Dominant negative toxoid antigen approach for prophylactic and post-infection treatment of swine against african swine fever virus with differentiating infected from vaccinated animals (DIVA) capability

Abstract

A composition including modified ASFV outer-membrane protein antigen mutants (termed dominant negative toxoid antigens) that exhibit non-binding affinity to RBCs while inducing an antibody-mediated response capable of neutralizing unmodified proteins found on infectious outer-membrane-laden ASFV virions. A method for the treatment and / or prevention of ASFV by administering a dominant negative toxoid antigenic composition to animals, thereby averting RBC aggregation caused by the antigen and concurrently treating and / or preventing ASFV. An ASFV vaccine composition including dominant negative toxoid antigens. A composition including dominant negative toxoid antigens in conjunction together and in conjunction with antigens derived from capsid-based proteins, which collectively target both lysogenic and lytic viral replication cycles, thereby achieving optimal immune stimulatory protection. Methods and compositions allowing for differentiation of infected from vaccinated animals (DIVA).

Description

Attorney Docket No.: 0431.00069DOMINANT NEGATIVE TOXOID ANTIGEN APPROACH FOR PROPHYLACTIC AND POSTINFECTION TREATMENT OF SWINE AGAINST AFRICAN SWINE FEVER VIRUS WITH DIFFERENTIATING INFECTED FROM VACCINATED ANIMALS (DIVA) CAPABILITYBACKGROUND OF THE INVENTION1. TECHNICAL FIELD

[0001] The present invention relates to compositions and methods for treating / vaccinating and preventing African Swine Fever Virus (ASFV), and methods differentiating infected from vaccinated animals (DIVA) for diagnostic testing.2. BACKGROUND ART

[0002] African Swine Fever Virus is a large double-stranded DNA virus of the Asfarviridae family that primarily infects domestic pigs, wild boars, warthogs, and bush pigs. It also resides in soft ticks, thereby acting as an infectious vector. ASFV primarily infects the monocytes and macrophages, although, at acute infection many other cell types can be infected. ASFV causes high fever, hemorrhagic lesions, cyanosis, anorexia, and fatalities in these animals. There is no vaccine or treatment for this virus, and the only way to currently prevent its spread is culling animals.

[0003] Development of a vaccine and / or treatment for ASFV is a highly active endeavor across multiple laboratories in China, the United States and Europe. There have been many approaches to develop such vaccines / treatments, however, all of them have fallen short of curative / prophylactic expectations.

[0004] Some of these approaches have included the engineering of various types of attenuated viruses, immune stimulation using viral antigens in various and multiple combinations, gene editing, and antibody neutralization therapies, to name a few.

[0005] Traditional vaccine and therapeutic development protocols mostly take into consideration viral proteins that are exposed on the surface of the virion as potential targets for immune neutralization or as antigens to stimulate the immune system. Other approaches can target viral machinery that is biochemically active within the infected host cell, with small molecule, RNA interfering, or CRISPR gRNA inhibition methods. Further, more exotic approaches such as gene editing (CRISPR for example) can be utilized to alter the viral genome once it has taken root in the infected host cell (Hubner et al., Borca, et al., Wozniakowski et al.). Finally, training CD8 / CD4 T-cells, prior to infection by using viral peptide antigen-based stimulating strategies, to rapidly kill newly infected cells has also been gaining traction.

[0006] In the context of ASFV, many approaches are either unrealistic or perform poorly due to limited functional, structural, and viral replication cycle information, as well as highAttorney Docket No.: 0431.00069 production costs. For example, subunit or whole protein antigen-based vaccine approaches often fail to account for complex cellular-to-viral interactions, making them less effective than intended. The lack of protein structural data further complicates these challenges, leaving researchers to guess about domain functions and sites of post-translational modification. Additionally, these approaches do not consider the nuances of timing and spatial administration governed by ASFV’s replication cycle, which are critical to addressing key points of the cycle and stopping the virus before it becomes a problem. RNA and CRISPR gene-editing strategies hold promise for effective treatment, but their exorbitantly high costs make them commercially unviable, especially when considering the relatively low capital value of individual livestock animals. Finally, newer approaches, such as T-cell training using viral peptide ligands that are broadly dominant and expressed on MHC Class molecules, fail to consider viral replication mechanisms that immediately shut down antigen expression in infected cells.

[0007] Most of the failures of vaccine or therapeutic design can be attributed to a lack of deep understanding of viral protein function, structure and the viral replication cycle. Recently, the structure of ASFV has been defined (Liu, et al.). It has since become clear that the virus exists in two forms: 1 ) a virion capsid surrounded by an outer lipid bilayer and, 2) a naked virion encompassing a capsid only (FIGURE 1 A). The outer lipid bilayer membrane has been shown to contain human proteins, thereby strongly suggesting that the capsid-based virion buds from the infected cell. This property is mostly associated with the lysogenic replication cycle that is observed in most other viruses. Further, it has been reported that ASFV capsid-based virions (without the outer lipid bilayer membrane) increase in the circulating serum at later stages of infection, strongly correlating to the dynamics of the lytic cycle of most other viruses (see description of lysogenic and lytic ASFV in PCT / US20 / 50939 to Chen, et al.). Taking these structural components into consideration and thereby defining them in the context of an early lysogenic versus late lytic replication cycle, has allowed Applicants to pursue a novel vaccine and therapeutic development program based on temporal and physical viral properties (PCT / US20 / 50939, U.S. Patent Application No. 17 / 535,545) (FIGURE 1 B and 10).

[0008] Such a strategy takes into consideration the necessity for an immediate neutralization of early infectious membrane bound virions that are derived from a lysogenic cycle, followed by the rapid neutralization of capsid-based virions that exist in low quantities prior to a lytic cycle switch.

[0009] Through this strategy, Applicants have tested several known outer-membranebound proteins as potential targets for both antibody therapeutic production and antigen-based vaccines. These targets include the outer-membrane proteins EP402R (CD2v), and EP153R, theAttorney Docket No.: 0431.00069 outer-membrane and inner envelope protein 016R (p12), the inner envelope and capsid spanning protein E183L (p54) as well as several main capsid proteins including B646L (p72), E120R (p14.5), B438L (p49) and the inner envelope protein CP204L (p30), although research is not limited to these proteins (FIGURES 2 and 3). It has been shown that immune responses against outer-membrane proteins (either as a protein subunit antigen, full protein antigen, or expressed on the surface of an attenuated virus) produce very weak immune responses in swine, likely due to their ability to bind to RBCs, burrow deep within them and cause them to agglutinate, thus affectively hiding from B-cell stimulation. Furthermore, wildtype EP402R has been shown to be toxic in that it binds to the CD58 receptor of macrophage and triggers cellular apoptosis (Chaulagain et al). Therefore, this agglutination ability of outer membrane proteins and toxic effect must be eliminated in order to expose the surfaces of outer-membrane antigens including EP402R, EP153R and 016R (but not limited to these protein antigens) to the swine immune system to mount a strong and neutralizing immune reaction (FIGURES 4A-4B), while simultaneously eliminating subsequent apoptotic toxicities. The altering of the function of these viral protein antigens to stimulate an effective and safe immune response that will recognize the original (wildtype) viral protein, cannot be easily accomplished without knowledge of the protein’s structure and the aid of computational intelligence. The Applicants have defined the full protein and dominant negative toxoid antigen structures of EP402R, EP153R, and CP204L (FIGURES 11A-11 C, 19, and 26A-26B) using computational intelligence. The structures of the remaining proteins have yet to be determined due to the absence of likely and unknown viral chaperone proteins that may assist in their correct folding. These variables have yet to be empirically and biochemically determined to allow for computational intelligence calculations to obtain correctly folded protein structures.

[0010] The capsid-based proteins have thus far not been shown to cause agglutination in RBCs, although the Applicants have observed a mild swine RBC agglutination effect that is species-specific with the DBD containing and capsid interacting subunit of the inner envelope and capsid spanning protein E183L (p54) (FIGURE 5B), an effect that may be due to improperly folded protein, as the structure cannot be determined computationally with the current information. Nonetheless, E183L mutations may be necessary to block its ability and expose this antigen effectively to the swine immune system to mount a strong neutralizing effect.

[0011] Since the capsid-based proteins have not been shown to possess agglutination properties, dominant negative mutations to achieve strong neutralizing effects may not be necessary, as they are likely exposed to the swine’s immune system. In fact, several groups have demonstrated that different types of capsid proteins produce a strong B-cell-stimulated immuneAttorney Docket No.: 0431.00069 response; however, the protection afforded by these responses is temporary, most likely due to the overwhelming number of viral particles that instantaneously erupt from infected host cells during a lysogenic-to-lytic switch. However, combining capsid antigens with other dominantnegative toxoid viral antigens will likely enhance and provide lasting immunogenicity against both types of ASF virions (membrane-containing and capsid) at different stages of the ASFV replication cycle. This supports the Applicants' thesis that targeting both lysogenic cycle proteins (mainly outer membrane proteins) and lytic cycle proteins (mainly capsid-based proteins) is necessary to effectively protect swine from ASFV infection. This approach is crucial, as small amounts of lytic cycle virions are likely present during the lysogenic stage due to 1. primary infection and 2. the release of virions into circulation from cells in the lysogenic stage that die and rupture.

[0012] Not to be bound by theory, the dominant negative toxoid approach to vaccine development for stronger immune reactions has been achieved previously by similar approaches such as the tetanus and diphtheria toxoids. These toxoids are inactivated forms of the toxic proteins for tetanus and diphtheria that produce an effective immune response (Rosengard et al).

[0013] By creating dominant negative toxoid protein subunit versions of the wildtype ASFV outer membrane protein antigens typically used in vaccines that cause agglutination, the agglutination will not occur, thus exposing the antigens to the immune system (FIGURES 4A-4B).

[0014] Creating dominant-negative toxoid protein subunits from the wild-type ASFV outer membrane protein antigens typically used in vaccines prevents the toxic receptor-driven apoptotic effects seen in monocytes, macrophage, and other cell types. This approach enhances safety, efficacy, and immune responses, rather than inhibiting them.

[0015] Applicants’ latest research has shown that the extraluminal subunits of the outermembrane target EP402R are most likely responsible for binding the virus to swine red blood cells (RBCs) causing them to agglutinate in species-specific manner (compared to human, murine, and bovine RBCs) (shown in FIGURE 5A). Furthermore, it has recently been shown that EP402R extraluminal domains are responsible for CD58 receptor binding on monocytes and macrophage and trigger toxic apoptosis events (Chaulagain et al). Additionally, the outermembrane protein EP153R has previously been shown to co-facilitate the agglutination of RBCs alongside EP402R in attenuated viral strains (Petrovan et al). Applicants observe a faint EP153R subunit-driven agglutination of RBCs. The aggregated RBCs then trigger macrophage to initiate RBC aggregate destruction through a yet to be determined mechanism. As the RBCs attract macrophage, the virions that are buried within the RBC aggregate(s) are also internalized into the macrophage, the choice cell for early infection. There is likely a mechanism that links one of the outer-membrane proteins to RBC aggregation and the internalization into macrophage. SinceAttorney Docket No.: 0431.00069ASFV induces hemagglutination and the subsequent hemadsorption to the surface of macrophage prior to infection via rosette-like structures, the role of these proteins in an orchestrated internalization process is highly probable (Malmquist, et al., Yang, et al.). To date, there has never been an ASFV receptor identified on the surface of macrophage that would facilitate a receptor mediated internalization of the virus. However, the uptake of capsid-based virion has been shown through the far less efficient macropinocytosis entry pathway (Sanchez, et al.). For capsid-based virions to efficiently infect macrophage via macropinocytosis on a systemic and rapid level, there must be copious amount of virus circulating in the swine. This likely occurs after the lysogenic replication phase reaches a critical point, and switches to a lytic phase where large amounts of virus are instantaneously released from the cell. Thus, by neutralizing the proteins responsible for RBC aggregation and preventing the lysogenic cycle, any remaining capsid-based virus is limited to macrophage entry via macropinocytosis and subsequently locked into an early lysogenic phase that are neutralized upon viral budding as its predecessors (FIGURE 6). Finally, remaining capsid-based virions can be neutralized by antibodies derived from capsidbased targets, such as B646L (p72), E120R (p14.5), B438L (p49) and the inner envelope protein CP204L (p30), thereby eliminating even the smallest threat (FIGURE 6 and 7 - replication cycles and strategy overview).

[0016] Applicants’ previous data has shown that polyclonal antibodies raised against the outer-membrane protein EP402R prevents and destabilizes RBC aggregates when the polyclonal antibodies are added to the soluble EP402R protein prior to incubation with RBCs (FIGURE 8A) and in a species-specific manner (FIGURES 8B-8C). This antibody mediated disruption of the RBC aggregate likely prevents the internalization of these proteins into the macrophage. However, when EP402R protein is added to RBCs prior to polyclonal treatment, EP402R-RBC complexes are not disrupted, suggesting that the polyclonal antibodies do not bind to EP402R because it is burrowed in the RBCs and hidden from interaction (data not shown). Therefore, injection of wildtype EP402R antigen into swine will likely cause hemagglutination and poor immune response.

[0017] As mentioned above, there have been many attempts to use ASFV protein antigens to stimulate an internal antibody response in swine. These attempts have failed to produce any long lasting or meaningful protective measures against the virus in swine challenge models. For example, nearly every capsid protein has been explored as a potential vaccine candidate. With new knowledge of the replication cycles and structure of ASFV (FIGURES 1A- 1 C and 7), it is not surprising that these approaches have failed. Once the virus switches to a lytic cycle (a critical and populous point after the lysogenic cycle), the immune system has alreadyAttorney Docket No.: 0431.00069 been severely compromised and cannot keep up with the overwhelming amount of capsid-based virus. Even the introduction of large quantities of antibodies or small-molecule inhibitors in an attempt to neutralize the virus proves futile, as the sheer number of viral particles is too great to overcome. Furthermore, targeting the outer-membrane proteins for antigen-based vaccines has also failed, likely due to their ability to aggregate RBCs and ‘hide’ from B and T-cell responses. For example, EP402R (termed HA in early publications) and EP153R have been shown to facilitate the binding of virus to RBCs, allowing the virus to tunnel / burrow into the RBC membranes. This allows the virus to hide by preventing the exposure of critical surface epitopes from a strong and sustained antibody mediated immune response (Qunitero, et al., Ruiz-Gonzalvo et al.). Several of the outer-membrane proteins including EP153R also have immune suppressive properties against T-cell responses (Teklue, et al., Petrovan, et al.).

[0018] In addition to hemagglutination, EP153R has been shown to sequester MHC class I complexes from being expressed on the surface of T-cells. The amino acid at position Arg133 is critical for the sequestering of MHC Class I complexes after macrophage infection. Although it is highly unlikely to occur, it is precautionary to develop a dominant negative toxoid antigen derivative of EP153R that contains a structurally silent mutation in Arg133, so that the antigen vaccine does not inadvertently become internalized into a host cell and cause secondary MHC sequestering.

[0019] These observations have recently been accentuated by previous data showing that most of the outer-membrane proteins in attenuated viruses or antigens have relatively weak immune stimulation properties, where exposed capsid proteins have relatively high immune stimulatory characteristics but fail to immunize. Therefore, Applicants’ observation that the subunits of the outer-membrane proteins EP402R (CD2v), EP183L (p54) (to a lesser degree), and EP153R (to a much lesser degree) cause RBC aggregation, and previous observations in the literature where EP153R causes lymphocyte neutralizations, occurs specifically in swine and no other species, likely contributes to this synopsis. Furthermore, it is likely that antigen-based vaccine approaches to stimulate an immune response to outer-membrane protein targets fail due to their RBC agglutination properties allowing them to hide from the immune response. Indeed, it is observed that the extraluminal domains of these outer-membrane proteins retain these RBC binding properties (Figures 5A and 5B).

[0020] Another major issue that arises from the vaccination of swine herd populations is to determine which animals have been vaccinated / treated and which animals have not been vaccinated or treated. Differentiating Infected from Vaccinated Animals or (DIVA) is a challenge with other types of vaccines such as RNA / DNA vaccines or attenuated virus. Lengthy and / orAttorney Docket No.: 0431.00069 complicated engineering of these approaches may be necessary to achieve DIVA.

[0021] Based on the above, there remains a critical need to develop dominant negative toxoid antigen-based vaccines using ASFV outer-membrane protein antigens that do not cause RBC aggregation and therefore do not ‘hide’ from eliciting a strong antibody neutralizing immune response. There remains a critical need to develop dominant negative toxoid antigen-based vaccines using ASFV outer membrane protein antigens that do not cause receptor-driven apoptotic toxicities.

[0022] There also remains a need to develop dominant negative toxoid antigen-based vaccines by determining critical structural domains and post-translational modifications and altering these domains and / or post-translational modifications to disrupt immune-suppressing functions while retaining the ability to stimulate an effective immune response (such a B-cell stimulation). There further remains a need to develop these dominant negative toxoid antigen vaccines in manner that gives them rapid DIVA capability - i.e., tag identification.SUMMARY OF THE INVENTION

[0023] The present invention provides for a composition of a vaccine for ASFV, including a dominant negative toxoid antigen in a vaccine.

[0024] The present invention provides for a composition including engineered ASFV outer-membrane protein antigen mutants (dominant negative toxoids) that do not bind to RBCs yet elicit an antibody response that neutralizes wildtype proteins present on infectious outermembrane containing ASFV virions.

[0025] The present invention provides for a method of treating and / or preventing ASFV by administering a composition of a dominant negative toxoid antigen to an animal, preventing RBC aggregation from the antigen, and treating and / or preventing ASFV.

[0026] The present invention provides for a composition including engineered ASFV outer-membrane protein antigen mutants (dominant negative toxoids) that do not trigger toxic apoptotic events in monocytes or macrophage through via interaction with the CD58 cellular receptor.

[0027] The present invention provides for a method of treating and / or preventing ASFV by administering a composition of a dominant negative toxoid antigen to an animal, preventing toxic apoptotic events in monocytes and / or macrophage, and treating and / or preventing ASFV.

[0028] The present invention provides for a composition including engineered ASFV outer-membrane protein antigen mutants (dominant negative toxoids) that do not suppress the immune response through the death of macrophage, prevent the suppression of MHC complexes, and enhance the clearance of infected cells through Antibody Dependent Cellular CytotoxicityAttorney Docket No.: 0431.00069(ADCC).

[0029] The present invention provides for a method treating and / or preventing ASFV by administering a composition of a dominant negative toxoid antigen to an animal that prevents immune suppression by either T-cell inhibition, B-cell stimulation, or macrophage destruction.

[0030] The present invention provides for a method treating and / or preventing ASFV by administering a composition of a dominant negative toxoid antigen to an animal that enhances ADCC in infected cells.

[0031] The present invention provides for a composition including engineered ASFV capsid-based protein antigens (and subunits and variations thereof) that stimulate the B-cell response to create antibodies that neutralize early capsid forms of ASFV. These capsid forms of ASFV can arise from initial and low-level infection, or from infected cells in the lysogenic stage that die and release capsid virions (prior to a lytic stage).

[0032] The present invention provides for a method of treating and / or preventing ASFV by administering a composition of a capsid-based antigen (or subunits and variations thereof) to an animal that prevents early stage infection of low-level capsid-based virions that occurs through pinocytosis.

[0033] The present invention provides for a composition including dominant negative toxoid antigens in combination (either separately or linked / tethered) with antigens derived from capsid-based proteins, to address both the lysogenic and lytic viral replication cycles to achieve maximum immune stimulatory protection and prevent immune suppression that may be observed with wildtype antigens.

[0034] The present invention provides for a composition including dominant negative toxoid antigens in combination (either separately or linked / tethered) with antigens derived from other outer membrane or viral proteins, and in combination with capsid-based proteins, to address both the lysogenic and lytic viral replication cycles to achieve maximum immune stimulatory protection.

[0035] The present invention provides for a method of using artificial intelligence and deep learning systems to construct ASFV viral antigen protein structures and derive from them dominant negative toxoid antigens in combination (either separately or linked / tethered) with antigens derived from other outer membrane or viral proteins, and in combination with capsidbased protein antigens, to address both the lysogenic and lytic viral replication cycles to achieve maximum immune stimulatory protection.

[0036] The present invention provides for a composition derived from the above method.

[0037] The present invention provides for a method of tagging or linking toxoid or capsid-Attorney Docket No.: 0431.00069 based antigens with protein markers enabling the detection of the tagged antigen in samples collected in vivo, and providing the ability to differentiate infected from vaccinated animals (DIVA).

[0038] The present invention provides for a composition of a capsid-based antigen tagged or linked with protein markers that enables detection of the tagged antigen.

[0039] The present invention provides for a method of detecting, via diagnostic assays, the immune response stimulated by these dominant negative toxoid antigens in swine after they have been vaccinated, and providing the ability to achieve DIVA.

[0040] The present invention provides for a diagnostic assay including a mechanism for detecting an immune response stimulated by dominant negative toxoid antigens in swine after they have been vaccinated.DESCRIPTION OF THE DRAWINGS

[0041] Other advantages of the present invention are readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

[0042] FIGURE 1 A shows the structure of ASFV derived from the lysogenic replication cycle (left) and the subsequent lytic replication cycle(right), FIGURE 1 B shows why current neutralizing strategies do not work for both replication cycles, and FIGURE 10 shows the major defined proteins that exist in the membrane of ASFV virions derived from the lysogenic replication cycle (top) vs. the major defined capsid proteins from virions derived from the lytic replications cycle (bottom);

[0043] FIGURE 2 shows the legend of polyclonal antibodies that have been raised against the parallel ASFV proteins for pre-clinical studies;

[0044] FIGURE 3 shows the overall strategy for neutralizing each type of ASFV (with or without membrane) by neutralizing antibodies produced after antigen injection;

[0045] FIGURE 4A is a representation showing wildtype protein or subunit antigens versus FIGURE 4B using dominant negative antigens for immune system exposure;

[0046] FIGURE 5A shows how ASFV protein EP402R specifically causes porcine RBC aggregation, and FIGURE 5B shows how ASFV protein E183L causes porcine RBC aggregation;

[0047] FIGURE 6 is a detailed representation of the ASFV infection and replication cycle via RBC-mediated macrophage entry;

[0048] FIGURE 7 is a detailed representation of strategy to block the cycle(s) shown in FIGURE 6;

[0049] FIGURE 8A shows graphs showing ASFV proteins EP402R and E183L (p54) mediated porcine RBC aggregation is disrupted by O-EP402R and modestly by E183L (p54)Attorney Docket No.: 0431.00069 polyclonal antibodies in serum, FIGURE 8B is a graph showing how polyclonal antibodies raised against EP402R and E183L (p54) block these proteins from causing RBC aggregation, and FIGURE 8C is a graph showing EP402R binding inhibition on murine RBCs;

[0050] FIGURE 9A shows graphs showing the effect of the deglycosylation of EP402R by PNGase F on porcine RBC aggregation and FIGURE 9B shows the deglycosylation of EP402R and by PNGase F on a Coomassie stained gel;

[0051] FIGURE 10A shows the protein sequence for EP402R and the respective N- glycosylation sites that can be used for mutational analysis and dominant negative toxoid antigen development, and FIGURE 10B shows the orientation of EP402R in the outer membrane of the virus;

[0052] FIGURE 1 1 A shows the 3-dimensional structure of EP402R, the structure reveals an extraluminal domain with two lobes, a hydrophobic N-glycosylation signal peptide at the N- terminus, and a hydrophobic membrane spanning a-helix at the C-terminus, FIGURE 11 B shows that by eliminating the signal peptide, one can eliminate the N-glycosylations - an alternative to individual mutations at each site, and FIGURE 11 C shows that by eliminating the c-terminal alpha helix, the remaining protein’s hydrophobicity (solubility) increases (the sequence for each protein antigen variant is highlighted below the structures );

[0053] FIGURES 12A-12C show the hydropathy plot for each of the protein structures shown in FIGURES 11A-11 C, respectively, with FIGURE 12A showing EP402R domain with signal peptide, FIGURE 12B showing EP402R long domain, and FIGURE 12C showing EP402R short domain;

[0054] FIGURE 13A shows the sequence map of N-glycosylation sites on EP402R in relation to the signal peptide at the N-terminus and the hydrophobic anchor peptide at the C- terminus, and FIGURE 13B shows the highlighted N-glycosylation sites ranked by jury score for N-glycosylation probability where a score of 9 is the most probable for the NXS / T consensus and scores under 6 are highly improbable in TABLE 1 ;

[0055] FIGURE 14A shows continued mapping of N-glycosylation sites on EP402R in relation to structurally highlighted sequences such as p-strand, a-helices and coiled peptides, and FIGURE 14B shows that these alignments agree with the computationally derived 3-dimensional structure and the subsequent N-glycosylation sites;

[0056] FIGURE 15A shows the N-glycosylation sites superimposed on the 3-Dimensional structure of EP402R, the left lobe of the extraluminal domain appears to contain the most probable N-glycosylation sites, while the right lobe which is closer to the hydrophobic membrane anchoring peptide have a low probability of modification at the N-glycosylation sites, this suggests in parallelAttorney Docket No.: 0431.00069 with previous biochemical data, that the localized glycosylation sites in the left lobe are likely responsible for receptor binding, and FIGURE 15B shows an EP402R functional N-glycosylation model;

[0057] FIGURES 16A-16E show mutations at various N-glycosylation sites throughout the EP402R protein reveal a stable and resilient protein structure and mutations at a structurally significant site using amino acids known to cause structural alterations show that negligible epitope changes occur, FIGURE 16A shows the area of the mutations, FIGURE 16B shows a first mutation, FIGURE 16C shows a second mutation, FIGURE 16D shows a third mutation, and FIGURE 16E shows a fourth mutation;

[0058] FIGURES 17A-17E show a strategy to screen for the optimal antigen that induces the most robust antibody response against the full-length glycosylated EP402R protein, based on the empirical and structural data for EP402R shown in above figures, with FIGURE 17A showing EP402R left and right lobe minus the signal peptide and plus the C-terminal hydrophobic helix, FIGURE 17B showing EP402R left and right lobe minus the signal peptide and minus the C- terminal hydrophobic helix, FIGURE 17C showing EP402R left lobe minus the signal peptide, FIGURE 17D showing EP402R right lobe minus the signal peptide and plus the C-terminal hydrophobic helix, and FIGURE 17E showing EP402R right lobe minus the signal peptide and minus the C-terminal hydrophobic helix;

[0059] FIGURE 18A shows sequence and structure diagrams of the proposed regions and amino acid sequences of EP153R where dominant negative mutations can be made and screened to prevent T-cell interactions resulting in the prevention of immune suppression, Arg133 is also highlighted as a potential mutation to prevent inadvertent sequestering that can occur with wildtype sequencing, each is derived from reference sequence China_AnhuiXCGQ_2018, and FIGURE 18B is a representation of the orientation of folded full length EP153R in the outer membrane of ASFV;

[0060] FIGURE 19 shows the EP153R full length outer membrane protein (left) that contains a hydrophobic transmembrane region at the N-terminus, followed by a linker domain that is connected to a hydrophilic domain structured with multiple a-helices and -sheet motifs pictured on the right;

[0061] FIGURE 20A shows that EP153R amino acid sequence analysis matches the 3- dimensional structure of the outer membrane domain shown in FIGURE 20B with high confidence and reveals many exposed soluble residues that are accessible to antibody recognition;

[0062] FIGURE 21 A shows the sequence map of N-glycosylation sites on EP153R in relation to the N-terminus anchor and FIGURE 21 B shows the highlighted N-glycosylation sitesAttorney Docket No.: 0431.00069 are ranked by jury score for N-glycosylation probability in TABLE 2 where a score of 9 is the most probable for the NXS / T consensus, scores under 6 are highly improbable;

[0063] FIGURE 22A shows continued mapping of N-glycosylation sites on EP153R in relation to structurally highlighted sequences such as p-strand, a-helices and coiled peptides, and these alignments agree with the computationally derived 3-dimensional structure and the subsequent N-glycosylation sites as shown in FIGURE 22B;

[0064] FIGURE 23A shows the wild-type ASFV EP153R full-length and FIGURE 23B shows the extraluminal subunit structural analysis using hydropathy plots reveals that structural integrity is maintained (in parallel with computational 3-dimensional data) when the anchoring a- helix is deleted, the remaining protein is highly hydrophilic;

[0065] FIGURE 24A shows sequence and structure diagrams of the proposed regions and amino acid sequences of E183L where dominant negative mutations will be made in the DBD region, while retaining the highly conserved C-terminal peptide that is exposed on the surface of the outer membrane, these mutations likely prevent E183L binding to receptor proteins and alter its function by preventing immune suppression and potentially its capsid-facilitated binding to RBCs, and FIGURE 24B shows the orientation of E183L in relation to the inner envelope and the capsid, each is derived from reference sequence China_AnhuiXCGQ_2018;

[0066] FIGURE 25A shows CP204L protein sequence and structure, and its orientation in relation to capsid layers shown in FIGURE 25B, each is derived from reference sequence China_AnhuiXCGQ_2018;

[0067] FIGURE 26A shows that most of p30 full length protein had poor folding in computational model prediction software, likely due to the absence of factors that are necessary for the protein’s folding - a chaperone or concerted folding with other capsid proteins; however, the C-terminus (from amino acid position -120 to 194) shows a high degree of predictive folding and hydrophilic properties; furthermore, based on computational screening, amino acids from -124 to -142 are highly conserved across genotypes / serotypes (highlighted in red). In addition to this sequence, an a-helical bundle was chosen that contains a portion of the capsid layer and externally exposed peptides for further antigen development (highlighted in blue), FIGURE 26B shows the 3-dimensional computational structure for these regions; and

[0068] FIGURE 27 shows EP153R C-terminal domain linked to the p30 N-terminal domain with a [glycine(4) serine] non-immunogenic peptide.DETAILED DESCRIPTION OF THE INVENTION

[0069] The present invention provides generally for compositions and methods of treating and / or preventing ASFV. The compositions include engineered ASFV outer-membrane proteinAttorney Docket No.: 0431.00069 antigen mutants (dominant negative toxoids) that do not bind to RBCs, do not suppress T-cell immunity, enhance Antibody Dependent Cell Cytotoxicity (ADCC) of infected cells, yet elicit antibody responses that neutralize wildtype proteins present on infectious outer-membrane containing ASFV virions as well as capsid-based ASFV virions. More specifically, dominant negative toxoid antigens are derived from a protein of outer membrane protein EP402R (CD2v), outer membrane protein EP153R, protein EP183L (p54), and inner envelop protein CP204L, and can optionally be in combination with additional wild type viral proteins such as p72 and p49.

[0070] The present invention also provides for combinations of antigens to stimulate an immune response to effectively neutralize ASFV infection in swine. As further described below, capsid-based antigens can be used with dominant negative toxoid antigens derived from outer membrane proteins or inner envelope and capsid spanning proteins. The antigens can be used separately or tethered together as described below.

[0071] The present invention also provides for methods of determining DIVA by adding a protein tag to the dominant negative toxoid antigen that produces a mild yet detectable immune response in swine.

[0072] “Animal” as used herein refers to any non-human species of animal.

[0073] “Porcine” or “swine” as used herein, can be a domestic pig, wild boar, warthog, or bush pig.

[0074] The term “vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors. An “expression vector” is a vector that includes a regulatory region. Vectors are also further described below.

[0075] The term “antibody” as used herein refers to a blood protein produced in response to and counteracting a specific antigen. Antibodies combine chemically with substances which the body recognizes as alien, such as bacteria, viruses, and foreign substances in the blood.

[0076] The term “mRNA” as used herein refers to a type of RNA in cells that carries genetic information required to make proteins.

[0077] The term “dominant negative toxoid” antigen for ASFV is used to describe a change to the native ASFV protein, so that the protein becomes exposed to the swine’s immune system resulting in a strong immune response that can effectively inactivate the natural proteins on the virus.

[0078] Dominant negative toxoid antigens can be engineered several ways:

[0079] 1 ) Mutations in the N- or O-glycosylation sites of each of the proteins EP402R(CD2v), EP153R, and EP183L (p54), respectively, to prevent receptor-mediated binding to RBCs or to prevent receptor-mediated binding to T-cells. Applicants’ data has shown thatAttorney Docket No.: 0431.00069 deglycosylation with PNGase F partially inhibits EP402R protein-mediated RBC aggregation (FIGURES 9A-9B). Therefore, silent mutations in these sites or sites that effect glycosylation that flank these sites, or multiple sites that prevent RBC-antigen receptor interactions can be implemented to create a dominant negative form of the antigen (FIGURES 10A-10B, 1 1A-11 C, and 12A-12C).

[0080] 2) The dominant negative toxoid antigens can be engineered by deleting various parts of the protein structure to create a domain, sub domain, or peptide antigen that retains its structural features yet remains necessary for B-cell stimulation to produce antibodies that recognize the wildtype viral protein antigen. Recent research has identified that the EP402R protein contains functionally distinct domains, with the N-terminal immunoglobulin-like domain (left lobe) mediating both erythrocyte binding and CD58 interactions that contribute to macrophage apoptosis, while the C-terminal domain (right lobe) maintains structural integrity for antibody recognition. A specific mutation (E99R) in the left lobe has been demonstrated to abrogate erythrocyte binding and hemagglutination. Additionally, the left lobe's interaction with CD58 has been shown to induce macrophage death through specific binding sites that remain to be fully characterized.

[0081] To address potential functional concerns while maintaining immunogenicity, multiple toxoid strategies can be employed: (1 ) deletion of the entire left lobe to eliminate both RBC binding and CD58-mediated cytotoxicity while preserving the right lobe as an immunogenic toxoid that retains structural features necessary for antibody production against the wildtype protein; (2) alternatively, a left lobe-only toxoid can be created incorporating both the E99R mutation to prevent RBC binding and additional mutations targeting CD58 interaction sites to eliminate macrophage cytotoxicity while maintaining the immunogenic properties of this domain; and (3) full-length protein toxoids incorporating the E99R mutation and CD58-binding site mutations to preserve overall protein structure while eliminating harmful interactions.

[0082] The Applicants have produced computationally derived structures of the proteins and dominant negative toxoid antigens for EP402R, EP153R and CP204L and have developed strategies for toxoid development (FIGURES 11A-1 1C, 17A-17E, 19, 26A-26B, and 27)

[0083] The dominant negative toxoid antigens (whole protein, domain, sub domains, peptides) can be engineered such that they contain a signal peptide that allows for N- Glycosylation, thereby mimicking the modifications that occur on the wildtype protein. The Applicants have made such modifications to various antigens derived from EP402R, such that each domain and sub domain (lobe) can contain a signal peptide at its N-terminus (FIGURES 17A-17E). The purpose of this addition is to ensure that each domain or sub domain is N-Attorney Docket No.: 0431.00069 glycosylated in a manner similar to the wildtype protein, thus triggering an immune response (antibodies via B-cell stimulation) that will recognize the wildtype protein on the virus during infection (which is N-glycosylated). The use of EP402R antigens without the N-glycosylation signal peptide are also useful in that an immune response against them can produce neutralizing antibodies against ‘exposed’ epitopes of the antigen, thus have neutralizing capabilities that are more specific towards the wildtype viral protein. The Applicants have also designed / created a dominant negative toxoid antigen for CP204L (p30) that includes the latter portion of the protein sequence at the C-Terminus, which they have termed the ‘predictable domain’ due to its high folding predictability and conserved sequence, compared to the N-terminal majority of the protein which likely requires chaperones or alternate modifications for correct folding. Thus, the C- terminal portion was chosen due to its highly conserved amino acid sequence between residues -124 to -142, high folding predictability, and hydrophilic properties for better solubility. CP204L (p30) is a protein that is expressed very early in the infection cycle of ASFV and has been predicted to be involved with viral processing. p30 has been shown to be expressed on the surface of infected cells and therefore is a formidable target for antibody directed cell cytotoxicity. Therefore, by creating toxoid derivative of p30 as described above, the Applicants can stimulate a B-cell response and produce antibodies that would recognize the p30 protein on the surface of the infected cells and ‘enhance’ ADCC.

[0084] 3) The dominant negative toxoid antigens can also be engineered by introducing mutations within interactive / modifiable amino acids such as serine, threonine, tyrosine and histidine (and combinations thereof) to prevent or alter phosphorylation patterns, or amino acids that are key for or associated with protein-protein interactions such as the Dynein Binding Domain (DBD) of E183L. The Applicants have produced a toxoid for EP153R by introducing a mutation in the Arg 133 site in order to prevent off-targeting related to MHC Class I sequestering. The Arg 133 can be replaced with Lysine, Glutamine or Cysteine, but not limited to these amino acid replacements.

[0085] The dominant negative toxoid antigens can be engineered in a manner that tethers them to one another with a non-immunogenic linker peptide of various length with or without adjuvant-stimulation properties. The toxoids can be tethered with a single tether or multiple tethers to achieve a link between two or more dominant negative toxoid antigens. The purpose of tethering dominant negative toxoid antigens with a linker (described in detail below) is to maintain consistent dosing levels, improve efficacy and reduce cost treatment.

[0086] The negative toxoid antigen can include a modification to the protein that prevents binding, such as; i) a structural component that sterically hinders the RBC, T-cell orAttorney Docket No.: 0431.00069 monocyte / macrophage interaction yet leaves critical amino residues exposed for immune recognition, ii) an alternate glycosylation (branched carbohydrate) that prevents binding, ill) a pegylated residue, iv) and other type of conjugated ligand that interferes with RBC, T-cell or monocyte / macrophage binding without disrupting the structure of the protein so it maintains relevant immunoreactive epitopes that cross react with wildtype virus.

[0087] The composition can include any degree of modification such as, but not limited to, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the protein peptide structure or the post-translational modification (glycosylation site, phosphorylation site, but not limited to these).

[0088] A dominant negative toxoid antigen is necessary for a robust and efficacious antigen vaccine approach since the wildtype antigens cause / lead to RBC aggregation and ‘hide’ from the immune response or bind to T-cells and suppress the immune system, or cause cellular toxicities that lead to apoptosis. Since capsid-based ASFV does not cause aggregation of RBCs, the use of dominant negative toxoid antigen(s) is not necessary at this stage.

[0089] The present invention provides for a composition including engineered ASFV outer-membrane protein antigen mutants (dominant negative toxoids) that do not trigger toxic apoptotic events in monocytes or macrophage through via interaction with the CD58 cellular receptor.

[0090] The present invention provides for a composition including engineered ASFV outer-membrane protein antigen mutants (dominant negative toxoids) that do not suppress the immune response through the death of macrophage, prevent the suppression of MHC complexes, and enhance the clearance of infected cells through ADCC.

[0091] The present invention provides for a composition including engineered ASFV capsid-based protein antigens (and subunits and variations thereof) that stimulate the B-cell response to create antibodies that neutralize early capsid forms of ASFV. These capsid forms of ASFV can arise from initial and low-level infection, or from infected cells in the lysogenic stage that die and release capsid virions (prior to a lytic stage).

[0092] The present invention provides for a method of treating and / or preventing ASFV infection by administering a composition of a dominant negative toxoid antigen to an animal, preventing RBC aggregation from the antigen, preventing immune suppression, preventing monocyte / macrophage apoptosis, and enhancement of ADCC in infected cells, and treating and / or preventing ASFV at the early stages of infection.

[0093] The present invention provides for a method of treating and / or preventing ASFV by administering a composition of a dominant negative toxoid antigen to an animal, preventingAttorney Docket No.: 0431.00069 toxic apoptotic events in monocytes and / or macrophage, and treating and / or preventing ASFV.

[0094] The present invention provides for a method treating and / or preventing ASFV by administering a composition of a dominant negative toxoid antigen to an animal that prevents immune suppression by either T-cell inhibition, B-cell stimulation, or macrophage destruction.

[0095] The present invention provides for a method treating and / or preventing ASFV by administering a composition of a dominant negative toxoid antigen to an animal that enhances ADCC in infected cells.

[0096] The present invention provides for a method of treating and / or preventing ASFV by administering a composition of a capsid-based antigen (or subunits and variations thereof) to an animal that prevents early stage infection of low-level capsid-based virions that occurs through pinocytosis.

[0097] The present invention provides for a method of treating and / or preventing ASFV infection by administering a composition of a dominant negative toxoid antigen in combination with a wildtype capsid protein or capsid subunit / domain / peptide to address and treat both the lysogenic and lytic viral replication cycles, respectively. The dominant negative toxoid antigen can also be tethered to the capsid protein by linker peptide.

[0098] The present invention provides for a composition and method of treating and / or preventing ASFV infection by tethering a dominant negative toxoid antigen to another antigen such as a capsid protein (p72, p49, or subunits / domain / peptide thereof) or a dominant negative toxoid antigen derivative of the inner envelope protein (CP204L or subunits / domain / peptide thereof) with a non-immunogenic linker peptide that can include four glycine residues followed by a serine residue in a repeating pattern [Glycine(4) Serine] (G4Sn). It should be understood that the linker peptide is not limited to this type of amino acid sequence, i.e., any other suitable linker can be used with any suitable amino acids. The linker peptide can be of various lengths where n can equal 1 , 2, 3, 4, 5, 6, 7, 8, 9...100 repeats. The antigens can be linked together in various orientations to one another, including N-Terminal linked to C-terminal, N-Terminal linked to N- Terminal, C-Terminal linked to C-Terminal, and C-Terminal linked to N-Terminal. The antigens can also be linked together in orientations where the linker is conjugated to an internal and modified protein residue, in a manner that allows the terminal ends to be free, with the purpose to expose certain epitopes at the terminals for optimal immune stimulation, when applicable. The purpose of tethering dominant negative toxoid antigens and other antigens with a linker (described in detail below) is to maintain consistent dosing levels, improve efficacy by stimulating simultaneous and strong immune responses, and reduce cost of treatment by reducing cost of manufacturing.Attorney Docket No.: 0431.00069

[0099] Therefore, the present invention provides for a composition including dominant negative toxoid antigens in combination (either separately or linked / tethered) with antigens derived from capsid-based proteins, to address both the lysogenic and lytic viral replication cycles to achieve maximum immune stimulatory protection and prevent immune suppression that may be observed with wildtype antigens.[000100] The present invention provides for a composition including dominant negative toxoid antigens in combination (either separately or linked / tethered) with antigens derived from other outer membrane or viral proteins, and in combination with capsid-based proteins, to address both the lysogenic and lytic viral replication cycles to achieve maximum immune stimulatory protection.[000101] The present invention provides for a composition and method of monitoring ASFV infection by ‘tagging’ each of the dominant negative toxoid antigens with another protein with the goal to be able to differentiate between animals that have vaccinated versus animals that have not been vaccinated (DIVA). For example, a Glutathione S-Transferase (GST) tag can be added to the dominant negative toxoid antigen(s). Once the animal is treated, it produces an immune response against the GST tag. There are two modes of detection: 1. Direct detection based on the enzyme’s activity, or 2. Indirect detection with a diagnostic assay such a rapid ELISA detecting antibodies against the GST tag. Other tags can include, but are not limited to, polyhistidines, green fluorescent protein (GFP), immunogenic peptide flags, c-Myc, maltose binding protein (MBP), streptavidin / biotin, SUMO, V5, T7, S-Tag, but not limited to these tags. Each of the above tags can be linked to the dominant negative toxoid antigen at either the N-terminus or C-terminus of the protein, or the tag itself. Each of the above protein tags can be detected by various means including the many forms of ELISA assay. The detection can be as simple as a rapid point of care diagnostic or a more complex assay involving plate reader readout data. The dominant negative toxoid antigens can also be tagged with barcoding nanobeads or nanochips that can be detected via scanning or transmission collection measures, respectively. Each of these approaches establishes rapid DIVA.[000102] Therefore, the present invention provides for a method of tagging or linking toxoid or capsid-based antigens with protein markers enabling the detection of the tagged antigen in samples collected in vivo, and providing the ability to differentiate infected from vaccinated animals (DIVA).[000103] The present invention provides for a composition of a capsid-based antigen tagged or linked with protein markers that enables detection of the tagged antigen.[000104] The present invention provides for a method of detecting, via diagnostic assays,Attorney Docket No.: 0431.00069 the immune response stimulated by these dominant negative toxoid antigens in swine after they have been vaccinated, and providing the ability to achieve DIVA.[000105] The present invention provides for a diagnostic assay including a mechanism for detecting an immune response stimulated by dominant negative toxoid antigens in swine after they have been vaccinated. The diagnostic assay can be an ELISA assay with antigens attached to a solid substrate as described above, and include any necessary reagents, sampling supplies, and instructions for use as needed. Samples can be blood serum or any other suitable sample. In general, an ELISA assay can detect antibodies that are raised in a swine against any of the above protein tags that are linked to the toxoid antigens. An indirect ELISA can be preferred, which uses two antibodies (a primary antibody that binds to the antigen of interest and a secondary antibody that binds to the primary antibody and is linked to an enzyme for detection). The ELISA assay can be prepared as follows. Microplate wells can be coated and immobilized with the specific antigen that was injected into the swine. A blocking buffer (BSA, non-fat dry milk) can be added to block any remaining non-specific binding sites on the well surface. In order to perform the assay, serum from the swine is added to the wells, and if the swine’s immune system has produced antibodies against the injected protein (i.e., if the swine has been vaccinated), the antibodies bind to the coated antigen. The wells are then washed to remove unbound antibodies and other proteins. A secondary antibody that specifically recognizes the swine’s antibodies (typically an anti-species antibody, such as anti-rabbit IgG for rabbit serum) is added. The secondary antibody is conjugated to an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The wells are washed again to remove unbound secondary antibody. A substrate specific to the enzyme on the secondary antibody is added, and the enzyme catalyzes a reaction that produces a detectable signal (such as a color change). The intensity of the signal (color change) is measured with a spectrophotometer and the intensity is proportional to the amount of antibody bound to the antigen, indicating the level of the immune response. Therefore, the assay can easily show which swine have been vaccinated and show an immune response and which can still be infected with ASFV. The indirect ELISA assay can be preferred as it is highly sensitive, allows for detection of different types of antibodies by choosing an appropriate secondary antibody, and it is more economical than other ELISA methods since only the secondary antibody needs to be enzyme-labeled.[000106] The composition can be provided in a vaccine such as by 1 ) an mRNA or selfamplifying RNA (saRNA) vaccine for delivery to APCs and B-cells so that the mRNA expresses the antigen internally to create an immune response, 2) a direct antigen injection, or 3) a DNA vaccine (which sends instructions for making the antigen from DNA). Therefore, the presentAttorney Docket No.: 0431.00069 invention provides for a composition of a vaccine for ASFV, including a dominant negative toxoid antigen in a vaccine.[000107] There can be additional applications for dominant negative toxoid proteins for viruses, bacteria, fungus, or parasites binding to reticulocytes or RBCs, which can be masked in other tissues. Hemagglutination is a tactic used by a handful of viruses to enhance their infectivity into target cells. For example, HIV-1 and HIV-2 bind to Duffy Antigen Receptor for Chemokines (DARC), a receptor on RBCs. The binding causes hemagglutination that in turn increases viral infectivity up to approximately 100-fold higher than unbound and circulating virus (Lachgar, et al., He and Neil, et al., Beck, et al.), the degree of which depends on the blood type of the infected individual (Abdulazeez, et al.). On the other hand, HIV-1 has also been shown to bind to Pkreceptor of PBMCs to block competing viruses (Lund, et al.). Noroviruses also cause hemagglutination by selectively binding to group A, H, and / or difucosylated Lewis blood groups via Receptor / Lebligand interactions (Nillson, et al., Shirato-Horikoshi, et al.). Other examples of viruses that bind to RBCs and cause hemagglutination include Bovine Corona Virus (BCV), Porcine Hemagglutinating Encephalomyelitis Virus (PHEV), Influenza C, and Toroviruses (Zeng, et al.). Each of these RNA viruses cause hemagglutination by the binding of their Hemagglutinin Esterase (HE) protein to sialic acid containing receptors on RBCs. The HE protein has two functions: 1 ) binding to sialic acid containing receptors to cause agglutination. The aggregated RBCs then transport the virus to the target cells more efficiently than unbound and circulating virus. Once in contact with the target cell the HE protein 2) undergoes conformational change to activate its target cell receptor destroying properties, allowing the RBC to release its viral payload for cellular entry. However, these RNA viruses do not contain an outer-membrane or outer coat like ASFV. There is no outer-membrane to protect the inner core of the virus from an immune response. For example, PHEV is dominated by spike proteins that protrude from a single membrane that also contains the HE and other membrane proteins. Therefore, when the virus binds to RBCs via its HE proteins, its spike proteins are still exposed to antibody responses. There is no or little epitope hindrance. As such, antibodies against major PHEV proteins have been shown to be strongly neutralizing until the spike proteins (or others) mutate. Further, PHEV does not differentiate between species. It causes hemagglutination in mice, rats, chickens, and several other animals. Therefore, even though the agglutination of RBCs is a common tactic by some viruses to increase their infectivity to targeted cells, the modes of action are considerably different. ASFV is a DNA virus with much slower mutation rates caused by genetic drift or environmental mutagens, as opposed to many RNA viruses with higher mutations rates caused by lack of replicative proofreading. RNA viruses may not be masked as well by agglutinated RBCs due toAttorney Docket No.: 0431.00069 their lack of a protective outer coat, but they make up for the difference by rapidly mutating to allow immune evasion. Taking into account these differences (especially the lack of species specificity in HE-containing RNA viruses), it is highly likely that ASFV binds specifically to swine RBCs by a different receptor, sialic acid motif, or carbohydrate than HE-containing RNA viruses. [000108] FIGURE 13A is a sequence map of N-glycosylation sites on EP402R in relation to the signal peptide at the N-terminus and the hydrophobic anchor peptide at the C-terminus. FIGURE 13B shows the highlighted N-glycosylation sites ranked by jury score for N-glycosylation probability where a score of 9 is the most probable for the NXS / T consensus and scores under 6 are highly improbable in TABLE 1. FIGURE 14A shows continued mapping of N-glycosylation sites on EP402R in relation to structurally highlighted sequences such as -strand, a-helices and coiled peptides, and FIGURE 14B shows that these alignments agree with the computationally derived 3-dimensional structure and the subsequent N-glycosylation sites.[000109] FIGURE 15A shows the N-glycosylation sites superimposed on the 3-Dimensional structure of EP402R, the left lobe of the extraluminal domain appears to contain the most probable N-glycosylation sites, while the right lobe which is closer to the hydrophobic membrane anchoring peptide have a low probability of modification at the N-glycosylation sites. This suggests in parallel with previous biochemical data, that the localized glycosylation sites in the left lobe are likely responsible for receptor binding. FIGURE 15B shows an EP402R functional N-glycosylation model.[000110] FIGURES 16A-16E show mutations at various N-glycosylation sites throughout theEP402R protein reveal a stable and resilient protein structure and mutations at a structurally significant site using amino acids known to cause structural alterations show that negligible epitope changes occur. FIGURE 16A shows the area of the mutations, FIGURE 16B shows a first mutation, FIGURE 16C shows a second mutation, FIGURE 16D shows a third mutation, and FIGURE 16E shows a fourth mutation. These observations suggest that post-translational modifications, such as N-glycosylations, have little effect on peptide / structural topography. A similar analysis was conducted on all N-glycosylation sites, including hydropathy and bulkiness plots (not shown).[000111] FIGURES 17A-17E show a strategy to screen for the optimal antigen that induces the most robust antibody response against the full-length glycosylated EP402R protein, based on the empirical and structural data for EP402R shown in above figures. FIGURE 17A shows EP402R left and right lobe minus the signal peptide and plus the C-terminal hydrophobic helix, FIGURE 17B shows EP402R left and right lobe minus the signal peptide and minus the C-terminal hydrophobic helix, FIGURE 17C shows EP402R left lobe minus the signal peptide, FIGURE 17DAttorney Docket No.: 0431.00069 shows EP402R right lobe minus the signal peptide and plus the C-terminal hydrophobic helix, and FIGURE 17E shows EP402R right lobe minus the signal peptide and minus the C-terminal hydrophobic helix. The deglycosylated protein, when compared to the glycosylated protein, exhibits common exposed regions that could potentially mitigate any masking effects caused by the carbohydrates.[000112] FIGURE 18A shows sequence and structure diagrams of the proposed regions and amino acid sequences of EP153R where dominant negative mutations can be made and screened to prevent T-cell interactions resulting in the prevention of immune suppression, Arg133 is also highlighted as a potential mutation to prevent inadvertent sequestering that can occur with wildtype sequencing, each is derived from reference sequence China_AnhuiXCGQ_2018. FIGURE 18B is a representation of the orientation of folded full length EP153R in the outer membrane of ASFV.[000113] FIGURE 19 shows the EP153R full length outer membrane protein (left) that contains a hydrophobic transmembrane region at the N-terminus, followed by a linker domain that is connected to a hydrophilic domain structured with multiple a-helices and -sheet motifs pictured on the right.[000114] FIGURE 20A shows that EP153R amino acid sequence analysis matches the 3- dimensional structure of the outer membrane domain shown in FIGURE 20B with high confidence and reveals many exposed soluble residues that are accessible to antibody recognition. EP153R does not bind to DNA or RNA but has a large protein binding region in its center - likely for sequestering MHO Class I molecules.[000115] FIGURE 21 A shows the sequence map of N-glycosylation sites on EP153R in relation to the N-terminus anchor and FIGURE 21 B shows the highlighted N-glycosylation sites are ranked by jury score for N-glycosylation probability in TABLE 2 where a score of 9 is the most probable for the NXS / T consensus, scores under 6 are highly improbable. Although there is a weak signal peptide prediction, it is likely that EP153R may not be glycosylated.[000116] FIGURE 22A shows continued mapping of N-glycosylation sites on EP153R in relation to structurally highlighted sequences such as p-strand, a-helices and coiled peptides, and these alignments agree with the computationally derived 3-dimensional structure and the subsequent N-glycosylation sites as shown in FIGURE 22B.[000117] FIGURE 23A shows the wild-type ASFV EP153R full-length and FIGURE 23B shows the extraluminal subunit structural analysis using hydropathy plots reveals that structural integrity is maintained (in parallel with computational 3-dimensional data) when the anchoring a- helix is deleted, the remaining protein is highly hydrophilic.Attorney Docket No.: 0431.00069[000118] FIGURE 24A shows sequence and structure diagrams of the proposed regions and amino acid sequences of E183L where dominant negative mutations will be made in the DBD region, while retaining the highly conserved C-terminal peptide that is exposed on the surface of the outer membrane, these mutations likely prevent E183L binding to receptor proteins and alter its function by preventing immune suppression and potentially its capsid-facilitated binding to RBCs, and FIGURE 24B shows the orientation of E183L in relation to the inner envelope and the capsid, each is derived from reference sequence China_AnhuiXCGQ_2018.[000119] FIGURE 25A shows CP204L protein sequence and structure, and its orientation in relation to capsid layers shown in FIGURE 25B, each is derived from reference sequence China_AnhuiXCGQ_2018.[000120] FIGURE 26A shows that most of p30 full length protein had poor folding in computational model prediction software, likely due to the absence of factors that are necessary for the protein’s folding - a chaperone or concerted folding with other capsid proteins; however, the C-terminus (from amino acid position -120 to 194) shows a high degree of predictive folding and hydrophilic properties. Furthermore, based on computational screening, amino acids from -124 to -142 are highly conserved across genotypes / serotypes (highlighted in red). In addition to this sequence, an o-helical bundle was chosen that contains a portion of the capsid layer and externally exposed peptides for further antigen development (highlighted in blue). FIGURE 26B shows the 3-dimensional computational structure for these regions.[000121] FIGURE 27 shows EP153R C-terminal domain linked to the p30 N-terminal domain with a [glycine(4) serine] non-immunogenic peptide. This toxoid protein is capable of neutralizing the lysogenic form of early outer membrane-containing ASFV as well as early staged capsid viral particles that mainly depend on macro / micropinocytosis for macrophage entry and subsequent infection.[000122] The compositions described herein can be dosed in animals according to knowledge of those skilled in the art. The compositions can be in a concentration of 1-100% and include any suitable excipients such as further described below. Delivery routes can include, but are not limited to, epicutaneous, intradermal, subcutaneous, transdermal, intramuscular, intravenous, oral, transcorneal, intraocular, intracerebral, epidural, intrathecal, intraperitoneal, intraosseous, intranasal (such as into a snout of a pig), intratracheal, as well as other routes described further below.[000123] The composition can be stored in various states that allow for shelf stability (one month to years) of proteins at various temperatures (4 degrees C, -20 degrees C, -20 to -80 degrees C), such as a lyophilized dry powder, aqueous solution, tris or phosphate buffers, or aAttorney Docket No.: 0431.00069 solution including 25-50% glycerol or ethylene glycol that act as cryoprotectants.[000124] The compound of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual animal, the site and method of administration, scheduling of administration, animal age, sex, body weight and other factors known to medical practitioners. The pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.[000125] In the method of the present invention, the compound of the present invention can be administered in various ways. It should be noted that it can be administered as the compound and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, adjuvants, and vehicles. The compounds can be administered orally, subcutaneously, or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, intratonsillar, and intranasal administration as well as intrathecal and infusion techniques. Implants of the compounds are also useful. The patient being treated is a warmblooded animal and, in particular, mammals including man. The pharmaceutically acceptable carriers, diluents, adjuvants, and vehicles as well as implant carriers generally refer to inert, nontoxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention.[000126] The doses can be single doses or multiple doses over a period of several days. The treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated.[000127] When administering the compound of the present invention parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.[000128] Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Nonaqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used asAttorney Docket No.: 0431.00069 solvent systems for compound compositions. Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds. [000129] Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.[000130] A pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres. Examples of delivery systems useful in the present invention include: 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art.[000131 ] The present invention provides for a method of using artificial intelligence and deep learning systems to construct ASFV viral antigen protein structures and derive from them dominant negative toxoid antigens in combination (either separately or linked / tethered) with antigens derived from other outer membrane or viral proteins, and in combination with capsidbased protein antigens, to address both the lysogenic and lytic viral replication cycles to achieve maximum immune stimulatory protection.[000132] The present invention provides for a composition derived from the above method.[000133] Throughout this application, various publications, including United States patents, are referenced by author and year and patents by number. Full citations for the publications are listed below. The disclosures of these publications and patents 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 invention pertains.[000134] The invention has been described in an illustrative manner, and it is to beAttorney Docket No.: 0431.00069 understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.[000135] Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention can be practiced otherwise than as specifically described.Attorney Docket No.: 0431.00069REFERENCES1 . Chen et al. A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs. Sci. China Life Sci. 2020, 63, 623-634.2. Borca etal. Development of a highly effective African swine fever virus vaccine by deletion of the I177L gene results in sterile immunity against the current epidemic Eurasia strain. J. Virol. 2020, 94, e02017-19.3. Teklue etal, Generation and evaluation of an African swine fever virus mutant with deletion of the CD2v and UK genes. Vaccines 2020, 8, 763.4. Liu et al. Cryo-EM structure of the African swine fever virus. Cell Host Microbe 2019, 26, 836-843.5. Hubner et al. Efficient inhibition of African swine fever virus replication by CRISPR / Cas9 targeting of the viral p30 gene (CP204L). Sci Rep. 2018 Jan 23;8(1):1449.6. Borca et al. CRISPR / Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus. Bio Protoc. 2018 Aug 20;8(16):e2978.7. Wozniakowski et al. Attempts at the Development of a Recombinant African Swine Fever Virus Strain with Abrogated EP402R, 9GL, and A238L Gene Structure using the CRISPR / Cas9 System. J Vet Res. 2020 Jun 3;64(2):197-205.8. Petrovan et al. Role of African swine fever virus (ASFV) proteins EP153R and EP402R in reducing viral persistence in blood and virulence in pigs infected with BenindeltaDP148R. J Virol. 2021 Oct 13:JVI0134021.9. Yang et al. Identification of a new cell-penetrating peptide derived from the african swine fever virus CD2v protein. Drug Deliv. 2021 Dec;28(1 ):957-962.10. Chen et al. Porcine Immunoglobulin Fc Fused P30 / P54 Protein of African Swine Fever Virus Displaying on Surface of S. cerevisiae Elicit Strong Antibody Production in Swine. Virol Sin. 2021 Apr;36(2):207-219.11 . Sanchez et al. African swine fever virus uses macropinocytosis to enter host cells. PLoS Pathog. 2012;8(6):e1002754.Attorney Docket No.: 0431.0006912. Lachgar et al. Binding of HIV-1 to RBCs involves the Duffy antigen receptors for chemokines (DARC). Biomed Pharmacother. 1998;52(10):436-9.13. He etal, Duffy antigen receptor for chemokines mediates trans-infection of HIV-1 from red blood cells to target cells and affects HIV-AIDS susceptibility. Cell Host Microbe. 2008 Jul 17;4(1):52-62.14. Beck et al. Human erythrocytes selectively bind and enrich infectious HIV-1 virions. PLoS One. 2009 Dec 14;4(12):e8297.15. Abdulazeez et al. Carriage rate of Human Immunodeficiency Virus (HIV) infection among different ABO and Rhesus blood groups in Adamawa state, Nigeria. Biomedical Research (2008) Volume 19, Issue 1 . Department of Biological Sciences, Federal University of Technology, Yola, Nigeria.16. Lund et al. The human P(k) histo-blood group antigen provides protection against HIV-1 infection. Blood. 2009 May 14;113(20):4980-91.17. Nilsson et al. Norwalk virus-like particles bind specifically to A, H and difucosylated Lewis but not to B histo-blood group active glycosphingolipids. Glycoconj J. 2009 Dec;26(9):1171-80.18. Shirato-Horikoshi et al. Binding activity of norovirus and sapovirus to histo-blood group antigens. Arch Virol. 2007;152(3):457-61 .19. Zeng et al. Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution. Proc Natl Acad Sci U S A. 2008 Jul1 ;105(26):9065-9.20. Rosengard et al. Lichen planus following tetanus-diphtheria-acellular pertussis vaccination: A case report and review of the literature. SAGE Open Med Case Rep. 2018 Jan 4;6:2050313X17750335.

Claims

Attorney Docket No.: 0431.00069CLAIMSWhat is claimed is:1 . A composition comprising a dominant negative toxoid antigen derived from African Swine Fever Virus (ASFV) protein, wherein said dominant negative toxoid antigen does not bind to red blood cells (RBCs), causes T-cell immunosuppression, initiates monocyte / macrophages apoptosis and elicits an antibody response that neutralizes wildtype proteins present on infectious outer-membrane containing ASFV virions and antibodies that enhance antibody dependent cellular cytotoxicity (ADCC) of wildtype proteins expressed on a surface of infected cells.

2. The composition of claim 1 , wherein said dominant negative toxoid antigen is derived from a protein chosen from the group consisting of outer membrane protein EP402R (CD2v), outer membrane protein EP153R, protein EP183L (p54), and inner envelop protein CP204L.

3. The composition of claim 1 , wherein said dominant negative toxoid antigen is tethered at least a second dominant negative toxoid antigen.

4. The composition of claim 1 , further including a capsid protein chosen from the group consisting of protein p72 and protein p49.

5. The composition of claim 1 , wherein said composition is tagged with an identifying protein chosen from the group consisting of polyhistidines, green fluorescent protein (GFP), immunogenic peptide flags, c-Myc, maltose binding protein (MBP), streptavidin / biotin, SUMO, V5, T7, S-Tag, and glutathione S-transferase (GST), in order to establish differentiating infected from vaccinated animals (DIVA).

6. The composition of claim 1 , wherein said composition is tagged with an identifying device chosen from the group consisting of barcoding nanobeads that can be detected via scanning and nanochips that can be detected via transmission collection measures, to establish rapid differentiating infected from vaccinated animals (DIVA).

7. The composition of claim 1 , further including a modification that prevents binding, chosen from the group consisting of i) a structural component that sterically hinders RBC interaction yet leaves critical amino residues exposed for immune recognition, ii) an alternate glycosylation (branched carbohydrate) that prevents binding, ill) a pegylated residue, iv) and a conjugated ligand that interferes with RBC binding without disrupting the structure of the protein so it maintains immunoreactive epitopes that cross react with wildtype virus.

8. The composition of claim 1 , further including a composition chosen from the group consisting of wildtype capsid protein, capsid subunit, domain, and peptide tethered to said dominant negative toxoid antigen.

9. A method of treating and / or preventing African Swine Fever Virus (ASFV), including theAttorney Docket No.: 0431.00069 steps of: administering a composition of a dominant negative toxoid antigen to an animal; preventing red blood cell (RBC) aggregation from the dominant negative toxoid antigen; preventing T-cell suppression from the dominant negative toxoid antigen; preventing receptor- mediated monocyte / macrophage apoptosis from the dominant negative toxoid antigen; enhancing antibody dependent cellular cytotoxicity (ADCC) from the dominant negative toxoid antigen; and treating and / or preventing ASFV.

10. The method of claim 9, wherein the dominant negative toxoid antigen is derived from a protein chosen from the group consisting of outer membrane protein EP402R (CD2v), outer membrane protein EP153R, protein EP183L (p54), and inner envelop protein CP204L.

11. The method of claim 10, further including a capsid protein chosen from the group consisting of protein p72 and protein p49.

12. The method of claim 9, further including the step of preventing early stage infection of low- level capsid-based virions that occurs through pinocytosis.

13. The method of claim 9, further including the step of administering a composition chosen from the group consisting of wildtype capsid protein, capsid subunit, domain, and peptide and treating both lysogenic and lytic viral replication cycles.

14. The method of claim 13, wherein the composition is tethered to the dominant negative toxoid antigen.

15. The method of claim 9, further including the step of monitoring ASFV infection by detecting a tag on antigens from a sample of the animal and differentiating infected and vaccinated animals.

16. The method of claim 9, wherein the animal is chosen from the group consisting of domestic pig, wild boar, warthog, and bush pig.

17. A method of engineering a dominant negative toxoid antigen, including the steps of: engineering mutations in N- or O-glycosylation sites of each of the proteins EP402R(CD2v), EP183L (p54), and EP153R of a viral African Swine Fever Virus (ASFV); and producing a dominant negative antigen.

18. The method of claim 17, wherein preventing receptor- mediated binding to red blood cells (RBCs), monocyte / macrophage apoptosis, and T-cell suppression whereby silent mutations in the N- or O-glycosylation sites create a dominant negative toxoid antigen form of the viral ASFV antigen for exposing to an immune system of an animal for B-cell stimulation and antibody production.Attorney Docket No.: 0431.0006919. The method of claim 17, wherein said engineering mutations step is further defined as deglycosylation with PNGase F.

20. The method of claim 17, further including the step of adding a signal peptide to the dominant negative toxoid antigen that allows for N-glycosylation.

21. The method of claim 17, further including the step of tethering at least two dominant negative toxoid antigens with a non-immunogenic linker peptide.

22. The method of claim 17, further including the step of adding a capsid protein chosen from the group consisting of protein p72 and protein p49.

23. A method of engineering a dominant negative toxoid antigen, including the steps of: creating a protein composition chosen from the group consisting of a domain, a sub domain, and a peptide antigen that produces antibodies that recognize a wildtype African Swine Fever Virus (ASFV) viral protein antigen; and producing a dominant negative antigen.

24. The method of claim 23, wherein the antigen is chosen from the group consisting of EP402R, EP153R, and CP204L.

25. The method of claim 23, further including the step of adding a signal peptide to the protein composition that allows for N-glycosylation.

26. The method of claim 23, further including the step of tethering at least two dominant negative toxoid antigens with a non-immunogenic linker peptide.

27. The method of claim 23, wherein said creating step is further defined as a step chosen from the group consisting of deleting an entire left lobe eliminating both red blood cell (RBC) binding and CD58-mediated cytotoxicity, creating a left lobe-only toxoid incorporating an E99 mutation to prevent RBC binding and mutations targeting CD58 interaction sites, and creating E99R mutations and CD58-binding site mutations.

28. The method of claim 23, further including the step of adding a capsid protein chosen from the group consisting of protein p72 and protein p49.

29. A method of engineering a dominant negative toxoid antigen, including the steps of: introducing mutations within an amino acid of an African Swine Fever Virus (ASFV) viral protein antigen for chosen from the group consisting of interactive / modifiable amino acids of an antigen and amino acids associated with protein-protein interactions: and producing a dominant negative antigen.

30. The method of claim 29, wherein the interactive / modifiable amino acids are chosen from the group consisting of serine, threonine, tyrosine, histidine, and combinations thereof and further including the step of preventing and / or altering phosphorylation patterns.Attorney Docket No.: 0431.0006931. The method of claim 29, wherein the amino acids associated with protein-protein interactions is further defined as the Dynein Binding Domain (DBD) of E183L.

32. The method of claim 29, wherein the dominant negative antigen produced is EP153R with a mutation in the Arg133 site.

33. The method of claim 32, wherein the mutation is chosen from the group consisting of lysine, glutamine, and cysteine.

34. The method of claim 33, further including the step of tethering at least two dominant negative toxoid antigens with a non-immunogenic linker peptide.

35. The method of claim 34, further including the step of adding a capsid protein chosen from the group consisting of protein p72 and protein p49.

36. A composition of a vaccine for African Swine Fever Virus (ASFV), comprising a dominant negative toxoid antigen in a vaccine.

37. The composition of claim 36, wherein said vaccine is in a form chosen from the group consisting of an mRNA vaccine, a self-amplifying RNA vaccine, and a DNA vaccine.

38. The composition of claim 36, wherein said dominant negative toxoid antigen is derived from a protein chosen from the group consisting of outer membrane protein EP402R (CD2v), outer membrane protein EP153R, protein EP183L (p54), and inner envelop protein CP204L.

39. The composition of claim 38, further including a capsid protein chosen from the group consisting of protein p72 and protein p49.

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