSPTITYAAYYQNGSRLTKAAYFLNHNLSKLGPGPGDLLEIINNLA AYLTICQNIAKAAYEGAVYTLVDPFGRPIGGSGGSVPTIVMVDAYKPTK SEQ ID NO: 2

[0144] TYQLVYSRNR

[0145] SEQ ID NO: 3

[0146] SPPPKPCPPPKPCPP

[0147] SEQ ID NO: 4

[0148] SVDSPTITY

[0149] SEQ ID NO: 5

[0150] LTICQNIAK

[0151] SEQ ID NO: 6

[0152] AGKQDITPITDATY

[0153] SEQ ID NO: 7

[0154] EGAVYTLVDPFGRPI

[0155] SEQ ID NO: 8

[0156] FLNHNLSKL

[0157] SEQ ID NO: 9

[0158] DLLEIINNL

[0159] SEQ ID NO: 10

[0160] SFLNLTKLYHHHSHY

[0161] SEQ ID NO: 11

[0162] NRKKSHYTDLLFICS

[0163] SEQ ID NO: 12

[0164] YQNGSRLTK SEQ ID NO: 13 CD2v

[0165] MIILIFLIFSNIVLSIDYWVSFNKTIILDSNITNDNNDINGVSWNFFNNSFNTLATCGKAGN

[0166] FCECSNYSTSIYNITNNCSLTIFPHNDVFDTTYQVVWNQIINYTIKLLTPATPPNITYNCTN FLITCKKNNGTNTNIYLNINDTFVKYTNESILEYNWNNSNINNFTATCIINNTISTSNETTL INCTYLTLSSNYFYTFFKLYYIPLSIIIGITISILLISIITFLSLRKRKKHVEEIESPPPESNEEEQ CQHDDTTSIHEPSPREPLLPKPYSRYQYNTPIYYMRPSTQPLNPFPLPKPCPPPKPCPPPKP CPPPKPCPSAESYSPPKPLPSIPLLPNIPPLSTQNISLIHVDRII

[0167] SEQ ID NO: 14 EP153R

[0168] MFSNKKYIGLINKKEGLKKKIDDYSILIIGILIGTNILSLIINIIGEINKPICYQNDDKIFYCPK DWVGYNNVCYYFGNEEKNYNNASNYCKQLNSTLTNNNTILVNLTKTLNLTKTYNHES NYWVNYSLIKNESVLLRDSGYYKKQKHVSLLYICSK

[0169] SEQ ID NO: 15 P72

[0170] MASGGAFCLIANDGKADKIILAQDLLNSRISNIKNVNKSYGKPDPEPTLSQIEETHLVHF NAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQ

[0171] DAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNL VYYCEYPGERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSV EGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDI TPITDATYLDIRRNVHYSCNGPQTPKYYQPPLALWIKLRFWFNENVNLAIPSVSIPFGERF ITIKLASQKDLVNEFPGLFVRQSRFIAGRPSRRNIRFKPWFIPGVINEISLTNNELYINNLFV TPEIHNLFVKRVRFSLIRVHKTQVTHTNNNHHDEKLMSALKWPIEYMFIGLKPTWNISD

[0172] QNPHQHRDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISDISPVTYPITLPIIKN ISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITFALKPREEYQPSGHINV SRAREFYISWDTDYVGSITTADLVVSASAINFLLLQNGSAVLRYST

[0173] SEQ ID NO: 16 PIO

[0174] MPTKAGTKST ANKKTTKGS SKSGS SRGHTGKTHAS S SMHSGMLYKDMVNIARSRGIPI YQNGSRLTKSELEKKIKRSK

[0175] SEQ ID NO: 17 CP530R

[0176] MPSNMKQFCKISVWLQQHDPDLLEIINNLCMLGNLSAAKYKHGVTFIYPKQAKIRDEIK KHAYSNDPSQAIKTLESLILPFYIPTPAEFTGEIGSYTGVKLEVEKTEANKVILKNGEAVL VPAADFKPFPDRRLAVWIMESGSMPLEGPPYKRKKEGGGNDPPVPKHISPYTPRTRIAIE VEKAFDDCMRQNWCSVNNPYLAKSVSLLSFLSLNHPTEFIKVLPLIDFDPLVTFYLLLEP YKTHGDDFLIPETILFGPTGWNGTDLYQSAMLEFKKFFTQITRQTFMDIADSATKEVDVP ICYSDPETVHSYANHVRTEILHHNAVNKVTTPNLVVQAYNELEQTNTIRHYGPIFPESTI NALRFWKKLWQDEQRFVIHGLHRTLMDQPTYETSEFAEIVRNLRFSRPGNNYINELNIT SPAMYGDKHTTGDIAPNDRFAMLVAFINSTDFLYTAIPEEKVGGNETQTSSLTDLVPTRL HSFLNHNLSKLKILNRAQQTVRNILSNDCLNQLKHYVKHTGKNEILKLLQE

[0177] SEQ ID NO: 18 E199L

[0178] MSCMPVSTKCNDIWVDFSCTGPSISELQKKEPKAWAAILRSHTNQQTAEDDNIIGSICDK QGLCSKDEYAYSQYCACVNSGTLWAECAFAPCNGNKNAYKTTEQRNILTNKQCPSGL TICQNIAEYGGSGNISDLYQNFNCNS VINTFLINVMNHPFLTLILIILILIIIYRLMS S SGGKH NDDKLPPPSLIFSNLNNF

[0179] REFERENCES

[0180] 1. Salguero FJ. Comparative Pathology and Pathogenesis of African Swine Fever Infection in Swine. Front Vet Set. 2Q2Q,'T.2'&2. Published 2020 May 19.

[0181] 2. Sanchez-Cordon PJ, Montoya M, Reis AL, Dixon LK. African swine fever: A re- emerging viral disease threatening the global pig industry. Vet J. 2018;233:41-48.

[0182] 3. Subedi D, Subedi S, Karki S. First outbreak of African swine fever in Nepal. Transbound EmergDis. 2022;69(5):e3334-e3335.

[0183] 4. Jia J, Zhang Y, Xin Y, Jiang C, Yan B, Zhai S. Interactions Between Nanoparticles and Dendritic Cells: From the Perspective of Cancer Immunotherapy. Front Oncol. 2018;8:404. Published 2018 Sep 25.

[0184] 5. Dhakal S, Renu S, Ghimire S, et al. Mucosal Immunity and Protective Efficacy of Intranasal Inactivated Influenza Vaccine Is Improved by Chitosan Nanoparticle Delivery in Pigs. Front Immunol. 2018;9:934. Published 2018 May 2.

[0185] 6. Carroll, E. C., L. Jin, A. Mori, N. Munoz-Wolf, E. Oleszycka, H. B. T. Moran, S. Mansouri, C. P. McEntee, E. Lambe, E. M. Agger, P. Andersen, C. Cunningham, P. Hertzog, K. A. Fitzgerald, A. G. Bowie and E. C. Lavelle "The Vaccine Adjuvant Chitosan Promotes Cellular Immunity via DNA Sensor cGAS-STING-Dependent Induction of Type I Interferons." Immunity 44(3): 597-608.

[0186] 7. Carroll, E. C., L. Jin, A. Mori, N. Munoz-Wolf, E. Oleszycka, H. B. T. Moran, S. Mansouri, C. P. McEntee, E. Lambe, E. M. Agger, P. Andersen, C. Cunningham, P. Hertzog, K. A. Fitzgerald, A. G. Bowie and E. C. Lavelle (2016). "The Vaccine Adjuvant Chitosan Promotes Cellular Immunity via DNA Sensor cGAS-STING- Dependent Induction of Type I Interferons." Immunity 44(3): 597-608.

[0187] 8. Dhakal, S., S. Renu, S. Ghimire, Y. Shaan Lakshmanappa, B. T. Hogshead, N. Feliciano- Ruiz, F. Lu, H. HogenEsch, S. Krakowka, C. W. Lee and G. J. Renukaradhya (2018). "Mucosal Immunity and Protective Efficacy of Intranasal Inactivated Influenza Vaccine Is Improved by Chitosan Nanoparticle Delivery in Pigs." Front Immunol 9: 934.

[0188] 9. Foged, C., B. Brodin, S. Frokjaer and A. Sundblad (2005). "Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model." Int J Pharm 298(2): 315-322.

[0189] 10. Heit, A., F. Schmitz, T. Haas, D. H. Busch and H. Wagner (2007). "Antigen coencapsulated with adjuvants efficiently drive protective T cell immunity." Eur J Immunol 37(8): 2063-2074.

[0190] 11. Islam, N., I. Dmour and M. O. Taha (2019). "Degradability of chitosan micro / nanoparticles for pulmonary drug delivery." Heliyon 5(5): e01684.

[0191] 12. Islam, N. and V. Ferro (2016). "Recent advances in chitosan-based nanoparticulate pulmonary drug delivery." Nanoscale 8(30): 14341-14358.

[0192] 13. Kantak, M. N. and S. S. Bharate (2022). "Analysis of clinical trials on biomaterial and therapeutic applications of chitosan: A review." Carbohydr Polym 278: 118999.

[0193] 14. Malik, A., M. Gupta, R. Mani, H. Gogoi and R. Bhatnagar (2018). "Trimethyl Chitosan Nanoparticles Encapsulated Protective Antigen Protects the Mice Against Anthrax." Front Immunol 9: 562.

[0194] 15. Renu, S., N. Feliciano-Ruiz, V. Patil, J. Schrock, Y. Han, A. Ramesh, S. Dhakal, J. Hanson, S. Krakowka and G. J. Renukaradhya (2021). "Immunity and Protective Efficacy of Mannose Conjugated Chitosan-Based Influenza Nanovaccine in Maternal Antibody Positive Pigs." Front Immunol 12: 584299.

[0195] 16. Schliehe, C., C. Redaelli, S. Engelhardt, M. Fehlings, M. Mueller, N. van Rooijen, M. Thiry, K. Hildner, H. Weller and M. Groettrup (2011). "CD8- dendritic cells and macrophages cross-present poly(D,L-lactate-co-glycolate) acid microsphere- encapsulated antigen in vivo." J Immunol 187(5): 2112-2121.

[0196] 17. Shim, S. and H. S. Yoo (2020). "The Application of Mucoadhesive Chitosan Nanoparticles in Nasal Drug Delivery." Mar Drugs 18(12).

[0197] 18. Tokatlian, T., B. J. Read, C. A. Jones, D. W. Kulp, S. Menis, J. Y. H. Chang, J. M. Steichen, S. Kumari, J. D. Allen, E. L. Dane, A. Liguori, M. Sangesland, D. Lingwood, M. Crispin, W. R. Schief and D. J. Irvine (2019). "Innate immune recognition of glycans targets HIV nanoparticle immunogens to germinal centers." Science 363(6427): 649-654. Turley, J. L., H. B. T. Moran, C. P. McEntee, K. O'Grady, N. Munoz-Wolf, L. Jin, F. Follmann, P. Andersen, M. Andersson and E. C. Lavelle (2021). "Chitin-derived polymer deacetylation regulates mitochondrial reactive oxygen species dependent cGAS-

[0198] STING and NLRP3 inflammasome activation." Biomaterials 275: 120961. Woodrow, K. A., K. M. Bennett and D. D. Lo (2012). "Mucosal vaccine design and delivery." Annu Rev Biomed Eng 14: 17-46. Zhuo, S. H., J. J. Wu, L. Zhao, W. H. Li, Y. F. Zhao and Y. M. Li (2022). "A chitosan- mediated inhalable nanovaccine against SARS-CoV-2." Nano Res 15(5): 4191-4200.

Claims

CLAIMSWhat is claimed is:

1. A vaccine composition, wherein the vaccine is effective against African Swine Fever virus (ASFV), wherein the vaccine comprises at least one immunogenic peptide, wherein said immunogenic peptide comprises CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

2. The vaccine of claim 1, wherein the vaccine comprises two or more peptides comprising CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

3. The vaccine of claim 1, wherein the vaccine comprises three or more peptides comprising CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

4. The vaccine of claim 1, wherein the vaccine comprises four or more peptides comprising CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

5. The vaccine of claim 1, wherein the vaccine comprises five or more peptides comprising CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

6. The vaccine of claim 1, wherein the vaccine comprises each of the following peptides: CD2v, EP153R, P72, plO, CP530R, and E199L, or a functional variant or functional fragment thereof.

7. The vaccine of any one of claims 1-6, wherein the vaccine further comprises additional immunogenic peptides.

8. The vaccine of claim 7, wherein said additional immunogenic peptides are derived from ASF virus.

9. The vaccine of claim 7, wherein said additional immunogenic peptides are derived from one or more viruses other than ASFV.

10. The vaccine of any one of claims 2-9, wherein flexible amino acid spacer sequences separate immunogenic peptides.

11. The vaccine of any one of claims 1-10, wherein said vaccine is associated with a nanoparticle.

12. The vaccine of claim 11, wherein the nanoparticle comprises mannose conjugated chitosan.

13. The vaccine of claim 11 or 12, wherein the immunogenic peptide or peptides are entrapped within the nanoparticle.

14. The vaccine of claim 11 or 12, wherein the immunogenic peptide is associated with the nanoparticle.

15. The vaccine of claim 1, wherein the vaccine is a synthetic polypeptide comprising SEQ ID NO: 1, or a functional variant thereof with 90% or more identity to SEQ ID NO: 1.

16. The vaccine of any one of claims 1-15, wherein the vaccine comprises an adjuvant.

17. The vaccine of claim 16, wherein the adjuvant comprises ADU-S100, Mycobacterium smegmatis whole cell lysate, CPD-ODN, or Poly (EC).

18. The vaccine of any one of claims 1-17, wherein the vaccine is formulated for intranasal, intradermal, intramuscular, or subcutaneous delivery.

19. The vaccine of claim 18, wherein the vaccine is formulated for delivery as a drop.

20. The vaccine of claim 18, wherein the vaccine is formulated for delivery as a mist.

21. The vaccine of any one of claims 1-20 in a pharmaceutically acceptable carrier.

22. A method of eliciting or enhancing an immune response to African Swine Fever virus (ASFV) in a subject, the method comprising administering to the subject a vaccine, wherein the vaccine comprises at least one immunogenic peptide, wherein said immunogenic peptide comprises CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

23. The method of claim 22, wherein the subject is swine.

24. The method of claim 22 or 23, wherein the vaccine is administered intranasally, intradermally, intramuscularly, or subcutaneously.

25. The method of any one of claims 22-24, wherein the vaccine comprises two or more peptides comprising CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

26. The method of any one of claims 22-24, wherein the vaccine comprises three or more peptides comprising CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

27. The method of any one of claims 22-24, wherein the vaccine comprises four or more peptides comprising CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

28. The method of any one of claims 22-24, wherein the vaccine comprises five or more peptides comprising CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

29. The method of any one of claims 22-24, wherein the vaccine comprises each of the following peptides: CD2v, EP153R, P72, plO, CP530R, and E199L, or a functional variant or functional fragment thereof.

30. The method of any one of claims 22-29, wherein the vaccine further comprises additional immunogenic peptides.

31. The method of claim 30, wherein said additional immunogenic peptides are derived from ASF virus.

32. The method of claim 30, wherein said additional immunogenic peptides are derived from one or more viruses other than ASFV.

33. The method of any one of claims 22-32, wherein flexible amino acid spacer sequences separate immunogenic peptides.

34. The method of any one of claims 22-33, wherein said vaccine is associated with a nanoparticle.

35. The method of claim 34, wherein the nanoparticle comprises mannose conjugated chitosan.

36. The method of claim 34 or 35, wherein the immunogenic peptide or peptides are entrapped within the nanoparticle.

37. The method of claim 34 or 36, wherein the immunogenic peptide is associated with the nanoparticle.

38. The method of claim 22, wherein the vaccine is a synthetic polypeptide comprising SEQ ID NO: 1, or a functional variant thereof with 90% or more identity to SEQ ID NO: 1.

39. The method of any one of claims 22-38, wherein the vaccine comprises an adjuvant.

40. The method of claim 39, wherein the adjuvant comprises ADU-S100.

41. The method of any one of claims 22-40, wherein the vaccine is administered to the subject more than once.

42. A method of preventing or lessening the severity of symptoms associated with African Swine Fever virus (ASFV) infection in a subject, the method comprising administering to the subject a vaccine, wherein the vaccine comprises at least one immunogenic peptide, wherein said immunogenic peptide comprises CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

43. The method of claim 42, wherein the subject is swine.

44. The method of claim 42 or 43, wherein the vaccine is administered intranasally, intradermally, intramuscularly, or subcutaneously.

45. The method of any one of claims 42-44, wherein the vaccine comprises two or more peptides comprising CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

46. The method of any one of claims 42-44, wherein the vaccine comprises three or more peptides comprising CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

47. The method of any one of claims 42-44, wherein the vaccine comprises four or more peptides comprising CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

48. The method of any one of claims 42-44, wherein the vaccine comprises five or more peptides comprising CD2v, EP153R, P72, plO, CP530R, E199L, or a functional variant or functional fragment thereof.

49. The method of any one of claims 42-44, wherein the vaccine comprises each of the following peptides: CD2v, EP153R, P72, plO, CP530R, and E199L, or a functional variant or functional fragment thereof.

50. The method of any one of claims 42-44, wherein the vaccine further comprises additional immunogenic peptides.

51. The method of claim 50, wherein said additional immunogenic peptides are derived from ASF virus.

52. The method of claim 50, wherein said additional immunogenic peptides are derived from one or more viruses other than ASFV.

53. The method of any one of claims 42-52, wherein flexible amino acid spacer sequences separate immunogenic peptides.

54. The method of any one of claims 42-53, wherein said vaccine is associated with a nanoparticle.

55. The method of claim 54, wherein the nanoparticle comprises mannose conjugated chitosan.

56. The method of claim 54 or 55, wherein the immunogenic peptide or peptides are entrapped within the nanoparticle.

57. The method of claim 54 or 55, wherein the immunogenic peptide is associated with the nanoparticle.

58. The method of claim 42, wherein the vaccine is a synthetic polypeptide comprising SEQ ID NO: 1, or a functional variant thereof with 90% or more identity to SEQ ID NO: 1.

59. The method of any one of claims 42-58, wherein the vaccine comprises an adjuvant.

60. The method of claim 59, wherein the adjuvant comprises ADU-S100.

61. The method of any one of claims 42-60, wherein the vaccine is administered to the subject more than once.

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