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

Vaccines and immunoglobulins targeting African swine fever virus and methods of making and using the same

technical field

The present disclosure generally relates 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 broad-spectrum immunity to ASFV-infected or susceptible ASFV-infected pigs and wild boars.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of US Provisional Application 62/906,357, filed September 26, 2019, which is incorporated herein by reference in its entirety.

sequence listing

This application contains a Sequence Listing electronically filed in ASCII format and incorporated herein by reference in its entirety. Said ASCII copy created on September 24, 2020 is named Seq_Listings_for_1401870-00006.txt and is 8,548 bytes in size.

Background technique

ASF is a highly contagious bleeding disorder caused by ASFV (USDA Surveillance Program, p. 3). ASF affects porcine mammals, including domestic pigs, feral pigs, and Eurasian wild pigs (USDA Surveillance Program, p. 3). The virus, first identified in East Africa in the early 1900s, spread from native warthogs to domestic pig populations in most sub-Saharan African countries (Sánchez-Cordón et al., African Swine Fever: A Reemerging Virus Threatening the Global Pig Industry) Sexual diseases (African swinefever: A re-emerging viral disease threatening the global pig industry), 233Vet.J.41,41(2018)). African warthogs and bush pigs are natural reservoir hosts for ASFV, showing few clinical signs and persistent infection (Dixon et al., African swine fever virusevasion of host defences, 266Virus Res.25 , 25 (2019)). In contrast, infection of domestic, feral or feral pigs results in acute hemorrhagic fever with high mortality (Dixon et al., p. 25).

ASFV spread to Europe in the late 1950s and later to South America and the Caribbean (Sánchez-Cordón et al., p. 41). With no effective vaccine, methods for controlling the spread of the virus are limited to the isolation and slaughter of infected and exposed pigs (Netherton et al., Identification and Immunogenicity of African Swine Fever Virus Antigens). Antigens), 10 Front. Immun. 1, 1 (2019)). In the mid-1990s, ASF was successfully eradicated outside Africa, but by 2007 the virus again experienced a second transcontinental spread to Georgia and Eastern Europe (Sánchez-Cordón et al., p. 41). Recently, outbreaks of ASF have been reported in China, Vietnam, Mongolia, Cambodia and South Korea (FAO website; ASF situation update). The spread of ASF to China is of particular concern as China is the largest hog producer in the world (Netherton et al., p. 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). More than 20 genotypes have been recorded by various research groups, and at least 8 serotypes have been identified. Kolbasov et al., Comparative Analysis of African Swine Fever Virus Genotypes and Serogroups, 21Emerg . Infect. Dis. 312, 312 (2015)). Traditional inactivated vaccines have not been successful, and live attenuated vaccines have also failed to produce the desired efficacy (Sánchez-Cordón et al., p. 44). The challenges associated with developing a successful ASF vaccine are believed to be due to a lack of understanding of how the virus modulates the host's response to infection and the lack of identification of protective antigens (Sánchez-Cordón et al., p. 44).

SUMMARY OF THE INVENTION

The present inventors have developed a method for isolating live ASFV and viral components to prepare an ASFV vaccine comprising intact viral particles, individual viral structural proteins, and viral components involved in aggravating infection Substances include, but are not limited to, immunosuppressive factors and/or host immune factors. This 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 species other than pigs or wild boars, such as birds, cattle, rabbits, goats, donkeys or horses, to generate resistance to ASFV Broad-spectrum specific polyclonal immunoglobulin. In a preferred embodiment, egg-laying birds, such as chickens, are vaccinated with the ASFV vaccine, and antibodies or antibody fractions can then be extracted and purified from egg yolks. The produced egg-laying avian antibodies can be used to prevent virus adhesion, virus transmission, treatment of ASF, and prevention of ASF. Antibodies from avian or avian IgY isotypes are particularly useful in these applications.

The ASFV-specific immunoglobulins can be administered for acute treatment of ASFV-infected pigs or wild boars. The acute treatment may include parenteral and/or oral administration of the immunoglobulin, eg, by intraperitoneal or even intramuscular injection and/or in food compositions. Additionally or alternatively, the immunoglobulin may be administered as prophylactic treatment by the same route of administration. In one embodiment, the ASFV-specific immunoglobulin can be in the form of a liquid or lyophilized powder, which can be injected intraperitoneally or intramuscularly after reconstitution, preferably at about 0.5 to about 1.0 mg per kg body weight. Doses are injected twice weekly for one or more weeks, eg, into one or more ASFV-infected or exposed pigs or wild boars. Alternatively, the ASFV-specific immunoglobulin may be administered orally at an oral dose of about 1.0 mg per kg body weight, eg, added to feed, once a day for about 5 to about 7 consecutive days, eg, for administration to one or more ASFV infections or Exposed pigs or wild boars.

In one embodiment, disclosed herein is a method of treating 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 compound comprising a group specific for ASF virus The composition of divided immunoglobulins.

Also disclosed herein are the methods of treating ASFV infection in infected pigs or wild boars, wherein the composition is administered in an amount that provides the immunoglobulin specific for ASF virus components at a dose of the Infected pigs or wild boars are about 0.5 mg to about 1.0 mg per kg body weight.

In another exemplary embodiment, the time period for administration of the composition comprising immunoglobulin specific for an ASF viral component comprises at least once a week or 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 an orally administered food product.

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

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

In another aspect, the time period for administration of the composition comprising immunoglobulins specific for ASF viral components includes at least once a week or 7 consecutive days.

It should also be understood that the present disclosure contemplates that the compositions comprising immunoglobulins specific for components of the ASF virus may be administered parenterally.

In another aspect, the composition comprising immunoglobulins specific for ASF viral components is an orally administered food product.

Another embodiment disclosed herein is a method of producing ASFV-specific immunoglobulins, wherein an ASFV vaccine comprising intact ASF virions, viral components and/or immunosuppressive protein factors is administered for the production of ASFV-specific immunoglobulins Non-porcine species hosts for sexual immunoglobulins.

In an exemplary embodiment, the host is an egg-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 and/or reducing the severity of ASF virus infection in an at-risk pig or wild boar, the method comprising administering to the pig or wild boar an effective amount of an ASF-containing ASFV vaccine composition of viral components.

In one aspect, the ASF viral component is inactive.

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

An exemplary embodiment is also disclosed, wherein the administered amount of the ASFV vaccine composition provides a dose of the ASF viral component of about 0.05 mg to about 1.0 mg 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 mononuclear cells (SMNC), ASF-infected peripheral blood mononuclear cells (PBMC), and/or ASF-infected primary alveolar macrophages (PAM).

On the other hand, the ASF viral components are inactivated.

In another aspect, the ASFV vaccine is used to treat and/or prevent ASF infection in at-risk pigs or wild boars.

One embodiment is an immunoglobulin specific for ASF viral components for use in the treatment and/or prevention of ASF infection in at-risk pigs or wild boars.

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

Description of drawings

Figure 1 shows an exemplary embodiment of a method of manufacturing an ASFV vaccine, an embodiment of a method of actively immunizing a pig or wild boar by administering the ASFV vaccine, immunization or vaccination for the production of ASFV-specific immunoglobulin Embodiments of a method of a non-porcine or non-susceptible species host, and of a method of passively immunizing a pig or wild boar by administering said ASFV-specific immunoglobulin.

Figure 2 shows an exemplary implementation of active immunization by administering the ASFV vaccine to pigs or wild boar (Figure 2A) or non-porcine or non-susceptible species hosts for the production of ASFV-specific immunoglobulins (Figure 2B). Way.

Figure 3 shows qPCR results of an exemplary embodiment of an ASFV-specific immunoglobulin composition. The ASFV-specific immunoglobulin compositions and 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 was free of 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 with 2 different ASFV vaccine compositions and saline as a control (no ASFV vaccine) Spend. Eggs laid by vaccinated hens were collected, immunoglobulin extracted, and coated with recombinant ASFV major capsid protein p72 (ASFV p72; NP_042775) on days 14 (Fig. 4A) and 28 (Fig. 4B) .1; SEQ ID NO: 2) Enzyme-linked immunosorbent assay (ELISA) plates to assess ASFV-specific antibody titers.

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

Detailed ways

definition

Some definitions are provided below. 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.

when used in this article. "About", "approximately" and "substantially" are understood to mean within a range of values, such as the range of -10% to +10% of the reference number, preferably -5% to +5% of the reference number , more preferably in the range of -1% to +1% of the reference number, most preferably in 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. Furthermore, these numerical ranges should be construed as supporting claims for any number or subset of numbers within the range. For example, a disclosure from 1 to 10 should be interpreted as supporting ranges from 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

When used in this disclosure and the claims, the singular includes the plural 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. Likewise, the terms "including", "containing" and "having" should all be construed as inclusive unless the context clearly prohibits such a construction. Furthermore, in this regard, these terms specify the presence of stated features, but do not exclude the presence of additional or other features.

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

The term "and/or" 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 "for example," especially when followed by a list of terms, are exemplary and illustrative only and should not be considered exclusive or comprehensive.

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

The term "pig" refers to domestic pig, wild boar or feral pig.

The term "swine" refers to domestic, wild or feral pigs.

The term "avian" refers to wild or domestic laying birds such as chickens, ducks, swans, geese, turkeys, peacocks, guinea fowls, ostriches, pigeons, quails, pheasants or doves.

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

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

The terms "antigen" or "immunogen" or "hapten" are substances or structures or small molecules that are or are considered foreign to the body and that elicit an immune response either alone or in complexes with larger molecules . The terms "antigen," "immunogen," or "hapten" are used interchangeably in this disclosure.

The term "passive immunization" refers to immunization resulting from the introduction into a subject of antibodies from another human, animal, species, or other external source.

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 "immune adjuvant" refers to a substance that can be added to a vaccine to stimulate a response from the immune system of a subject.

The terms "immunosuppressive protein factor" and/or "host hyperreactive immune factor" are meant to include, but are not limited to, cytokines (eg, cytokines of the TNF family), pro-inflammatory cytokines (eg, IL-17F and/or interferon) and/or down-regulated anti-inflammatory cytokines (eg IL-10). The terms "immunosuppressive protein factor" and/or "host hyperresponsive immune factor" are used interchangeably herein and generally refer to factors that evade innate and/or adaptive immune responses.

The term "treatment" includes both prophylactic or preventive treatment (stopping and/or slowing the development of the target pathological condition, infection, disorder or disease) and curative, therapeutic or disease-modifying treatment, including curing, slowing the diagnosis of pathological conditions, infections, disorders or diseases, treatments to alleviate their symptoms and/or prevent their progression; and subjects at risk of or suspected of having a disease or infection and subjects who are ill or have been diagnosed with a pathological condition, infection , treatment of a subject of a disorder or disease. The term "treatment" does not necessarily imply treatment of a subject until full recovery. The term "treating" also refers to maintaining and/or promoting health in individuals who do not have a pathological condition, infection, disorder or disease, but may be susceptible to developing the pathological condition, infection, disorder or disease. The term "treating" is also intended to include enhancing or otherwise enhancing one or more primary preventive or therapeutic measures. By way of non-limiting example, treatment can be performed by a physician, healthcare professional, veterinarian, veterinary professional, animal handler, or other person.

As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for a subject, each unit containing a predetermined quantity of this text in association with a pharmaceutically acceptable diluent, carrier or vehicle Compositions are disclosed in amounts sufficient to produce the desired effect. The size of the unit dosage form depends on the particular compound employed, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

The term "sterile" should be understood to be free of any bacteria or other living microorganisms.

As used herein, the term "pharmaceutically acceptable" refers to a substance that does not substantially cause an adverse allergic or immune response 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 pH measured at about 25°C using standard equipment. "Ambient temperature" or "room temperature" is between about 15°C and about 25°C, and the ambient pressure is about 100 kPa.

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

The terms "substantially free," "substantially free," or "substantially free," when used to refer to a particular component, mean that any component is present at no more than about 3.0 wt %, such as no more than about 2.0 wt % %, up to about 1.0% by weight, preferably up to about 0.5% by weight, or more preferably up to about 0.1% by weight.

The terms "food", "food" and "food composition" mean a product or composition intended for ingestion by animals, including humans, and to provide at least one nutrient to said animals. Preferred embodiments of the food product include at least one of proteins, carbohydrates, lipids, vitamins or minerals.

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

Implementation

ASFV vaccine

The present disclosure generally relates to an ASFV vaccine comprising intact live ASFV particles in combination with naturally expressed ASF virus components, optionally diluted in sterile buffer, eg, sterile saline solution to about 10%. The ASFV vaccine can be used to actively immunize or vaccinate a non-susceptible species host for the production of ASFV-specific immunoglobulin. A non-susceptible species host can be a non-swine mammalian host, such as 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 antigens are obtained from ASF-infected pigs or wild boars. In one embodiment, blood can be drawn from the ASF-infected pig or wild boar and collected in a blood collection tube with anticoagulant. The blood collection tube can be centrifuged, for example, at about 4°C at about 1,500 xg for about 15 minutes to obtain a buffy coat. Alternatively, plasma-containing peripheral blood mononuclear cells (PBMCs) can be isolated from blood by standard gradient centrifugation on Ficoll or other methods known to those skilled in the art. Additionally, any red blood cells (RBCs) can be lysed with a solution containing about 0.83% _NH4Cl or by any other method known to those skilled in the art.

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

In another embodiment, the ASFV vaccine can be obtained from an ASFV-infected lymphoid organ such as the spleen. Spleens can be harvested from ASFV-infected pigs or wild boars and dissected into multiple tissue sections. Preferably, the dissection is performed immediately after harvesting. The tissue sections can be added to buffer and homogenized on ice. The homogenized tissue mixture can be centrifuged to produce a single cell suspension, eg, at about 800 xg for about 15 minutes at a predetermined time and a predetermined temperature, eg, at about 4°C. The single cell suspension may contain RBCs and splenic mononuclear cells (SMNC). The RBCs can be cleaved using a solution containing about 0.83% _NH4Cl or by any other method known to those skilled in the art. SMNC can be collected and lysed 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 SMNCs can be collected by Ficoll gradient centrifugation. The supernatant and SMNC can be collected and subjected to one or more freeze-thaw cycles, wherein the mixture can be reduced to a low temperature, such as in a dry ice ethanol bath (about -70°C) for a first predetermined period of time, followed by left at room temperature for a second predetermined period of time. The mixture of supernatant and disrupted SMNCs can be centrifuged at about 800 x g for about 15 minutes at about 4°C. The supernatant can be collected and diluted one- or multiple-fold, eg, 10-fold, with a buffer of predetermined pH, eg, sterile saline buffer. The resulting ASFV vaccine can be stored in one or more portions at a temperature below room temperature, eg, in about 1 ml aliquots at or below about -20°C, preferably about -70°C. In another exemplary embodiment, the protein content and/or viral titer in the supernatant can 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 and grown overnight in complete medium containing fetal bovine serum (FBS). After about 24 hours, the cell monolayer can be washed, and medium containing serum infected with the ASFV stock can be added to the culture. The ASF-infected PAM can be cultured until at least about 75% of the cytopathic effect is observed in culture, eg, after about 5 to about 7 days after ASFV infection. The PAM and culture supernatant can be harvested, collected and subjected to one or more freeze-thaw cycles, wherein the PAM mixture can be reduced to a low temperature, for example, in a dry ice ethanol bath (about -70°C) for a first predetermined time period, and then placed at room temperature for a second predetermined period of time. The mixture of supernatant and disrupted PAM can be centrifuged at about 800 x g for about 15 minutes at about 4°C. The supernatant can be collected and diluted one- or multiple-fold, eg, 10-fold, with a buffer of predetermined pH, eg, sterile saline buffer. The resulting ASFV vaccine can be stored in one or more portions at a temperature below room temperature, eg, in about 1 ml aliquots at or below about -20°C, preferably about -70°C. In another exemplary embodiment, the protein content and/or viral titer in the supernatant can 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 envisaged herein that the ASFV vaccine compositions contain a broad range of ASFV antigens (ie, comprehensive ASFV proteins). It should be understood that the proteins or antigens that may be included in the ASFV vaccine compositions may include whole, 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 can be selected for use in producing the ASFV vaccine composition by first testing infected pigs. Additionally or alternatively, the methods of ASFV treatment disclosed herein may provide cross-protection against closely related virus strains, ASFV genotypes and/or ASFV serotypes.

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

Additionally or alternatively, non-porcine mammalian hosts such as birds, horses, cattle, donkeys, goats or rabbits can be vaccinated or immunized with non-irradiated ASFV vaccines, eg, for the production of ASFV-specific immunoglobulins.

ASFV-specific immunoglobulin

Another aspect of the present disclosure relates generally to methods of immunizing or vaccinating a non-susceptible species host to produce ASFV-specific immunoglobulins. An ASFV vaccine containing whole virions, viral components, immunosuppressive protein factors, and host hyperreactive immune factors, such as an aliquot (e.g., about 1 mL of about 10% ASFV vaccine in sterile saline buffer), can be administered. ) to a predetermined temperature, vortexed, and injected intramuscularly into a non-porcine mammalian host such as avian, equine, bovine, donkey, goat or rabbit. Following initial and optional re-immunization, samples of 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 vaccinated or vaccinated laying birds such as chickens. An ASFV vaccine containing whole virions, viral components and immunosuppressive protein factors, eg, an aliquot (eg, about 1 mL) of about 10% ASFV vaccine in sterile saline buffer, can be thawed to room temperature, vortexed Shake and inject intramuscularly into laying birds. Preferably, the ASFV vaccine is divided into equal fractions (approximately 100 μg protein content/fraction), one fraction is injected into the left breast of the hen and a second fraction is injected into the right breast of the hen, Optionally approximately equal volumes are used, eg about 500 ml in the right breast and about 500 ml in the left breast. Additionally or alternatively, the ASFV vaccine may be emulsified in complete Freund's adjuvant (CFA) at a ratio of about 1:1 prior to injection into the hens. In another embodiment, subsequent immunizations may include an ASFV vaccine composition comprising an about 1 : 1 solution of ASFV vaccine and incomplete Freund's adjuvant (IFA).

After the initial immunization, the hens may be re-vaccinated, eg, 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-immunizations (eg, about 27 days after the initial immunization), eggs laid by vaccinated hens can be collected one or more days for purification of IgY antibodies. Alternatively, eggs can be collected continuously during immunization. The IgY antibody can be obtained from the collected egg yolk by a water-soluble fraction. One or more egg yolks can be combined and diluted about 10-fold with cold 3 mM HCl to give a final pH of about 5 suspension (adjusted with about 10% acetic acid). The suspension can be frozen, eg, at about -20°C overnight. After thawing to a predetermined temperature, the mixture can be centrifuged at about 13,000 x g for about 15 minutes at about 4°C, and the IgY immunoglobulin-containing supernatant can be collected. The IgY immunoglobulins can be further purified by various precipitation methods known to those of ordinary skill in the art, such as using ammonium sulfate or biocompatible sodium chloride (see Hodek, P. et al., "Providing Electrophoretic Homogeneous Preparations. "Optimized Protocol of Chicken Antibody (IgY) Purification Providing Electrophoretically Homogenous Preparations", 8 Int. J. Electrochem. Sci. 113, 113-124 (2013)). Optionally, the IgY immunoglobulin can be obtained from the egg white fraction.

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

In certain embodiments of the antibody composition, the IgY is concentrated, isolated or purified from egg constituents. This can be accomplished by a variety of different methods, such as those known to those of ordinary skill in the art. If desired, the titer of IgY antibodies can be determined by immunoassays such as ELISA.

In certain embodiments of the antibody composition, the composition is prepared by a method comprising obtaining eggs from birds previously actively vaccinated against ASFV, and isolating the antibody fraction from the yolk. The poultry is preferably poultry. The poultry can be chickens, ducks, swans, geese, turkeys, peacocks, guinea fowls, ostriches, pigeons, quails, pheasants, doves or other poultry. The poultry is preferably chicken. The poultry is more preferably a domestic chicken raised mainly for egg or meat production.

In certain embodiments of the antibody composition, the antibody composition is prepared by a method comprising actively vaccinating hens against ASFV, collecting eggs from the hens after the immunization period, and removing eggs from the hens. Antibody fractions were isolated from egg yolk. Optionally, egg collection from the hens can be performed continuously after the immunization period.

Other methods of producing IgY with specific targets are known to those skilled in the art, although it is unknown whether these methods have been successfully used to produce antibodies against ASFV before. 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 (ie, immunoglobulins) in general. Such antibodies bind to the surface of an antigen-bearing virus (eg, ASFV), preventing the initial contact phase between the virus and potential host cells. As explained elsewhere in this disclosure, preventing the initial adhesion phase between virus and host cells has numerous applications, including the treatment of viral diseases and the prevention of viral diseases.

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

Methods are provided to prevent the virus from adhering to cells. The first step in the infection of a cell by a virus is the contact and adhesion between the virus and the cell. Although this step is critical for the establishment of the infection, there are few ways to stop the infection at this early stage. More commonly, techniques such as active vaccination are used to combat viral infections, which cause the body to produce antibodies that neutralize the virus. If active vaccination is not feasible, viral diseases are most often treated only symptomatically. The methods described herein provide an effective means of preventing this early step in the infection process without necessarily requiring administration prior to subject exposure to pathogens, as is required by active vaccination.

Antibodies can work by binding to the virus and interfering with the virus's ability to bind to its target membrane receptors, preventing the virus from sticking to the cell. Compared to mammalian antibodies, avian antibodies (eg, IgY) have distinct advantages in this application, especially when the subject is a mammal. As stated above, advantages of IgY antibodies include that IgY antibodies are more specific, more stable, and elicit fewer forms of unwanted immune responses than mammalian antibodies. IgY antibodies are also readily and inexpensively obtained from eggs.

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

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

treatment method

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

In one embodiment, the ASFV vaccine and/or 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: The Science and Practice of Pharmacy (20th Edition, edited 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. A therapeutically effective amount of the antibody can be an amount effective to inhibit adhesion and/or an amount effective to generate passive immunity in a subject (ie, pig or wild boar). Additionally or alternatively, such ASFV vaccine compositions contain a therapeutically effective amount of ASFV antigens (eg, ASFV virions and/or viral components) in order to form pharmaceutically acceptable compositions suitable for administration. A therapeutically effective amount of an irradiated ASFV antigen can be an amount effective to generate protective immunity in a subject (ie, pig or wild boar).

The pharmaceutical compositions of the present disclosure can be used in the methods of treatment and prevention of the present disclosure. Such compositions are administered to pigs or wild boars in amounts sufficient to deliver a therapeutically effective amount of ASFV-specific immunoglobulin or ASFV vaccine to be effective in the methods of treatment and prevention disclosed herein. The therapeutically effective amount can vary depending on a variety of 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 can be provided to a subject by 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 can be accomplished by addition to the subject's feed (solid or liquid).

The compositions of the present disclosure can be administered to a subject only once or more than once. In addition, when the composition is administered to a subject more than once, a variety of different schedules can be used, such as, but not limited to, once a day, once a week, once a month, or once a year. The composition can 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 ASFV vaccine composition can 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 can be administered separately to separate subjects. Additionally or alternatively, the ASFV-specific immunoglobulin composition and ASFV vaccine composition can be co-administered to an individual subject in need thereof in a variety of different therapeutic regimens. In addition, co-administration or sequential administration of other agents may be desirable.

The compositions of the present disclosure can be administered systemically, eg, by intraperitoneal, intravenous, or intramuscular administration.

The compositions of the present disclosure may also include agents that improve the solubility, half-life, absorption, etc. of the antibody. In addition, the compositions of the present disclosure may also include agents that attenuate unwanted side effects and/or reduce toxicity of the antibody. Examples of such agents are described in various textbooks, such as, but not limited to, Remington The Science and Practice of Pharmacy (20th Edition, Lippincott, Williams & Wilkins, eds. Daniel Limmer).

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, injectable solutions, lyophilized powders, or granules.

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, disintegrating agents and carriers. Generally, 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 can include polymers and polymer matrices. The nature of the pharmaceutically acceptable carrier may vary depending upon the particular dosage form employed and other characteristics of the composition.

For example, for oral administration of ASFV-specific immunoglobulin in solid forms such as, but not limited to, powders or granules, the antibody may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier, such as, but not limited to, inert Fillers, suitable binders, lubricants, disintegrants and adjuvants. 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, carboxymethyl cellulose, polyamides. Ethylene glycol, wax, etc. 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, methylcellulose, agar, bentonite, xanthan gum, and the like.

Formulations suitable for parenteral administration include aqueous isotonic sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic agents and solutes to render the formulation isotonic with the blood of the subject, as well as suspending agents, Aqueous suspension of solubilizers, thickeners, stabilizers and preservatives. The compositions can be administered in physiologically acceptable diluents, such as sterile liquids or liquid mixtures, including water, saline, aqueous dextrose and related sugar solutions.

Oils that can 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 parenteral formulations include polyethylene sorbitan fatty acid esters such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with hydrophobic bases, which are Condensation of oxypropane 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 parenteral formulations include fatty alkali metal, ammonium and triethanolamine salts, and suitable detergents include (a) cationic detergents such as dimethyldialkylammonium halides and alkylpyridiniums 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-alanine and 2-alkylimidazoline quaternary ammonium salts, and (e) their mixture.

Suitable preservatives and buffers can be used 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 can also be coupled with soluble polymers as targetable drug carriers. Such polymers may include, but are not limited to, polyvinylpyrrolidone, pyran copolymers, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethylene oxide polylysine substituted with palmitoyl residues acid. In addition, the antibodies of the present invention can be conjugated to a class of biodegradable polymers that can be used to achieve controlled release of drugs, such as polylactic acid, polyε caprolactone, polyhydroxybutyric acid, polyorthoester, polyacetal , crosslinked or amphiphilic block copolymers of polydihydropyrans, polycyanoacrylates and hydrogels.

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

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 can be stored in any pharmaceutically acceptable form, including aqueous solutions, refrigerated aqueous solutions, lyophilized powders, or any other form described herein.

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

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

The non-limiting example of the pharmaceutically acceptable ASFV vaccine composition preferably also comprises a lyophilized powder composition, eg for long term storage and/or transport. The lyophilized vaccine can be reconstituted to approximately the original volume in a solution such as saline prior to use in immunization or vaccination.

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

One aspect of the present disclosure is a method of treating ASFV-infected or exposed pigs or wild boars by administering a composition comprising ASFV-specific immunoglobulins. The ASFV-specific immunoglobulin can be added to the feed at a dose of about 1.0 mg per kg body weight, orally administered about once a day for about 5 to about 7 consecutive days to ASFV-infected or exposed pigs or wild boars in need thereof.

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

Non-limiting examples of methods of treatment include increasing the dose of ASFV-specific immunoglobulin administered parenterally or orally, in combination or alternatively with increased dosing frequency. One aspect of the present disclosure is a preferred method of treating ASFV-infected or exposed swine or wild boar pregnant sows, fetuses of sows, and/or piglets. ASFV-specific immunoglobulins are administered to the pregnant sows by methods discussed elsewhere in this disclosure. The piglets and/or fetuses receive the ASFV vaccine directly or indirectly during gestation and/or lactation.

Another aspect of the present disclosure is a preferred method of prophylactic treatment for swine or wild boar susceptible to ASF infection (FIG. 1). Irradiated ASFV vaccine compositions can preferably be administered to subjects (ie, pigs or wild boars), including but not limited to subjects who have been exposed to ASFV, subjects susceptible to ASF infection, and/or subjects infected with ASFV. The ASFV vaccine composition may contain additional components, such as pharmaceutical components discussed elsewhere in this disclosure. The ASFV vaccine composition can 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 weighing about 20 kg (ie, non-old pigs) . Preferably, the ASFV vaccine composition is administered in a dose of about 100 μg. Additionally or alternatively, the ASFV vaccine composition can be administered to an individual subject more than once. For example, a booster immunization may be given 14 days after the first or primary immunization. In addition, a third immunization can be given on the 21st day after the first or primary immunization.

Disclosed herein is the exemplary embodiment illustrated in Figure 2A, in particular a method of treating a subject comprising administering a first dose of an ASFV vaccine composition to CFA in a 1:1 ratio. Then after about two weeks, a second dose of the ASFV vaccine composition and IFA can be administered to the subject in a 1:1 ratio. About four weeks after the first or primary immunization, the pig or wild boar can be subjected to ASFV challenge to determine whether the immunized subject can survive a lethal ASF infection. The dose of the irradiated _ASFV vaccine may range from a live virus equivalent to about 104 ^HAD50 ( ₅₀ % erythrocyte adsorption dose) to about 105 ^HAD50 .

Example

The following non-limiting examples support the concept of using pharmaceutically acceptable ASF vaccine compositions to generate antibodies useful for treating or preventing infection in swine and/or wild boars.

Example 1

On days 1, 14 and 28, three groups of chickens (n=3 per group) were immunized with the ASFV vaccine. Group 1 received saline (no vaccine) as a control, Group 2 received ASFV vaccine formulation 1 containing intact virions and immunosuppressive protein factors, and Group 3 received ASFV vaccine formulation 1 containing intact virions, viral components and immunosuppressive proteins Factor ASFV vaccine formulation 2. After the second and third immunization, blood samples were obtained and ASFV-specific antibody titers were assessed using recombinant ASFV major capsid protein p72-coated ELISA plates (SEQ ID NO: 2). The results of Example 1 demonstrate that the ASFV vaccine is immunogenic and immunization is achieved. Furthermore, Example 1 demonstrated that the ASFV vaccine-induced antibody pools had 14 days ( FIG. 4A ) and 28 days ( FIG. 4B ) to ASF virus components such as the ASFV major capsid protein p72 (SEQ ID NO: 2) Comprehensive specificity.

Example 2

ASFV vaccine compositions were prepared from homogenates of ASFV-infected spleens from ASFV-infected pigs and ASFV-infected buffy coat containing PBMCs. The PBMC mixture was frozen in a dry ice ethanol bath and thawed to room temperature. The freeze-thaw procedure was repeated twice. The ASFV vaccine compositions were evaluated 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 a control or one of two different ASFV vaccine formulations. Group 1 received saline as a control (no vaccine), group 2 received ASFV vaccine formulation 1 (prepared from SMNC), and group 3 received ASFV vaccine formulation 2 (prepared from SMNC and PBMC). The hens were actively immunized on days 1, 14 and 3 by administering the ASFV vaccine (by intramuscular injection) or administering a control. Eggs were collected daily after the third immunization. Immunoglobulins were extracted from egg yolks using a simple water dilution method. After the second immunization, blood samples were obtained from the chickens on day 14 and no viral shedding was determined by qPCR.

Eggs collected from hens receiving Formulation 2 were used to produce ASFV-specific immunoglobulins. Eggs from hens receiving saline or Formulation 1 were not used to produce ASFV-specific immunoglobulins. The ASFV-specific immunoglobulin composition was analyzed using qPCR, and it was determined that there was no active ASFV (ie, the ASFV-specific immunoglobulin composition did not contain ASFV DNA) (Figure 3). The specificity of the IgY antibodies of the ASFV-specific immunoglobulin compositions was also assessed. ELISA was used to detect antibodies specific for recombinant ASFV capsid protein p72 (SEQ ID NO: 2) and ASFV p72-specific IgY was detected at a level of 6 log ₂ (Figure 5).

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

Clinical observations revealed that 4 days after ASFV exposure, all 3 pigs in Group C exhibited initial ASF symptoms including low activity (ie, lethargy) and exhibited reduced food intake. All 3 pigs in group C stopped eating 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 exhibited decreased appetite (ie, decreased food intake) and dark yellow urine, but no other signs or symptoms of ASF were observed, No signs of disease were observed. The results show that administration of ASFV-specific immunoglobulin compositions either before or after ASFV exposure successfully resulted in passive immunity.

sequence listing

<110>

IGY Immune Technologies and Life Sciences Inc.

<120> Vaccines and immunoglobulins targeting African swine fever virus and methods for their preparation and use

<130> 1401870.00006

<160> 2

<170> PatentIn version 3.5

<210> 1

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Claims (as amended by Article 19 of the Treaty)

1. A method of treating 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, the The composition comprises IgY immunoglobulins specific for (a) naturally expressed ASF viral components, (b) intact live ASF virions, and/or (c) immunosuppressive protein factors.

2. The method of claim 1, wherein the administration amount of the composition provides the specificity for (a) naturally expressed ASF viral components, (b) intact live ASF virions and/or (c) The dose of IgY immunoglobulin of the immunosuppressive protein factor is from about 0.5 mg to about 1.0 mg per kg body weight of the infected pig or wild boar.

3. The method of claim 1 , wherein the comprising specificity for (a) naturally expressed ASF viral components, (b) intact live ASF virions and/or (c) immunosuppressive protein factors The time period for administration of the composition of IgY immunoglobulin includes at least once a week or 7 consecutive days.

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

5. The method of claim 1 , wherein the comprising specificity for (a) naturally expressed ASF viral components, (b) intact live ASF virions and/or (c) immunosuppressive protein factors Compositions of IgY immunoglobulins are food products for oral administration.

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

7. The method of claim 6, wherein the administered amount of the composition provides the specificity for (a) naturally expressed ASF viral components, (b) intact live ASF virions and/or (c) The dose of IgY immunoglobulin of the immunosuppressive protein factor is from about 0.5 mg to about 1.0 mg per kg body weight of the pig or wild boar at risk.

8. The method of claim 6, wherein the method comprising specificity for (a) naturally expressed ASF viral components, (b) intact live ASF virions and/or (c) immunosuppressive protein factors The time period for administration of the composition of IgY immunoglobulin includes at least once a week or 7 consecutive days.

9. The method of claim 6, wherein the method comprising specificity for (a) naturally expressed ASF viral components, (b) intact live ASF virions and/or (c) immunosuppressive protein factors Compositions of IgY immunoglobulins are administered parenterally.

10. The method of claim 6, wherein the comprising specificity for (a) naturally expressed ASF viral components, (b) intact live ASF virions and/or (c) immunosuppressive protein factors Compositions of IgY immunoglobulins are food products for oral administration.

11. A method of producing ASFV-specific immunoglobulins, wherein an ASFV vaccine comprising intact live ASF virions, naturally expressed ASF virus components and/or immunosuppressive protein factors is administered to the production of ASFV-specific Non-porcine species hosts for sexual immunoglobulins.

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

13. A unit dosage form comprising a therapeutically or prophylactically effective amount comprising specific for (a) naturally expressed ASF viral components, (b) intact live ASF virions and/or (c) immunosuppressive protein factors The composition of IgY immunoglobulin.

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 reducing the severity of ASF virus infection in at-risk pigs or wild boars, said method comprising administering to said pigs or wild boars an effective amount of an amount comprising (a) ASFV vaccine compositions of naturally expressed ASF virus components, (b) intact live ASF virions and/or (c) immunosuppressive protein factors.

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

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

20. The unit dosage form of claim 19, wherein the naturally expressed ASF viral components and intact live ASF viral particles are derived from ASF-infected spleen mononuclear cells (SMNC), ASF-infected peripheral blood mononuclear cells cells (PBMC) and/or ASF-infected primary alveolar macrophages (PAM).

21. The unit dosage form of 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) naturally expressed ASF virus components, (b) intact live ASF virions and/or (c) immunosuppressive protein factors, wherein The ASFV vaccine described is for the treatment and/or prevention of ASF infection in at-risk pigs or wild boars.

23. A composition comprising IgY immunoglobulins specific for (a) naturally expressed ASF virus components, (b) intact live ASF virions and/or (c) immunosuppressive protein factors, wherein The composition is used for the treatment and/or prevention of ASF infection in at-risk pigs or wild boars.

24. The method of claim 1, wherein the naturally expressed ASF viral components, intact live ASF virions and/or immunosuppressive protein factors are derived from cells, tissues and / or organs.

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

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

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

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

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

30. The unit dosage form of claim 19, wherein the naturally expressed ASF viral components, intact live ASF virions and/or immunosuppressive protein factors are derived from ASF-infected splenic mononuclear cells (SMNCs) 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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