WO2023017536A1 - Immunogenic compositions for sars- cov-2 - Google Patents

Abstract

The present invention provides a stable immunogenic composition and a process for preparation thereof. The immunogenic composition comprises recombinant proteins suitable for SARS-CoV-2. More specifically, the present invention provides a composition comprising a receptor binding domain protein and a nucleocapsid protein.

Description

TITLE OF THE INVENTION:

IMMUNOGENIC COMPOSITIONS FOR SARS-CoV-2

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims benefit of the filing date of Indian Provisional Patent Application No. 202121036444 filed on Aug 12, 2021, which is entirely incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compositions for use in the treatment of severe acute respiratory syndrome (SARS) coronavirus-associated diseases or disorders. Particularly, the present invention provides an immunogenic composition for SARS-CoV-2 and a process for preparation thereof. The immunogenic composition comprises proteins from severe acute respiratory syndrome (SARS) Virus.

BACKGROUND OF THE INVENTION

Coronaviruses are a large family of viruses, and it contains a single-stranded positive-sense RNA genome encapsulated within a membrane envelope. While coronavirus infects in both human and certain animals, in humans, SARS-CoV-2 attacks the lower respiratory system to cause viral pneumonia, but it may also affect the gastrointestinal system, heart, kidney, liver, and central nervous system leading to multiple organ failure. Coronaviruses consists of four structural proteins - Spike protein (S) which is required for viral attachment and entry, Envelope protein (E) which plays a critical role in viral infection and pathogenesis, the Membrane Protein (M) which gives a defined shape to the viral envelope and the Nucleocapsid protein (N) which binds to the viral genome and is involved in viral replication cycle and host cellular response to the viral infection.

Structure Receptor Binding Domain: The SARS-CoV-2 receptor binding domain (RBD) forms a part of the SI subunit of the homo-trimeric spike protein and has a predicted non-glycosylated molecular weight of 25 kDa. It consists of a twisted five-stranded antiparallel P sheet with short connecting helices and loops that form the core of the protein. The core contains the Receptor Binding Motif (RBM) through which the virus attaches to the ACE2 receptor on the target cell. RBD contains 9 cysteine residues, out of which 8 form disulphide bonds. Out of the four pairs, three pairs are present in the core region to stabilize the P sheet while the remaining pair connects the loops in the distal end of RBM.

The spike protein plays an important role in viral attachment and fusion of the viral and host cellular membranes. The SI subunit of the spike protein binds to the ACE2 receptor through RBD which causes a conformational change in the S2 subunit. The S2 subunit then transforms from a metastable pre-fusion stage to a more stable post-fusion stage, thus initiating the fusion of viral and target cell membrane. This indicates that RBD can be a critical target for development of potential vaccine candidates that will prevent the entry of the SARS-CoV-2 virus in the host cell.

Nucleocapsid protein: The nucleocapsid protein is a highly immunogenic and abundantly expressed protein during infection. The N protein is an important structural protein for the coronaviruses and is being used as an alternative target antigen in vaccine development and serological assays.

Structure of Nucleocapsid protein: The N protein is an important antigen for coronavirus, which participate in RNA package and virus particle release. It is composed of 419 amino acids with a theoretical molecular weight of 45.6 kDa. SARS-CoV-2 N protein contains two distinct RNA- binding domains (the N-terminal domain [NTD] and the C-terminal domain [CTD]) linked by a poorly structured linkage region (LKR). Both of the NTD and CTD of SARS-CoV-2 N protein are rich in P-strands while CTD has some short helices. The coronavirus N protein is an important viral structural protein, which plays an important role in promoting of genome packaging, RNA chaperoning, intracellular protein transport, DNA degradation, interference in host translation, and restricting host immune responses. N proteins of many coronaviruses are highly immunogenic and are expressed abundantly during infection. High levels of IgG antibodies against N protein are usually detected in sera from SARS patients and T-cells isolated from convalescent patients exhibit strong reactivity to peptides from N protein indicating an important role of these N epitopes in driving cellular immune responses during SARS-CoV-2 infection.

Because of the conservation of the N protein sequence and its strong immunogenicity the N protein of SARS-CoV-2 can be strongly considered as a vaccine candidate for SARS-CoV-2. As per current WHO covid vaccine landscape, there are many organisations working towards covid vaccines. Specifically, few of them has tried recombinant protein subunit as a main drug substance which has been adjuvanted or formulated to provide a stable composition.

US20210000942 provides for a conjugate mixture of RBD protein with peptide conjugated with a virus particle. CN111450244 claims the use of Nucleocapsid for coronavirus infection by loading onto dendritic cells. CN111518175 provides for the recombinant SARS-COV-2 antigen polypeptide and its recombinant adeno-associated virus and application in preparing vaccine.

The receptor binding domain (RBD) of SARS-CoV-2 Spike protein binds to the ACE2 receptor and mediates the entry of the virus into the host cell. Immunization with recombinant RBD protein would stimulate an immune response and production of antibodies, some of which would block the interaction between the SARS-CoV-2 Spike protein and the ACE2 receptor effectively neutralizing viral entry into the host cell. SARS-CoV-1 virus neutralization has been demonstrated by sera from animal models immunized with RBD expressed in mammalian as well as yeast platforms (Du et al. Viral Immunology, 2010, Vol 23, Pg 211-219 and Chen et al. Human Vaccines & Immunotherapeutics, 2014, Vol 10, Pg 648-658). Recently, similar results were also demonstrated by Ravichandran et al., Science Translational Medicine, 2020, Vol 12, eabc3539 using sera from animals immunized with RBD from SARS-CoV-2.

Reports have indicated strong stimulation of cytotoxic T cells from convalescent patients by nucleocapsid protein peptides indicating that nucleocapsid may play an important role in enhancing cell mediated immunity and long-term protection (Le Bert et al. Nature, 2020, Vol 584, Page 457- 462 and Grifoni et al. Cell, 2020, Cell Vol 181, Pg 1489-1501). Further, there have been apprehensions and reports that have linked Adenovirus (Ad) vaccines a rare side effect called vaccine induced thrombotic thrombocytopenia (VITT) resulting in blood clots with thrombocytopenia in vaccine recipients especially in the under 45 age group. Clotting in the brain can be fatal. Due to safety concerns, adenovirus vaccines are not recommended in young adults in many countries in Europe and also not used in children. Thus, there is a strong and urgent need in the art for effective vaccines against coronaviruses, and accordingly, the present invention aims to provide a stable immunogenic composition which can be scaled up to meet the demands. Specifically, the present invention aims to provide a stable immunogenic composition which employs a combination of RBD and N protein of SARS-CoV-2. The rationale behind using this combination is that it would provide protection against viral infection through generation of neutralizing antibodies as well as stimulation of T cell memory and enhanced cytotoxic T cell responses upon viral infection. Additionally, the present invention envisages that this combination of RBD and N protein would provide broader protection against potential immune escape variants with mutations in Spike RBD wherein immune memory against N will provide protection from these immune escape variants. Thus, the present invention provides an immunogenic composition which provides a long-lasting immunity and better T cell memory. Further, the composition disclosed in the present invention aims to address the safety concerns that could be recommended for all age groups. The combination of RBD and nucleocapsid subunit proteins would provide a favourable safety profile that could be recommended for all age groups.

Also, the aim/objective of the present invention is to ensure quality, safety and efficacy of target antigens towards generation of antibodies as well as stimulation of cellular immunity against each of subunit which ultimately neutralizes the infectivity of virus and clears virus infected cells preventing further spread.

SUMMARY OF THE INVENTION

In one aspect of the present invention, there is provided a stable immunogenic composition for SARS-CoV-2, the composition comprising of:

(i) a receptor binding domain protein; and

(ii) a nucleocapsid protein.

In an embodiment of the present invention, there is provided an immunogenic composition, wherein the composition comprises the receptor binding domain and nucleocapsid protein, each present in the range of 1 pg - 50 pg.

In another embodiment of the present invention, there is provided an immunogenic composition, wherein the receptor binding domain protein is having an amino acid sequence of SEQ ID NO. 1. In one embodiment the present invention comprises a receptor binding protein having an amino acid sequence which is 90-100% identical to the amino acid sequence of SEQ.ID. No. 1

In yet another embodiment of the present invention, there is provided an immunogenic composition, wherein the nucleocapsid protein is having an amino acid sequence of SEQ ID NO.2.

In one embodiment the present invention comprises a nucleocapsid protein having an amino acid sequence which is 90-100% identical to the amino acid sequence of SEQ.ID. No. 2

In still another embodiment of the present invention, there is provided an immunogenic composition, wherein the composition optionally comprises of an adjuvant, a stabiliser, and a preservative.

In an embodiment of the present invention, there is provided an immunogenic composition, wherein the adjuvant is present in the range of 0.1 - 0.5 mg, and preservative is present in the range of 1.0 - 5.0 mg and suspended in a buffer.

In another embodiment of the present invention, there is provided an immunogenic composition, wherein the adjuvant is selected from aluminium salts, inulin, algammulin, combination of inulin and aluminium hydroxide, monophosphoryl lipid A (MPL), resiquimoid, muramyl dipeptide (MDP), N-glycolyl dipeptide (GMDP), poly IC, CpG oligonucleotides, resiquimod, aluminium hydroxide with MPL any water in oil emulsion, any oil in water emulsion that contains one or more of the following constituents: squalene or its analogues or any pharmaceutically acceptable oil, tween-80, sorbitan trioleate, alpha-tocopherol, cholecalciferol or any of the analogues and derivatives of the molecules thereof, or calcium phosphate and mixtures thereof.

In yet another embodiment of the present invention, there is provided an immunogenic composition, wherein the adjuvant is selected from Alum, CpG or mixture of both.

In still another embodiment of the present invention, there is provided an immunogenic composition, wherein the preservative is selected from phenol, 2-phenoxy ethanol benzethonium chloride, thiomersol or mixtures thereof.

In an embodiment of the present invention, there is provided an immunogenic composition, wherein the preservative is 2-Phenoxy ethanol. In another embodiment of the present invention, there is provided an immunogenic composition, wherein the preservative is dissolved in a buffer selected from phosphate, citrate and histidine or mixtures thereof.

In yet another embodiment of the present invention, there is provided an immunogenic composition, wherein the buffer is phosphate buffer.

In still another embodiment of the present invention, there is provided an immunogenic composition, wherein the stabiliser is selected from arginine, methionine, polysorbate, sucrose or mixture thereof.

In an embodiment of the present invention, there is provided an immunogenic composition, wherein the composition is in a form of a single dose or multi dose.

In another embodiment of the present invention, there is provided an immunogenic composition, wherein the composition is useful for the treatment of SARS-CoV-2.

In another aspect of the present invention, there is provided a process for preparing immunogenic composition comprising of: (i) a receptor binding domain protein; and (ii) a nucleocapsid protein, wherein the process comprises of: (i) admixing purified RBD and nucleocapsid protein in a buffer optionally comprising of stabilisers and preservatives, (ii) filtering the solution followed by (iii) addition of adjuvants:

These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided to introduce a selection of concepts in a simplified form.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. Figure 1 illustrates schematic representations of RBD, and N protein regions expressed. RBD - receptor binding domain extends from Arg 319 to Phe 541 as expressed. M - Transmembrane domain and IC - intracellular domain. For N protein, full length protein is expressed.

Figure 2 illustrates vector used for the expression of RBD protein.

Figure 3 illustrates vector used for the expression of Nucleocapsid protein.

Figure 4 illustrates representative SDS-PAGE and Western blot images for the purified RBD and N proteins.

Figure 5 illustrates effect of adjuvants on immunogenicity of RBD and N proteins in mice. 1 Opg of each antigen formulated with or without adjuvants in a total volume of lOOpL per was administered 14 days apart. The bars and the numbers indicate the geometric mean titer (GMT) for each group and error bars represent the 95% confidence interval (CI) for that group. Statistical significance was determined using unpaired, two-tailed T test with p- values <0.05 considered significant (*p value < 0.05, ** p value < 0.005, *** p value < 0.0005 and NS is not significant).

Figure 6 illustrates antibody response to I pg and lOpg doses of antigens in mice. Weekly antibody responses to I pg and lOpg doses of antigens with adjuvants in a total volume of lOOpL per dose were tested. Two doses were administered 14 days apart (Day 1 and Day 15) and antibody titers were monitored on days 7, 14, 21 and 28. The bars and the numbers indicate the normalized geometric mean titer (GMT) for each group and error bars represent the 95% confidence interval (CI) for that group. Statistical significance was determined using unpaired, two-tailed T test with p- values <0.05 considered significant (*p value < 0.05, ** p value < 0.005, *** p value < 0.0005 and NS is not significant).

Figure 7 illustrates time course of normalized antibody titer in mice against RBD and N after 5 pg dose with adjuvants in a total volume of l OOpL per dose. Two doses were administered 14 days apart (Day 1 and Day 15) and antibody titers were monitored from day 14 to day 76. The bars and the numbers indicate the normalized geometric mean titer (GMT) for each group and error bars represent the 95% confidence interval (CI) for that group. Titers for test group was normalized to GMT of placebo group run alongside the test groups. Statistical significance was determined using unpaired, two-tailed T test with p-values <0.05 considered significant (*p value < 0.05, ** p value < 0.005, *** p value < 0.0005).

Figure 8 illustrates immunogenicity of two different formulations of RBD and N protein with adjuvants and preservative in multi-dose presentations in mice. Two doses (200pL each) were administered 14 days apart and antibody titers were assessed on day 28. The 4pg dose included 200pg of alum and 80pg of CpG oligonucleotide as adjuvants and 2mg of 2-PE as a preservative while the lOpg dose included 500 pg of alum and 200 pg of CpG oligonucleotide as adjuvants and 5 mg of 2-PE as a preservative. The bars and the numbers indicate the geometric mean titer (GMT) for each group and error bars represent the 95% confidence interval (CI) for that group. Statistical significance was determined using unpaired, two-tailed T test with p-values <0.05 considered significant (*p value < 0.05, ** p value < 0.005, *** p value < 0.0005 and NS is not significant).

Figure 9 illustrates neutralization of two strains of SARS-CoV-2 virus using composition immunized mice sera. SARS-CoV-2 neutralizing endpoint titers (PRNT50) for 3 mouse serum pools (4 mice per pool) were determined. The serum pools were prepared from the same serum samples tested in Fig. 8. The bars and the numbers indicate the GMT for each group and error bars represent the 95% CI for that group. LLOD - lower limit of dilution, ULOD - upper limit of dilution. Statistical significance was determined using unpaired, two-tailed T test with p-values <0.05 considered significant (*p value < 0.05, ** p value < 0.005, *** p value < 0.0005).

Figure 10 illustrates cellular immune responses stimulated by immunogenic composition in mice determined using the IFN-y ELISPOT. Mice received the formulation containing lOpg of each antigen with 500pg of alum and 200pg of CpG oligonucleotide added as adjuvants and 5 mg of 2- PE added as preservative in a 200pL dose volume. No antigen control (200pL) was used as placebo. Two doses were administered 14 days apart and 3 mice from each group were sacrificed day 29 and day 30 respectively. Splenocytes were harvested after sacrifice for ELISpot assay. Cellular immune responses as measured by IFN-y ELISpot of splenocytes from immunized mice when stimulated with different antigens. The bars and the numbers indicate the mean number of IFN-y spot forming cells (SFCs) per million splenocytes for each group and error bars indicate the standard error of mean (SEM). Statistical significance was determined using unpaired, two-tailed T test with p-values <0.05 considered significant (*p value < 0.05, ** p value < 0.005, *** p value < 0.0005). Figure 11 illustrates in vivo viral challenge studies using the Golden Syrian Hamster model indicating body weight variations and lung viral load post SARS-CoV-2 intranasal inoculation. The hamsters were immunized on day 0 and day 14 and challenged with SARS-CoV-2 USA WA1/2020 intranasally at 10⁵ pfu on day 29. Animals were monitored daily for body weight and signs of illness. Animals were sacrificed 4 days post infection and lung were harvested and analysed by RTPCR for viral load relative to the uninfected control. The bars indicate the mean viral load, and the error bars indicate the standard error of mean (SEM). Kruskal-Wallis one way analysis of variance test was performed for significance and the comparison between groups is performed by Dunn’s multiple comparisons test, *p value < 0.05, ** p value < 0.005.

Figure 12 illustrates histological analysis of Golden Syrian Hamster lungs after SARS-CoV-2 viral challenge. The bars indicate the mean score, and the error bars indicate the standard error of mean (SEM). Kruskal -Wallis one way analysis of variance test was performed for significance and the comparison between groups is performed by Dunn’s multiple comparisons test, *p value < 0.05, ** p value < 0.005.

Figure 13 illustrates disease score for Golden Syrian hamsters challenged with SARS-CoV-2 Delta strain.

Figure 14 illustrates body weight variation for Golden Syrian hamsters challenged with SARS- CoV-2 Delta strain. Day 0 is the day of challenge. The mean body weight over time is indicated by the points and error bars indicate the standard deviation (SD). Statistical significance was determined using unpaired, two-tailed T test with p-values <0.05 considered significant (*p value < 0.05, ** p value < 0.005, *** p value < 0.0005).

Figurel5 illustrates lung weights for Golden Syrian hamsters challenged with SARS-CoV-2 Delta strain. Animals were sacrificed 4 days post infection and lung were harvested. The bars indicate the mean weight, and the error bars indicate the the standard deviation (SD). Statistical significance was determined using unpaired, two-tailed T test with p-values <0.05 considered significant (*p value < 0.05, ** p value < 0.005, *** p value < 0.0005).

Figure 16 illustrates lung viral load for Golden Syrian hamsters challenged with SARS-CoV-2 Delta strain. Animals were sacrificed 4 days post infection and lung were harvested and viral load was expressed as tissue culture infectivity dose 50 percent (TCID50) per gram of lung. The bars indicate the mean viral load and error bars indicate the standard deviation (SD). Statistical significance was determined using unpaired, two-tailed T test with p-values <0.05 considered significant (*p value < 0.05, ** p value < 0.005, *** p value < 0.0005).

Figure 17 illustrates lung histological analysis for Golden Syrian hamsters challenged with SARS- CoV-2 Delta strain Arrow heads indicate mild mononuclear cell vascular and perivascular inflammation while arrows indicate mild to moderate alveolar haemorrhage.

DETAILED DESCRIPTION OF THE INVENTION

For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected 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.

Definition'.

For the purposes of this invention, the following terms will have the meaning as specified therein:

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.

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”.

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.

The term “including” is used to mean “including but not limited to” “including” and “including but not limited to” are used interchangeably. As used herein, an “immunogenic composition” is a composition that comprises an antigen where administration of the composition to a subject result in the development in the subject of a humoral and/or a cellular immune response to the antigen.

As used herein, a “subunit” composition, for example a vaccine, that includes one or more selected antigens but not all antigens from a pathogen. Such a composition is substantially free of intact virus or the lysate of such cells or particles and is typically prepared from at least partially purified, often substantially purified immunogenic polypeptides from the pathogen. The antigens in the subunit composition disclosed herein are typically prepared recombinantly.

As used herein “RBD” means the recombinant protein receptor binding domain of SARS-CoV-2 spike protein that binds to Ace2 receptor on host cells and enables viral entry into host cells to infect humans. Herein RBD refers to receptor binding domain amino acid sequence of SEQ ID. No. 1 from the original SARS-CoV-2 strain discovered in Wuhan (L) and subsequent strains that have emerged since but not limited to B.1.1.7, B.1.351, P.l, P.2, B.l.617. l, B.l.617.2, B.1.529.1 (BA.1), B.1.529.2 (BA.2), B.l.529.3 (BA.3), B.l.529.4 (BA.4), B.l.529.5 (BA.5) and XE or the alpha, beta, gamma, delta, epsilon, kappa, iota and omicron variants as per WHO nomenclature showing specific mutations in the amino acid sequence of the receptor binding domain and is having at least 90% sequence identity or is 90-100% identical to the amino acid sequence of SEQ. ID. No. 1. The nucleotide sequence encoding RBD may be the native sequence or maybe codon optimized as per the host expression system.

As used herein “N” or “nucleocapsid” means the recombinant nucleocapsid protein of SARS-CoV2 that binds to viral RNA and assembles in the core of the mature virion. Herein nucleocapsid refers to nucleocapsid amino acid sequence from the original SARS-CoV-2 strain discovered in Wuhan (L) and subsequent strains that have emerged since but not limited to B.l.1.7, B.l.351, P.l, P.2, B.l.617.1, B.l.617.2, B.l.529.1 (BA.l), B.l.529.2 (BA.2), B.l.529.3 (BA.3), B.l.529.4 (BA.4), B. l.529.5 (BA.5) and XE or the alpha, beta, gamma, delta, epsilon, kappa, iota and omicron variants as per WHO nomenclature showing specific mutations in nucleocapsid amino acid sequence and is having at least 90% sequence identity or is 90-100% identical to the amino acid sequence of SEQ. ID. No. 2. The nucleotide sequence encoding N gene may be the native sequence or maybe codon optimized as per the host expression system. As used herein, the term refers to an “immunogenic composition” or “vaccine”, refers to a composition of an immunogen derived from a pathogen, which is used to induce an immune response against the pathogen that provides protective immunity (e.g., immunity that protects a subject against infection with the pathogen and/or reduces the severity of the disease or condition caused by infection with the pathogen). The protective immune response may include formation of antibodies and/or a cell-mediated response. Depending on context, the term “vaccine” may also refer to a composition of an immunogen that is administered to a subject to produce protective immunity.

As used herein an “effective dose” or “effective amount” refers to an amount of an immunogen sufficient to induce an immune response that reduces at least one symptom of pathogen infection. An effective dose or effective amount may be determined e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by plaque reduction neutralization test (PRNT), complement fixation, enzyme-linked immunosorbent (ELISA), or microneutralization assay.

As used herein, the term “adjuvant” refers to a compound that, when used in combination with an immunogen, augments or otherwise alters or modifies the immune response induced against the immunogen. Modification of the immune response may include intensification or broadening the specificity of either or both antibody and cellular immune responses.

As used herein the term “drug substance” refers to the recombinant antigen component which can be Receptor Binding protein as a part of SI subunit of spike protein of Novel Sars-Cov2 virus and nucleocapsid protein.

As used herein the term “coronavirus” includes SARS-Cov-2 and all its possible strains or mutations that have emerged and are emerging since the outbreak was declared a global pandemic in March 2020 by the World Health Organization.

The present invention provides immunogenic compositions for SARS-CoV-2, a two antigen adjuvanted subunit vaccine candidate comprising of the RBD and N proteins of SARS-CoV-2 virus. The composition provides protection against viral infection through generation of neutralizing antibodies as well as stimulation of T cell memory and enhanced cytotoxic T cell responses upon viral infection. Accordingly, the immunogenic composition disclosed in the present invention provides a long-lasting immunity and better T cell memory.

Thus, in accordance with the present invention, there is provided a stable immunogenic composition for SARS-CoV-2, the composition comprising of: a receptor binding domain protein; and a nucleocapsid protein.

In an embodiment of the present invention, there is provided an immunogenic composition, wherein in the composition the receptor binding domain and nucleocapsid protein, each are present in the range of 1 pg - 50 pg.

In another embodiment of the present invention, there is provided an immunogenic composition, wherein the receptor binding domain protein is having an amino acid sequence of SEQ ID NO. 1. and is having at least 90% sequence identity to the amino acid sequence of SEQ. ID. No. 1.

In an embodiment of the present invention, there is provided an immunogenic composition, wherein the nucleocapsid protein is having an amino acid sequence of SEQ ID NO.2 and having at least 90% identity to the amino acid sequence of SEQ. ID. No. 2.

In another embodiment of the present invention, there is provided an immunogenic composition, wherein the composition optionally comprises adjuvants, stabiliser, and preservative.

In an embodiment of the present invention, there is provided an immunogenic composition, wherein the adjuvant is present in the range of 0.1 - 0.5 mg, and preservative is present in the range of 1.0 - 5.0 mg in a buffer.

In another embodiment of the present invention, there is provided an immunogenic composition, wherein the adjuvant is selected from aluminium salts, inulin, algammulin, combination of inulin and aluminium hydroxide, monophosphoryl lipid A (MPL), resiquimoid, muramyl dipeptide (MDP), N-glycolyl dipeptide (GMDP), poly IC, CpG oligonucleotides, resiquimod, aluminium hydroxide with MPL any water in oil emulsion, any oil in water emulsion that contains one or more of the following constituents: squalene or its analogues or any pharmaceutically acceptable oil, tween-80, sorbitan trioleate, alpha-tocopherol, cholecalciferol or any of the analogues and derivatives of the molecules thereof, or calcium phosphate and mixtures thereof. In an embodiment of the present invention, there is provided an immunogenic composition, wherein the adjuvant is selected from Alum, CpG or mixture of both.

In another embodiment of the present invention, there is provided an immunogenic composition, wherein the preservative is selected from phenol, 2-phenoxy ethanol benzethonium chloride and Thiomersol.

In an embodiment of the present invention, there is provided an immunogenic composition, wherein the preservative is 2-Phenoxy ethanol.

In another embodiment of the present invention, there is provided an immunogenic composition, wherein the preservative is dissolved in a buffer selected from phosphate, citrate and histidine.

In an embodiment of the present invention, there is provided an immunogenic composition, wherein the buffer is phosphate buffer.

In another embodiment of the present invention, there is provided an immunogenic composition, wherein the stabiliser is selected from arginine, methionine, polysorbate, sucrose or mixture thereof.

In an embodiment of the present invention, there is provided an immunogenic composition, wherein the composition is in the form of a single dose or multi dose.

In another embodiment of the present invention, there is provided an immunogenic composition used in the treatment of SARS-CoV-2.

In another aspect of the present invention, there is provided a process for preparing immunogenic composition as defined in claims 1-14, the process comprising of:

(i) adding preservative to a buffer in a container with constant mixing to obtain a solution;

(ii) adding CpG adjuvant to the solution of step (i) followed by addition of RBD and N protein and mixing it uniformly;

(iii) filtering the solution of step (ii) through a sterile 0.22 pl filter; and (iv) adding 2% Alhydrogel adjuvant to the solution obtained in step (iii) followed by mixing and keeping the solution with gentle mixing on a magnetic stirrer or rocker for a duration of 6 - 12 hours to obtain the immunogenic composition.

EXAMPLES

The following Examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the Examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

The present invention is directed to an immunogenic composition against SARS-CoV-2 virus. The composition includes an antigen Receptor Binding Protein (RBD) as a part of the SI subunit of spike protein of the novel SARS-CoV-2 virus. It binds to the target cells through ACE2 (Angiotensin-Converting Enzyme 2) receptors and mediates entry of virus into the cell by triggering fusion of viral and cellular membranes. The other antigen is the nucleocapsid protein (N) which binds to viral genome and is involved in viral replication cycle and host cellular responses to viral infections. These two antigens were manufactured by recombinant DNA technology. The RBD protein of SEQ ID NO. 1 and variant thereof having 90-100% sequence similarity was manufactured in CHO as host system (Figure 2). The nucleocapsid protein of SEQ ID NO. 2 and variant thereof having 90-100% sequence similarity was manufactured using E.coli as an expression host (Figure 3). Post purification of these proteins, initial characterization has been performed to study protein sequence, post-translational modifications and to confirm its structural characteristics.

Example 1: Purification of RBD and Nucleocapsid Protein

RBD protein: RBD protein is translocated to the extracellular environment during cell culture fermentation and is accumulated in the cell culture medium during the entire duration of fermentation process. The cell culture process was continuously monitored for cell viability and other parameters. The harvest from the cell culture fermentation was processed to purify RBD by multiple chromatography, viral inactivation and filtration steps.

These steps include affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and Gel permeation chromatography. The chromatography resins used in the process are not limiting to Blue sepharose, Q-Sepharose, Butyl 650M, Superdex S75 and Etoxiclear resins. Further, during the downstream purification, process and product related impurities are reduced in the subsequent chromatography operations. Optimized purification protocol demonstrated overall purity of >95% by SE-HPLC.

Nucleocapsid protein: SARS CoV-2 N protein was produced in E.coli cells as a soluble intracellular protein. The harvest was subjected to cell lysis for isolation of protein. The cell lysate was directly taken up for processing. The fermentation broth was harvested and was subjected to centrifugation to obtain the cell mass. The cells were lysed by homogenizer and the lysate was processed on multiple chromatography steps not limited to DEAE sepharose, poros XS, Sephdex G25 facilitating buffer exchange and Etoxiclear for removal of bacterial endotoxins. During the down streaming, the process and product related impurities were reduced in the subsequent chromatography operations. Optimized purification protocol demonstrated overall purity of >90% by RP-HPLC.

Example 2: Details of Adjuvant and Excipient Used for Preparation of Immunogenic Composition

Aluminium hydroxide (Alum): Aluminium hydroxide is the most commonly used chemical as adjuvant. The mechanism of how aluminium hydroxide-based adjuvants exert their beneficial effect is still not fully understood. The current understanding for mode of action includes repository effect, phagocytic effect and activation of pro-inflammatory pathway. All this together stimulate innate as well as acquired immune response and activate the complement system.

Unmethylated CpG DNA: Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs trigger cells that express Toll-like receptor 9 (including human plasmacytoid dendritic cells and B cells) to mount an innate immune response characterized by the production of Thl and pro-inflammatory cytokines. As vaccine adjuvants, CpG oligodeoxynucleotides improves presentation of antigen thereby generating humoral and cellular vaccine specific immune response.

Preservative (2-phenoxy ethanol): Compounds commonly used as preservative in commercial vaccines are phenol, 2-phenoxy ethanol benzethonium chloride and Thiomersol. The concentrations necessary to induce significant killing of bacterial cells were significantly higher for all preservatives except 2-phenoxy ethanol. The current approach in the industry is to develop thiomersol free products and hence 2-phenoxy ethanol was selected as a preservative for multi-dose formulation.

Example 3: Preparation of Immunogenic Composition

In the present invention, SARS-CoV-2 RBD and nucleocapsid proteins are used as candidates against the novel coronavirus strain, causing the COVID 19 disease. The composition will be available as a combination of SARS-CoV-2 RBD and SARS-CoV-2 N protein as part of a multi- antigenic composition. The immunogenic composition can be made available in multiple presentations: a single dose presentation in 1.0 mL type I glass syringes with a fill volume of 0.5 mb which is equivalent to a single human dose, multi-dose presentation in 2 mL type-I glass vial with a fill volume of 4.0 mL equivalent to four human doses and a multi-dose presentation in 5 mL type I glass vial with a fill volume of 5.0 mL which is equivalent to ten human doses.

The composition for both single dose and multi dose presentations for RLS SARS-CoV-2 comprises 1 pg - 50pg of RBD and nucleocapsid and 0.1 to 0.5mg of adjuvants such as Alum and CPG along with lmg-5 mg of preservative such as 2-Phenoxyethanol in a buffer preferably phosphate buffer. Single dose formulation would not include preservative.

The SARS-CoV-2 immunogenic composition is manufactured by adsorption of SARS-CoV-2 RBD and SARS-CoV-2 nucleocapsid on Aluminium hydroxide in the presence of CpG as adjuvants. The entire formulation process is carried out in Grade A classified area. The manufacturing process involves addition of RBD and nucleocapsid purified proteins to the formulation buffer along with CpG. For multi-dose presentation, 2-PE is added as a preservative. After mixing the components for complete dissolution, the solution is sterile filtered through 0.22pm filter. The entire process is carried out in Grade A classified area. After filtration, 0.5 mg per dose of 2 % Alum is added to the sterile filtered solution and kept for gentle rocking overnight for mixing and adsorption. The following day, the formulated bulk is taken forward for filling.

Example 4: Preparation of Composition for Single Dose Presentation

The protein concentration of SARS-CoV-2 RBD and nucleocapsid is determined by UV 280nm. Based on the protein concentration and the number of syringes required, the total quantity of each antigen that is required for the production of the sterile formulated bulk is calculated and transferred from storage to the laminar air flow (LAF). Initially after formulation, the drug substance for both RBD and N is stored at -70 ± 5°C. The real time temperature for the storage would be proposed after the results of the stability study. The drug substance is transferred from -70 ± 5°C to the LAF where it is thawed at room temperature.

The required amount of drug substance for both RBD and N is added to the required amount of 0.22pm sterile filtered formulation buffer in a plastic container. This is followed by addition of the CpG to the buffered solution. The contents of the container are gently swirled for mixing and then filtered through 0.22pm filter. 2% Alum in required amount is added to the sterile filtered buffer solution containing the protein and CpG and the solution is then kept for gentle rocking overnight for mixing and adsorption. The following day, the formulated bulk is filled into sterile 1.0 mL glass syringes at a target fill volume of 0.55 mL including a 10% overage.

Example 5: Preparation of Composition for Multi Dose Presentation

The protein concentration of SARS-CoV-2 RBD and nucleocapsid in the respective drug substances is determined by UV 280nm. Based on the protein concentration and the number of vials required, the total quantity of drug substance that is required for production of the sterile formulated bulk was calculated and transferred from storage to the laminar air flow (LAF). Initially after formulation, the drug substance for both RBD and N is stored at -70 ± 5°C. The real time temperature for the storage would be proposed after the results of the stability study. The drug substance is transferred from -70 ± 5°C to the LAF where it is thawed at room temperature.

The entire formulation process is carried out in a LAF under sterile conditions. The required amount of the preservative, 2-Phenoxyethanol or 2-PE is added to the required amount of 0.22pm sterile filtered formulation in a plastic container and mixed by swirling for complete dissolution. The required amount of drug substance for both RBD and N is added to the 2-PE containing formulation buffer. This is followed by addition of the CpG to the buffered solution. The contents of the container are gently swirled for mixing and then filtered through 0.22pm filter. 2% Alum in required amount is added to the sterile filtered buffer solution containing the protein and CpG and the solution is kept for gentle rocking overnight for mixing and adsorption. The following day, the formulated bulk is filled into sterile 2R or 5 R glass vials at a target fill volume of 2.5 mL and 5.5 mL respectively to include a 10% overage for 4 doses and 10 doses vials.

Example 6: Evaluating Stability of Immunogenic Composition

Stability studies on three batches of both SARS-CoV-2 RBD and Nucleocapsid at storage temperatures of -70±5°C, -20±3°C and 5±3°C and three batches each of single dose and multi-dose presentations at storage temperatures of 5±3°C, 25±3°C and 40±3°C for a duration of 24 months has been ongoing. The samples were tested at intermittent time points at 3 months, 6 months and 9 months. The compositions were found stable without any significant variation in the test parameters based on WHO and ICH guidelines.

Example 7: Analysis/ In Vitro Testing of Immunogenic Composition

The composition was assessed for adsorption of RBD and N on Alum. The composition was centrifuged, and the supernatant was collected. The supernatant was assessed for unbound RBD and N by ELISA developed in-house to detect and quantify RBD and N proteins. The bound proteins were desorbed by addition of 250pL of 1.1 M solution of disodium hydrogen phosphate and 0.1% v/v Tween 20 to 500 pL of composition followed by incubation with rocking at room temperature for 24 hours. The supernatant post centrifugation was assessed by ELISA to determine desorbed proteins. In addition, the supernatant and drug substance were also evaluated by western blotting to estimate the extent of adsorption.

Example 8: In Vivo and Ex Vivo Analysis of Immunogenic Composition

Animal studies: The study was carried out to assess the dose response of the immunogenic composition. BALB/c mice were administered different doses of composition through intramuscular route. Immunogenicity and T-cell responses were tested. Each test group had 6 mice, and these were administered fixed dose of antigens ( I Ogg) with different combination of adjuvants. Dose was administered intramuscularly with a constant volume of 100 pL/mouse (50 pL per site) on days 1 and 15. Blood was collected from the retro-orbital plexus on days 0, 14 and 28 (Figure 5). In a second experiment, each test group had 6 mice and a log difference in dose of antigens was tested (1 pg and 10 pg). The dose was administered intramuscularly with a constant volume of 100 pL/mouse (50 pL per site) on days 1 and 15. Blood was collected from the retro-orbital plexus on days 0, 7, 14, 21 and 28 (figure 6). In a third experiment, each test group had 6 mice and a fixed dose of antigen (5 pg) was administered intramuscularly with a constant volume of 100 pL/mouse (50 pL per site) on days 1 and 15. Blood was collected from the retro-orbital plexus on days 0, 14, 28, 35, 49, 62 and 76 (figure 7). In a fourth experiment, an optimized multi-dose formulation with preservative was administered to mice and each test group had 12 animals with two different doses of antigen (4pg and 10pg). Dose was administered intramuscularly with a constant volume of 200 pL/mouse (100 pL per site) on days 1 and 15. Blood was collected from the retro-orbital plexus on days 0, 14 and 28 (Figure 8). Although, oedema was observed at site of injection, no adverse clinical signs were seen throughout the study. No mortality or morbidity was noticed throughout the experiment. All the blood samples were centrifuged at 3000 rpm for 15 minutes to separate the serum. In a fifth experiment, each test group having 6 mice each were dosed with the composition/ formulation. Dose was administered intramuscularly with a constant volume of 200 pL/mouse (100 pL per site) on days 1 and 15. Three mice from each group were sacrificed on day 29 and 30 and the spleens were collected (Figure 10).

Immunogenicity assessment in mice by ELISA: Anti-RBD and anti-NC antibody titers in immunized mice sera were determined by ELISA using the end point titer method.

Required number of plates were coated with RBD or NC in IX phosphate buffered saline (PBS). Plates were covered and incubated overnight at 4°C. Plates were washed once with phosphate buffered saline with tween (PBST) and 300pl of blocking buffer (3% bovine serum albumin or BSA in PBST) was added to all wells and incubated for 1 hr at room temperature. Blocking buffer was discarded and the plates were dried by dabbing on absorbent paper. Serum samples were appropriately diluted in diluent buffer (0.1% BSA in PBST) and added to the first row of the ELISA plate in duplicate. These were then serially diluted 2-fold down each column. Plates were incubated at 37°C for 2 hours and washed 3 times with PBST. An appropriate dilution of anti-mouse IgGHRP conjugated antibody in diluent was added to coated plates and incubated at 37 °C for 1 hr and plates were washed 3 times. IX TMB solution was added to each well and plate was incubated in the dark for 30 min. Optical density at 450 nm and reference wavelength 620 nm was measured using an ELISA plate reader. For end point titers, cut-off OD (OD450nm- OD620nm) was determined as the mean OD + 3 SD of the l:200-fold dilution of the placebo mouse group. Reciprocal of dilution for each mouse having OD above cut-off was determined and the geometric means for each group with standard deviation was used to plot the bar graphs.

Anti-RBD and anti-N antibody titers were significantly higher after booster dose. The combination of aluminium hydroxide gel and CpG oligonucleotide adjuvants gave highest titers for both anti- RBD and anti-N antibodies. (See Figure 5)

A dose of l Opg of each antigen produced higher overall anti-RBD and anti-N antibody titers compared to a dose of I pg but statistically the difference was significant on day 14 but not on day 21 (a week after booster dose) (See Figure 6)

Anti-RBD and anti-N antibody titers were assessed after immunization with 2 doses (day 1 and day 15) of 5 pg of each antigen over a duration of 76 days. Peak anti-RBD antibody titers were observed on day 28 and peak anti-N antibody titers were observed on days 35 and 49. A decline from peak levels was seen by day 76, however, both anti-RBD and anti-N titers were significantly higher than after single dose and more than 300-fold over placebo levels. (See Figure 7).

An optimized formulation with 2-PE as preservative gave similar titers on day 28 for anti-RBD and anti-N antibodies for both doses of each antigen (4pg and lOpg) indicating the dose sparing effect of this composition. (See Figure 8).

Virus neutralization assay: Plaque reduction neutralization test (PRNT50) was performed on serum pools of mice immunized with the composition with two virus strains that include B.1.1.306 and B.1.617.2 (Delta strain). Serum from 4 mice was pooled in serial order to obtain 3 pools for each dose (4 pg and 10 pg) and placebo groups. For this assay, Vero cells were seeded in 24 well plates and grown to about 90% confluency prior to starting the assay. Test serum samples along with controls were heat inactivated at 56°C for 30 mins. Serial dilution of serum samples was then performed. The viral stocks were diluted and added to the wells containing serum dilutions (1: 1 ratio at 60 to 100 pfu viral titer per well) and incubated for Ihour at 37°C and 5% CO2. After incubation, these were transferred in duplicate to the 24 well plate containing Vero cells after removing growth media incubated for 1 hour at 37°C and 5% CO2. One mb of overlay media (MEM + 2% FBS + 1% carboxy methyl cellulose) was added to the wells the plates were incubated for 5 days at 37°C and 5% CO2. Wells were washed with PBS and fixed with formaldehyde solution. After washing, 1% crystal violet solution was added to the wells. Plates were washed with distilled water, dried and the plaques were counted using the CTL Immunospot analyzer. The mean number of plaques from the virus only control wells was used to determine the cutoff for desired level of infection reduction. PRNT50 was reported as the highest dilution of test serum for which virus infectivity is reduced by 50%. A standard logical regression model was followed for determination of PRNT50 and cut-off values for determination of valid data points were calculated using the NIH LID Statistical web tool

Strong neutralization of both viral strains was observed by test serum pools at both 4pg and 1 Opg antigen doses was observed relative to placebo immunized mice serum pools. A dose of lOpg of each antigen led to significantly higher neutralization of the delta strain compared to a dose of 4 pg. (See Figure 9).

T cell response by ELISPOT: To determine the number of antigen reactive T cells, we performed ELISPOT using antigen-specific IFN-y- ELISPOT kits as per the manufacturer’s instructions. Briefly, spleens from mice were individually crushed using sterile frosted slides in petri dishes containing complete RPMI, passed through a 40 pm cell strainer and the cells were collected in sterile centrifuge tubes and pelleted. The cells were resuspended in ACK lysing buffer and incubated for 5 mins at room temperature followed by addition of 10% FBS in PBS. The cells were further washed with 10% FBS in PBS, resuspended in complete RPMI, counted and plated in IFN-y mAb coated plates at 1 million cells per well and 0.5 million cells per well. The cells were stimulated by RBD protein alone, N protein alone and RBD protein plus N protein together at 10 pg/mL final concentration. ConA (10 pg/mL) was added as positive control and media as unstimulated control for each group. The plates were incubated at 37°C for 40 to 42 hours. Subsequently, the plates were washed and incubated with a biotinylated detection antibody, followed by Streptavidin-HRP. The plates were developed with the TMB substrate as per the manufacturer’s instructions until distinct spots emerged. The plates were imaged using ImmunoSpot reader (Cellular Technologies Ltd) and number of spots per well were counted manually using Biospot 5.0 software.

Results showed significantly higher number of IFN-y releasing cells in the test group compared to the placebo group across all treatment conditions. In test mice, N and RBD + N treatment resulted in significantly higher number of IFN-y producing cells compared to RBD treatment alone, given higher number of antigen specific IFN-y ELISPOTS when compared to RBD alone. Between the two antigens, RBD and N, N is responsible for a higher number of antigen specific IFN-y ELISPOTS which suggest that presence of N would be responsible for a better T cell response. (See Figure 10).

Protection of Golden Syrian hamsters from SARS-CoV-2 viral challenge D614G strain (USA- WA1/2020): Golden Syrian hamsters aged 6-8 weeks were immunized intramuscularly (n=6 animals/ group) on with 3 different doses of immunogenic composition or placebo on day 0 followed by a booster dose on day 14. Thereafter, the animals were shifted to ABSL3 facility and challenged intranasally on day 29 with live SARS-CoV2 105 PFU Isolate USA-WA1/2020 at 50pl/nostril (lOOpl/animal) under mild anesthetized condition induced by using ketamine (150mg/kg) and xylazine (lOmg/kg) intraperitoneal injection SARS-CoV2 challenge. Post challenge the animals were monitored daily for the changes in body weight, activity and physical signs of illness. All infected animals were euthanized on 4 days post infection at ABSL3. Post sacrifice, the excised lungs and spleen of the animals were imaged for gross morphological changes. Right lower lobe of the lung was fixed in 10% neutral formalin solution and used for histological analysis. The complete left lobe of the lung was homogenized in 2 ml Trizol solution for viral load estimation. The tissue samples in trizol were stored immediately at -80 °C till further use. All the experimental protocols involving the handling of virus culture and animal infection were approved by RCGM, institutional biosafety and IAEC animal ethics committee.

RNA was isolated from homogenized lung samples using Trizol chloroform method as per the manufacturer’s protocol. A total of 1 pg of RNA was then reverse transcribed to cDNA. Diluted cDNAs (1:5) was used for qPCR. The CDC-approved SARS-CoV-2 N gene primers: 5'- GACCCCAAAATCAGCGAAAT-3' (Forward), 5'-TCTGGTTACTGCCAGTTGAATCTG-3' (Reverse) was used for viral load calculation. The relative expression of each gene was expressed as fold change and was calculated by subtracting the cycling threshold (Ct) value of hypoxantine- guanine phosphoribosyl transferase (HGPRT-endogenous control gene) from the Ct value of target gene (ACT). Fold change was then calculated according to the previously described formula POWER (2, -ACT).

For histological analysis, fixed lungs were processed, and paraffin wax embedded blocks were transverse sectioned and stained with hematoxylin and eosin. The H & E stained lung sections was then quantitatively examined under the microscope for pneumonitis and disease index score on the scale of 0-5 by expert histologist. Images of the HE stained lungs sections were acquired at 40X.

Results indicate that all test doses of the immunogenic composition were able to protect golden Syrian hamsters against the body weight loss as compared to the unvaccinated infected control hamster group receiving placebo. Reduction in relative lung viral load was observed for all the composition dose groups studied as compared to the infected control group. The most dramatic virus clearance was observed in 10 pg and 20 pg dose groups (See Figure 11).

Consistently, lungs isolated from the euthanized animals on day 4 post challenge showed significantly lesser regions of pneumonitis and inflammation in the 10 pg and 20 pg immunized hamsters as compared to the infected control hamsters receiving placebo (See Figure 12).

Protection of Golden Syrian hamsters from SARS-CoV-2 viral challenge Delta strain (B.l.617.2): The study design was similar to the previous study except that the unvaccinated unchallenged control received a placebo control, and the unvaccinated challenge control received no treatment. Sedated hamsters were intranasally inoculated with 0.1 ml of SARS-CoV-2 Delta variant acquired from BEI Resources. The results indicated that the disease score was lower in the treated group compared with the challenged control (Figure 13).

Decrease in body weight was significantly lower in the treated groups compared with the control animals (Figure 14). Lung weight in challenged control was significantly higher than the unchallenged normal hamsters. The lung weights of mice were lower in treated groups. This could be due to decreased alveolar oedema in treated groups compared with control group (Figure 15).

A dose dependent decrease in viral load was observed in all treatment groups indicating protection conferred to vaccinated hamsters in a composition dose dependent manner (Figure 16). Histological analysis showed that there were no microscopic findings in unvaccinated/non- challenged SARS-CoV-2 animals. The infected control group animals displayed lungs that were poorly inflated with collapsed airways and appeared artifactually consolidated. Mild mononuclear cell vascular/perivascular inflammation (arrowhead), mild to moderate mixed cell inflammation (arrows), minimal to moderate alveolar hemorrhage, minimal alveolar edema, and minimal alveolar/bronchiolo-alveolar hyperplasia was observed. Lungs of composition treated animals showed minimal to mild mononuclear cell perivascular inflammation (arrowheads), minimal to moderate mixed cell inflammation (arrows), minimal to mild alveolar hemorrhage, and minimal alveolar/bronchiolar alveolar hyperplasia. Histologically, decreased severity and incidence of pathological abnormalities were seen in vaccinated animals compared with unvaccinated animals (Figure 17).

Toxicity assessment of Immunogenic Composition in preclinical models: A 28 Day repeated dose toxicity study of SARS-CoV-2 composition was conducted in New Zealand White Rabbits and Wistar rates by Intramuscular Route. Rats and rabbits were administered doses at three dose levels (5, 10 and 20 pg dose per animal) 3 times (day 1, 15 and 28) during the treatment period. To assess reversal of toxicity, if any, high dose recovery group was used. For comparison, control groups (Control and control recovery) were housed.

None of the treatment groups showed signs of morbidity or mortality during the study. No treatment related toxicologically relevant changes were observed in clinical signs, body weight, feed consumption, ophthalmology, hematology, clinical chemistry, urinalysis and organ weight.

Site of injection indicated local reaction in both rats and rabbits. Oedema was observed at site of injection in rat study. In internal gross pathology, most rats and a few rabbits showed changes at the site of injection. Histology indicated changes at the site of injection. The changes at the site of injection completely recovered in rabbits and showed a trend towards recovery in rats.

The changes at the site of injection are expected for a composition as the components of the formulation (Aluminium Hydroxide, CpG and proteins of the composition) causes immunostimulation. Antibodies against both RBD and N were observed in all animals, indicating seroconversion. Based on the findings, the NOAEL in both rat and rabbit studies were fixed at 20 pg/dose/ animal for the immunogenic composition. Table 1 summarizes the observations from the toxicity studies.

Table 1: Assessment of toxicity of immunogenic composition in rabbits and rats at 3 dose levels of 5 pg, 1 Opg and 20pg with 3 dose regimen. Various parameters recorded are summarized in the table below.

References:

Ahlen G., Frelin L. et al. The SARS-CoV-2 N Protein Is a Good Component in a Vaccine. J Virol 94, e01279-20 (2020). https://doi.org/10.1128/JVI.01279-20.

J., Ge, J., Yu, J. et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215-220 (2020). htps://doi.org/10.1038/s41586-020-218Q-5).

Kang S, Yang M. et al. Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites. Acta Pharm Sin B 10, 1228-38 (2020) htps://doi.Org/10.1016/i.apsb.2020.04.009.

Lan, J., Ge, J., Yu, J. et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215-220 (2020). htps://doi.org/10.1038/s41586-020-218Q-5.

Le Bert N, Tan AT, Kunasegaran K, Tham CYL, Hafezi M, Chia A, et al. SARS-CoV- 2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature 584, 457-62 (2020). htps://doi.org/10.1038/s41586- 020-2550-z.

Tai, W., He, L., Zhang, X. et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol 17, 613-620 (2020). htps://doi.org/10.1038/s41423-020-040Q-4).

Thus, while we have described fundamental novel features of the invention, it will be understood that various omissions and substitutions and changes in the form and details may be possible without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, be within the scope of the invention.

Claims

We Claim: . A stable immunogenic composition for SARS-CoV-2, the composition comprising of:

(i) a receptor binding domain protein; and

(ii) a nucleocapsid protein. . The immunogenic composition as claimed in claim 1, wherein the receptor binding domain and the nucleocapsid protein, each is in the range of 1 pg - 50pg. . The immunogenic composition as claimed in claim 1 , wherein the receptor binding domain protein is having an amino acid sequence which is 90-100% identical to SEQ ID NO. 1. . The immunogenic composition as claimed in claim 1, wherein the nucleocapsid protein is having an amino acid sequence which is 90-100% identical to SEQ ID NO 2. . The immunogenic composition as claimed in claim 1, wherein the composition optionally comprises an adjuvant, a stabiliser, and a preservative. . The immunogenic composition as claimed in claim 5, wherein the adjuvant is present in the range of 0.1 - 0.5 mg, and preservative is present in the range of 1.0 - 5.0 mg and suspended in a buffer. . The immunogenic composition as claimed in claim 5, wherein the adjuvant is selected from aluminium salts, inulin, algammulin, combination of inulin and aluminium hydroxide, monophosphoryl lipid A (MPL), resiquimoid, muramyl dipeptide (MDP), N-glycolyl dipeptide (GMDP), poly IC, CpG oligonucleotides, resiquimod, aluminium hydroxide with MPL, any water in oil emulsion, any oil in water emulsion that contains one or more of the following constituents: squalene or its analogues or any pharmaceutically acceptable oil, tween- 80, sorbitan trioleate, alpha-tocopherol, cholecalciferol or any of the analogues and derivatives of the molecules thereof, or calcium phosphate and mixtures thereof. . The immunogenic composition as claimed in claim 7, wherein the adjuvant is selected from Alum, CpG or mixture of both.

28 The immunogenic composition as claimed in claim 5, wherein the preservative is selected from phenol, 2-phenoxy ethanol benzethonium chloride, thiomersol or mixtures thereof. The immunogenic composition as claimed in claim 9, wherein the preservative is 2-Phenoxy ethanol. The immunogenic composition as claimed in claim 9, wherein the preservative is dissolved in a buffer selected from phosphate, citrate, histidine or mixtures thereof. The immunogenic composition as claimed in claim 11, wherein the buffer is phosphate buffer. The immunogenic composition as claimed in claim 5, wherein the stabiliser is selected from arginine, methionine, polysorbate, sucrose or mixture thereof. The immunogenic composition as claimed in claim 1, wherein the composition is in a form of a single dose or multi dose. The immunogenic composition as claimed in claim 1, wherein the composition is useful for the treatment of SARS-CoV-2. A process for preparing immunogenic composition as defined in claims 1-15, the process comprising of: (i) admixing purified RBD and nucleocapsid protein in a buffer optionally comprising of stabilisers and preservatives, (ii) filtering the solution followed by (iii) addition of adjuvants.