WO2022224271A1

WO2022224271A1 - Polyclonal antibodies against sars-cov-2 and implementations thereof

Info

Publication number: WO2022224271A1
Application number: PCT/IN2022/050371
Authority: WO
Inventors: Aldon FERNANDES; John Kaundinya; Kripa MURZELLO
Original assignee: Bharat Serums And Vaccines Limited
Priority date: 2021-04-19
Filing date: 2022-04-18
Publication date: 2022-10-27
Also published as: AR125373A1

Abstract

The present invention discloses polyclonal antibody F(ab')2 fragments against spike protein or variants thereof of SARS-CoV-2 and a process for obtaining them from hyperimmunized equines. The disclosed polyclonal antibody F(ab')2 fragments exhibit better neutralizing activity against different SARS-CoV-2 RBD variants and can be used for the treatment of coronavirus disease in a subject.

Description

POLYCLONAL ANTIBODIES AGAINST SARS-COV-2 AND

IMPLEMENTATIONS THEREOF

FIELD OF INVENTION

[001] The present disclosure broadly relates to the field of immunology and vaccines and particularly refers to a process for obtaining polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2.

BACKGROUND OF INVENTION

[002] Coronaviruses have caused three large-scale outbreaks over the past two decades: severe acute respiratory syndrome (SARS), Middle Eastern respiratory syndrome (MERS), and now COVID- 19. SARS-CoV-2, is the causative agent of coronavirus disease 2019 (COVID-19) and currently causing a global pandemic. Currently, worldwide approximately 105 million people have been infected, with over 2 million deaths and numbers are increasing daily ( Covidl9.who.int. 2021. WHO Coronavirus Disease (COVID-19) Dashboard, [online] Available at: <https://covidl9.who.int/> [Accessed 10 February 2021]). To find solutions to contain this raging pandemic, global research efforts have been quickly mobilized such as the access to COVID-19 Tools (ACT) Accelerator, a ground-breaking global collaboration to accelerate development, production, and equitable access to COVID- 19 tests, treatments, and vaccines.

[003] There are currently more than 50 COVID- 19 vaccine candidates in trials and vaccine candidates by Pfizer-BioNTech, Modema, AstraZeneca-Oxford and Bharath Biotech have received emergency approval in multiple countries. These candidates include various type of possible ways to target the virus such as live attenuated or inactivated virus, DNA and mRNA vaccines, viral vectors expressing SARS-CoV-2 genes, and recombinant proteins or protein fragments. Despite these multiple efforts, finding, producing and distributing a safe, cheap and efficacious vaccine to protect those most in need, worldwide, remains a formidable challenge. SUMMARY OF THE INVENTION

[004] In an aspect of the present disclosure, there is provided a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2.

[005] In another aspect of the present disclosure, there is provided a method for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, wherein said method comprises obtaining at least one biological sample from an equine immunized with spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2; collecting and purifying the polyclonal antibody F(ab')₂ fragments from said biological sample.

[006] In another aspect of the present disclosure, there is provided a polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2 obtained by the process as described herein.

[007] In another aspect of the present disclosure, there is provided polyclonal antibody F(ab')₂ fragments for use in the treatment of a coronavirus disease in a subject in need thereof. [008] In another aspect of the present disclosure, there is provided a composition comprising: (a) polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2; (b) at least one excipient; and (c) at least one adjuvant, wherein said polyclonal antibody F(ab')₂ fragments are obtained from a process as described herein.

[009] In another aspect of the present disclosure, there is provided a method for the treatment of a coronavirus disease in a subject in need thereof, comprising administering to said subject the polyclonal antibody F(ab')₂ fragment or the composition as described hereinabove.

[0010] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0011] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0012] Figure 1(A) depicts the representative structure of severe acute respiratory syndrome coronavirus 2 and its components and Figure 1(B) depicts the representative spike protein on the surface of the severe acute respiratory syndrome coronavirus 2, comprising receptor binding domain (RBD) and S1 and S2 glycoprotein, in accordance with an embodiment of the present disclosure.

[0013] Figure 2 depicts the magnified and detailed structure of the spike protein in severe acute respiratory syndrome coronavirus 2, in accordance with an embodiment of the present disclosure.

[0014] Figure 3 depicts the structure of the gene encoding various components of the spike protein in accordance with an embodiment of the present disclosure. [0015] Figure 4 depicts the schematic representation and the structure of the spike protein of severe acute respiratory syndrome coronavirus 2 comprising wherein ; (a) the overall topology of the spike protein monomer wherein FP refers to fusion peptide; HR1 refers to heptad repeat 1; HR2 refers to heptad repeat 2; IC refers to intracellular domain; NTD refers to N-terminal domain; SD1 refers to subdomain 1; SD2 refers to subdomain 2; TM refers to transmembrane region; (b) the secondary structure of the spike protein with representative a helix and β sheets corresponding to the amino acid sequence of the spike protein, the receptor binding motif (RBM) sequence is shown in red; and (c) the quaternary structures of the receptor binding domain (RBD) bound to angiotensin converting enzyme 2 (ACE2), in accordance with an embodiment of the present disclosure. In Figure 4(c), ACE2 is shown in green, the RBD core is shown in cyan and RBM is shown in red, the disulfide bonds are shown as sticks and indicated by arrows and the N-terminal helix of ACE2 responsible for binding is labelled.

[0016] Figure 5 is a schematic representation of the process of obtaining purified equine anti-serum containing polyclonal antibody against severe acute respiratory syndrome coronavirus 2, in accordance with an embodiment of the present disclosure. [0017] Figure 6 depicts the neutralizing activity of the equine fragment F(ab')2 polyclonal antibody against different S ARS-CoV-2 RBD variants, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions

[0019] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0020] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0021] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0022] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

Sequences:

[0023] SEQ ID NO: 1 depicts the amino acid sequence encoding spike protein registered as uniprot accession number P0DTC2

[0024] The SEQ ID NO: 1 hereinabove represents the sequence of the entire spike protein. The sequence can be segregated as below:

SEQ ID NO: 2: represents the ECD region of the spike protein

I

SEQ ID NO: 3: represents the RBD region of the spike protein

SEQ ID NO: 4: represents the SI region of the spike protein

[0025] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0026] For the purposes of the present document, the term “SARS-CoV-2” refers to severe acute respiratory syndrome coronavirus 2. The term “ COVID- 19” refers to coronavirus diseases 2019.

[0027] The term “vaccine candidate” refers to a protein or polypeptide fragment that can be potentially used in a vaccine composition.

[0028] The term “polyclonal antibody” refers to a collection of immunoglobulin molecules released by the B cells in the mammalian body that react against a specific antigen, each identifying a different epitope of the antigen. For the purposes of the present invention, the term “polyclonal antibody” and “polyclonal antibody F(ab')₂ fragment” have been used interchangeably.

[0029] The term “spike protein” refers to the S glycoprotein or the variants of the S glycoprotein on the surface of the severe acute respiratory syndrome coronavirus 2, the causative agent of COVID 19 and can be used to incite a humoral immune response. The variants include:

[0030] The term “RBD” or “receptor binding domain”, is a polypeptide domain of the spike protein of severe acute respiratory syndrome coronavirus 2. It is also a poorly conserved polypeptide domain between SARS -coronaviruses and other pathogenic human coronaviruses. In the present disclosure, “RBD” and “receptor binding domain” have been used interchangeably. In the present disclosure, the RBD is represented by SEQ ID NO: 3

[0031] The term “subject” as used herein refers to any vertebrate animal and does not merely cover equines. Equine subjects have been used to exemplify the invention but, said exemplification should not be considered in any way limiting to the scope of the subject matter as covered under the term subject. Other possible subjects include sheep, camel, llama, and chicken.

[0032] Current approaches for producing a vaccine against SARS-CoV-2 suffer from various problems as described below.

[0033] Messenger RNA (mRNA) vaccines: In this approach, a formulation of the mRNA encoding the antigens of interest is used. The mRNA which is a highly charged molecule has to be delivered into the target cells. For targeted delivery, the mRNA molecule is delivered in liposomes or by coating the mRNA molecule with a lipid molecule. The mRNA molecule has to be translated into protein and then be either exported outside the cell or processed inside the cell to stimulate humoral or cellular immunity respectively. Pfizer/BioNTech and Modema have formulated mRNA vaccine candidates that have received regulatory approval in many regions and are currently being administered. However, scaling up and effective distribution remain a primary concern. The vaccine released by Pfizer/BioNTech further needs to be maintained at low temperatures due to un-stability at room temperature. Other drawbacks include the requirement of two doses for effective immune response and the high amount of vaccine in each dose.

[0034] DNA vaccines: In this approach, instead of mRNA, DNA is used for preparing the composition. Similar to the mRNA, the DNA has to enter the cell nucleus, undergo transcription and translation to yield the antigens of interest. While this approach works well in mice, immunogenicity in humans for DNA vaccines is typically not very high and there is a small but non-zero chance of genomic integration. There is also currently no DNA vaccine that has been approved for human use. However, ZyCov-D a DNA vaccine candidate developed by Zydus Cadila has been approved to proceed to Phase HI trial.

[0035] Viral vectors: In this approach, the gene(s) of interest are incorporated into a non-pathogenic virus capable of infecting cells. This may be either a replicating or non-replicating vector, typically the latter are preferred. Upon infection the genetic material is replicated, and any encoded protein antigens are expressed as with the mRNA and DNA vaccines discussed above. An advantage with this approach is that viral infection is very efficient, the disadvantage is that anti- vector immunity arises rapidly and so only a limited number of boosting immunizations are possible. Currently the Oxford/Astrazeneca vaccine candidate is using adenoviral vectors using this approach, however due to the rise of more infectious variants of SARS COV 2, the vaccine may not prove as effective.

[0036] Live attenuated virus: In this approach, an attenuated (weakened) form of the virus is used. In the case of SARS-CoV-2, a process called codon-deoptimization is being used to generate such a weakened virus. This process takes time and extensive safety testing will be required for a highly pathogenic, novel virus such as in the present instance. [0037] Inactivated virus: This is standard methodology for many vaccines. However, large amounts of pathogenic virus may need to be handled and some earlier studies with S ARS-CoV have suggested the possibility of immune enhancement of infection when the inactivated virus was used as a vaccine modality. For example, Bharat Biotech.

[0038] Further, there are several reports on the use of convalescent plasma or purified immunoglobulin for emerging viral infections in a scenario when there is no drug or vaccine is available. The administration of such plasma is fraught with risks due to the risks associated with the viral safety and may also preclude delivery of an effective dose of specific viral antibodies. Scale up of convalescent plasma is impractical due to the lack of sufficient number of high titre donors. Therefore, to overcome the problems associated with the conventional approaches as discussed above, the present disclosure provides a process of obtaining polyclonal antibodies and in particularly provides equine polyclonal antibody F(ab')₂ fragments.

[0039] The polyclonal antibody F(ab')₂ fragment that can be raised in equines are obtained by the process of the present disclosure. Polyclonal antibodies recognize multiple epitopes of the spike S glycoprotein of the severe acute respiratory syndrome, the causative agent of COVID 19 and can be used to incite a humoral immune response. The polyclonal antibody F(ab')₂ fragments obtained by the process of the present disclosure recognize multiple epitopes, whereas monoclonal antibodies only recognize one epitope. The polyclonal antibodies obtained by the method of the present disclosure have also proved to be highly cost-efficient as compared to monoclonal antibodies. Another disadvantage of using monoclonal antibodies is their susceptibility to variants if there are changes in the epitopes of the antigen.

[0040] According to the process of the present disclosure, the polyclonal antibodies or equine anti-serum are obtained within a very short period of time. The equines can subsequently produce very high titers against the target antigen by administration of booster doses. The dosage for every batch can be standardized resulting in batch-to- batch consistency of potency.

[0041] The present disclosure also described the manufacturing process for production of purified and concentrated therapeutic polyclonal antibody F(ab')₂ fragment from hyperimmunized equine plasma. In the present disclosure, nanofiltration, was used to minimise the risk of any other zoonotic transmissions. The filtration method is used for obtaining the purified and concentrated therapeutic polyclonal antibody F(ab')₂ fragments from hyperimmunized equine plasma. The results demonstrate consistency in the quality and safety of such equine products. With the process of the present disclosure, the production of equine polyclonal antibodies can be scaled up very rapidly.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0043] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0044] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood;-e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2. In one of the embodiments, the spike protein or variants thereof is at least 250 contiguous amino acid residues from SEQ ID NO: 1. In another embodiment, the spike protein or variants thereof is at least 270 contiguous amino acid residues from SEQ ID NO: 1. In yet another embodiment, the spike protein or variants thereof is at least 272 contiguous amino acid residues from SEQ ID NO: 1.

[0045] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2 as described herein, wherein the spike protein has an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the spike protein has an amino acid sequence with at least 92% identity to the amino acid sequence of SEQ ID NO: 1. In yet another embodiment, the spike protein has an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In one of the embodiments, the spike protein has an amino acid sequence with at least 98% identity to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the spike protein has an amino acid sequence with at least 99% identity to the amino acid sequence of SEQ ID NO: 1. In yet another embodiment, the spike protein has an amino acid sequence as set forth in SEQ ID NO: 1.

[0046] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 22-29 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2. In another embodiment of the present disclosure, immunizing the equine with one or more booster dose for a time period in the range of 23-28 days, or 24-27 days, or 25-26 days.

[0047] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-13 days after each booster dose, and isolating plasma from the blood;-e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2. In another embodiment, SEQ ID NO: 1 comprises SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4 and SEQ ID NO:5.

[0048] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein the adjuvant is selected from the group consisting of freund's complete adjuvant (FCA), freund's incomplete adjuvant (FIA), carbopol based adjuvant, aluminium based adjuvants, oil-based adjuvants, surfactant adjuvants, micro emulsions, and immunostimulatory adjuvants. [0049] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti- serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein purifying the equine anti-serum mixture by chromatography selected from the group consisting of anion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, and cation exchange chromatography. In another embodiment of the present disclosure, purifying the equine anti-serum mixture is done by anion exchange chromatography.

[0050] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein purifying the equine anti- serum mixture by chromatography selected from the group consisting of anion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, and cation exchange chromatography, and wherein the diafiltration is performed using tangential flow filtration.

[0051] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibodies against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein immunizing the equine with at least three immune booster doses.

[0052] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibodies against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein immunizing the equine with three to five immune booster doses.

[0053] In an embodiment of the present disclosure, there is provided a a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein immunizing the equine with one or more booster dose in the range of 100-550μg. In another embodiment of the present disclosure, immunizing the equine with one or more booster dose in the range of 150-250μg. In yet another embodiment of the present disclosure, immunizing the equine with one or more booster dose in the range of 180-230μg. In one another embodiment of the present disclosure, immunizing the equine with one or more booster dose in the range of 190-210μg.

[0054] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein the spike protein is a homo- trimeric class I fusion protein that allows a viral membrane of severe acute respiratory syndrome coronavirus 2, to fuse with a host cell membrane.

[0055] In an embodiment of the present disclosure, there is provided a a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein the spike protein is a sequence deposited at uniprot under accession number P0DTC2 having amino acid sequence as set forth in SEQ ID NO: 1.

[0056] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein the protease is pepsin.

[0057] In an embodiment of the present disclosure, there is provided a a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein the polyclonal antibody F(ab')₂ fragments has a molecular weight in the range of 100-120 kDa.

[0058] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster for a time period in the range of 21- 30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein the polyclonal antibody F(ab')₂ fragments have a titre value in the range of 1: 90,000-1:120,000. In another embodiment the polyclonal antibodies have a titre value of 1:100,000.

[0059] In an embodiment of the present disclosure, there is provided a process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein the spike protein has an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In another embodiment of the present disclosure, the spike protein has an amino acid sequence with 90-99% identity, or 92-98% identity, or 94-97% identity, or 95-96% identity, to the amino acid sequence of SEQ ID NO: 1.

[0060] In an embodiment of the present disclosure, there is provided a method for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, wherein said method comprises: (a) obtaining at least one biological sample from an equine immunized with spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2; (b) collecting and purifying the polyclonal antibody F(ab')₂ fragments from said biological sample.

[0061] In an embodiment of the present disclosure, there is provided a polyclonal antibody F(ab')₂ fragment against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2 obtained by the process as described herein.

[0062] In an embodiment of the present disclosure, there is provided a composition comprising polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2; and at least one excipient, wherein said polyclonal antibodies are obtained from a process, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1 ; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2.

[0063] In an embodiment of the present disclosure, there is provided a composition comprising polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2; and at least one excipient, wherein said polyclonal antibody F(ab')₂ fragments are obtained from a process, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein the spike protein is a homo-trimeric class I fusion protein that allows a viral membrane of severe acute respiratory syndrome coronavirus 2, to fuse with a host cell membrane.

[0064] In an embodiment of the present disclosure, there is provided a composition comprising polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2; and at least one excipient, wherein said polyclonal antibody F(ab')₂ fragments are obtained from a process, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein the adjuvant is selected from the group consisting of freund's complete adjuvant (FCA), freund's incomplete adjuvant (FIA), carbopol based adjuvant, aluminium based adjuvants, oil-based adjuvants, surfactant adjuvants, micro emulsions, and immunostimulatory adjuvants.

[0065] In an embodiment of the present disclosure, there is provided a composition comprising polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2; and at least one excipient, wherein said polyclonal antibodies are obtained from a process, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1 ; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein the excipient is selected from the group consisting of sodium chloride, and glycine.

[0066] In an embodiment of the present disclosure, there is provided a composition comprising polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2; and at least one excipient, wherein said polyclonal antibody F(ab')₂ fragments are obtained from a process, said process comprising: a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1; b) immunizing an equine with the antigen formulation; c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days; d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood; e) treating the plasma with saline to obtain a treated plasma; f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma; g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture; h) centrifuging and filtering the equine anti-serum mixture using diafiltration; i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2, wherein the polyclonal antibody against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2 is in the range of 20-30mg/mL. In another embodiment of the present disclosure the polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2 is 25 mg/mL.

[0067] In an embodiment of the present disclosure, there is provided a polyclonal antibody F(ab')₂ fragment as described herein for use in the treatment of a coronavirus disease in a subject in need thereof.

[0068] In an embodiment of the present disclosure, there is provided a method for the treatment of a coronavirus disease in a subject in need thereof, comprising administering to said subject the polyclonal antibody F(ab')₂ fragment specific for the coronavirus.

[0069] In an embodiment of the present disclosure, there is provided a method for the treatment of a coronavirus disease in a subject in need thereof, comprising administering to said subject the composition as described herein.

[0070] In an embodiment of the present disclosure, there is provided a composition as described herein for use in treatment of a coronavirus disease in a subject in need thereof.

[0071] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the specification.

[0072] EXAMPLES

[0073] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Example 1

Selection, transportation, and housing of Eouines

[0074] Locally available non-descript ponies were selected. The physical eligibility criteria for the selected equines were determined based on a physical examination wherein the selected healthy equines were male, in the age bracket of 5 to 8 years, weighed at least 150kg and were at least 115cm tall. The selected equines were screened using a diagnostic blood test to check for complete blood count, liver and kidney function tests and test for diagnosis of glander, equine infectious anaemia, strangles, trypanosomiasis, and babesiosis. The selected equines were transported to the farm.

[0075] The selected animals were transported to the farm in animal transport vehicles by maintaining proper restraining conditions and grass bedding. Adequate feed and water were arranged so as to make the transportation process comfortable for the equines.

[0076] Post transportation, the animals were checked and isolated in a designated quarantine area for a minimum of 21 days. During the quarantine period, the animals were kept under observation by monitoring and recording the health parameters and given prophylactic vaccination for trypanosomiasis, tetanus and rabies. During quarantine period, the animals were tested for various diseases and dewormed by proper anthelmintic, and their stool samples were checked for worm load.

Example 2

Hyper Immunization of the Eouines against severe acute respiratory syndrome coronavirus 2 [0077] The region around the neck was selected as the immunization site and was washed with sufficient amount of water, shaved and disinfected. Disposable syringes and needles were used to avoid contamination.

(a) Primary Immunization:

[0078] The “spike protein” comprises multiple epitopes of the spike S glycoprotein of the severe acute respiratory syndrome coronavirus 2, the causative agent of COVID 19 and can be used to incite a humoral immune response. The diagrammatic representation of the structure of severe acute respiratory syndrome coronavirus 2, and the corresponding spike protein on its surface have been depicted in Figure 1. Further, the spike protein and the components of the spike protein as well the gene arrangement of the spike protein structure are depicted in Figure 2 and Figure 3 of the present disclosure (Lan et al., 2020. Structure of the SARS-CoV-2 spike receptor- binding domain bound to theACE2 receptor. Nature, 581(7807), pp.215-220). It can include variants of the spike protein as well.

[0079] The animals were immunized as per the schedule disclosed in Table 1. As shown in Table 1, during the primary immunization, the animals were immunized with an antigenic formulation, wherein the antigen formulation comprises spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, having the amino acid sequence as set forth in SEQ ID NO: 1, and at least one adjuvant. After immunization, the equines were kept under observation for any untoward reactions for a period of 72 hours. If any of the subjects displayed symptoms such as high temperature, sweating, salivation and respiratory distress, they were immediately treated.

[0080] Table 1

[0081] As indicated in Table 1, post immunization, the equine was given a rest for

21 days. A booster dose was administered post 21 days (time range of 21-30 days) and the equine was bled to perform subsequent analysis. Approximately 10ml of blood (Test Bleed) was collected from each immunized animal at periodic intervals in order to monitor the titre. The titres were assessed using ELISA and based on the results, the animals were either taken up for additional boosters or for blood collection or plasmapheresis. The selected animals were screened based on physical health and haematological test results. Equines with hematocrit or packed cell volume less than 30% and haemoglobin less than 10g/dL were not considered for blood collection.

(b) Blood Collection:

[0082] Prior to blood collection, health parameters such as temperature, respiration and heart rate were monitored and if the results were in the normal range, the bleeding of animal was commenced. The animal was bled by venepuncture of the jugular vein using sterilized bleeding sets. The area near the site was thoroughly cleaned shaved and disinfected before bleeding.

[0083] The blood was collected in sterilized and graduated blood collection bottles filled with anticoagulant citrate-dextrose solution. The bottle was labelled with the identifiers such as designated ID number of the selected equine, date, and signature of the veterinarian. During bleeding a constant flow of blood was ensured, and the bottles were shaken gently to ensure proper mixing of the collected blood with anticoagulant citrate-dextrose solution. The total time for blood collection was approximately 10 to 15 minutes per animal and varied depending upon the volume of blood to be collected. The quantity of blood collected was 1.5 % of the body weight (example for an equine having body weight 200kg - blood collected was 2.5 Litres). The equine was monitored during the blood collection process and the process was discontinued in case the animal showed any signs of discomfort.

[0084] Post blood collection as per the schedule indicated in Table 1, the needle was withdrawn, and continuous pressure was applied immediately to the puncture site till the blood flow stopped. Heparin was applied to prevent the inflammation of the jugular vein. The equines were kept under observation for at least 24 hours post blood collection. The sterilized and graduated blood collection bottles were cleaned properly using 70% isopropyl alcohol and then transferred to cold room at 2 to 8° C for at least 6 hours and not more than 24 hours to facilitate settling of blood cells and separation from plasma. [0085] The stored blood bottles were transferred for plasma separation through dynamic pass box. The plasma separation from whole blood was performed using sterilized separation sets, under a laminar air flow unit with grade C background. A vacuum pump was used for suction of the separated plasma and the plasma was collected aseptically in sterilized tanks and labelled with the plasma lot number, group number of animals bled and the date of plasma separation. The collected plasma was transported at 2 to 8°C to Ambemath Factory.

[0086] Post plasma separation, the blood cells were reconstituted with normal saline injection to compensate the volume of plasma removed. The bottles were then transferred to the water bath with shaker until the temperature of the blood was 37°C. After attaining the temperature, the bottles were transferred through pass box to the infusion area for re-infusion of blood cells in equines.

(c) Re-infusion of blood cells:

[0087] The health parameters of the equines such as temperature, respiration and heart rate were monitored and if they were in the normal range re-infusion as commenced. The blood cells were re-infused into the animal through venepuncture using sterilized transfer sets after ensuring that the ID number of the animal matched the ID number of the blood collection bottle. During the re-infusion process, the equine was continuously monitored, and the process was discontinued if any equine displayed signs of discomfort. Post re-infusion, the needle was withdrawn, and continuous pressure was applied to the puncture site till the blood flow stopped. Heparin sodium was applied at the jugular vein to prevent inflammation. Post re- infusion the animals were kept under observation for at least 48 hours. The details and anomalies if any during the process of immunization, blood collection and re- infusion were recorded.

[0088] Quality checks were performed on the recorded data using the soundness of examination record, quarantine record, diagnostic test reports, livestock register with animal details, prophylactic vaccination details, weighing record, grooming record, hoof care record, immunization, bleeding and re-infusion (plasmapheresis) record for individual animal, post bleeding and re-infusion (plasmapheresis) health monitoring record, master plasma record, sick animal treatment record, periodic blood testing reports, cleaning of stables record and approval from committee for the purpose of control and supervision of experiments on animals (CPCSEA) and institutional animal ethics committee (IAEC). Periodic assessments of all farm related documents was performed to ensure smooth functioning of the farms.

Results:

[0089] A total of 19 equines were immunized as shown in Table 2. Among the 19, 3 equines were immunized with vaccine against the spike protein, 4 were immunized with spike protein against severe acute respiratory syndrome coronavirus 2 from GenScript and 12 animals were immunized with antigen comprising a spike protein or variant thereof of SARS COV 2. As shown in Table 2, two assays were conducted to determine the viral quantification referred to the UTMB neutralization assay also referred to as micro neutralization assay and ELISA (ePass™ kit).

[0090] In the micro neutralization assay, the number of infectious virus particles was quantified by using the Median Tissue Culture Infectious Dose (TCID 50) assay. The Vero E6 cell monolayer was initiated by seeding to a 96-well culture plate with a well-characterized, low-passage cell stock of 2 x 10⁴ cells per well. Cells were incubated at 37 °C in 5% CO₂ incubator for 16-24 hours or until cell confluency of at least 90% was achieved. The serum samples were initially diluted in the ratio 1:25 (1mg/mL) in dilution medium (Minimal Essential Medium/2% heat-inactivated fetal bovine serum [HI-FBS]), up to 1: 54,675 following 3-fold serial dilution. The viral stock was added at two virus concentrations: 1,000 and 10,000 TCID50/mL (100 and 1,000 TCID50/sample well, respectively). One of the samples was tested against 1,000 TCID50/mL (100 TCID50 per sample well) only. The serum-virus sample (0.1 mL per well) was overlayed on the monolayer, each plate having a separate negative and positive control. Culture plates were incubated at 37°C in 5% CO₂ incubator for 72 hours after which cytopathic effect (CPE) was observed microscopically. The results were used to calculate the 50% neutralization titre and concentration as described Reed, L. And Muench, H., 1938. A Simple Method Of Estimating Fifty Per Cent Endpointsll. American Journal Of Epidemiology, 27(3), Pp.493-497.

[0091] In the second assay, ELISA was performed using the ePass™ technology for the rapid detection of total neutralizing antibodies in a sample by mimicking the interaction between the virus and the host cell. The test was performed using SARS- CoV-2 surrogate virus neutralization test kit from GenScript, USA. In a separate tube, the positive and negative control diluted with sample dilution buffer (1:10) and the test samples were diluted separately. The diluted controls and test samples were then mixed in equal volume with HRP-RBD solution, further diluted with HRP dilution buffer and incubated at 37°C for 30 minutes. These mixtures were separately added (100 μL) in the corresponding wells of the 96-well capture plates. The plates were incubated at 37°C for 15 minutes and washed four times with IxWash Solution.

Further, 100 μL of TMB solution was added in each well of each plate and incubated for 15 at 20-25°C in dark. The reaction was quenched using stop solution (50 μL) and the results were obtained at 450 nm in ELISA plate reader. The IC₅₀ (50% Inhibitory Concentration) was calculated to evaluate the neutralizing antibody potency.

[0092] The results of the receptor binding domain to ePass™ ratio for the two clinical batches, an average of the two batches results were used for calculating the final ratio of neutralising antibodies to the total number of polyclonal antibodies of 1:10.

[0093] Table 2

[0094] In Table 2 RBD titre indicates the total amount of antibodies present in the sample. The microneutralization assay and ELISA were used to determine the amount of neutralising Antibodies in the sample. Post the determination of the two values, the ratio of neutralising antibodies to total antibodies was calculated from the two data sets.

[0095] The neutralising antibodies are virus specific and play a key role in the effectiveness of convalescent plasma by reduction in viral replication and increasing the viral clearance. The neutralising antibodies determined using the results in Table 2 recognizes the receptor-binding domain on the spike protein of severe acute respiratory syndrome coronavirus 2 and blocks the entry of the virus. The neutralising antibodies specific to spike proteins or variants thereof may contribute to the functional neutralisation of the virus.

[0096] In humans, it has been observed that only 3% of the total antibodies produced by immune responses are neutralizing antibodies. As indicated in Table 2, equines immunized with a vaccine against spike protein of severe acute respiratory syndrome coronavirus 2, the GenScript Antigen and the antigen comprising a spike protein or variants thereof of SARS COV 2, it was seen that the ratio of neutralizing antibodies to total antibodies ranged from 8% to 25%.

[0097] Further, it has been observed that equines immunized with a vaccine against spike protein, showed a very low titre value of receptor binding domain as well as neutralization antibodies. In contrast, equines immunized with the antigen from GenScript and antigen comprising a spike protein or variants thereof of SARS COV 2, showed a high titre value of total polyclonal antibody F(ab')₂ fragments against the receptor binding domain of the spike protein as well as and virus specific neutralizing antibodies.

Example 3

Purification of protein

[0098] The protein purification process has been clearly depicted in the flow chart in Figure 5. As per Figure 5, post re-infusion, the equine anti-serum plasma from different animals were pooled to reach a standardized batch size. The equine anti- serum plasma was diluted using saline to obtain the treated plasma. The treated plasma was further subjected to enzymatic digestion which involved the addition of pepsin (protease). The enzymatic digestion of pepsin on the anti-serum results in the cleavage of the immunoglobulin G (IgG) below the hinge region, present in the anti- serum, that further results in the formation of two fragments. The resulting fragments were Fc fragment of 50 kDa and F(ab')₂ fragment of 100 kDa and F(ab')₂ fragment retained the original specificity of the parent immunoglobulin, eliciting a lower immunogenic response.

[0099] The pepsin digested plasma was purified using a precipitation step by an octanoic acid such as caprylic acid. Saturated fatty acids such as octanoic acid and its salts act as preservatives in the production of human biological products such as human albumin. The simple precipitation step allowed the immunoglobulin (IgG) and its fragments to remain in liquid phase and precipitated most of the plasma proteins thereby enhancing the purity and recovery. The resulting fractionated equine anti- serum mixture contains immunoglobulin fragments which were superior in terms of yield, turbidity, protein aggregate and potency.

[00100] The fractionated equine anti-serum mixture was centrifuged at 25- 26 °C at 800 rpm for 3 to 4 hrs and the post centrifuge clarified product was dialyzed and concentrated using a tangential flow filtration step. A polyethylene sulfone 50 KDa membrane was used for the tangential flow filtration, which retained the equine antibodies against severe acute respiratory syndrome coronavirus 2, while permitting passage of low molecular weight proteins and octanoic acid to permeate. The resultant filtrate was free from low molecular weight proteins and octanoic acid and the concentration step minimized the volume load on the next chromatographic polishing step. For further purification of the filtrate, the filtrate was subjected to anion exchange chromatography followed by nanofiltration using commercially available membranes such as hollow fibre type, tubular type, capillary type, flat sheet type, spirally wound that were available from manufacturers such as Asahi Kasei, Merck, Pall Corporation, Cytiva and Sartorius Corporation. Nanofiltration is a robust process employed for virus removal. This step involved nanofiltration using an Asahi filter of the equine anti-serum filtrate obtained post anion exchange chromatography at 6 mg/ml protein concentration, the pH of the filtrate was adjusted to 6.70-6.90 before nanofiltration. The purified equine anti-serum was finally filtered by repeating the concentration step using a fresh tangential flow filtration polyethylene sulfone membrane (50kDa). The concentrated filtrate was further subjected to nanofiltration. This step was used to collect the purified bulk into a formulation buffer condition.

[00101] The purified equine anti-serum bulk as collected, was added to a formulation buffer comprising excipients such as sodium chloride and glycine. Potency of the purified equine anti-serum bulk was adjusted using a dilution buffer and protein of the bulk was adjusted to 25 mg/ml using equine normal anti-serum bulk comprising the fragment F(ab’)2. The final formulated bulk (formulation) was sterile filtered using 0.22μm filter and collected in a pre-sterilized vessel under a laminar air flow hood.

Example 4

Functional characteristics of the polyclonal antibodies F(ab')₂ fragments against

SARS-COV-2 RBD variants

[00102] The present example demonstrates the effect of the polyclonal antibodies obtained by the method of the present disclosure. The polyclonal antibody described herein is fragment F(ab')₂ polyclonal antibody. The fragment F(ab')₂ polyclonal antibody is further formulated in a composition that is used in the form of a vaccine. In earlier studies (Zylberman V, Sanguined S, Pontoriero AV, Higa SV, Cerutti ML, Morrone Seijo, et al. Development of a hyperimmune equine serum therapy for COVID-19 in Argentina. Medicina (B Aires). 2020;80 Suppl 3:1-6. English. PMID: 32658841; and Pan X, Zhou P, Fan T, Wu Y, Zhang J, Shi X, et al. Immunoglobulin fragment F(ab’)2 against RBD potently neutralizes SARS-CoV-2 in vitro. Antiviral Research, 2020; 182, 104868), it was seen that the receptor binding domain (RBD as represented by SEQ ID NO: 3) from the viral Spike glycoprotein (as represented by SEQ ID NO: 1) elicits high titers of neutralizing antibodies against SARS-CoV-2 when used as an immunogen in horses. RBD triggered high-titer neutralizing antibodies in vivo, and polyclonal immunoglobulin fragment F(ab’)2 was prepared in BSV (Bharat Serums and Vaccines Limited) from horse antisera through removal of the Fc region. The antibody was screened for its binding capacity to the Wild Type Virus and later to the Variants (Alpha, Beta, Gamma, Delta and Omicron). Figure 6 depicts the neutralizing activity of the equine fragment F(ab’)2 polyclonal antibody against different S ARS-CoV-2 RBD variants. The antibody was found to bind to the Wild type Strain and its Variants at a titre concentration of 0.51 ng/ml for Wild type, 0.58 ng/ml for Alpha, 0.76 ng/ml for Beta, 0.64 ng/ml for Gamma, 0.57 ng/ml for Delta and 0.84 ng/ml for Omicron.

[00103] The loss of binding and neutralizing ability of Antibodies against Omicron was expected due to more than 30 mutations in the Omicron “spike” protein, which is the antibody target, on the surface of the virus. However, the Equine Antibody of the present disclosure demonstrated significant binding to the Omicron variant. Table 3 shows the EC₅₀ values and titre values against different SARS-CoV-2 RBD variants.

Table 3

[00104] From Figure 6 and Table 3, it can be observed that the equine fragment F(ab')₂ polyclonal antibody exhibited potent neutralizing activity with EC₅₀ values ranging from 1.07 to 1.38ng/ml against different SARS-CoV-2 RBD variants.

[00105] Comparison of EC₅₀ values between fragment F(ab’)2 polyclonal antibody and monoclonal antibodies.

[00106] The neutralizing activity of the polyclonal antibody F(ab')₂ fragments of the present disclosure with different EC₅₀ values were evaluated against SARS-CoV- 2 virus, and compared with the EC₅₀ value of the monoclonal antibodies known in the art: (a) Monoclonal antibody Al of Vanderbilt (Seth J. Z, Pavlo G, Potently neutralizing human antibodies that block SARS-CoV-2 receptor binding and protect animals bioRxiv preprint doi: https://doi.org/10.1101/2020.05.22.111005); and (b) Monoclonal antibody A2 of Rockefeller (Alexandra S , Frauke M, Julio L. Antibody potency, effector function and combinations in protection from SARS-CoV-2 infection in vivo. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.15.298067). The comparison of EC₅₀ values between the polyclonal antibody F(ab')₂ fragments of the present disclosure and monoclonal antibodies (Al and A2) is provided in Table 4.

[00107] Table 4

antibody F(ab')₂ Monoclonal fragments antibody

1.07 Wild 2 Al of Vanderbilt

1.13 Alpha 6.9 A2 of Rockefeller

1.20 Beta

1.17 Gamma

1.12 Delta

1.38 Omicron

[00108] Referring to Table 4, it can be observed that the polyclonal antibody F(ab')₂ fragments of the present disclosure exhibited higher potency and better neutralizing activity against different SARS-CoV-2 RBD variants, as compared to that of the monoclonal antibody (Al and A2), wherein the EC₅₀ value of the polyclonal antibody F(ab')₂ fragments ranged between 1.07 to 1.38ng/ml, and EC₅₀ value of the monoclonal antibody Al and A2 was 2ng/ml and 6.9ng/ml, respectively.

Advantages of the present disclosure

[00109] The present disclosure discloses the process of obtaining purified and concentrated therapeutic polyclonal antibody F(ab')₂ fragments from hyperimmunized equines. The process can be used to produce very high titres against the target antigen i.e., spike protein or variants thereof, in a quick and efficient manner by administration of booster doses. The dosage for every batch can be standardized, resulting in batch-to-batch consistency of potency. The results demonstrate consistency in the quality and safety of such equine products. The polyclonal antibody of the present disclosure exhibits potent neutralizing activity with EC₅₀ values ranging from 1.07 to 1.38 ng/ml against different SARS-CoV-2 RBD variants.

[00110] Overall, the present disclosure provides a cost-effective and time-efficient process that provides the polyclonal antibody F(ab')₂ fragments on a large scale. The present disclosure further provides the use of polyclonal antibody F(ab')₂ fragments for treating or preventing coronavirus disease in the subject. The composition comprising polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2 is also disclosed herein.

[00111] The process as described herein will be applicable for production of the antibodies against the variants of the cov-2 antigen as described hereinabove.

Claims

I/We Claim:

1. A process for obtaining polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, said process comprising:

(a) obtaining an antigen formulation comprising spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one adjuvant, wherein the spike protein or variants thereof is at least 200 contiguous amino acid residues from SEQ ID NO: 1;

(b) immunizing an equine with the antigen formulation;

(c) immunizing the equine with one or more booster dose for a time period in the range of 21-30 days;

(d) collecting blood from the equine after 10-14 days after each booster dose, and isolating plasma from the blood;

(e) treating the plasma with saline to obtain a treated plasma;

(f) digesting the treated plasma of step (e) with a protease, to obtain protease digested plasma;

(g) precipitating the protease digested plasma of step (f) with octanoic acid to obtain an equine anti-serum mixture;

(h) centrifuging and filtering the equine anti-serum mixture using diafiltration;

(i) purifying the equine anti-serum mixture by chromatography to obtain a filtrate; and

(j) purifying the filtrate of step (i) by nanofiltration to obtain polyclonal antibody F(ab')₂ fragments against severe acute respiratory syndrome coronavirus 2.

2. The process as claimed in claim 1, wherein the adjuvant is selected from the group consisting of freund's complete adjuvant (FCA), freund's incomplete adjuvant (FIA), carbopol based adjuvant, aluminium based adjuvants, oil- based adjuvants, surfactant adjuvants, micro emulsions, and immunostimulatory adjuvants.

3. The process as claimed in claim 1, wherein purifying the equine anti-serum mixture by chromatography selected from the group consisting of anion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, and cation exchange chromatography.

4. The process as claimed in claim 1, wherein the diafiltration is performed using tangential flow filtration.

5. The process as claimed in claim 1, wherein purifying the equine anti-serum mixture by anion exchange chromatography.

6. The process as claimed in claim 1, wherein immunizing the equine with one or more booster dose in the range of 100-550 μg, preferably in the range of 150- 250 μg.

7. The process as claimed in claim 1, wherein immunizing the equine with at least three immune booster doses.

8. The process as claimed in claim 1, wherein immunizing the equine with three to five immune booster doses.

9. The process as claimed in claim 1, wherein SEQ ID NO: 1 comprises SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4 and SEQ ID NO:5.

10. The process as claimed in claim 1, wherein the spike protein is a homo-trimeric class I fusion protein that allows a viral membrane of severe acute respiratory syndrome coronavirus 2, to fuse with a host cell membrane.

11. The process as claimed in claim 1, wherein the protease is pepsin.

12. The process as claimed in claim 1, wherein the polyclonal antibodies F(ab')₂ fragment has a molecular weight in the range of 100-120 kDa.

13. A polyclonal antibody F(ab')₂ fragment against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2 obtained by the process as claimed in claim 1.

14. A polyclonal antibody F(ab')₂ fragment as claimed in claim 13 for use in the treatment of a coronavirus disease in a subject in need thereof.

15. A method for obtaining a polyclonal antibody F(ab')₂ fragment against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2 as claimed in claim 13, wherein said method comprises: (a) obtaining at least one biological sample from an equine immunized with spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2; and

(b) collecting and purifying the polyclonal antibody F(ab')₂ fragments from said biological sample.

16. A composition comprising polyclonal antibody F(ab')₂ fragments against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2, and at least one excipient, wherein said polyclonal antibody F(ab')₂ fragment are obtained from the process as claimed in claim 1.

17. The composition as claimed in claim 16, wherein the spike protein is a homo- trimeric class I fusion protein that allows a viral membrane of severe acute respiratory syndrome coronavirus 2, to fuse with a host cell membrane.

18. The composition as claimed in claim 16, wherein the excipient is selected from the group consisting of sodium chloride, and glycine.

19. The composition as claimed in claim 16, wherein the polyclonal antibody F(ab')₂ fragment against spike protein or variants thereof of severe acute respiratory syndrome coronavirus 2 is in the range of 20-30 mg/mL.

20. A method for the treatment of a coronavirus disease in a subject in need thereof, comprising administering to said subject the polyclonal antibody F(ab')₂ fragment as claimed in claim 14 or the composition as claimed in claim 16.