CN114746110A - Vaccine and immunoglobulin targeting African swine fever virus, and methods of making and using the same - Google Patents

Abstract

The present disclosure provides a method of isolating and preparing live African Swine Fever (ASF) virus (ASFV) and ASFV vaccines comprising intact ASF virions, viral components and/or immunosuppressive protein factors. The ASFV vaccine may be used for immunization of pigs and boars, or may be used for immunization of species other than pigs or boars, such as poultry, cattle, goats, rabbits, donkeys or horses, to produce polyclonal immunoglobulins with broad specificity for ASFV. The ASFV-specific immunoglobulin may then be extracted and purified. The ASFV-specific immunoglobulin may provide an acute treatment for a pig or boar infected with ASF, or a prophylactic treatment for a pig or boar at risk for ASF, e.g. a subject that may have been exposed to ASFV or ASFV infection.

Description

Vaccine and immunoglobulin targeting African swine fever virus, and methods of making and using the same

Technical Field

The present disclosure relates generally to compositions for use in active and/or passive immunization for the treatment and prevention of African Swine Fever (ASF) virus (ASFV) infection. The present disclosure also relates to methods of isolating and preparing combinations of ASF whole virions and ASF individual viral components for use as vaccines in porcine and/or non-porcine species hosts for the production of ASFV-specific immunoglobulins. The ASFV-specific immunoglobulins disclosed herein provide a broad spectrum of immunity to pigs and wild boars infected with or susceptible to ASFV infection.

Cross reference to related applications

This application claims the benefit of U.S. provisional application 62/906,357 filed 2019, 26, month 9, which is incorporated herein by reference in its entirety.

Sequence listing

This application contains a sequence listing submitted electronically in ASCII format and incorporated by reference herein in its entirety. The ASCII copy created on 24.9.2020 is named Seq _ Listings _ for _1401870-00006.txt, size 8,548 bytes.

Background

ASF is a highly infectious hemorrhagic disease caused by ASFV (USDA supervisory program, page 3). ASF affects porcine mammals, including domestic pigs, wild pigs and european asian wild pigs (USDA supervisory program, page 3). The virus was first discovered in the east Africa in the beginning of the 1900S, and spread from local warts to domestic swine in most sub-Saharan countries (S a nchez-Cord Lolo, et al, African swine fever: a recurring viral disease threatening the global swine industry (African swine farm: A re-emergent treatment of the viral pig industry), 233vet.J.41,41 (2018)). Verrucous and jungle pigs are natural storage hosts for ASFV, show few clinical signs and persist in infection (Dixon et al, host defense evasion of African swine fever Virus, 266Virus res.25,25 (2019)). In contrast, infection of domestic, wild or wild pigs leads to acute hemorrhagic fever with high mortality (Dixon et al, page 25).

ASFV has spread in Europe later in the 1950S, and later in south America and the Caribbean (S a nchez-Cord Lo, et al, p.41). Without an effective vaccine, the methods used to control the spread of the Virus are limited to isolation and slaughter of infected and exposed pigs (Netherton et al, Identification and Immunogenicity of African Swine Fever Virus Antigens, 10front. immune.1, 1 (2019)). In the mid 1990S ASF was successfully eradicated from areas outside africa, but by 2007 the virus again experienced a second transcontinental spread to grurgia and eastern europe (S a nchez-corp, et al, p 41). Recently, ASF outbreaks (FAO site; ASF location update) have been reported in China, Vietnam, Mongolia, Cambodia, and Korea. The spread of ASF to china is particularly alarming as china is the world's largest pig producing country (Netherton et al, page 1).

ASFV itself is a large, complex double-stranded DNA virus that replicates in the cytoplasm of macrophages, monocytes and dendritic cells (Dixon et al, p.25). Over 20 Genotypes have been recorded by each study group and a Comparative Analysis of at least 8 serotypes, Kolbasov et al, African Swine Fever Virus Genotypes and Serogroups has been identified (comprehensive Analysis of African Swine Fever viruses genotyps and Serogroups), 21emerg. Conventional inactivated vaccines have been unsuccessful and attenuated live vaccines have not yet yielded the desired efficacy (S-nchez-Cord Lo n et al, page 44). The challenges associated with the development of a successful ASF vaccine are believed to be due to a lack of understanding of how the virus modulates the host response to infection and the lack of identification of protective antigens (S < nchez-corp, et al, page 44).

Disclosure of Invention

The present inventors have developed a method of isolating live ASFV and viral components to make an ASFV vaccine comprising intact viral particles, single viral structural proteins and viral components involved in exacerbation of infection, including but not limited to immunosuppressive factors and/or host immune factors. The ASFV vaccine can be used for active immunization or vaccination of pigs, wild boars or other species susceptible to ASF infection after gamma-ray irradiation. Additionally or alternatively, live or gamma-irradiated ASFV vaccines can be used to actively immunize or vaccinate a species other than swine or wild boar, such as avian, bovine, rabbit, goat, donkey, or horse, to produce polyclonal immunoglobulins with broad-spectrum specificity for ASFV. In a preferred embodiment, the ASFV vaccine is used to vaccinate laying birds, such as chickens, and the antibodies or antibody fractions may then be extracted and purified from the egg yolk. The produced egg-laying fowl antibody can be used for preventing virus adhesion and virus transmission, treating ASF, and preventing ASF. Antibodies of the IgY isotype from birds or avians are particularly useful in these applications.

The ASFV-specific immunoglobulin may be administered for acute treatment of ASFV-infected pigs or boars. The acute treatment may comprise parenteral and/or oral administration of the immunoglobulin, e.g. by intra-peritoneal occasionally intramuscular injection and/or in a food composition. Additionally or alternatively, the immunoglobulin may be administered as a prophylactic treatment by the same route of administration. In one embodiment, the ASFV-specific immunoglobulin may be in the form of a liquid or lyophilized powder, which may be injected intraperitoneally or intramuscularly after reconstitution, preferably twice weekly for one or more weeks at an injection dose of about 0.5 to about 1.0mg per kg body weight, for example to one or more ASFV infected or exposed pigs or boars. Alternatively, the ASFV-specific immunoglobulin may be administered orally, e.g. added to feed, at an oral dose of about 1.0mg per kg body weight, once a day for a total of about 5 to about 7 days, e.g. to one or more ASFV-infected or exposed pigs or boars.

In one embodiment, disclosed herein is a method of treating an ASFV infection in an infected pig or wild boar, the method comprising administering to the infected pig or wild boar an effective amount of a composition comprising immunoglobulins specific for an ASF viral component.

Also disclosed herein are methods of treating an ASFV infection in an infected pig or wild boar, wherein the composition is administered in an amount to provide a dose of the immunoglobulin specific for an ASF viral component of about 0.5mg to about 1.0mg per kg body weight of the infected pig or wild boar.

In another exemplary embodiment, the period of time for which the composition comprising an immunoglobulin specific for an ASF viral component is administered comprises at least once per week or for 7 consecutive days.

In another aspect, the composition comprising immunoglobulins specific for ASF viral components is administered parenterally by intramuscular or intraperitoneal injection.

In another aspect, the composition comprising immunoglobulins specific for ASF viral components is a food product for oral administration.

Another embodiment is a method of preventing, reducing the incidence of and/or reducing the severity of an ASF virus infection in a pig or wild boar at risk comprising administering to the pig or wild boar an effective amount of a composition comprising immunoglobulins specific for ASF virus components.

In another aspect, the composition is administered in an amount to provide a dose of the immunoglobulin specific for an ASF viral component of about 0.5mg to about 1.0mg per kg body weight of the at-risk pig or boar.

In another aspect, the period of time for which the composition comprising immunoglobulins specific for ASF viral components is administered comprises at least once per week or for 7 consecutive days.

It is also to be understood that the present disclosure contemplates that the composition comprising an immunoglobulin specific for an ASF viral component may be administered parenterally.

In another aspect, the composition comprising an immunoglobulin specific for an ASF viral component is a food product for oral administration.

Another embodiment disclosed herein is a method of producing an ASFV-specific immunoglobulin, wherein an ASFV vaccine comprising intact ASF virions, viral components, and/or immunosuppressive protein factors is administered to a non-porcine species host for producing an ASFV-specific immunoglobulin.

In an exemplary embodiment, the host is an laying bird.

Another exemplary embodiment disclosed herein is a unit dosage form comprising a therapeutically or prophylactically effective amount of a composition comprising an immunoglobulin specific for an ASF viral component.

In another embodiment, the composition is a food product formulated for oral administration.

Also disclosed herein is a method of preventing, reducing the incidence of, and/or reducing the severity of an ASF virus infection in a pig or wild boar at risk comprising administering to the pig or wild boar an effective amount of an ASFV vaccine composition comprising an ASF virus component.

In one aspect, the ASF viral component is inactive.

In another aspect, the ASFV vaccine composition is administered parenterally by intramuscular or intraperitoneal injection.

Also disclosed is an exemplary embodiment wherein the ASFV vaccine composition is administered in an amount that provides a dose of the ASF viral component of about 0.05mg to about 1.0mg per pig or wild boar.

Another embodiment is a unit dosage form comprising an effective amount of an ASFV vaccine composition comprising an ASF viral component.

In one aspect, the ASF viral component is derived from ASF-infected Spleen Monocytes (SMNC), ASF-infected Peripheral Blood Mononuclear Cells (PBMC), and/or ASF-infected Primary Alveolar Macrophages (PAM).

In another aspect, the ASF viral component is inactivated.

In another aspect, the ASFV vaccine is for use in the treatment and/or prevention of ASF infection in a pig or a wild boar at risk.

One embodiment is an immunoglobulin specific for an ASF viral component for use in the treatment and/or prevention of an ASF infection in a pig or boar at risk.

It is understood and contemplated herein that the ASFV vaccine may be useful in the prophylactic treatment of ASF infection in pigs or wild boars. In another embodiment, the ASFV vaccine and the ASFV-specific immunoglobulin may be used in combination in a therapeutic regimen and/or administered together to a pig or a boar.

Drawings

Fig. 1 shows exemplary embodiments of a method of manufacturing an ASFV vaccine, an embodiment of a method of actively immunizing a pig or a wild boar by administering the ASFV vaccine, an embodiment of a method of immunizing or vaccinating a non-porcine or non-susceptible species host for producing ASFV-specific immunoglobulins, and an embodiment of a method of passively immunizing a pig or a wild boar by administering the ASFV-specific immunoglobulins.

Figure 2 shows an exemplary embodiment of active immunization by administering the ASFV vaccine to a pig or wild boar (figure 2A) or a non-pig or non-susceptible species host for the production of ASFV-specific immunoglobulins (figure 2B).

Figure 3 shows qPCR results for an exemplary embodiment of an ASFV-specific immunoglobulin composition. The ASFV-specific immunoglobulin composition and the three controls were analyzed for the presence of ASFV p72 DNA (NC-001659.2; SEQ ID NO: 1). The qPCR results confirmed that the ASFV-specific immunoglobulin composition does not contain ASFV p72 DNA (SEQ ID NO: 1).

Figure 4 shows the titers of ASFV-specific antibodies in 3 groups of hens immunized on days 1, 14 and 28 using 2 different ASFV vaccine compositions and saline as a control (no ASFV vaccine). Eggs laid by the immunized hens were collected, immunoglobulin was extracted, and titers of ASFV-specific antibodies were evaluated using recombinant ASFV major capsid protein p 72-coated (ASFV p 72; NP-042775.1; SEQ ID NO: 2) enzyme-linked immunosorbent assay (ELISA) plates on days 14 (FIG. 4A) and 28 (FIG. 4B).

Figure 5 shows the titers of ASFV-specific antibodies in 3 groups of hens immunized on days 1, 14 and 28 using 2 different ASFV vaccine compositions and saline as a control (no ASFV vaccine). The eggs laid by the immunized hens were collected, immunoglobulins were extracted, and the titers of ASFV-specific antibodies were evaluated on day 28 using ELISA plates coated with recombinant ASFV major capsid protein p72(SEQ ID NO: 2).

Detailed Description

Definition of

Some definitions are provided later herein. However, definitions may also be located in the "embodiments" section below, and the above heading "definitions" does not imply that such disclosure in the "embodiments" section is not a definition.

As used herein. "about", "about" and "substantially" are understood to mean a number within a certain range of values, for example, within the range of-10% to + 10% of the reference number, preferably within the range of-5% to + 5% of the reference number, more preferably within the range of-1% to + 1% of the reference number, and most preferably within the range of-0.1% to + 0.1% of the reference number.

All numerical ranges herein should be understood to include all integers or fractions within the range. Further, these numerical ranges should be construed as providing support for declaration of any number or subset of numbers within the range. For example, a disclosure from 1 to 10 should be interpreted to support a range of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, and so forth.

As used in this disclosure and the claims, the singular form includes the plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component" includes two or more components.

The word "comprising" should be interpreted inclusively rather than exclusively. Also, the terms "comprising," "including," and "having" are to be construed as inclusive unless the context clearly dictates otherwise. Further, in this regard, the terms specify the presence of stated features, but do not preclude the presence of additional or other features.

However, the compositions and methods disclosed herein may lack any elements not specifically disclosed herein. Thus, a disclosure of an embodiment using the term "comprising" is (i) a disclosure of an embodiment having the specified components or steps and additional components or steps, (ii) a disclosure of an embodiment consisting essentially of the specified components or steps, and (iii) a disclosure of an embodiment consisting of the specified components or steps. Any embodiment disclosed herein may be combined with any other embodiment disclosed herein.

The term "and/or" as used in the context of "X and/or Y" should be interpreted as "X" or "Y" or "X and Y". Similarly, "at least one of X or Y" should be interpreted as "X" or "Y" or "X and Y.

As used herein, the terms "example" and "such as," particularly when followed by a listing of terms, are merely exemplary and illustrative, and should not be deemed exclusive or comprehensive.

The "subject" or "individual" is a mammal, preferably a pig or a boar. As used herein, an "effective amount" is an amount that prevents an infection, treats a disease or medical condition, or more generally, reduces symptoms, manages disease progression, or attenuates a viral infection for a period of time in an individual.

The term "pig (pig)" refers to a domestic, wild or wild pig.

The term "pig (swine)" refers to a domestic, wild or wild pig.

The term "bird" refers to a wild or domesticated laying bird such as a chicken, duck, swan, goose, turkey, peacock, guinea fowl, ostrich, pigeon, quail, pheasant or dove.

The term "non-susceptible species" or "non-susceptible host" refers to a species that is not susceptible to infection by ASFV or ASF in general.

The term "immunoglobulin" or "antibody" refers to a glycoprotein molecule produced by leukocytes and lymphocytes and is involved in the body's immune system and immune response by specifically recognizing and binding to specific antigens and aiding in their neutralization.

The term "antigen" or "immunogen" or "hapten" is a substance or structure or small molecule that is or is considered foreign to the body and evokes an immune response, either alone or upon forming a complex with a larger molecule. The terms "antigen", "immunogen" or "hapten" are used interchangeably in this disclosure.

The term "passive immunity" refers to immunity resulting from the introduction of an antibody from another person, animal, species, or other external source into a subject.

The term "active immunity" refers to immunity resulting from the natural and/or artificial introduction of an antigen into a subject.

The term "adjuvant" or "immunological adjuvant" refers to a substance that can be added to a vaccine to stimulate a response of the immune system of a subject.

The term "immunosuppressive protein factor" and/or "host hyper-reactive immune factor" refers to factors that may include, but are not limited to, cytokines (e.g., cytokines of the TNF family), pro-inflammatory cytokines (e.g., IL-17F and/or interferons), and/or down-regulated anti-inflammatory cytokines (e.g., IL-10). The terms "immunosuppressive protein factor" and/or "host hyper-reactive immune factor" are used interchangeably herein and generally refer to a factor that evades an innate and/or adaptive immune response.

The term "treatment" includes both prophylactic or preventative treatment (i.e., arresting and/or slowing the progression of the targeted pathological condition, infection, disorder or disease) and curative, therapeutic or disease-modifying treatment, including any treatment that cures, slows, reduces the symptoms of, and/or arrests the progression of the diagnosed pathological condition, infection, disorder or disease; and treatment of subjects at risk of or suspected of having a disease or infection as well as subjects who are diseased or have been diagnosed as having a pathological condition, infection, disorder or disease. The term "treatment" does not necessarily imply that the subject is treated until complete recovery. The term "treating" also refers to maintaining and/or promoting health in an individual who does not have a pathological condition, infection, disorder, or disease, but may be predisposed to developing the pathological condition, infection, disorder, or disease. The term "treating" is also intended to include enhancing or otherwise potentiating one or more primary prophylactic or therapeutic measures. By way of non-limiting example, treatment can be by a doctor, healthcare professional, veterinarian, veterinary professional, animal care professional, or other person.

As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined quantity of a composition disclosed herein, in association with a pharmaceutically acceptable diluent, carrier or vehicle, in an amount sufficient to produce the desired effect. The specifications for the unit dosage form depend on the particular compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

The term "sterile" should be understood as not containing any bacteria or other living microorganisms.

As used herein, the term "pharmaceutically acceptable" refers to a material that does not substantially cause an adverse allergic or immune reaction when administered to a subject.

All percentages expressed herein are by weight of the total weight of the composition, unless otherwise indicated. When referring to pH herein, the value corresponds to the pH measured using standard equipment at about 25 ℃. "ambient temperature" or "room temperature" is between about 15 ℃ to about 25 ℃, and ambient pressure is about 100 kPa.

As used herein, the term "mM" refers to the molar concentration unit of an aqueous solution, which is mmol/L. For example, 1.0mM equals 1.0 mmol/L.

When used to refer to a particular component, the terms "substantially free", "essentially free", or "substantially free" mean that no component present comprises no more than about 3.0 wt%, such as no more than about 2.0 wt%, no more than about 1.0 wt%, preferably no more than about 0.5 wt%, or more preferably no more than about 0.1 wt%.

The terms "food", "foodstuff" and "food composition" mean a product or composition intended for ingestion by an animal, including a human, and which provides at least one nutrient to the animal. Preferred embodiments of the food product comprise at least one of a protein, a carbohydrate, a lipid, a vitamin or a mineral.

In the present disclosure, the terms "immunization" or "vaccination" are used interchangeably.

Detailed description of the preferred embodiments

ASFV vaccine

The present disclosure generally relates to an ASFV vaccine comprising intact live ASFV particles in combination with naturally expressed ASF viral components, and optionally diluted in a sterile buffer, e.g., to about 10% in sterile saline solution. The ASFV vaccine may be used for active immunization or vaccination of a non-susceptible species host for the production of ASFV-specific immunoglobulins. The non-susceptible species host may be a non-porcine mammalian host, such as an avian, equine, bovine, donkey, goat or rabbit.

Another aspect of the present disclosure relates generally to a method of producing an ASFV vaccine. In a preferred embodiment, the ASFV antigen is obtained from an ASF-infected pig or boar. In one embodiment, blood may be drawn from the ASF-infected pig or boar and collected in a blood collection tube with an anticoagulant. The blood collection tube can be centrifuged, for example, at about 1,500x g for about 15 minutes at about 4 ℃ to obtain a buffy coat. Alternatively, plasma-containing Peripheral Blood Mononuclear Cells (PBMCs) can be separated from blood by standard gradient centrifugation on Ficoll or other methods known to those skilled in the art. In addition, any Red Blood Cell (RBC) may be treated with a composition containing about 0.83% NH₄A solution of Cl or by any other method known to those skilled in the art.

The collected and/or isolated PBMCs may be disrupted and/or lysed by one or more freeze-thaw cycles, for example, by being placed in a dry ice ethanol (about-72 ℃) bath for a first predetermined period of time and then placed at room temperature for a second predetermined period of time. This process may be repeated one or more times. The disrupted PBMCs may be centrifuged in a second centrifugation step, for example at about 800x g for about 15 minutes at about 4 ℃. The supernatant, which preferably contains intact virions, viral components, immunosuppressive protein factors and host hyper-reactive immune factors, can be collected and diluted one or more times, e.g. 10 times, with a buffer of predetermined pH, e.g. sterile saline buffer. The resulting ASFV vaccine may be stored in one or more portions at a temperature below room temperature, e.g., in about 1ml aliquots at or below about-20 ℃. In an exemplary embodiment, the protein content and/or viral titer of the supernatant may be assessed prior to freezing and storage.

In another embodiment, the ASFV vaccine may be obtained from an ASFV-infected lymphoid organ, such as the spleen. The spleen may be harvested from an ASFV-infected pig or boar and dissected into multiple tissue sections. Preferably, the dissection is performed immediately after harvesting. The tissue slices can be added to buffer and homogenized on ice. The homogenized tissue mixture may be centrifuged to produce a single cell suspension, for example at about 800x g for about 15 minutes at a predetermined time and a predetermined temperature, for example at about 4 ℃. The single cell suspension may contain RBCs and splenic mononuclear cells (SMNC). The RBC may be used containing about 0.83% NH₄A solution of Cl or by any other method known to those skilled in the art. SMNC can be collected and cleaved by any method known to those skilled in the art. Cell debris can be removed by centrifugation, and the supernatant can be collected.

The supernatant preferably contains intact virions, viral components, immunosuppressive protein factors, and SMNC can be collected by Ficoll gradient centrifugation. The supernatant and SMNC may be collected and subjected to one or more freeze-thaw cycles, wherein the mixture may be lowered to a low temperature, for example, placed in a dry ice ethanol bath (about-70 ℃) for a first predetermined period of time and then placed at room temperature for a second predetermined period of time. The mixture of supernatant and disrupted SMNC may be centrifuged at about 800x g for about 15 minutes at about 4 ℃. The supernatant may be collected and diluted one or more times, e.g., 10 times, with a buffer of a predetermined pH, e.g., a sterile saline buffer. The resulting ASFV vaccine may be stored in one or more portions at a temperature below room temperature, e.g., in aliquots of about 1ml at or below about-20℃, preferably about-70℃. In another exemplary embodiment, protein content and/or viral titer in the supernatant may be assessed prior to freezing and storage.

In another embodiment, fresh Primary Alveolar Macrophages (PAM) are collected from healthy pigs and plated in cell culture flasks, cultured overnight using complete medium containing Fetal Bovine Serum (FBS). After about 24 hours, the cell monolayer may be washed, and the culture may be supplemented with medium containing serum infected with the ASFV stock. The ASF-infected PAM may be cultured until at least about 75% of the cytopathic effect is observed in the culture, for example, after about 5 to about 7 days post-ASFV infection. The PAM and culture supernatant may be harvested, collected and subjected to one or more freeze-thaw cycles, wherein the PAM mixture may be lowered to a low temperature, for example placed in a dry ice ethanol bath (about-70 ℃) for a first predetermined period of time and then placed at room temperature for a second predetermined period of time. The mixture of supernatant and fragmented PAM can be centrifuged at about 800x g for about 15 minutes at about 4 ℃. The supernatant may be collected and diluted one or more times, e.g., 10 times, with a buffer of a predetermined pH, e.g., sterile saline buffer. The resulting ASFV vaccine may be stored in one or more portions at temperatures below room temperature, for example in aliquots of about 1ml at or below about-20 ℃, preferably about-70 ℃. In another exemplary embodiment, protein content and/or viral titer in the supernatant may be assessed prior to freezing and storage.

In a preferred embodiment, the ASFV vaccine composition comprises a mixture of proteins, viral particles, and viral components from one or more of SMNC, PBMC, and/or PAM.

It is understood and contemplated herein that the ASFV vaccine composition contains a broad range of ASFV antigens (i.e., complete ASFV proteins). It is to be understood that the proteins or antigens that may be included in the ASFV vaccine composition may include complete, intact ASFV proteins, and/or may also include portions or fragments of the disclosed ASFV proteins.

It will also be appreciated that a particular genotype or serotype of ASFV may be selected for use in producing the ASFV vaccine composition by first testing the infected swine. Additionally or alternatively, the ASFV treatment methods disclosed herein may provide cross-protection against closely related virus strains, ASFV genotypes, and/or ASFV serotypes.

Also disclosed herein are methods for inactivating an ASFV vaccine composition prior to use. In one exemplary embodiment, the ASFV vaccine composition may be irradiated using a gamma irradiator at a dose of about 30 kGy. ASFV DNA is damaged at a dose of about 30kGy, while viral morphology and viral protein integrity are generally preserved.

Additionally or alternatively, non-porcine mammalian hosts, such as avian, equine, bovine, donkey, goat or rabbit may be vaccinated or immunized with a non-irradiated ASFV vaccine, for example for the production of ASFV-specific immunoglobulins.

ASFV-specific immunoglobulin

Another aspect of the disclosure relates generally to methods of immunizing or vaccinating a host of a non-susceptible species to produce ASFV-specific immunoglobulins. An aliquot (e.g., about 1mL) of an ASFV vaccine comprising intact virions, viral components, immunosuppressive protein factors, and host hyper-reactive immune factors, e.g., about 10% ASFV vaccine in sterile saline buffer, can be thawed to a predetermined temperature, vortexed, and injected intramuscularly into a non-porcine mammalian host, e.g., avian, equine, bovine, donkey, goat, or rabbit. After the initial and optional re-immunization, a sample of the host venous blood can be collected by a variety of different methods known to those of ordinary skill in the art.

In a preferred embodiment, the anti-ASFV immunoglobulin is an IgY antibody produced by an immunized or vaccinated laying bird, such as a chicken. An aliquot (e.g., about 1mL) of an ASFV vaccine comprising intact virions, viral components, and immunosuppressive protein factors, e.g., about 10% ASFV vaccine in sterile saline buffer, can be thawed to room temperature, vortexed, and injected intramuscularly into laying birds. Preferably, the ASFV vaccine is divided into equal fractions (about 100 μ g protein content/fraction), one fraction is injected into the left chest of a hen and the second fraction is injected into the right chest of the hen, optionally in approximately equal volume amounts, e.g., about 500ml in the right chest and about 500ml in the left chest. Additionally or alternatively, the ASFV vaccine may be emulsified with Complete Freund's Adjuvant (CFA) in a ratio of about 1:1 prior to injection into the hens. In another embodiment, the subsequent immunization may comprise an ASFV vaccine composition comprising an about 1:1 solution of an ASFV vaccine and Incomplete Freund's Adjuvant (IFA).

After the initial immunization, the hens may be re-immunized, for example, about 7 days after the initial immunization and/or about 14 days after the initial immunization and/or about 28 days after the initial immunization. After the initial immunization and any re-immunization (e.g., about 27 days after the initial immunization), the eggs laid by the immunized hens may be collected for purification of the IgY antibodies on one or more days. Alternatively, eggs may be collected continuously during immunization. The IgY antibody may be obtained from collected egg yolk through a water-soluble fraction. One or more egg yolks may be combined and diluted about 10-fold with cooled 3mM HCl to give a suspension with a final pH of about 5 (adjusted with about 10% acetic acid). The suspension may be frozen overnight, for example, at about-20 ℃. After thawing to a predetermined temperature, the mixture may be centrifuged at about 13,000x g for about 15 minutes at about 4 ℃, and the supernatant containing IgY immunoglobulin may be collected. The IgY immunoglobulins can be further purified by various precipitation methods known to those of ordinary skill in the art, for example, using ammonium sulfate or biocompatible sodium chloride (see Hodek, P. et al, "optimization scheme for Purification of Chicken antibodies (IgY) to provide an Electrophoretically homogeneous preparation" (Optimized Protocol of Chicken Antibody (IgY) Purification schemes, 8int. J. electrochem. Sci.113,113-124 (2013)). Optionally, the IgY immunoglobulin may be obtained from an egg white fraction.

In certain embodiments, the ASFV-specific immunoglobulin composition comprises egg yolk or any IgY antibody-containing fraction thereof. Egg yolk is a preferred part of eggs, since egg yolk generally contains a much higher concentration of IgY than egg white. However, egg white may also contain a sufficient concentration of IgY for certain applications.

In certain embodiments of the antibody composition, the IgY is concentrated, isolated or purified from the constituent components of an egg. This can be achieved by a variety of different methods, such as those known to those of ordinary skill in the art. If desired, the titer of the IgY antibody can be determined by an immunoassay, for example ELISA.

In certain embodiments of the antibody composition, the composition is prepared by a method comprising obtaining eggs laid by birds that have previously been actively vaccinated against ASFV, and separating an antibody fraction from the egg yolk. The poultry is preferably poultry. The poultry may be chicken, duck, swan, goose, turkey, peacock, guinea fowl, ostrich, pigeon, quail, pheasant, dove or other poultry. The poultry are preferably chickens. The poultry is more preferably domestic chickens raised primarily for the production of eggs or meat.

In certain embodiments of the antibody composition, the antibody composition is prepared by a method comprising actively vaccinating a hen against ASFV, collecting eggs from the hen after the immunization period, and separating an antibody fraction from the egg yolk. Optionally, egg collection from the hens may be performed continuously after the immunization period.

Other methods for producing IgY with a specific target are known to those skilled in the art, although it is not known whether these methods have been used successfully before for the production of antibodies against ASFV. The antibodies disclosed in this section are suitable for use in any of the methods and compositions described in this disclosure.

IgY antibodies from avian eggs have been found to be a cost-effective and abundant source of viral adhesion inhibitors (i.e., immunoglobulins) in general. Such antibodies bind to the surface of the virus (e.g., ASFV) bearing the antigen, thereby preventing the initial contact phase between the virus and the potential host cell. As explained elsewhere in this disclosure, preventing the initial adhesion stage between the virus and the host cell has a number of applications, including the treatment of viral diseases and the prevention of viral diseases.

In certain embodiments of the inhibitor, the inhibitor comprises a component of an avian egg, wherein the avian egg comprises ASFV-specific IgY in an amount effective to inhibit adhesion. The egg constituents may be any constituent of an antibody composition described as suitable in this disclosure.

Methods of preventing viral adhesion to cells are provided. The first step in the infection of cells by viruses is the contact and adhesion between the virus and the cell. Although this step is critical for the establishment of infection, there are few ways to prevent infection at this early stage. More commonly, techniques such as active vaccination are used to combat viral infections, which results in the body producing antibodies that neutralize the virus. Viral diseases are most often only treatable symptomatically if active vaccination is not feasible. The methods described herein provide an effective means of preventing this early step in the infection process without having to be administered prior to exposure of the subject to the pathogen, as required for active vaccination.

Antibodies can act by binding to the virus and interfering with the ability of the virus to bind to its target membrane receptor to prevent adhesion between the virus and the cell. Avian antibodies (e.g., IgY) have significant advantages in this application over mammalian antibodies, particularly when the subject is a mammal. As stated above, advantages of IgY antibodies include that IgY antibodies are more specific, more stable, and cause fewer forms of unwanted immune responses than mammalian antibodies. IgY antibodies can also be obtained easily and inexpensively from eggs.

In one embodiment of the method, the method comprises administering to the subject an amount of a viral adhesion inhibitor effective to inhibit adhesion. The viral adhesion inhibitor may be any embodiment of an ASFV-specific immunoglobulin composition disclosed herein. In certain embodiments of the methods, the viral adhesion inhibitor comprises a component of an avian egg comprising ASFV-specific IgY in an amount effective to inhibit adhesion. The constituent may be any constituent disclosed herein as a suitable antibody composition.

In certain embodiments of the methods, the ASFV-specific immunoglobulin composition is a pharmaceutical product comprising avian egg contents comprising an effective amount of ASFV-specific IgY. The pharmaceutical product may comprise additional components as discussed herein. The pharmaceutical product may be administered by any method known in the art or described herein.

Method of treatment

Yet another aspect of the present disclosure relates generally to ASFV vaccines and pharmaceutically acceptable compositions of ASFV-specific immunoglobulins that can be administered to ASFV-infected or exposed pigs or boars. Additionally or alternatively, the ASFV vaccine may be administered to a non-porcine mammalian host as described previously.

In one embodiment, the ASFV vaccine and/or the ASFV-specific immunoglobulin is in the form of a composition, such as, but not limited to, a pharmaceutical composition. The disclosed compositions may comprise one or more of such compositions disclosed above in combination with a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation can be found in Remington pharmaceutical sciences and practices (Remington: The Science and Practice of Pharmacy) (20 th edition, ed. by Lippincott, Williams & Wilkins, Daniel Limmer). Such ASFV-specific immunoglobulin compositions contain a therapeutically effective amount of the antibody in order to form a pharmaceutically acceptable composition suitable for administration. The therapeutically effective amount of the antibody can be an amount effective to inhibit adhesion and/or an amount effective to produce passive immunity in a subject (i.e., a pig or boar). Additionally or alternatively, such ASFV vaccine compositions contain a therapeutically effective amount of an ASFV antigen (e.g., ASFV virions and/or viral components) in order to form a pharmaceutically acceptable composition suitable for administration. A therapeutically effective amount of an irradiated ASFV antigen may be an amount effective to produce protective immunity in a subject (i.e., a pig or a boar).

The pharmaceutical compositions of the present disclosure may be used in the treatment and prevention methods of the present disclosure. Such compositions are administered to pigs or wild boars in an amount sufficient to deliver a therapeutically effective amount of an ASFV-specific immunoglobulin or an ASFV vaccine so as to be effective in the treatment and prevention methods disclosed herein. The therapeutically effective amount may vary depending on various factors, such as, but not limited to, the condition, weight, sex, and age of the subject. Other factors include the mode and site of administration. The pharmaceutical composition may be provided to a subject in any method known in the art. Exemplary routes of administration include, but are not limited to, intraperitoneal, intramuscular, subcutaneous, intravenous, topical, epidermal, oral, intraosseous, intranasal. Oral administration of the ASFV-specific immunoglobulin may be achieved by addition to the feed (solid or liquid) of the subject.

The compositions of the present disclosure may be administered to a subject only once or more than once. In addition, when the composition is administered to a subject more than once, various regimens may be used, such as, but not limited to, once per day, once per week, once per month, or once per year. The composition may also be administered to the subject more than once per day. A therapeutically effective amount and suitable dosing regimen of the ASFV-specific immunoglobulin composition and/or the ASFV vaccine composition may be identified by routine testing in order to obtain optimal activity while minimizing any potential side effects. The ASFV-specific immunoglobulin composition and the ASFV vaccine composition may be administered separately to separate subjects. Additionally or alternatively, the ASFV-specific immunoglobulin composition and the ASFV vaccine composition may be co-administered to an individual subject in need thereof in a variety of different treatment regimens. In addition, co-administration or sequential administration of other agents may be desirable.

The compositions of the present disclosure may be administered systemically, for example, by intraperitoneal, intravenous, or intramuscular administration.

The compositions of the present disclosure may also comprise agents that improve the solubility, half-life, absorption, etc., of the antibodies. In addition, the compositions of the present disclosure may also comprise agents that attenuate and/or reduce the toxicity of unwanted side effects of the antibodies. Examples of such agents are described in various textbooks such as, but not limited to, Remington's pharmaceutical sciences and practices (20 th edition, Lippincott, Williams & Wilkins, Daniel Limmer eds.).

The compositions of the present disclosure can be administered in a wide variety of dosage forms for administration. For example, the composition may be administered in the form of, for example, but not limited to, an injection, a lyophilized powder, or a granule.

In the present disclosure, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, vehicles, adjuvants, suspending agents, inert fillers, diluents, excipients, wetting agents, binders, buffers, disintegrants and carriers. Typically, the pharmaceutically acceptable carrier is chemically inert to the active antibody and has no deleterious side effects or toxicity under the conditions of use. The pharmaceutically acceptable carrier may include a polymer and a polymer matrix. The nature of the pharmaceutically acceptable carrier can vary depending upon the particular dosage form employed and other characteristics of the composition.

For example, for oral administration of an ASFV-specific immunoglobulin in solid form such as, but not limited to, a powder or granules, the antibody may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as, but not limited to, an inert filler, a suitable binder, a lubricant, a disintegrant, and an adjuvant. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

Formulations suitable for parenteral administration include aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the subject, and aqueous suspensions which may contain suspending agents, solubilizers, thickeners, stabilizers and preservatives. The compositions can be administered in a physiologically acceptable diluent, such as a sterile liquid or liquid mixture, including water, saline, aqueous dextrose and related sugar solutions.

Oils that may be used in parenteral formulations include petroleum, animal, vegetable or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral oil. Suitable fatty acids for use in parenteral formulations include polyethylene sorbitan fatty acid esters such as sorbitan monooleate, and the high molecular weight adducts of ethylene oxide with a hydrophobic base formed by condensation of propylene oxide with propylene glycol, oleic acid, stearic acid and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium and triethanolamine salts, and suitable detergents include (a) cationic detergents such as dimethyl dialkyl ammonium halides and alkyl pyridinium halides, (b) anionic detergents such as alkyl, aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates and sulfosuccinates, (c) nonionic detergents such as fatty amine oxides, fatty acid alkanolamides and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as alkyl beta-aminopropionates and 2-alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.

Suitable preservatives and buffers may be employed in such formulations. To minimize or eliminate irritation at the injection site, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of from about 12 to about 17.

The compositions of the present disclosure may also be coupled with soluble polymers as targetable drug carriers. Such polymers may include, but are not limited to, polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide phenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxide polylysine substituted with palmitoyl residues. Furthermore, the antibodies of the invention may be conjugated to a class of biodegradable polymers useful for achieving controlled release of a drug, such as polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphiphilic block copolymers of hydrogels.

The pharmaceutical compositions of the present disclosure may be modified to prevent adverse reactions in a subject. Such potential adverse reactions include host recognition, allergies, local inflammation and other forms of allergic reactions.

Adverse reactions to immunoglobulin compositions are more common in heterogeneous antibody therapy than in homogeneous antibody therapy, although the advantages of IgY antibodies in this regard have been explained. In certain embodiments of the pharmaceutical composition, the antibody is modified to alter the Fc region of the molecule. In other embodiments, the antibody is treated to prevent binding between the Fc region of the antibody and the Fc receptor of the cell.

The pharmaceutical formulations of the present disclosure may be stored in any pharmaceutically acceptable form, including aqueous solutions, frozen aqueous solutions, lyophilized powders, or any other form described herein.

Non-limiting examples of the pharmaceutically acceptable ASFV-specific immunoglobulin composition and/or the ASFV vaccine composition preferably further comprise an anti-inflammatory agent.

Non-limiting examples of such pharmaceutically acceptable ASFV-specific immunoglobulin compositions preferably further comprise an antigen-binding fragment of an antibody, such as a Fab or Fab2 fragment, which may be substituted for the antibody. For example, the antigen-binding fragment may be any fragment comprising the antigen-binding region of the original IgY. In certain embodiments of the compositions and methods, the IgY antibodies may be replaced with modified versions of IgY antibodies, so long as the antigen-binding regions of the IgY antibodies retain their ability to recognize ASFVs.

Non-limiting examples of such pharmaceutically acceptable ASFV vaccine compositions preferably further comprise a lyophilized powder composition, e.g. for long term storage and/or transport. The lyophilized vaccine can be reconstituted to about the original volume in a solution, such as saline, prior to use for immunization or vaccination.

One aspect of the present disclosure is a preferred method of treating an ASFV infected or exposed pig or boar, the method comprising generating passive immunity in an ASFV infected or exposed pig or boar (fig. 1). The ASFV-specific immunoglobulin composition may comprise additional components as pharmaceutical components discussed elsewhere in the disclosure. The ASFV-specific immunoglobulin composition may be administered to a pig or boar infected or exposed with ASFV in need thereof by intraperitoneal or intramuscular injection at a dose of about 0.5 to about 1.0mg per kg body weight twice a week for one or more weeks.

One aspect of the present disclosure is a method of treating an ASFV-infected or exposed pig or boar by administering a composition comprising ASFV-specific immunoglobulins. The ASFV-specific immunoglobulin may be added to feed at a dose of about 1.0mg per kg body weight for about once a day for about 5 to about 7 consecutive days for oral administration to a pig or boar in need thereof infected with or exposed to ASFV.

Such oral administration methods for ASFV-specific immunoglobulins further comprise oral administration of uncooked egg yolk or egg yolk fractions, alone or in combination with egg white. Oral administration of raw egg yolk or egg yolk fractions may be carried out, for example, by feeding the egg yolk fraction. The egg yolk fraction may be administered in combination with other ingredients to make it more palatable or nutritious. Thus, the egg yolk fraction can be ingested by a subject as a food; alternatively, the egg yolk fraction may be ingested as part of a pharmaceutical composition. It is preferably an uncooked or very lightly cooked egg yolk fraction, since cooking inactivates the antibodies.

Non-limiting examples of methods of treatment include increasing the dosage of ASFV-specific immunoglobulin by parenteral or oral administration, and administering in combination or alternatively with an increased frequency of administration. One aspect of the present disclosure is a preferred method of treating pregnant sows, gilts of sows and/or piglets of ASFV infected or exposed pigs or boars. ASFV-specific immunoglobulin is administered to the pregnant sow by methods discussed elsewhere in the disclosure. The piglets and/or the foetal pigs receive the ASFV vaccine directly or indirectly during pregnancy and/or lactation.

Another aspect of the present disclosure is a preferred prophylactic treatment method for pigs or boars susceptible to ASF infection (fig. 1). The irradiated ASFV vaccine composition may preferably be administered to a subject (i.e., a pig or boar), including but not limited to a subject that has been exposed to ASFV, a subject susceptible to ASF infection, and/or a subject infected with ASFV. The ASFV vaccine composition may comprise additional components, such as pharmaceutical components discussed elsewhere in the disclosure. The ASFV vaccine composition may be administered to a subject by intraperitoneal, subcutaneous, or intramuscular injection at a dose of about 0.05 mg/dose to about 1.0 mg/dose for piglets (i.e., non-older pigs) weighing about 20 kg. Preferably, the ASFV vaccine composition is administered at a dose of about 100 μ g. Additionally or alternatively, the ASFV vaccine composition may be administered to an individual subject more than once. For example, 14 days after the first or primary immunization, a booster immunization can be performed. In addition, a third immunization may be performed 21 days after the first or primary immunization.

Disclosed herein is the exemplary embodiment illustrated in fig. 2A, in particular a method of treating a subject comprising administering a first dose of an ASFV vaccine composition to CFA in a ratio of 1: 1. A second dose of the ASFV vaccine composition may then be administered to the subject with IFA at a ratio of 1:1 after about two weeks. About four weeks after the first or primary immunization, the swine or wild boar can be subjected to an ASFV challenge to determine whether the immunized subject can survive a lethal ASF infection. The dose of the irradiated ASFV vaccine may be equivalent to about 10⁴HAD₅₀(50% erythrocyte adsorbing amount) to about 10⁵HAD₅₀Is in the range of live viruses.

Examples

The following non-limiting examples support the concept of using pharmaceutically acceptable ASF vaccine compositions to generate antibodies that can be used to treat or prevent infection of infected pigs and/or wild boars.

Example 1

Three groups of chickens (n-3 per group) were immunized with the ASFV vaccine on days 1, 14 and 28. Group 1 received saline (no vaccine) as a control, group 2 received ASFV vaccine formulation 1 containing intact virions and immunosuppressive protein factors, group 3 received ASFV vaccine formulation 2 comprising intact virions, viral components and immunosuppressive protein factors. After the second and third immunizations, blood samples were taken and ASFV-specific antibody titers were evaluated using ELISA plates (SEQ ID NO: 2) coated with recombinant ASFV major capsid protein p 72. The results of example 1 demonstrate that the ASFV vaccine is immunogenic and achieves immunity. Furthermore, example 1 demonstrates that the ASFV vaccine induces a pool of antibodies with full specificity for an ASF viral component such as ASFV major capsid protein p72(SEQ ID NO: 2) after 14 days (FIG. 4A) and after 28 days (FIG. 4B).

Example 2

An ASFV vaccine composition is prepared from a homogenate of ASFV-infected spleen from an ASFV-infected pig and ASFV-infected PBMC-containing buffy coat. The PBMC mixture was frozen in a dry ice ethanol bath and thawed to room temperature. The freeze-thaw procedure was repeated twice. Evaluating the ASFV vaccine composition for active ASFV using qPCR. The results confirmed that the ASFV vaccine composition did not contain ASFV DNA. Three groups of laying hens (n-3/group) were dosed with either a control or one of two different ASFV vaccine formulations. Group 1 received saline as a control (no vaccine), group 2 received ASFV vaccine preparation 1 (prepared from SMNC), and group 3 received ASFV vaccine preparation 2 (prepared from SMNC and PBMC). The hens were actively immunized on days 1, 14 and 3 by administration of ASFV vaccine (by intramuscular injection) or control. Eggs were collected daily after the third immunization. Immunoglobulin was extracted from egg yolk using a simple water dilution method. After the second immunization, blood samples were taken from the chickens at day 14 and no virus shedding was confirmed by qPCR.

Eggs collected from hens receiving formulation 2 were used to produce ASFV-specific immunoglobulin. Eggs from hens that received saline or formulation 1 were not used to produce ASFV-specific immunoglobulins. The ASFV-specific immunoglobulin composition was analyzed using qPCR, and no active ASFV was determined (i.e., the ASFV-specific immunoglobulin composition did not contain ASFV DNA) (fig. 3). The specificity of the IgY antibodies of the ASFV-specific immunoglobulin composition was also evaluated. ELISA was used to detect antibodies specific for recombinant ASFV capsid protein p72(SEQ ID NO: 2) and a level of 6log of ASFV p72 specific IgY was detected₂(FIG. 5).

The 3-component year pigs were designated A, B and C. Group a consisted of 6 adult pigs (approximately 20kg each) and received 100mg of ASFV-specific immunoglobulin composition one day prior to exposure to ASFV. Group B consisted of 3 adult pigs, which received 100mg of ASFV-specific immunoglobulin composition one day after exposure to ASFV. Finally, group C consisted of 3 adult pigs exposed to ASFV and did not receive the ASFV-specific immunoglobulin composition.

Clinical observations revealed that all 3 pigs in group C showed initial ASF symptoms including low activity (i.e. lethargy) and showed reduced food intake at 4 days post-ASFV exposure. All 3 pigs in group C stopped feeding 6 days after ASFV exposure. Pigs in group a continued to appear normal and healthy 6 days after ASFV exposure, while animals in group B showed reduced appetite (i.e. reduced food intake) and dark yellow urine, but no other signs or symptoms of ASF and no signs of disease were observed. The results indicate that administration of an ASFV-specific immunoglobulin composition either before or after ASFV exposure successfully produced passive immunity.

Sequence listing

<110>

IGY immuno-technology and Life Sciences Inc. (IGY Immune Technologies and Life Sciences Inc.)

<120> vaccine and immunoglobulin targeting african swine fever virus, methods of making and methods of use thereof

<130> 1401870.00006

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<170> PatentIn version 3.5

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The claims (modification of treaty clause 19)

1. A method of treating an African Swine Fever (ASF) virus (ASFV) infection in an infected pig or wild boar, the method comprising administering to the infected pig or wild boar an effective amount of a composition comprising IgY immunoglobulins specific for (a) a naturally expressed ASF viral component, (b) intact live ASF viral particles, and/or (c) an immunosuppressive protein factor.

2. The method of claim 1, wherein said composition is administered in an amount to provide a dose of said IgY immunoglobulin specific for (a) a naturally expressed ASF viral component, (b) intact live ASF viral particles, and/or (c) an immunosuppressive protein factor of from about 0.5mg to about 1.0mg per kg body weight of said infected pig or boar.

3. The method of claim 1, wherein the period of time for which the composition comprising IgY immunoglobulin specific for (a) a naturally expressed ASF viral component, (b) intact live ASF viral particles, and/or (c) an immunosuppressive protein factor is administered comprises at least once a week or for 7 consecutive days.

4. The method of claim 1, wherein the composition comprising IgY immunoglobulins specific for (a) naturally expressed ASF viral components, (b) intact live ASF viral particles, and/or (c) immunosuppressive protein factors is administered parenterally by intramuscular or intraperitoneal injection.

5. The method of claim 1, wherein the composition comprising IgY immunoglobulins specific for (a) naturally expressed ASF viral components, (b) intact live ASF viral particles, and/or (c) immunosuppressive protein factors is a food product for oral administration.

6. A method of preventing, reducing the incidence and/or reducing the severity of ASF virus infection in a pig or boar at risk comprising administering to the pig or boar an effective amount of a composition comprising IgY immunoglobulins specific for (a) a naturally expressed ASF viral component, (b) intact live ASF viral particles and/or (c) an immunosuppressive protein factor.

7. The method of claim 6, wherein said composition is administered in an amount that provides a dose of said IgY immunoglobulins specific for (a) naturally expressed ASF viral components, (b) intact live ASF viral particles and/or (c) immunosuppressive protein factors of from about 0.5mg to about 1.0mg per kg body weight of said at-risk swine or wild boar.

8. The method of claim 6, wherein the period of time for which the composition comprising IgY immunoglobulins specific for (a) naturally expressed ASF viral components, (b) whole live ASF virions, and/or (c) immunosuppressive protein factors is administered comprises at least once weekly or for 7 consecutive days.

9. The method of claim 6, wherein the composition comprising IgY immunoglobulins specific for (a) naturally expressed ASF viral components, (b) whole live ASF viral particles and/or (c) immunosuppressive protein factors is administered parenterally.

10. The method of claim 6, wherein the composition comprising IgY immunoglobulins specific for (a) naturally expressed ASF viral components, (b) whole live ASF virions, and/or (c) immunosuppressive protein factors is a food product for oral administration.

11. A method of producing ASFV-specific immunoglobulins wherein an ASFV vaccine comprising intact live ASF virions, naturally expressed ASF viral components and/or immunosuppressive protein factors is administered to a non-porcine species host for production of ASFV-specific immunoglobulins.

12. The method of claim 11, wherein the host is an laying bird.

13. A unit dosage form comprising a therapeutically or prophylactically effective amount of a composition comprising IgY immunoglobulins that are specific for (a) a naturally expressed ASF viral component, (b) whole live ASF virions, and/or (c) immunosuppressive protein factors.

14. The unit dosage form of claim 13, wherein the composition is a food product formulated for oral administration.

15. A method of preventing, reducing the incidence of and/or lessening the severity of ASF virus infection in a pig or wild boar at risk comprising administering to the pig or wild boar an effective amount of an ASFV vaccine composition comprising (a) a naturally expressed ASF viral component, (b) intact live ASF viral particles and/or (c) an immunosuppressive protein factor.

16. The method of claim 15, wherein the naturally expressed ASF viral component and intact live ASF virions are inactivated by gamma-ray irradiation.

17. The method of claim 15, wherein the ASFV vaccine composition is administered parenterally by intramuscular or intraperitoneal injection.

18. The method according to claim 15, wherein the ASFV vaccine composition is administered in an amount that provides a dose of the naturally expressed ASF viral component of about 0.05mg to about 1.0mg per pig or wild boar.

19. A unit dosage form comprising an effective amount of an ASFV vaccine composition comprising a naturally expressed ASF viral component, intact live ASF virions, and/or immunosuppressive protein factors.

20. The unit dosage form according to claim 19, wherein the naturally expressed ASF viral component and intact live ASF virions are derived from ASF-infected Spleen Monocytes (SMNC), ASF-infected Peripheral Blood Mononuclear Cells (PBMC), and/or ASF-infected Primary Alveolar Macrophages (PAM).

21. The unit dosage form according to claim 19, wherein the naturally expressed ASF viral component is inactivated by gamma-ray irradiation.

22. An ASFV vaccine comprising IgY immunoglobulins specific for (a) a naturally expressed ASF viral component, (b) whole live ASF virions and/or (c) an immunosuppressive protein factor for use in the treatment and/or prevention of ASF infection in a pig or a wild boar at risk.

23. A composition comprising IgY immunoglobulins specific for (a) a naturally expressed ASF viral component, (b) whole live ASF virions and/or (c) an immunosuppressive protein factor for use in the treatment and/or prevention of ASF infection in a pig or a wild boar at risk.

24. The method of claim 1, wherein said naturally expressed ASF viral component, intact live ASF virions and/or immunosuppressive protein factors are derived from cells, tissues and/or organs of an ASFV-infected pig or boar.

25. The method of claim 1, wherein the naturally expressed ASF viral component and intact live ASF viral particles are inactivated by gamma-ray irradiation.

26. The method according to claim 6, wherein the naturally expressed ASF viral component, intact live ASF virions, and/or immunosuppressive protein factors are derived from cells, tissues, and/or organs of an ASFV-infected pig or boar.

27. The method of claim 11, wherein the naturally expressed ASF viral component, intact live ASF virions, and/or immunosuppressive protein factors are derived from cells, tissues, and/or organs of an ASFV-infected pig or boar.

28. The method of claim 13, wherein the naturally expressed ASF viral component, intact live ASF virions, and/or immunosuppressive protein factors are derived from cells, tissues, and/or organs of an ASFV-infected pig or boar.

29. The method of claim 13, wherein the naturally expressed ASF viral component and intact live ASF viral particles are inactivated by gamma-ray irradiation.

30. The unit dosage form according to claim 19, wherein the naturally expressed ASF viral component, intact live ASF virions and/or immunosuppressive protein factors are derived from ASF infected spleen mononuclear cells (SMNC) and/or ASF infected Peripheral Blood Mononuclear Cells (PBMC).

Claims (23)

1. A method of treating an African Swine Fever (ASF) virus (ASFV) infection in an infected pig or wild boar, the method comprising administering to the infected pig or wild boar an effective amount of a composition comprising immunoglobulins specific for an ASF viral component.

2. The method of claim 1 wherein said composition is administered in an amount to provide a dose of said immunoglobulin specific for an ASF viral component of about 0.5mg to about 1.0mg per kg body weight of said infected pig or boar.

3. The method of claim 1, wherein the period of time for which the composition comprising an immunoglobulin specific for an ASF viral component is administered comprises at least once per week or for 7 consecutive days.

4. The method of claim 1, wherein the composition comprising immunoglobulins specific for ASF viral components is administered parenterally by intramuscular or intraperitoneal injection.

5. The method of claim 1, wherein the composition comprising immunoglobulins specific for ASF viral components is a food product for oral administration.

6. A method of preventing, reducing the incidence and/or reducing the severity of an ASF virus infection in a pig or boar at risk comprising administering to the pig or boar an effective amount of a composition comprising immunoglobulins specific for an ASF virus component.

7. The method of claim 6, wherein the amount of the composition administered provides a dose of the immunoglobulin specific for an ASF viral component of about 0.5mg to about 1.0mg per kg body weight of the at-risk pig or boar.

8. The method of claim 6, wherein the period of time for which the composition comprising immunoglobulins specific for ASF viral components is administered comprises at least once weekly or for 7 consecutive days.

9. The method of claim 6, wherein the composition comprising immunoglobulins specific for ASF viral components is administered parenterally.

10. The method of claim 6, wherein the composition comprising immunoglobulins specific for an ASF viral component is a food product for oral administration.

11. A method for producing an ASFV-specific immunoglobulin, wherein an ASFV vaccine comprising intact ASF virions, viral components, and/or immunosuppressive protein factors is administered to a non-porcine species host for the production of an ASFV-specific immunoglobulin.

12. The method of claim 11, wherein the host is an laying bird.

13. A unit dosage form comprising a therapeutically or prophylactically effective amount of a composition comprising an immunoglobulin specific for an ASF viral component.

14. The unit dosage form of claim 13, wherein the composition is a food product formulated for oral administration.

15. A method of preventing, reducing the incidence and/or lessening the severity of ASF viral infection in a pig or boar at risk comprising administering to the pig or boar an effective amount of an ASFV vaccine composition comprising an ASF viral component.

16. The method of claim 15, wherein the ASF viral component is inactive.

17. The method of claim 15, wherein the ASFV vaccine composition is administered parenterally by intramuscular or intraperitoneal injection.

18. The method according to claim 15, wherein the ASFV vaccine composition is administered in an amount that provides a dose of the ASF viral component from about 0.05mg to about 1.0mg per pig or wild boar.

19. A unit dosage form comprising an effective amount of an ASFV vaccine composition comprising an ASF viral component.

20. The unit dosage form according to claim 19, wherein the ASF viral component is derived from ASF-infected Spleen Monocytes (SMNC), ASF-infected Peripheral Blood Mononuclear Cells (PBMC) and/or ASF-infected Primary Alveolar Macrophages (PAM).

21. The unit dosage form according to claim 19, wherein the ASF viral component is inactivated.

22. An ASFV vaccine for use in the treatment and/or prevention of ASF infection in a pig or boar at risk.

23. An immunoglobulin specific for an ASF viral component for use in the treatment and/or prevention of an ASF infection in a pig or wild boar at risk.

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