Dominant negative antigen method for prevention and post-infection treatment of pigs against African swine fever virus

Abstract

The disclosed invention relates to and encompasses a composition comprising a modified ASFV outer membrane protein antigen mutant (referred to as dominant negatives) that exhibits a non-binding affinity for RBCs while inducing an antibody-mediated response capable of neutralizing unmodified proteins found on infectious outer membrane loaded ASFV virions. Furthermore, a method for treating and / or preventing ASFV is provided, the method comprising administering to an animal a dominant negative antigen composition, thereby avoiding RBC aggregation caused by the antigen and concurrently treating and / or preventing ASFV. In addition, the present invention encompasses an ASFV vaccine composition containing a dominant negative antigen as an ingredient. Furthermore, the present invention encompasses formulations that bind these dominant negative antigens with antigens derived from capsid-based proteins, which co-target lyogenic and cleavage viral replication cycles, thereby achieving optimal immunostimulatory protection.

Description

Background of the Invention 1. Technical Field

[0002] The present invention relates to compositions and methods for treating and preventing African swine fever virus (ASFV). 2. Background Art

[0003] African swine fever virus is a large double-stranded DNA virus of the family Asfarviridae that mainly infects domestic pigs, wild boars, warthogs, and bushpigs. It also occurs in soft ticks, thereby serving as an infection vector. ASFV mainly infects monocytes and macrophages, but during acute infection, many other cell types can also be infected. ASFV causes high fever, hemorrhagic lesions, cyanosis, anorexia, and death in these animals. There is no vaccine or treatment for this virus, and currently the only way to prevent its spread is to cull animals.

[0004] The development of vaccines and / or treatment methods for ASFV is a highly active effort in multiple laboratories in China, the United States, and Europe. There are many approaches to developing such vaccines / treatment methods; however, all of these methods have failed to meet the desired treatment / prevention.

[0005] Some of these methods include the engineering of various types of attenuated viruses, immunostimulation using various and multiple combinations of viral antigens, gene editing, and antibody neutralization therapy, among others.

[0006] Traditional vaccine and treatment development programs mostly consider viral proteins exposed on the surface of virus particles as potential targets for immune neutralization or as antigens to stimulate the immune system. Other methods can use small molecules, RNA interference, or CRISPR gRNA inhibition methods to target the viral mechanisms with biochemical activity within infected host cells. In addition, once the viral genome takes root in an infected host cell, more exotic methods such as gene editing (e.g., CRISPR) can be used to alter the viral genome (Hübner et al., Borca et al., et al.).

[0007] In the context of ASFV, many of these methods are unrealistic because the cost of treating each livestock animal with a relatively low capital value is too high.

[0008] In addition to logical flaws, many failures in vaccine or treatment design can also be attributed to a lack of in-depth understanding of the virus structure and the viral replication cycle. Recently, the structure of ASFV has been defined (Liu et al.). Since then, it has become clear that the virus exists in two forms: 1) the virus particle capsid surrounded by an outer lipid bilayer, and 2) naked virus particles that only cover the capsid ( Figure 1A)。The outer lipid bilayer membrane has been shown to contain human proteins, strongly suggesting that capsid-based viral particles bud from infected cells. This property is mainly associated with the lysogenic replication cycle observed in most other viruses. Additionally, it has been reported that in the late stage of infection, capsid-based viral particles (without the outer lipid bilayer membrane) of ASFV increase in the circulating serum, closely related to the dynamics of the lytic cycle of most other viruses (see the description of lysogenic and lytic ASFV in PCT / US20 / 50939 by Chen et al.). Considering these structural components and defining them in the context of the early lysogenic versus late lytic replication cycles enables the applicant to pursue new vaccine and therapeutic development programs (PCT / US20 / 50939, US Patent Application No. 17 / 535,545)( Figure 1B and 1C )。

[0009] This strategy takes into account the need to immediately neutralize early infectious membrane-bound viral particles derived from the lysogenic cycle and then rapidly neutralize the small number of capsid-based viral particles present before the transition to the lytic cycle.

[0010] Through this strategy, the applicant has tested several known outer membrane-binding proteins as potential targets for antibody therapy production and antigen-based vaccines. These targets include outer membrane proteins EP402R (CD2v) and EP153R, outer and inner envelope protein O16R (p12), inner envelope and capsid transmembrane protein E183L (p54), and several major capsid proteins, including B646L (p72), E120R (p14.5), B438L (p49), and CP204L (p30), although the studies are not limited to these proteins (Figures 2 and 3). It has been shown that the immune response against outer membrane proteins (as protein subunit antigens, whole protein antigens, or expressed on the surface of attenuated viruses) generates a very weak immune response in pigs, probably because they can bind to RBCs, penetrate into the RBCs, and cause RBC agglutination. Therefore, this agglutination ability must be eliminated in order to expose the surface of outer membrane antigens (including EP402R, EP153R, and O16R, but not limited to these protein antigens) to the porcine immune system to generate a strong and neutralizing immune response (Figure 4).

[0011] To date, capsid-based proteins have not been shown to cause agglutination in RBCs, although the applicant has observed slight porcine RBC agglutination that is species-specific for subunits containing DBD and interacting with the capsid of the inner envelope and capsid transmembrane protein E183L (p54)( Figure 5B )。Therefore, an E183L mutation may be necessary to block this ability and enable effective exposure of this antigen to the porcine immune system to generate a strong neutralization.

[0012] Since capsid-based proteins have not been shown to possess these agglutination properties, dominant-negative mutations may not be required to achieve strong neutralization. However, the combination of capsid antigens with dominant-negative antigens may enhance immunogenicity against each stage of the ASFV replication cycle, thus strengthening the applicant's argument that targeting lysogenic cycle proteins (primarily outer membrane proteins) and the lytic cycle (primarily capsid-based proteins) is necessary for effective protection of pigs against ASFV infection. Therefore, capsid-based antigenic proteins will be combined with dominant-negative outer membrane proteins (such as EP402R, EP153R, and O16R) and inner envelope and capsid transmembrane proteins (such as E183L) to stimulate an immune response to effectively neutralize AFSV infection in pigs.

[0013] Without being bound by theory, the dominant-negative approach for vaccine development for stronger immune responses has been achieved previously by similar methods such as tetanus and diphtheria toxoids. These toxoids are inactivated forms of the toxic proteins for tetanus and diphtheria and can generate effective immune responses.

[0014] By creating a dominant-negative protein subunit version of the wild-type ASFV outer membrane protein antigen in a vaccine that is normally used to cause agglutination, agglutination does not occur, thus exposing the antigen to the immune system (Figure 4).

[0015] The applicant's latest research indicates that the subunit of the outer membrane target EP402R is most likely responsible for binding the virus to porcine red blood cells (RBCs), causing them to agglutinate in a species-specific manner (compared to human, murine, and bovine RBCs). ( Figure 5A)。In addition, the outer membrane protein EP153R has previously been shown to promote RBC aggregation in concert with EP402R. The applicant observed RBC aggregation driven by the EP153R subunit. However, enhanced aggregation occurs in the presence of the extracellular domain of EP402R. Petrovan et al. reported similar observations in 2021 using an attenuated virus. The aggregated RBCs then trigger natural macrophages, initiating the destruction of RBC aggregates by a mechanism that has not yet been determined. When RBCs attract macrophages, the virus particles buried within the RBC aggregates are also internalized into the macrophages (the selected cells for infection). There may be a mechanism that links one of the outer membrane proteins to RBC aggregation and internalization into macrophages. Since ASFV induces hemagglutination before infection and subsequent hemadsorption to the surface of macrophages, these proteins are likely to play a role in the well-orchestrated internalization process (Malmquist et al., Yang et al.). To date, no ASFV receptor capable of promoting receptor-mediated virus internalization has been found on the surface of macrophages. However, uptake of capsid-based virus particles has been shown to occur via the much less efficient macropinocytosis entry pathway (Sánchez et al.). For capsid-based virus particles to efficiently infect macrophages at the systemic and rapid levels via macropinocytosis, there must be a large amount of virus circulating in the pig. This may occur after the lysogenic replication phase reaches a critical point and switches to the lytic phase, where a large number of viruses are released from the cells. Thus, by neutralizing the proteins responsible for RBC aggregation and preventing the lysogenic cycle, any remaining capsid-based virus is limited to entering macrophages via macropinocytosis and is subsequently locked in the early lysogenic phase, which is neutralized upon virus budding, just like its predecessor ( Figure 6 )。Finally, the remaining capsid-based virus particles can be neutralized by antibodies derived from capsid-based targets such as B646L (p72), E120R (p14.5), B438L (p49), and CP204L (p30), thus eliminating even the slightest threat ( Figure 6 and 7 -Overview of the Replication Cycle and Strategies).

[0016] The applicant's previous data indicated that antibodies generated against the outer membrane protein EP402R can prevent and disrupt RBC aggregates (Figure 8A) and act in a species-specific manner (Figure 8B). This antibody-mediated disruption of RBC aggregates may prevent the internalization of these proteins into macrophages.

[0017] As described above, there have been many attempts to use ASFV protein antigens to stimulate an internal antibody response in pigs. These attempts have failed to produce any durable or meaningful protection against the virus in porcine challenge models. For example, almost every capsid protein has been explored as a potential vaccine candidate. With the new understanding of the replication cycle and structure of ASFV ( Figures 1A to 1C and 7), it is not surprising that these methods have failed. Once the virus switches to the lytic cycle (a critical and population-dense point after the lysogenic cycle), the immune system or any other protective measures cannot keep up with the large number of capsid-based viruses. In addition, targeting outer membrane proteins for antigen-based vaccines has also failed, probably because they are able to aggregate RBCs and "hide" from B-cell and T-cell responses. For example, EP402R (referred to as HA in earlier publications) and EP153R have been shown to promote the binding of the virus to RBCs, allowing the virus to penetrate / drill into the RBC membrane. This enables the virus to hide by preventing key surface epitopes from being exposed to a strong and sustained antibody-mediated immune response (Qunitero et al., Ruiz-Gonzalvo et al.). It should also be noted that some outer membrane proteins also have immunosuppressive 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 expressed on the surface of T cells. The amino acid at the Arg133 site is crucial for the sequestration of MHC class I complexes after macrophage infection.

[0019] These observations have recently been highlighted by the observation that most outer membrane proteins in attenuated viruses or antigens have relatively weak immunostimulatory properties, while exposed capsid proteins have relatively high immunostimulatory properties. Thus, we have observed that the outer membrane proteins EP402R (CD2v) and EP183L (p54) cause RBC aggregation, and previous observations in the literature (where EP153R causes lymphocyte neutralization) specifically occur in pigs and no other species, which may contribute to this summary. In addition, antigen-based vaccine approaches that stimulate an immune response against outer membrane protein targets may fail because their RBC agglutination properties allow them to evade the immune response. In fact, it has been observed that the minimal extracellular domains of these outer membrane proteins retain these RBC-binding properties ( Figure 5A and B).

[0020] Therefore, there remains an urgent need to develop an antigen-based vaccine using ASFV outer membrane protein antigens that do not cause RBC aggregation and thus do not "hide" from eliciting a strong antibody-neutralizing immune response. Summary of the Invention

[0021] The present invention provides a composition comprising an engineered ASFV outer membrane protein antigen mutant (dominant negative), which mutant does not bind to RBCs but elicits an antibody response that neutralizes the wild-type protein present on the infectious outer membrane containing ASFV virus particles.

[0022] The present invention provides a method of treating and / or preventing ASFV by administering a composition of a dominant negative antigen to an animal, preventing RBC aggregation from the antigen, and treating and / or preventing ASFV.

[0023] The present invention provides a composition for a vaccine against ASFV, the composition comprising a dominant negative antigen in the vaccine.

[0024] The present invention provides a composition for using these dominant negative antigens in combination with antigens derived from capsid-based proteins to address lysogenic and lytic viral replication cycles to achieve maximal immunostimulatory protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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 conjunction with the accompanying drawings, wherein:

[0026] Figure 1A shows the structure of ASFV derived from the lysogenic replication cycle (left) and the subsequent lytic replication cycle (right), Figure 1B shows why current neutralization strategies do not work for both replication cycles, and Figure 1C shows the major identified proteins present in the membrane of ASFV virus particles derived from the lysogenic replication cycle (top) and the major identified capsid proteins from virus particles derived from the lytic replication cycle (bottom);

[0027] Figure 2 shows a legend of polyclonal antibodies generated against parallel ASFV proteins for preclinical studies;

[0028] Figure 3 shows the overall strategy of neutralizing each type of ASFV (with or without membrane) by neutralizing antibodies generated after antigen injection;

[0029] Figure 4 is a diagram showing why dominant negative action is more effective than using wild-type proteins or subunit antigens. Immune system exposure;

[0030] Figure 5A shows how the ASFV protein EP402R specifically causes porcine RBC aggregation, and Figure 5B shows how the ASFV protein E183L causes porcine RBC aggregation;

[0031] Figure 6is a detailed illustration of the ASFV infection and replication cycle via RBC-mediated macrophage entry;

[0032] Figure 7 is a detailed illustration of strategies to block the cycle shown in Figure 6 ;

[0033] Figures 8A and 8B show how polyclonal antibodies raised against EP402R and E183L (p54) block RBC aggregation induced by these proteins;

[0034] Figure 9A is a graph showing the effect of PNGase F-mediated deglycosylation of EP402R on porcine RBC aggregation, and Figure 9B shows the deglycosylation of EP402R and PNGase F on a Coomassie-stained gel;

[0035] Figure 10A is a sequence and structure diagram showing the proposed regions and amino acid sequences of EP402R, where dominant negative mutations will be made and screened to prevent RBC penetration / binding leading to exposure to the immune system. Each is derived from the reference sequence China_AnhuiXCGQ_2018;

[0036] Figure 10B is an illustration of the orientation of full-length EP402R folded in the outer membrane of ASFV;

[0037] Figure 11A is a sequence and structure diagram showing the proposed regions and amino acid sequences of EP153R, where dominant negative mutations will be made and screened to prevent RBC penetration / binding leading to exposure to the immune system. Each is derived from the reference sequence China_AnhuiXCGQ_2018;

[0038] Figure 11B is an illustration of the orientation of full-length EP153R folded in the outer membrane of ASFV;

[0039] Figure 12A is a sequence and structure diagram showing the proposed regions and amino acid sequences of E183L, where dominant negative mutations will be made and screened to prevent RBC penetration / binding leading to exposure to the immune system. Each is derived from the reference sequence China_AnhuiXCGQ_2018;

[0040] Figure 12B is an illustration of the orientation of full-length E183L folded in the capsid and viral envelope of ASFV. DETAILED DESCRIPTION

[0041] The present invention generally provides compositions and methods for treating and / or preventing ASFV. The compositions include engineered ASFV outer membrane protein antigen mutants (dominant negatives) that do not bind to RBCs but elicit a neutralizing antibody response against wild-type proteins present on the infectious outer membrane containing ASFV virions.

[0042] As used herein, "animal" refers to any non-human species of animal.

[0043] As used herein, "Porcine" or "swine" can be domestic pigs, wild boars, warthogs or bushpigs.

[0044] The term "vector" includes cloning and expression vectors, as well as viral vectors and integration vectors. An "expression vector" is a vector that includes regulatory regions. Vectors are also further described below.

[0045] As used herein, the term "antibody" refers to a blood protein produced in response to and resistant to a specific antigen. Antibodies chemically bind to substances recognized by the body as foreign, such as bacteria, viruses, and foreign substances in the blood.

[0046] As used herein, the term "mRNA" refers to a type of RNA in cells that carries the genetic information required to make proteins.

[0047] The term "dominant negative" antigen for ASFV is used to describe an alteration to the native ASFV protein such that the protein is exposed to the porcine immune system, eliciting a strong immune response that can effectively inactivate the native protein on the virus.

[0048] Dominant negative antigens can be engineered in several ways:

[0049] 1) Mutations are made in the N- or O-glycosylation sites of each of the proteins pE402R (CD2v), EP153R, and EP183L (p54) respectively to prevent receptor-mediated binding to RBCs. The Applicant's data shows that deglycosylation with PNGase F partially inhibits RBC aggregation mediated by the EP402R protein (Figure 9A). Thus, silent mutations can be implemented at these sites or at sites that affect glycosylation flanking these sites or at multiple sites that prevent RBC-antigen receptor interaction to generate a dominant negative form of the antigen (Figures 10 to 12).

[0050] 2) Dominant negative antigens can be screened using a peptide library that defines the epitope regions responsible for RBC interaction. The epitopes responsible for binding can then be mutated to prevent binding. The mutations can be silent mutations to the structure.

[0051] 3) Dominant negative antigens can be screened using any type of suitable yeast two-hybrid screening platform. The two-hybrid library can constitute a reticulocyte-to-protein antigen bait-prey system.

[0052] 4) High-throughput sequencing of a library composed of fragments of yeast genes can identify polypeptide antigens with dominant negative properties (Reference - PMID: 30962621).

[0053] 5) By determining the receptors on RBCs that interact with outer membrane antigens, the exact amino acids involved in the interaction between the receptors within the antigen and the antigen can be resolved. RBC receptor resolution can be determined using a variety of methods, including two-hybrid systems, crystallography, ligand ligation systems, peptide interaction screening methods, and so on.

[0054] Dominant negative antigens can include modifications to the protein that prevent binding, such as; i) structural components that sterically hinder RBC interaction but expose key amino residues for immune recognition, ii) alternative glycosylation (branched carbohydrates) that prevent binding, iii) polyethylene glycolylated residues, iv) and other types of conjugated ligands that interfere with RBC binding without disrupting the protein structure, so it retains the relevant immunoreactive epitopes that cross-react with wild-type virus.

[0055] 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%.

[0056] Dominant negative antigens are required for a robust and effective antigen vaccine approach because wild-type antigens cause / lead to RBC aggregation and "evade" the immune response. Since capsid-based ASFV does not cause RBC aggregation, there is no need to use dominant negative antigens at this stage.

[0057] The present invention provides a method for treating and / or preventing ASFV infection by administering a composition of dominant negative antigens to an animal, preventing RBC aggregation from the antigen, and treating and / or preventing ASFV.

[0058] The present invention provides a method for treating and / or preventing ASFV infection by combining a composition of dominant negative antigens with wild-type capsid proteins or capsid subunits / domains / peptides to address the lysogenic and lytic viral replication cycles, respectively.

[0059] The composition can be provided in a vaccine, such as 1) an mRNA vaccine for delivery to APCs and B cells such that the mRNA expresses the antigen internally to generate an immune response, 2) direct antigen injection, or 3) a DNA vaccine (which sends instructions to make the antigen into DNA). Accordingly, the present invention provides a composition for a vaccine against ASFV, the composition comprising a dominant negative antigen in the vaccine.

[0060] For viruses, bacteria, fungi, or parasites that bind to reticulocytes or RBCs, dominant-negative proteins can have additional applications, and these proteins can be masked in other tissues. Hemagglutination is a strategy used by a few viruses to enhance their infectivity for target cells. For example, HIV-1 and HIV-2 bind to the receptor Duffy antigen chemokine receptor (DARC) on RBCs. This binding causes hemagglutination, which in turn makes the virus about 100 times more infectious than the unbound and circulating virus (Lachgar et al., He and Neil et al., Beck et al.), and the extent 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 the P k receptor on PBMCs to block competing viruses (Lund et al.). Norovirus also binds via the receptor / Le bLigand interactions selectively bind to group A, group H, and / or difucosylated Lewis blood types to cause hemagglutination (Nillson et al., Shirato-Horikoshi et al.). Examples of other viruses that bind to RBCs and cause hemagglutination include bovine coronavirus (BCV), porcine hemagglutinating encephalomyelitis virus (PHEV), influenza C, and torovirus (Zeng et al.). Each of these RNA viruses causes hemagglutination by binding, via its 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. Aggregated RBCs then transport the virus to target cells more efficiently than unbound and circulating virus. Once in contact with the target cell, the HE protein 2) undergoes a conformational change to activate its target cell receptor-destroying property, causing the RBC to release its viral payload to enter the cell. However, these RNA viruses do not have an outer membrane or envelope like ASFV. There is no outer membrane to protect the viral core from the immune response. For example, PHEV is dominated by spike proteins that project from a single membrane that also contains HE and other membrane proteins. Thus, when the virus binds to RBCs via its HE protein, its spike proteins remain exposed to the antibody response. There are no or few epitope hindrances. Thus, antibodies against the major PHEV proteins have been shown to be strongly neutralizing until the spike protein (or other proteins) mutates. In addition, PHEV does not distinguish between species. It causes hemagglutination in mice, rats, chickens, and several other animals. Thus, although the agglutination of RBCs is a common strategy for some viruses to increase their infectivity for target cells, the mode of action is very different. ASFV is a DNA virus with a much slower mutation rate due to genetic drift or environmental mutagens, while many RNA viruses have a higher mutation rate due to a lack of replication proofreading. Due to the lack of a protective outer layer, RNA viruses may not be masked by agglutinated RBCs, but they compensate for this difference by rapid mutation to allow immune evasion. Given these differences (especially the lack of species specificity of RNA viruses containing HE), ASFV most likely binds specifically to porcine RBCs via a different receptor, sialic acid motif, or carbohydrate than RNA viruses containing HE.

[0061] Based on the knowledge of those skilled in the art, the compositions described herein can be administered in animals. The concentration of the composition can be from 1% to 100%, and include any suitable excipients, such as those described further below. Delivery routes can include, but are not limited to, epidermal, intradermal, subcutaneous, transdermal, intramuscular, intravenous, oral, corneal, intraocular, intracerebral, epidural, intrathecal, intraperitoneal, intraosseous, intranasal (such as into the nose of a pig), intratracheal, and other routes described further below.

[0062] The composition can be stored in various forms that allow the protein to be stable during the shelf life (from one month to several years) at different temperatures (4 °C, -20 °C, -20 to -80 °C), such as freeze-dried dry powder, aqueous solution, tris(hydroxymethyl)aminomethane or phosphate buffer, or a solution containing 25% to 50% glycerol or ethylene glycol used as a cryoprotectant.

[0063] The compounds of the present invention are 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, the timing of administration, the age, sex, and body weight of the animal, and other factors known to the medical practitioner. Thus, a pharmaceutically "effective amount" for the purposes herein is determined by such considerations known in the art. This amount must be effective to achieve an improvement, including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms, and other metrics selected by those skilled in the art as appropriate measures.

[0064] In the methods of the present invention, the compounds of the present invention can be administered in various ways. It should be noted that it can be administered as a compound and can be administered alone or in combination with a pharmaceutically acceptable carrier, diluent, adjuvant, and excipient as an active ingredient. The compound can be administered orally, subcutaneously, or parenterally, including intravenous, intraarterial, intramuscular, intraperitoneal, intratonsillar, and intranasal administration, as well as intrathecal and infusion techniques. Implants of the compound are also useful. The patients to be treated are warm-blooded animals, and particularly mammals, including humans. Pharmaceutically acceptable carriers, diluents, adjuvants, and excipients, as well as implant carriers, generally refer to inert, non-toxic solid or liquid fillers, diluents, or encapsulating materials that do not react with the active ingredient of the present invention.

[0065] The dose can be a single dose or multiple doses over several days. The duration of treatment is generally proportional to the duration of the disease process, the effectiveness of the drug, and the species of the patient being treated.

[0066] When the compounds of the present invention are administered parenterally, they are usually formulated into a unit-dose injectable form (solution, suspension, emulsion). Pharmaceutical preparations 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 a dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils.

[0067] For example, by using coatings such as lecithin, in the case of a dispersion by maintaining the desired particle size and by using surfactants to maintain proper fluidity. Non-aqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower seed oil or peanut oil and esters such as isopropyl myristate can also be used as the solvent system for the compound composition. Additionally, various additives that enhance the stability, sterility and isotonicity of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents and buffers. The action of microorganisms can be ensured against by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. In many cases, it is desirable to include isotonic agents such as sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms can be achieved by using agents that delay absorption such as aluminum monostearate and gelatin. However, according to the present invention, any vehicle, diluent or additive used must be compatible with the compound.

[0068] Sterile injectable solutions can be prepared by incorporating the compounds useful in the practice of this invention in the required amounts into a suitable solvent and various other ingredients as desired.

[0069] The pharmaceutical formulations of the present invention can be administered to a patient in the form of injectable formulations that contain any compatible carrier such as various vehicles, adjuvants, additives and diluents; or, the compounds useful for the present invention can be administered parenterally to a patient in the form of a sustained release subcutaneous implant or a targeted delivery system such as monoclonal antibodies, carrier delivery, iontophoresis, polymer matrix, liposomes and microspheres. Examples of delivery systems useful for 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.

[0070] Throughout this application, various publications, including U.S. patents, are cited by author and year and patents by number. The complete citations for the publications are listed below. The disclosures of these publications and patents are hereby incorporated by reference in their entirety into this application to more fully describe the state of the art to which this invention pertains.

[0071] The invention has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be of a descriptive rather than a limiting nature.

[0072] Obviously, in view of the above teachings, many modifications and variations of the present invention are possible. Therefore, it is to be understood that within the scope of the appended claims, the present invention may be practiced in a manner different from that specifically described.

[0073] References

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Claims

1. A composition comprising an engineered ASFV outer membrane protein antigen mutant (dominant negative) that does not bind to RBCs and elicits a neutralizing antibody response against the wild-type protein present on the infectious outer membrane containing ASFV virions.

2. The composition according to claim 1, comprising the dominant negative, further defined as an antigen for ASFV, which is used to describe an alteration to the native ASFV protein such that the protein is exposed to the porcine immune system, eliciting a strong immune response that can effectively inactivate the native protein on the virus.

3. A method of treating and / or preventing ASFV, comprising the steps of: administering to an animal a composition of a dominant negative antigen; preventing RBC aggregation caused by the antigen; and treating and / or preventing ASFV.

4. The method according to claim 2, wherein the antigen defined as for ASFV is used to describe an alteration to the native ASFV protein such that the protein is exposed to the porcine immune system, eliciting a strong immune response that can effectively inactivate the native protein on the virus.

5. A method of engineering a dominant negative antigen by introducing engineered mutations into the N- or O-glycosylation sites of each of the proteins pE402R (CD2v), EP153R, and EP183L (p54), respectively, and preventing receptor-mediated binding to RBCs, such that silent mutations can be implemented at these sites to produce a dominant negative form of the antigen.

6. A method of engineering the dominant negative antigen by screening a peptide library defining the epitope regions responsible for RBC interaction so that the epitopes responsible for binding can be mutated to prevent binding.

7. The method according to claim 6, wherein the mutation can be a silent mutation to the structure.

8. A method of engineering a dominant negative antigen by screening a dominant negative antigen using any type of applicable yeast two-hybrid screening platform, the two-hybrid screening platform constituting a reticulocyte-to-protein antigen bait-prey system.

9. A dominant negative antigen comprising a modification to the protein that prevents binding, such as; i) a structural component that sterically hinders RBC interaction but exposes key amino residues for immune recognition, ii) alternative glycosylation (branched carbohydrates) that prevents binding, iii) polyethylene glycolylated residues, iv) and other types of conjugated ligands that interfere with RBC binding without disrupting the structure of the protein, and thus it retains the relevant immunoreactive epitopes that cross-react with the wild-type virus.

10. A composition for a vaccine against ASFV, the composition comprising the dominant negative antigen in the vaccine.

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