TW202203970A - Igy immunoglobulins targeting coronavirus, methods of preparing same, and methods of using same - Google Patents

Abstract

The present disclosure provides a method of preparing compositions or vaccines for immunizing or vaccinating egg-laying fowl such as hens against coronavirus. The eggs can be collected and the coronavirus-specific IgY antibodies can be extracted and purified. The coronavirus-specific IgY immunoglobulin composition can be used to treat individuals exposed to, susceptible to, or infected with coronavirus.

Description

IgY immunoglobulin targeting coronavirus, its preparation method, and its use method

The present invention generally relates to immunoglobulins from egg-laying poultry specific for coronaviruses. The present invention also relates to methods of immunization and methods of treating or preventing coronavirus infection. Immunoglobulins specific for the coronaviruses disclosed herein provide broad-spectrum immunity to individuals infected or susceptible to the coronaviruses. Cross-references to related applications

This application claims the benefit of US Provisional Application 63/008,298, filed April 10, 2020, which is incorporated herein by reference in its entirety. sequence listing

This application contains Sequence Listings, which have been submitted electronically in ASCII format and are incorporated herein by reference in their entirety. This ASCII copy was created on April 9, 2021, is a sequence listing named 1401870.00012.txt, and is 8,192 bytes in size.

Coronavirus disease 19 (COVID-19) is a highly contagious and pathogenic viral infection caused by severe acute respiratory coronavirus syndrome 2 (SARS-CoV-2) that originated in Wuhan, China and has spread worldwide. Genome analysis has shown that SARS-CoV-2 is phylogenetically related to severe acute respiratory syndrome (SARS-like) bat viruses, so bats are the main reservoir. The intermediate source of origin and transfer to humans is unknown, but rapid human-to-human spread is widely established. There are currently no clinically approved antiviral drugs or vaccines available for use against COVID-19. However, in clinical trials, few broad-spectrum antiviral drugs have been assessed for clinical recovery against COVID-19.

Coronaviruses belong to the Coronaviridae family, the order Nidovirales. "Corona" refers to the crown-like spines on the surface of the virus; hence the name coronavirus. Coronaviruses are tiny in size (65-125 nm in diameter) and contain single-stranded RNA as nucleic acid material, ranging in size from 26 to 32 kb in length. (Anthony R. Fehr et al, at 1 p.). The subgroups of the coronavirus family are alpha (alpha), beta (beta), gamma (gamma) and delta (delta) coronaviruses. Most coronaviruses cause infectious disease in humans and vertebrates. α-CoV and β-CoV mainly infect the respiratory tract, gastrointestinal tract, and central nervous system of humans and mammals, while γ-CoV and δ-CoV mainly infect birds (Stanley Perlman et al., at p. 1) (Yudong Yin et al., on page 132). Typically, several coronavirus members cause mild respiratory disease in humans; however, the outbreaks of SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2002-2003 and 2012, respectively, caused Fatal severe respiratory disease (Kathryn V. Holmes et al, p. 1949). SARS-CoV and MERS-CoV belong to β-CoV. 2019-nCoV is also a β-CoV according to a phylogenetic analysis based on the viral genome (Fan Wu et al., p. 267). Although the nucleotide sequence similarity is less than 80% between 2019-nCoV and SARS-CoV (about 79%) or MERS-CoV (about 50%), 2019-nCoV can also cause fatal infections and is more common than the two other coronaviruses The virus spreads faster (Peng Zhou et al., at p. 270).

Initially, interferon-alpha sprays, broad-spectrum antibiotics, and antiviral drugs were used to reduce viral load (Chen Seng Ng et al., p. 2), (Ben X Wnag et al., p. 1), (Manli Wang et al., p. 1). at p. 269), however, only remdesivir showed promising effects against the virus (Maria L. Agostini et al. at p. 1). Remdesivir alone and in combination with chloroquine or interferon beta significantly blocked SARS-CoV-2 replication, and patients declared clinical recovery (Timothy P. Sheahan et al., at p. 10), (Michelle L. Holshue et al. , at 931), (Manli Wang et al., at 270). Various other antiviral agents are currently being evaluated for infection. When testing Nafamostat, Nitazoxanide, Ribavirin, Penciclovir, Favipiravir against infection in patients and in vitro clinical isolates ), Ritonavir, AAK1, Baricitinib, and Arbidol showed moderate results (Timothy P. Sheahan et al., p. 10) , (Peter Richardson et al., at p. 30), (Manli Wang et al., at p. 270). Several combinations, such as combining antiviral agents or antibiotics with traditional Chinese medicines, were also evaluated for SARS-CoV-2-induced infection in humans and mice (Timothy P. Sheahan et al., at 3). Recently in Shanghai, physicians isolated plasma from clinically recovered patients with COVID-19 and injected it into infected patients who showed positive results, resulting in rapid recovery (Muhammad Adnam Shereen et al., p. 94). In a recent study, a monoclonal antibody (CR3022) was identified that binds to the spine RBD of SARS-CoV-2. This may be due to the fact that the epitopes of the antibody do not overlap with the divergent ACE2 receptor binding motif. CR3022 has the potential to be developed as a therapeutic candidate alone or in combination with other neutralizing antibodies for the prevention and treatment of COVID-19 infection (Muhammad Adnam Shereen et al., p. 93).

The present inventors have developed a method for producing coronavirus-specific IgY immunoglobulin using an adenovirus vector encoding a coronavirus antigen and/or a composition comprising a coronavirus antigen or a coronavirus recombinant protein. Adenoviral vectors encode coronavirus antigens and/or compositions coronavirus antigens or recombinant proteins and can be used alone or in combination to immunize or vaccinate egg-laying poultry, such as hens, to produce broad-spectrum coronavirus Specific polyclonal immunoglobulins. IgY immunoglobulins or antibodies can be extracted and purified from egg yolk fractions. The antibodies produced can be used to prevent virus adhesion, virus spread, treat coronavirus infection or prevent coronavirus infection. IgY isotype antibodies from poultry or birds are particularly suitable for these applications.

Coronavirus-specific immunoglobulins can be administered intranasally and/or orally for the treatment of individuals infected with coronavirus. Alternatively or additionally, immunoglobulins can be administered to individuals who have been previously exposed to or are otherwise susceptible to coronavirus infection. The coronavirus-specific immunoglobulin may be in liquid or lyophilized powder form, which is reconstituted prior to administration at a dose effective to reduce, alleviate or eliminate symptoms in an infected individual, or at a dose effective to prevent infection or reduce prior Dose for severity of infection or symptoms in individuals exposed or susceptible to coronavirus infection.

In one embodiment disclosed herein is a method of treating a coronavirus infection in an individual with a coronavirus infection, the method comprising administering to egg-laying poultry a first composition comprising encoding one or more coronaviruses one or more adenoviral vectors for viral antigens; and providing an individual suffering from a coronavirus infection with a second composition comprising a water-soluble portion of egg yolk from egg-laying poultry to which the first composition was administered.

Also disclosed herein are methods of treating a coronavirus infection, wherein the second composition is administered to a subject suffering from a coronavirus infection in an amount that provides the subject suffering from a coronavirus infection from about 0.25 mg to about 1.0 mg per kilogram of body weight Dosage of water-soluble portion of egg yolk.

In another illustrative embodiment, is a method of treating a coronavirus infection, wherein the second composition is administered intranasally.

In one aspect, a method of treating a coronavirus infection, wherein the second composition is administered to an individual suffering from a coronavirus infection more than once.

In one aspect, there is a method of treating a coronavirus infection, wherein the first composition is administered to egg-laying poultry more than once.

In another aspect, a method of treating a coronavirus infection, wherein the first composition is administered a second time to egg-laying poultry at least 14 days after administration of the first composition.

In one embodiment disclosed herein, is a method of treating a coronavirus infection, wherein an additional composition is administered to egg-laying poultry at least 14 days after administration of the first composition.

In another aspect, for a method of treating a coronavirus infection, the additional composition comprises a live or inactivated coronavirus, an adenovirus vector encoding a coronavirus antigen, a coronavirus recombinant protein, and/or a spine expressing an anchor Protein inactivation of Newcastle disease virus.

One embodiment disclosed herein is a method of preventing, reducing the incidence and/or reducing the severity of coronavirus infection in an individual at risk of coronavirus infection, the method comprising administering to egg-laying poultry a first composition , the first composition comprises one or more adenoviral vectors encoding one or more coronavirus antigens; and the individual is provided with a second composition comprising from the eggs that have been administered the first composition The water-soluble portion of the egg yolk of poultry.

In another embodiment disclosed herein, a method of preventing, reducing the incidence, and/or reducing the severity of coronavirus infection in an individual at risk of coronavirus infection, wherein the second composition is to provide the individual with each A dosage amount of about 0.25 mg to about 1.0 mg of the water-soluble portion of the egg yolk is administered to the subject per kilogram of body weight.

In another embodiment disclosed herein, for a method of preventing, reducing the incidence, and/or reducing the severity of coronavirus infection in individuals at risk for coronavirus infection, wherein the second composition is administered intranasally give.

In another aspect, a method of preventing, reducing the incidence, and/or reducing the severity of coronavirus infection in an individual at risk of coronavirus infection, wherein the second composition is administered to the individual more than once.

In another embodiment, a method of preventing, reducing the incidence, and/or reducing the severity of coronavirus infection in individuals at risk of coronavirus infection, wherein the first composition is administered to egg-laying poultry more than once.

In another aspect, for a method of preventing, reducing the incidence and/or reducing the severity of coronavirus infection in an individual at risk of coronavirus infection, wherein at least 14 days after administration of the first composition, the The first composition is administered to the laying poultry twice.

In another aspect, a method of preventing, reducing the incidence, and/or reducing the severity of coronavirus infection in an individual at risk of coronavirus infection, wherein at least 14 days after administration of the first composition, the Additional doses are administered to laying poultry.

In another embodiment disclosed herein, is a method of preventing, reducing the incidence and/or reducing the severity of coronavirus infection in individuals at risk of coronavirus infection, wherein the additional composition comprises live or non- Activated coronaviruses, adenovirus vectors encoding coronavirus antigens, recombinant coronavirus proteins, and/or inactivated Newcastle disease viruses expressing anchored spike proteins.

It should also be understood that the present invention encompasses a method of producing an intranasally administrable composition effective to treat, prevent, reduce the incidence and/or reduce the severity of a coronavirus infection in an infected individual, the method comprising adding The composition is formed by adding a water-soluble portion of egg yolk obtained from egg-laying poultry administered with one or more adenoviral vectors encoding one or more coronavirus antigens to the solution.

Another embodiment disclosed herein is a unit dosage form comprising a therapeutically or prophylactically effective amount of a composition comprising a water-soluble portion of egg yolk that has been administered since administration of one of the encoding one or more coronavirus antigens or multiple adenoviral vectors obtained from egg-laying poultry.

In another aspect, an immunoglobulin specific for a coronavirus for use in the treatment and/or prevention of infection by the coronavirus in individuals at risk of infection with the coronavirus.

definition

Some definitions are provided below. Nonetheless, definitions may be located in the "Examples" section below, and the heading "Definitions" above does not imply that such disclosures in the "Examples" section are not definitions.

As used herein, "about", "approximately" and "substantially" should be understood to refer to numbers within a numerical range, eg, -10% to +10% of the reference number , -5% to +5% of the preferred reference number, -1% to +1% of the better reference number, and -0.1% to +0.1% of the best reference number.

All numerical ranges herein are understood to include all integers, whole, or fractions within the range. Furthermore, these numerical ranges should be construed as supporting technical solutions involving any number or subset of numbers within the range. For example, the disclosure of 1 to 10 should be interpreted as supporting ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

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

The words "comprise", "comprises" and "comprising" are to be construed as inclusive and not exclusive. Likewise, the terms "include," "including," "containing," and "having" should be construed as inclusive unless the context clearly prohibits this construct. Furthermore, in this regard, these terms specify the presence of stated features, but do not exclude the presence of additional or other features.

Nonetheless, the compositions and methods disclosed herein may lack any element not specifically disclosed herein. Thus, disclosures of specific instances using the term "comprising" are (i) disclosures with specific instances of the identified component or step and additional components or steps, (ii) "substantially by" identified Disclosure of specific instances of a component or step "consisting essentially of", and (iii) "consisting of" an identified instance of a component or step "consisting of". Any specific example disclosed herein can be combined with any other specific example disclosed herein.

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

As used herein, the terms "example" and "such as", especially when followed by a list of terms, are merely exemplary and explanatory and should not be considered exclusive or comprehensive.

A "subject" or "individual" is a mammal, preferably a human.

As used herein, an "effective amount" is the amount over a period of time to prevent infection, treat a disease or medical condition in an individual, or, more generally, reduce symptoms, manage disease progression, or alleviate viral infection.

The term "fowl" means wild, domestic or domestic egg-laying poultry such as chickens or hens, ducks, swans, geese, turkeys, peacocks, guinea fowls, ostriches, pigeons, quails, pheasants, pigeons or other Domestic egg laying poultry. The terms "fowl", "egg-laying fowl", "chicken" and/or "hen" are used interchangeably herein.

The term "immunoglobulin" or "antibody" refers to glycoprotein molecules produced from leukocytes and lymphocytes that assist by specifically recognizing and binding to specific antigens It neutralizes and participates in the body's immune system and immune response. The terms "immunoglobulins", "antibodies" and "IgY" are used interchangeably herein.

The terms "antigen", "immunogen" or "hapten" refer to a substance or structure or small molecule which is foreign to the body or perceived as foreign and which alone Or elicit an immune response after complexing with larger molecules.

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

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

Within the present invention, the terms "immunize" or "vaccinate" are used interchangeably.

The term "adjuvant" or "immunologic adjuvant" refers to a substance that is usually added to a vaccine to stimulate an individual's immune system response.

The terms "treatment" and "treat" include prophylactic or prophylactic treatment (preventing and/or slowing the progression of a target pathological condition, infection, disorder or disease) and curative, therapeutic or disease-modifying treatment (including therapeutic measures to cure, slow, alleviate the symptoms of a diagnosed pathological condition, infection, disorder or disease and/or stop the progression of a diagnosed pathological condition, infection, disorder or disease). Treatment of individuals at or at risk of contracting a disease or suspected of having contracted a disease or being infected, as well as individuals suffering or diagnosed with a pathological condition, infection, disorder or disease is intended to be included. The terms "treatment" and "treat" do not necessarily mean treating an individual until full recovery. The terms "treatment" and "treat" also refer to maintaining and/or promoting the health of an individual who is not suffering from a pathological condition, infection, disorder or disease but may be susceptible to the pathological condition, infection, disorder or disease . The terms "treatment" and "treat" are 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, health care professional, or other person.

As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for an individual, each unit containing a predetermined quantity of a composition disclosed herein, such amount as pharmaceutically acceptable The combination of diluents, carriers or vehicles is sufficient to produce the desired effect. The size of the unit dosage form depends on the particular compound employed, the efficacy to be achieved, and the pharmacokinetics associated with each compound in the host.

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

As used herein, the term "pharmaceutically acceptable" refers to a substance that does not elicit a substantially harmful allergic or immune response when administered to an individual.

The terms "food," "food product," and "food composition" mean a product or composition that is intended to be ingested by an animal and that provides at least one nutrient to the animal. Preferred specific examples of the food include at least one of proteins, carbohydrates, lipids, vitamins or minerals. Foodstuffs may include macronutrients and/or micronutrients.

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

As used in reference to a particular component, the terms "substantially no", "essentially free" or "substantially free" mean any of the components present 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%.

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

Within the present invention, the terms "immunize" or "vaccinate" are used interchangeably.

The term "isolated" means changed or removed from the natural state. For example, a protein that occurs naturally in a living animal is not "isolated", but the same protein that is partially or completely separated from all or some of its coexisting substances in its natural state is "isolated."

The term "peptide", "protein" or "polypeptide" refers to a polymer of amino acid residues covalently linked by peptide bonds. The terms "peptides", "proteins" or "polypeptides" as used herein may also refer to polymers of amino acids, one or more of which may be modified residues , such as artificial amino acid mimetics or synthetic amino acid residues. The terms "peptide", "protein" or "polypeptide" are used interchangeably.

The present invention generally relates to IgY compositions comprising purified polyclonal IgY immunoglobulins or antibodies from egg-laying poultry. Compositions can be diluted in sterile buffers, such as saline. The IgY composition can be used to prevent or treat coronavirus infection and/or reduce or eliminate symptoms of coronavirus infection.

Some specific examples of the coronavirus-specific IgY immunoglobulin component of the IgY composition are obtained by a method comprising collecting eggs from poultry previously actively vaccinated or immunized against coronavirus, and The antibody fraction was isolated from the egg yolk fraction. The egg-laying poultry is preferably domesticated poultry. The domesticated poultry is preferably domesticated chicken. Domesticated chickens can be raised for egg or meat production.

Some specific examples relate to a method of preparing a first composition or vaccine to be administered to egg-laying poultry. The first composition or vaccine may comprise a live or inactivated coronavirus, an adenovirus vector encoding a coronavirus antigen, and/or a composition comprising a coronavirus antigen or a coronavirus recombinant protein. The first composition or vaccine can be used to actively vaccinate or immunize egg-laying poultry against the coronavirus. Eggs can be collected from vaccinated or vaccinated laying poultry, and IgY immunoglobulins or antibodies (ie, antibody fractions) can then be extracted and purified from egg yolks ( Figure 1 ).

Some specific examples provide a method of preparing a first composition or vaccine comprising preparing a compound to be administered to egg-laying poultry for the purpose of immunizing or vaccinating poultry to produce coronavirus-specific IgY immunoglobulins or antibodies Coronavirus antigen or recombinant protein. The adenovirus vector can be encoded using the nucleotide sequence of the severe acute respiratory syndrome (Severe acute respiratory syndrome, SARS) coronavirus 2 spike protein (that is, the coronavirus spike protein gene; Genbank accession number NC_045512.2; SEQ ID NO: 1) Coronavirus antigens. Additionally or alternatively, the nucleotide sequence of the coronavirus spike protein gene without the transmembrane domain (ie, the extracellular domain of the SARS-Cov-2 spike protein) can be codon-optimized for expression in domestic chickens (Genscript ). Selected nucleotide sequences can be cloned into pCI (Promega) between the immediate early enhancer/promoter from cytomegalovirus (CMV) and the terminator/polyadenylation signal from SV40. Plastid DNA can be purified using the NoEndo JETSTAR plastid kit (Genomed, Germany) or any other method known to those of ordinary skill in the art. Purified DNA (ie, plastids) can be suspended in a suitable buffer (eg, PBS) at pH about 7.4. This purified DNA (ie, plastids) can be used to produce coronavirus antigens or recombinant coronavirus proteins, or additionally or alternatively, as described below, the purified DNA (ie, plastids) can be used as DNA Vaccines are given as booster or re-immunization of laying poultry.

For the production of coronavirus antigens or recombinant proteins, approximately 62 µg to approximately 250 µg of purified DNA (i.e., plastids) can be combined with Lipofectin transfection reagent (Life Technologies, USA) as recommended by the manufacturer, or the DNA can be Combine with any comparable transfection reagent. DNA can be sub-colonized into pFastBacgp67 transfer plastids (GenScript) or any other comparable method or reagent. The resulting baculovirus transfer vector can then be transformed into competent DH 10Bac recombinant E. coli cells to produce recombinant baculovirus plastid DNA (Bacmid DNA). Recombinant baculovirus plastids can be transferred to Sf9 insect cells (such as but not limited to Spodoptera frugiperda ) or other comparable cells using transfection reagents, and the expressed coronavirus recombinant proteins or Coronavirus antigens.

Another embodiment provides a method of preparing a first composition or vaccine comprising one or more adenoviral vectors encoding coronavirus antigens for the purpose of immunizing or vaccinating hens to produce coronavirus-specific immunoglobulins. Purpose, the first composition or vaccine is to be administered to hens or egg-laying poultry. Adenoviral vectors encoding coronavirus antigens can be generated using the nucleotide sequence of the SARS coronavirus 2 spike protein (SEQ ID NO: 1). The gene can be cloned into pShuttle/CMV plastids to allow homologous recombination with the plastid encoding the adenovirus backbone in BJ5183 E. coli. Recombinant adenoviral vectors can be produced by transfecting plastids containing the bioengineered adenoviral genome encoding the transgene into 293 cells. The resulting produced vector (AdV-S) can be purified and titrated manually according to AdEasy or similar methods known to those of ordinary skill in the art. Alternatively, adenoviral vectors can be constructed using any commercially available system, such as the Ad-HQ system developed by Vector Biolabs.

Another embodiment provides a method of preparing and storing a first composition or vaccine comprising a live or inactivated coronavirus, an adenovirus vector encoding a coronavirus antigen and/or comprising a coronavirus antigen Or a composition of recombinant coronavirus protein in an aliquot (eg, about 1 ml) with saline or an equivalent solution at a concentration suitable for vaccination or immunization of hens via intramuscular injection.

Another aspect of the invention generally relates to a method of making a first composition or vaccine comprising a live or inactivated coronavirus, an adenovirus vector encoding a coronavirus antigen and/or comprising Compositions of coronavirus antigens or recombinant proteins emulsified in aliquots with adjuvant.

Another embodiment provides a method of administering to egg-laying poultry a first composition or vaccine comprising a live or inactivated coronavirus, an adenovirus vector encoding a coronavirus antigen, and /or a composition comprising a coronavirus antigen or a coronavirus recombinant protein. The composition or coronavirus vaccine can be administered intramuscularly to laying poultry or hens. In some embodiments, at least a portion (optimally, all) of the first composition or coronavirus vaccine is injected into the breast of the hen, eg, a first portion in the left breast of the hen and a second portion in the right breast of the hen , as appropriate, in approximately equal amounts, such as about 500 mL into the right mid-chest and about 500 mL into the left mid-thorax. The hens can be re-vaccinated after the initial immunization (eg, about 7 days after the initial immunization and/or about 14 days after the initial immunization). The re-immunization composition may be the same composition or vaccine that was originally used to immunize the egg-laying poultry or hens, or the composition or vaccine used for the re-immunization may contain live or inactivated coronavirus Different compositions of viruses, adenovirus vectors encoding coronavirus antigens and/or compositions comprising coronavirus antigens or recombinant proteins. Preferably, the immunization or repeat immunization should occur within about 7 days to about 28 days relative to the previous immunization. After the initial immunization and any re-immunizations (e.g., about twenty-seven days after the initial immunization), eggs from vaccinated hens can be collected one or more days for purification of coronavirus-specific IgY antibodies . Alternatively, eggs can be collected continuously during the immunization period.

Additionally or alternatively, the re-immunization composition administered to egg-laying poultry may be an inactivated Newcastle disease virus expressing the anchored spike protein (NDV-S; SEQ ID NO: 1).

Some embodiments provide a method of collecting, isolating and purifying coronavirus-specific IgY from eggs laid by vaccinated or vaccinated hens. Coronavirus-specific IgY antibodies can be obtained from egg yolk or water-soluble fractions. One or more egg yolks can be collected and diluted about 10-fold with cold HCl (eg, about 3 mM HCl), resulting in a suspension of about pH 5 (adjusted with approximately 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 approximately 13,000 xg for approximately 15 minutes at approximately 4°C, and the IgY antibody-containing supernatant can be collected. IgY antibodies 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., Optimized Protocol of Chicken Antibody ( IgY ) Purification Providing Electrophoretically Homogenous Preparations , 8 Int. J. Electrochem. Sci. 113, 113-124 (2013)).

In some embodiments, the IgY purification further comprises collecting eggs from hens vaccinated or immunized against the coronavirus. The egg yolk portion can be separated and diluted with a solution such as water. Lipids can be separated from water-soluble proteins using gravity. The water-soluble fraction can be further concentrated using an ultrafiltration (UF) system. Water-soluble proteins and IgY antibodies can then be separated by ion exchange. Corona-specific IgY antibodies can be further isolated using coarser UF filtration.

In some embodiments, the coronavirus-specific IgY composition comprises, in part, egg yolk or any portion thereof containing an IgY antibody. The yolk is the preferred part of the egg because the yolk generally contains a higher concentration of IgY than the white. However, the protein may contain sufficient concentrations of IgY for some applications.

In some embodiments, IgY is concentrated, isolated or purified from egg components. This can be accomplished by a variety of methods, such as those known to those of ordinary skill in the art. See Schwarzkopf and Thiele, Effectivity of Different Methods for the Extraction and Purification of IgY , ALTEX 13, 35-39 (1996), which is incorporated herein by reference in its entirety. If desired, the potency of the IgY antibody can be determined by immunoassays, such as enzyme-linked immunosorbent assay (ELISA).

In some embodiments, the antibody composition is prepared by a method comprising: actively vaccinating hens against the coronavirus, collecting eggs from the hens after the immunization period, and isolating the antibody fraction from the yolk. In some embodiments, egg collection from the hens can be performed continuously after the immunization period.

Other methods of generating IgY with specific targets are known to those of ordinary skill in the art, but it is not known that these methods have been successfully used to generate antibodies against coronaviruses previously. The antibodies disclosed in this section are suitable for use in any of the methods and compositions described in the present invention.

Some specific examples relate to pharmaceutically acceptable compositions of coronavirus-specific IgY immunoglobulin compositions that can be administered to individuals exposed to, susceptible to, or infected with coronaviruses.

One aspect of the invention relates generally to a method of administering an effective amount of coronavirus-specific IgY immunoglobulin at a dose of about 0.25 mg/kg body weight to about 1 mg/kg body weight. The coronavirus-specific IgY immunoglobulin can be administered to an individual more than once.

In a specific example, such antibodies are in the form of compositions, such as, but not limited to, pharmaceutical compositions. The disclosed compositions may comprise one or more of such antibody or immunoglobulin compositions disclosed above and a pharmaceutically acceptable carrier. Examples of such carriers and formulation methods can be found in Remington: The Science and Practice of Pharmacy ( 20th Edition, Lippincott, Williams & Wilkins, Daniel Limmer, eds., which is incorporated herein by reference in its entirety). To form pharmaceutically acceptable compositions suitable for administration, such compositions will contain a therapeutically effective amount of the antibody. The therapeutically effective amount can be an adhesion-inhibiting effective amount.

The pharmaceutical composition of the present invention can be used in the treatment and prevention methods of the present invention. Such compositions are administered to humans in amounts sufficient to deliver a therapeutically effective amount of the antibody for use in the methods of treatment and prevention disclosed herein. A therapeutically effective amount may vary depending on various factors, such as, but not limited to, the individual's condition, weight, sex, and age. Other factors include the mode and site of administration. Pharmaceutical compositions can be provided to an individual by any method known in the art.

The compositions of the present invention may be administered to a subject once or more than once. Furthermore, when the composition is administered to an individual, more than once, preferably less than two months. Various regimens may be used, such as, but not limited to, once a day, once a week, once a month, or once a year, or any variation in between. The composition may also be administered to a subject more than once per day. A therapeutically effective amount of the composition, antibody, and appropriate dosing regimen can be identified by routine testing in order to obtain optimal activity while minimizing any potential side effects. In addition, co-administration or sequential administration of other agents may be required.

The compositions of the present invention can be administered systemically, such as by intranasal and/or oral administration ( Figure 2 ). Preferred routes of administration include intranasal and oral. In some embodiments, the composition is administered intranasally. In some embodiments, the composition is administered orally.

Oral administration methods for coronavirus-specific immunoglobulins additionally include, for example, oral administration of uncooked egg yolks or egg yolk fractions, either alone or in combination with egg whites. Oral administration of a raw egg yolk or egg yolk portion can be performed, for example, by eating the egg yolk portion. The egg yolk fraction or the water-soluble egg yolk fraction can be administered in combination with other ingredients to make it more palatable or nutritious. Thus, the egg yolk portion may be consumed by a subject/individual as a food product; alternatively, the egg yolk portion may be consumed as part of a pharmaceutical composition. It is preferably the uncooked or very lightly cooked egg yolk portion since cooking inactivates the antibodies.

In some embodiments, the water-soluble portion of the egg yolk can be readily mixed with any type of food product or any edible ingredient for oral administration. Compositions may also be formulated to contain or provide a portion of the macronutrient and micronutrient requirements for an individual, and may be provided as a replacement or supplement to an individual's regular diet. The composition may be provided as, added to, or mixed with: snacks, beverages, foods, or other supplements to supplement normal intake of foods.

The composition of the present invention may further comprise additional agents to improve the solubility, half-life, absorption rate, etc. of the antibody. In addition, the compositions of the present invention may further comprise agents that reduce unwanted side effects and/or reduce the toxicity of the antibody. Examples of such agents are described in various texts, such as, but not limited to, Remington: The Science and Practice of Pharmacy ( 20th Edition, Lippincott, Williams & Wilkins, Daniel Limmer, eds., incorporated herein by reference in its entirety.

The compositions of the present invention can be administered in a wide variety of dosage forms for administration. For example, compositions can be administered in forms such as, but not limited to, injectable solutions, lyophilized powders, and/or granules.

In some embodiments, 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/or carriers. In general, pharmaceutically acceptable carriers are chemically inert to the active antibody and have no deleterious side effects or toxicity under the conditions of use. Pharmaceutically acceptable carriers can include polymers and polymer matrices. The nature of the pharmaceutically acceptable carrier may vary depending upon other characteristics of the particular dosage form and composition employed.

For example, for oral administration in solid form such as, but not limited to, powders or granules, the antibody may be combined with oral non-toxic pharmaceutically acceptable inert carriers such as, but not limited to, inert fillers, suitable binders agent, lubricant, disintegrant and/or adjuvant) combination. Suitable binders include, for example, but not limited to, starch, gelatin, natural sugars (such as glucose or beta-lactose), corn sweeteners, natural and synthetic gums (such as acacia, astragalus, or sodium alginate), carboxymethyl cellulose vegans, polyethylene glycols, waxes and the like. Lubricants used in these dosage forms include, for example, but not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and the like. Disintegrants include, for example, but not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like.

Formulations suitable for intranasal or oral 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 individual, and may include suspending agents, enhancing agents Aqueous suspensions of solvents, thickeners, stabilizers and/or preservatives. Antibodies may be in physiologically acceptable diluents such as sterile liquids or liquid mixtures, including water, saline, aqueous dextrose and/or related sugar solutions, or any other solution known to those of ordinary skill in the art cast.

Oils that can be used in intranasal or oral formulations include, for example, but not limited to, petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, paraffin fat, and mineral oil. Fatty acids suitable for use in intranasal or oral formulations include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and ethylene oxide and by propylene oxide and propylene glycol, oleic acid, A high molecular weight adduct of a hydrophobic matrix formed by the condensation of stearic acid and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Soaps suitable for use in intranasal formulations include fatty alkali metal, ammonium and triethanolamine salts, and suitable cleansers include: (a) cationic cleansers such as, but not limited to, dimethyldialkylammonium halides and alkyl halides (b) anionic detergents, such as, but not limited to, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates; and sulfosuccinates, (c) ) non-ionic detergents such as but not limited to fatty amine oxides, fatty acid alkanolamides and polyoxyethylene polypropylene copolymers; (d) amphoteric detergents such as but not limited to alkyl beta-alanine and 2-alkylimidazoline quaternary ammonium salts; and (e) mixtures thereof.

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-lipophile balance (HLB) of about 12 to about 17.

The antibodies of the present invention can also be coupled with soluble polymers as targeted drug carriers. Such polymers may include, but are not limited to, polyvinylpyrrolidone, pyran copolymers, polyhydroxypropylmethacrylate-amidophenol, polyhydroxyethylaspartamine, or substituted with palmitate residues The polyethylene oxide polylysine (polyethyl-eneoxidepolylysine). In addition, the antibodies can be coupled with a class of biodegradable polymers suitable for achieving controlled release of drugs, such as polylactic acid, poly-epsilon caprolactone, polyhydroxybutyric acid, polyorthoester, polyacetal, polydimethy Crosslinked or amphiphilic block copolymers of hydropyran, polycyanoacrylates and hydrogels.

Pharmaceutical compositions can be modified to prevent adverse reactions in an individual. Such potential adverse reactions include host cognition, anaphylaxis, local inflammation, and other forms of anaphylaxis. Adverse reactions are more common in heterologous antibody therapy than in homologous antibody therapy, but the advantages of IgY antibodies have been explained in this regard. In some embodiments of pharmaceutical compositions, 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.

In some embodiments, the pharmaceutical formulations of the present invention can 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 pharmaceutically acceptable coronavirus-specific IgY immunoglobulin compositions preferably further comprise anti-inflammatory.

A non-limiting example of a pharmaceutically acceptable coronavirus-specific IgY immunoglobulin composition preferably further comprises an antigen-binding fragment of an antibody, such as a Fab or Fab2 fragment, which can replace the antibody. For example, an antigen-binding fragment can be any fragment that includes the antigen-binding region of the original IgY. In some embodiments of compositions and methods, a modified version of an IgY antibody can be substituted for an IgY antibody, so long as the antigen-binding region of the IgY antibody retains its ability to recognize coronaviruses.

Non-limiting examples of pharmaceutically acceptable coronavirus-specific IgY immunoglobulin compositions preferably further comprise lyophilized powder compositions, such as for long-term storage and/or delivery. The lyophilized composition can be reconstituted into a solution, such as normal saline, to about the original volume prior to use in immunization, vaccination or treatment.

As can be seen from Figure 4, IgY antibodies are useful as virus-neutralizing inhibitors of viral adhesion. IgY antibodies from poultry eggs are an inexpensive and abundant source of viral adhesion inhibitors. Such antibodies bind to the surface of an antigen-bearing virus, such as a coronavirus, 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 many applications, including the treatment of viral infections and the prevention of viral infections.

In some embodiments of the viral adhesion inhibitor, the inhibitor comprises a component of poultry eggs, wherein the poultry eggs comprise an adhesion-inhibiting effective amount of IgY specific for a coronavirus. The components of poultry eggs can be any of those described in the present invention as suitable antibody compositions.

Some methods are provided for preventing viral adhesion to cells. The first step in the infection of cells by the virus is the contact and adhesion between the virus and the cell. Although this step is critical for establishing infection, there are few ways to prevent infection at this early stage. More typically, viral infections are countered using techniques such as active vaccination, which cause the body to produce antibodies that neutralize the virus. When active vaccination is not feasible, the most common viral diseases are symptomatic treatment only. The methods described herein provide an effective means to prevent this early step in the infection process without requiring sufficient administration before an individual is exposed to the pathogen, as is required for active vaccination.

Antibodies can be used to prevent adhesion between viruses and cells (ie, inhibitors of viral adhesion) by binding to the virus and interfering with the virus' ability to bind to its target membrane receptor. Immunoglobulins from egg-laying poultry, such as IgY antibodies, have distinct advantages over the mammalian antibodies of the present application, especially when the individual is a mammal. As noted above, advantages of IgY antibodies include that IgY antibodies are more specific, more stable, and elicit less undesirable forms of immune response than mammalian antibodies. IgY antibodies are also readily and inexpensively obtained from eggs.

In some embodiments, the method comprises administering to the individual an amount of a coronavirus-specific immunoglobulin effective to inhibit and/or prevent adhesion between the coronavirus and the individual's cells. In some embodiments of the method, the coronavirus-specific immunoglobulin or viral adhesion inhibitor comprises a poultry egg component comprising an adhesion-inhibiting effective amount of IgY specific for a coronavirus. The ingredients can be any of those disclosed herein as suitable antibody compositions.

In some embodiments, the coronavirus-specific immunoglobulin composition is a medicament comprising the contents of poultry eggs comprising an effective amount of IgY specific for the coronavirus. The medicament can include additional components as discussed herein. Medicines can be administered by any method known in the art or as described herein.

Some specific examples provide a method for treating an individual exposed to, susceptible to, or infected with a coronavirus. The coronavirus-specific IgY immunoglobulin composition may include other components as pharmaceutical components discussed elsewhere in this disclosure. The coronavirus-specific IgY immunoglobulin composition can be administered orally and/or intranasally. Oral administration can prevent or reduce transmission of virus in the gastrointestinal tract or can eliminate virus from the gastrointestinal tract.

Such oral administration methods additionally include oral administration of uncooked egg yolks or egg yolk fractions, alone or in combination with egg whites. Oral administration of a raw egg yolk or egg yolk portion can be performed, for example, by eating the egg yolk portion. The egg yolk portion can be administered in combination with other ingredients to make it more palatable or nutritious. Thus, the egg yolk portion can be consumed by an individual as a food product; alternatively, the egg yolk portion can be consumed as part of a pharmaceutical composition. It is preferably the uncooked or very lightly cooked egg yolk portion since cooking inactivates the antibodies.

Non-limiting examples of methods of treatment include administration of increased doses of coronavirus-specific IgY immunoglobulin compositions intranasally, orally, or in combination, or alternatively, at increased doses and/or frequency.

Some embodiments provide a method of treating an individual infected with a coronavirus, the method comprising administering to the infected individual a concentration or dose of a coronavirus-specific IgY immunoglobulin composition that is effective in reducing, ameliorating or Removing symptoms associated with coronavirus infection has implications.

Some embodiments provide a method of treating an individual previously exposed to or otherwise susceptible to coronavirus infection, the method comprising administering to the infected individual a concentration or dose of a coronavirus-specific IgY immunoglobulin composition substance, the concentration or dose has an effect on preventing or reducing the severity of coronavirus infection.

The coronavirus-specific IgY immunoglobulin composition can be administered intranasally and/or orally. The coronavirus-specific IgY immunoglobulin composition can be administered to an individual more than once. Example

The following non-limiting examples support the concept of using pharmaceutically acceptable coronavirus vaccine compositions to generate antibodies for treating infected humans or preventing human infection. Example 1

Three groups of chickens (n=3/group) were immunized on days 1, 7 and 14 with either coronavirus or adenovirus antigenic compositions. Group 1 received saline as a control (no antigenic composition), group 2 received a primary immunization with either a coronavirus antigenic composition or an adenovirus antigenic composition, followed by a booster immunization, and group 3 received a coronavirus antigenic composition 3 immunizations of the animals. Following the second and third immunizations, blood samples were collected and antibody titers were assessed using antigen-coated ELISA plates. Example 1 demonstrates that the antigen-induced antibody pool is globally specific for coronavirus and adenovirus after 14 and 28 days.

Example 2

Thirty-week-old laying hens were immunized intramuscularly with an adenovirus-vectored vaccine encoding the transmembrane domain-free SARS coronavirus 2 spike protein (SEQ ID NO: 1). Two weeks later, they are boosted or re-vaccinated with the same vaccine. Two weeks after re-immunization, laying hens were further boosted with a DNA vaccine (ie plastid) of SEQ ID NO: 1 containing either the full-length ectodomain sequence or with the CMV promoter (DNA-S).

Eggs were collected 5 weeks after the first immunization. IgY immunoglobulins were isolated from pooled egg yolks and purified using a water dilution method followed by polyethylene glycol (PEG) precipitation.

The specificity of purified coronavirus-specific immunoglobulins was determined by ELISA, compared with unvaccinated control hens ( Figure 3 ), and virus neutralization activity was assessed using a virus neutralization assay, compared with unvaccinated control hens The vaccinated and control groups were compared ( Figure 4 ). As shown in Figure 4 , the lowest concentration of IgY observed for virus neutralization was 0.3 mg/ml. Example 3

A composition comprising coronavirus-specific IgY immunoglobulin can be provided to the individual. Compositions comprising coronavirus-specific IgY immunoglobulin can be administered intranasally and/or orally for the treatment of individuals infected with coronavirus ( Figure 2 ). The composition can be administered to the subject only once or more than once.

without

[ Fig. 1 ] Shows a specific example of a method of making a coronavirus vaccine composition, a specific example of a method of immunizing or vaccinating egg-laying poultry such as hens to produce coronavirus-specific IgY immunoglobulin , and a specific example of a method for collecting and purifying IgY immunoglobulin from eggs laid by vaccinated or vaccinated hens.

[ FIG. 2] Shows a specific example of a method of treating individuals exposed to, susceptible to, or infected with coronavirus.

[ Figure 3] Shows the specificity of coronavirus-specific IgY immunoglobulin from vaccinated or vaccinated hens compared to unvaccinated or control hens (egg IgY).

[ Fig. 4] Shows the coronavirus-specific IgY immunoglobulin (AdV -S-induced IgY) virus neutralizing activity.

Claims (19)

A method of treating a coronavirus infection in an individual suffering from the coronavirus, the method comprising: administering to egg-laying poultry a first composition comprising one or more adenoviral vectors encoding one or more coronavirus antigens; and The individual suffering from the coronavirus infection is provided with a second composition comprising a water-soluble fraction from egg yolk of the egg-laying poultry to which the first composition is administered. The method of claim 1, wherein the second composition is administered to the coronavirus-infected individual in an amount that provides the coronavirus-infected individual in an amount ranging from about 0.25 mg to about 1.0 mg of the water-soluble portion of the egg yolk per kilogram of body weight. Administered to an infected individual. The method of claim 1, wherein the second composition is administered intranasally. The method of claim 1, wherein the second composition is administered to the subject with the coronavirus infection more than once. The method of claim 1, wherein the first composition is administered to the egg-laying poultry more than once. The method of claim 5, wherein the first composition is administered a second time to the egg-laying poultry at least 14 days after the first administration of the first composition. The method of claim 1, wherein an additional composition is administered to the egg-laying fowl at least 14 days after the first administration of the first composition. The method of claim 7, wherein the additional composition comprises a live or inactivated coronavirus, an adenovirus vector encoding a coronavirus antigen, a coronavirus recombinant protein, and/or an inactivated Newcastle disease expressing an anchored spike protein Virus. A method of preventing, reducing the incidence and/or reducing the severity of coronavirus infection in an individual at risk of coronavirus infection, the method comprising: administering to egg-laying poultry a first composition comprising one or more adenoviral vectors encoding one or more coronavirus antigens; and A second composition is provided to the individual, the second composition comprising a water-soluble portion of egg yolk from the egg-laying poultry to which the first composition has been administered. The method of claim 9, wherein the second composition is administered to the individual in an amount that provides the individual with a dose of about 0.25 mg to about 1.0 mg of the water-soluble portion of the egg yolk per kilogram of body weight of the individual. The method of claim 9, wherein the second composition is administered intranasally. The method of claim 9, wherein the second composition is administered to the individual more than once. The method of claim 9, wherein the first composition is administered to the egg-laying poultry more than once. The method of claim 13, wherein the first composition is administered a second time to the egg-laying poultry at least 14 days after administration of the first composition. 9. The method of claim 9, wherein an additional composition is administered to the egg-laying fowl at least 14 days after administration of the first composition. The method of claim 15, wherein the additional composition comprises a live or inactivated coronavirus, an adenovirus vector encoding a coronavirus antigen, a coronavirus recombinant protein, and/or an inactivated Newcastle disease expressing an anchored spike protein Virus. A method of producing an intranasally administrable composition effective for treating, preventing, reducing the incidence and/or reducing the severity of a coronavirus infection in an individual infected with a coronavirus, the method comprising applying a The composition is formed by adding a water-soluble portion of egg yolk from egg-laying poultry administered with one or more adenoviral vectors encoding one or more coronavirus antigens to the solution. A unit dosage form comprising a therapeutically or prophylactically effective amount of a composition comprising a water-soluble portion of egg yolk that has been administered from a lay egg encoding one or more adenovirus vectors for one or more coronavirus antigens obtained from poultry. An immunoglobulin specific for coronavirus for use in the treatment and/or prevention of coronavirus infection in individuals at risk of coronavirus infection.

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