Multicistron expression vector for covid-19 vaccine

Abstract

The present invention provides an expression vector comprises gene of interest encode more than one structural protein to enhance immune responses against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants. Further, the present invention the expression vector comprises spike protein of SARS-CoV-2 and one or more structural protein selected from membrane protein, envelope protein and nucleocapsid protein of SARS-CoV-2. The expression vector expressed mRNA capable to encode more than one structural protein to provide immune response against SARS-CoV-2 virus.

Description

[0001] MULTICISTRON EXPRESSION VECTOR FOR COVID-19 VACCINE

[0002] FIELD OF INVENTION:

[0003] The present invention provides an expression vector comprises gene of interest encode more than one structural protein to enhance immune responses against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0004] Furthermore, the expression vector construct comprises regulatory elements selected from promoter, internal ribosome entry site, untranslated regions, gene of interest which includes proteins of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) comprises selected from spike (S) and one or more structural protein selected from, envelope (E), membrane (M), and nucleocapsid (N).

[0005] In addition, the invention provides mRNA vaccine composition comprising mRNA capable to encode spike protein and one or more structural protein selected from envelope (E), membrane (M) and nucleocapsid (N) and pharmaceutically acceptable carrier.

[0006] BACKGROUND OF INVENTION

[0007] Vaccines are biological preparations required to initiate an immune response against antigens specifically provides prophylactic or therapeutic efficacy to disease.

[0008] Coronaviruses are a group of related RNA viruses causes mild to lethal respiratory tract infections in mouse, pig, cat, dog, birds and mammals. Coronaviruses from the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirales. Coronavirus is a singlestranded, positive-stranded RNA with a complete genome with its length between 26 and 32 kb. In the past years, human coronavirus vividly affected human population with a total of 7 coronaviruses i.e., Human coronavirus 229E (HCoV-229E) and HCoV-OC43 discovered in the 1960s, SARS-CoV that appeared in 2003, and HCoV-NL63 that was isolated in the Netherlands in 2004, HCoV-HKUl identified in Hong Kong in 2005, new middle east respiratory syndrome (Middle East respiratory syndrome virus, MERS) coronavirus MERS-CoV that appeared in the Middle East in 2012 and the global outbreak of SARS-CoV-2 (2019-nCoV) that appeared in 2019. The first genome sequence of a SARS-CoV-2 isolate (Wuhan-Hu-1) was released by investigators from the Chinese CDC in Beijing on January 10, 2020 at Virological, a UK-based discussion form for analysis and interpretation of virus molecular evolution and epidemiology. The sequence was then deposited in GenBank on January 12, 2020, having Genbank Accession number MN908947.1. On 26 November 2021, SARS-CoV-2 new variant with large number of mutations reported in South Africa named Omicron (B.1.1.529) a variant of concern, designated by WHO. The spike protein (S protein) of the Severe Acute Respiratory Syndrome Coronavirus (SARS- CoV-2 and SARS) recognizes the receptor protein ACE2 (angiotensin-converting enzyme 2) on the cell membrane finally mediates and promotes the fusion of the viral envelope and the cell membrane leads to highly pathogenic infection. The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2 and SARS) have a high degree of homology. The total length of SARS-CoV-2 S is 1273 aa and consists of a signal peptide (amino acids 1-13) located at the N- terminus, the SI subunit (14-685 residues), and the S2 subunit (686-1273 residues); the last two regions are responsible for receptor binding and membrane fusion, respectively. In the S 1 subunit, there is an N-terminal domain (14-305 residues) and a receptor-binding domain (RBD, 319-541 residues); the fusion peptide (FP) (788-806 residues), heptapeptide repeat sequence 1 (HR1) (912— 984 residues), HR2 (1163-1213 residues), TM domain (1213-1237 residues), and cytoplasm domain (1237-1273 residues) comprise the S2 subunit. The SARS-CoV-2 has four structural proteins which includes spike (S), envelope (E), membrane (M), and nucleocapsid (N). The surface protein, or spike protein of SARS-CoV-2 is the foremost with two subunits SI and S2, the SI includes mainly a Receptor Binding Domain (RBD) that recognizes cellular receptors. The SARS- CoV-2 have sixteen non-structural proteins (NSPs) involved in genome replication and early transcription regulation. The structural proteins also hold the potential to develop as vaccine candidate for generating neutralizing antibodies in subject.

[0009] The inactivated or traditional vaccine has fifty to seventy percent of effectiveness against variants of the virus. The advanced SARS-CoV-2 mRNA vaccine encodes a single protein of virus that isn’t effective against mutated strain therefore it may fail and its coverage for upcoming variants is also limited. The vaccine elicits an immune response in a specific manner toward its encoded protein. An emerging scope is warranted to advance messenger ribonucleic acid (mRNA) vaccine that can encode more than one protein in a single construct and maximize its potential to neutralize the virus and upcoming mutated variants by induing strong immune responses.

[0010] The present invention successfully developed an expression vector capable to express mRNA larger than 4kb comprising spike protein and one or more structural protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) suitable for the preparation of mRNA vaccine as prophylactic or therapeutic medium against viral infection caused by SARS-CoV-2.

[0011] SUMMARY OF INVENTION

[0012] In one aspect, the present invention discloses the novel expression vector construct to produce mRNA comprises a gene of interest encoding spike protein and more than one structural protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants. In another aspect, the present invention the capability of expression vector encoding structural proteins of SARS-COV- 2 to transcribe large fragments of DNA into mRNA higher than 4kb preferably selected from about 8kb, about 10 kb, and about 12 kb.

[0013] In an embodiment the present invention discloses the novel expression vector construct to produce mRNA comprises a gene of interest encoding spike protein and more than one structural protein selected from envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants suitable for the preparation of mRNA vaccine.

[0014] In an embodiment the present invention discloses the novel expression vector construct to produce mRNA comprises a gene of interest encoding more than one spike protein and more than one structural protein selected from envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants suitable for the preparation of mRNA vaccine

[0015] In an embodiment the present invention provides a novel expression vector construct comprising at least one spike protein of Omicron variant.

[0016] In an embodiment the present invention provides a novel expression vector construct comprising at least one spike protein of Omicron and one spike protein of Delta variant.

[0017] In an embodiment, the present invention discloses the novel expression vector construct to produce mRNA vaccine encoding spike (S), envelope (E), membrane (M), and nucleocapsid (N or NC) proteins of SARS-CoV-2.

[0018] In an embodiment, the present invention discloses the novel expression vector construct to produce mRNA vaccine encoding spike (S), receptor-binding domain (RBD), envelope (E), membrane (M), and nucleocapsid (N or NC) proteins of SARS-CoV-2 omicron variant.

[0019] In an embodiment, the present invention discloses the novel expression vector construct to produce mRNA vaccine encoding spike (S), receptor-binding domain (RBD), envelope (E), membrane (M), and nucleocapsid (N or NC) proteins of SARS-CoV-2 delta variant.

[0020] In an embodiment, the present invention comprises vector construct with regulatory and non- regulatory elements. In another embodiment, the present invention discloses an engineered expression vector comprising an origin of replication, a multicloning site, and a selectable marker. In an embodiment, the expression vector comprises elements selected from core promoters, proximal promoters, distal enhancers, silencers, insulators / boundary elements, and locus control regions.

[0021] In an embodiment, the invention related to the preparation of expression vector construct comprises; a) promoter sequence; b) untranslated regions (3’ UTR and 5’ UTR); c) more than one Internal Ribosome Entry Site (IRES) element; d) gene of interest encoding more than one structural protein of SARS-CoV-2; e) poly(A) tail.

[0022] In an embodiment, an expression vector encoding more than one structural protein of Severe Acute Respiratory Syndrome Coronavirus- 2 (SARS-CoV-2) comprising: a) suitable promoter; b) untranslated region; c) structural protein of SARS-CoV-2 comprising; i. spike protein and one or more structural protein selected from; ii. membrane protein; iii. envelope protein; and iv. nucleocapsid protein; d) more than one Internal ribosomal entry sites (IRES) element; e) untranslated region; f) and Polyadenylation (Poly A) tail.

[0023] In an embodiment, the expression vector is capable to encode mRNA having size more than 4 kb.

[0024] In an embodiment, the expression vector encoding more than one structural protein of SARS-CoV- 2 comprises; a) promoter sequence; b) 5’ untranslated region (5’ UTR); c) spike protein of SARS-CoV-2 delta variant. d) internal Ribosome Entry Site (IRES) element. e) spike protein of SARS-CoV-2 omicron variant. f) internal Ribosome Entry Site (IRES) element. g) membrane protein of SARS-CoV-2 omicron variant. h) internal Ribosome Entry Site (IRES) element. i) envelope protein of SARS-CoV-2 delta variant. j) internal Ribosome Entry Site (IRES) element. k) nucleocapsid protein of SARS-CoV-2 omicron variant; l) 3’ untranslated region (3’ UTR); m) polyadenylation poly(A) tail.

[0025] In an embodiment, the expression vector encoding at least one domain of spike protein and other structural proteins of SARS-CoV-2 in a construct comprises; a) promoter sequence; b) 5’ untranslated regions (5’ UTR); c) SI domain of spike protein of SARS-CoV-2 delta variant; d) internal Ribosome Entry Site (IRES) element; e) SI domain of spike protein of SARS-CoV-2 omicron variant; f) internal Ribosome Entry Site (IRES) element; g) membrane protein of SARS-CoV-2 omicron variant; h) internal Ribosome Entry Site (IRES) element; i) envelope protein of SARS-CoV-2 delta variant; j) internal Ribosome Entry Site (IRES) element; k) nucleocapsid protein of SARS-CoV-2 omicron variant; l) 3’ untranslated region (3’ UTR); m) polyadenylation poly(A) tail.

[0026] In an embodiment, the expression vector encoding more than one structural protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) comprising: a) T7 promoter; b) 5’ untranslated region (5’ UTR); c) spike protein of SARS-CoV-2 delta variant; d) internal Ribosome Entry Site (IRES) element; e) envelope protein of SARS-CoV-2 delta variant; f) internal Ribosome Entry Site (IRES) element; g) membrane protein of SARS-CoV-2 delta variant; h) internal Ribosome Entry Site (IRES) element; i) nucleocapsid protein of SARS-CoV-2 delta variant; j) 3’ untranslated region (3’ UTR); and k) polyadenylation (Poly A) tail.

[0027] In an embodiment, the mRNA vaccine composition comprising; a) a mRNA comprises a gene of interest encoding at least one spike protein and one or more structural protein selected from envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants; b) suitable pharmaceutical acceptable excipient.

[0028] BRIEF DESCTIPTION OF FIGURES

[0029] Figure 1: depicts schematic representation of gene construct that includes T7 promoter, 5' untranslated region (5’ UTR), spike protein of SARS-CoV-2 delta variant, spike protein of SARS- CoV-2 omicron variant, membrane protein of SARS-CoV-2 omicron variant, envelope protein of SARS-CoV-2 delta variant, nucleocapsid protein of SARS-CoV-2 omicron variant, Internal ribosomal entry sites (IRES) element, 3' untranslated region (3’ UTR) and Polyadenylation (Poly A) tail.

[0030] Figure 2: depicts schematic representation of gene construct that includes T7 promoter, 5' untranslated region (5’ UTR), SI domain of SARS-CoV-2 spike protein delta variant, SI domain of SARS-CoV-2 spike protein omicron variant, membrane protein of SARS-CoV-2 omicron variant, envelope protein of SARS-CoV-2 delta variant, nucleocapsid protein of SARS-CoV-2 omicron variant, Internal ribosomal entry sites (IRES) element, 3' untranslated region (3’ UTR) and Polyadenylation (Poly A) tail.

[0031] Figure 3: depicts schematic representation of multicistron vector map encoding spike protein of SARS-CoV-2 delta variant, IRES element, spike protein of SARS-CoV-2 omicron variant, IRES element, membrane protein of SARS-CoV-2 omicron variant, IRES element, envelope protein of SARS-CoV-2 delta variant, IRES element, nucleocapsid protein of SARS-CoV-2 omicron variant and other elements enclosed in construct.

[0032] Figure 4: depicts schematic representation of multicistron vector map encoding SI domain of SARS-CoV-2 spike protein delta variant, IRES element, S 1 domain of SARS-CoV-2 spike protein omicron variant, IRES element, membrane protein of SARS-CoV-2 omicron variant, IRES element, envelope protein of SARS-CoV-2 delta variant, IRES element, nucleocapsid protein of SARS-CoV-2 omicron variant and other elements enclosed in construct.

[0033] Figure 5: depicts schematic representation of gene construct that includes T7 promoter, 5' untranslated region (5’ UTR), spike protein of SARS-CoV-2 delta variant, envelope protein of SARS-CoV-2 delta variant, membrane protein of SARS-CoV-2 delta variant, nucleocapsid protein of SARS-CoV-2 delta variant, Internal Ribosome Entry Site (IRES) element, 3 ' untranslated region (3’ UTR) and Polyadenylation tail (Poly A).

[0034] Figure 6: depicts schematic representation of multicistron vector map encoding spike protein of SARS-CoV-2 delta variant, IRES element, envelope protein of SARS-CoV-2 delta variant, IRES element, membrane protein of SARS-CoV-2 delta variant, IRES element, nucleocapsid protein of SARS-CoV-2 delta variant and other elements enclosed in construct.

[0035] Figure 7: depicts schematic representation of gel electrophoresis 1) plasmid DNA (Supercoiled, ~11 kb) and 2) plasmid DNA (Linear, ~11 kb) and Marker.

[0036] Figure 8: in-vitro cell-based expression of delta spike in HEK 293 Cell line of mRNA (~8kb).

[0037] Figure 9: depicts surface expression of delta spike on HEK 293 cells in ~8 Kb mRNA by Flow cytometry.

[0038] Figure 10: depicts schematic representation of gel electrophoresis 1) plasmid DNA (Supercoiled, ~15 kb) and 2) plasmid DNA (Linear, ~15 kb) and Marker

[0039] Figure 11: depicts schematic representation of RNA Marker and IVT mRNA (8kb and 12kb) in agarose gel electrophoresis.

[0040] Figure 12: depicts in-vitro cell-based expression of spike protein ~12 kb mRNA in HEK 293 Cell line.

[0041] Figure 13: depicts surface expression of delta spike on HEK 293 cells in ~12 Kb mRNA by Flow cytometry.

[0042] Figure 14: depicts in-vitro cell-based expression of ~12 kb mRNA in HEK 293 Cell line 1) Standard Nucleocapsid Protein 2) Nucleocapsid Protein from 12 Kb mRNA from HEK 293 expression 3) Blank 4) Negative Control and 5) Marker

[0043] Figure 15: depicts ~12 kb mRNA in HEK 293 Cell line 1) Nucleocapsid Protein 2) Envelope Protein 3) Membrane Protein

[0044] DETAILED DESCRIPTION OF INVENTION

[0045] The present invention discloses the novel expression vector construct to produce mRNA vaccine comprises gene of interest encoding more than one structural protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants. The construct includes nucleic acid molecule as a part of vaccine composition which is useful to provide to trigger immune response. The present invention discloses the expression vector expresses at least one spike protein and one or more structural protein selected from membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0046] The present invention discloses the novel expression vector construct that expresses the mRNA capable to encode one or more structural protein provides immune response against SARS-CoV- 2 virus.

[0047] Expression vector construct

[0048] The term "expression vector construct" or "expression construct" or “expression vector” are interchangeable and refers to the nucleotide sequences of the invention containing the nucleotide sequences to be expressed. The restriction sites between the 5 'and 3' ends of the sequences allows insertion, removal of sequences in a vector. The expression vector construct includes nucleotide sequence, transcriptional and translational control sequences, such as a promoter and / or termination sequences, operably linked to the nucleotide sequence and allowing expression in a host cell.

[0049] “Plasmid” or “Plasmid DNA vector” or “Plasmid DNA” used in mRNA vaccine production as are easy to replicate (copy) and reliable containing the target gene sequence. Plasmid DNA vector comprises a multiple cloning site, an RNA promoter sequence, optionally a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication. The plasmid backbone is pHSG298 in the context of the present invention.

[0050] The "multicistron or multici stronic expression vector” comprising a nucleotide sequence as mRNA molecule that encodes more than polypeptide or protein and other regulatory elements. In an embodiment, the term “about” for the expression vector is capable to encode mRNA size of about more than 4kb. In certain embodiment, the mRNA size is about 8kb, about lOkb, about 1 Ikb, and about 12kb. In an embodiment, the expression vector expresses at least one spike protein and one or more structural protein selected from envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants. In another embodiment, the expression vector expresses at least more than one spike protein preferably two spike protein and one or more structural protein selected from envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants. In another embodiment, the expression vector expresses at least one spike protein and one or more structural protein selected from envelope (E) and membrane (M) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants. In another embodiment, the expression vector expresses at least one spike protein and one or more structural protein selected from envelope (E) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0051] In another embodiment, the expression vector expresses at least one spike protein and one or more structural protein selected from membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0052] The present expression construct should not be limited to the expression construct disclosed in the present application, any skilled person in the art can select the mentioned of structural protein from any variant of existing or future variant of SARS-CoV-2 which should be considered in the scope of the present invention.

[0053] Promoter

[0054] The term “promoter” in the present invention refers to a sequence of nucleic acid required to turn a gene on or off. The RNA polymerase After the recognition of the promoter region by an RNA polymerase, the binding of the RNA polymerase becomes an initiation complex. The promoter sequence selected from simian vacuolating virus 40 (SV40), cytomegalovirus (CMV), elongation factor (EF)-l, lactase LAC4, pPolh, trp, Z.PL, A0X1, GALI, GAL10, nmtl, nmt42, nmt81 and glyceraldehyde- 3 -phosphate dehydrogenase (GAP). The promoter for controlling RNA in vitro transcription based on DNA dependent RNA polymerases selected from T3,T4, T7, and SP6 RNA polymerases. The regulatory promoter sequence T7 promoter with a sequence of 18 base pairs long up nucleotides to transcription start site recognized by T7 RNA polymerase used to regulate gene expression of recombinant proteins,

[0055] IRES

[0056] The term “IRES” or “internal ribosome entry site” refers to an RNA element that allows for translation initiation in a cap-dependent manner and cap-independent manner, as part of the greater process of protein synthesis. A vector construct can contain a multiple IRES element.

[0057] UTR

[0058] The term “untranslated region” or “UTR” refers to a part of a nucleic acid molecule typically located 5’ or 3’ located of a coding sequence.

[0059] 5’ UTR

[0060] The term “5’ untranslated region” or “5’ UTR” refers as a specific part of messenger RNA (mRNA). The 5 ‘UTR is located 5’ of the open reading frame of the mRNA. The 5’ UTR can contain elements for controlling gene expression. The 5’UTR begins at the transcription start site and ends one nucleotide before the start codon of the open reading frame. The 5 ‘UTR can be modified by adding a cap after transcription and / or during co-transcription.

[0061] 3' UTR

[0062] The term “3 ' untranslated region” or “3 ' UTR” refers to a mRNA that is located between the region encoding the protein and the poly (A) sequence. The 3 ’UTR sequences transcribed into their respective mRNAs encoded by genes during the gene expression process and not further translated into an amino acid sequence. The 3 UTR sequence can be an RNA sequence, or a DNA sequence.

[0063] Capping

[0064] The term “capping” or “capping enzyme” or “Co-transcriptional cap” or “cap” refers to a cap analog introduced into the IVT reaction along with NTPs, RNA polymerase etc in a single reaction vessel, resulting in mature mRNA. The cap analog protects the newly synthesized mRNA from degradation, gene expression and overall mRNA stability. It also assists in ribosome binding to help initiating translation.

[0065] Polyadenylation

[0066] The term “Polyadenylation” or “Poly(A) tail” or “polyadenylation poly (A) tail” refers to addition of multiple adenosine monophosphates as a poly(A) tail to an RNA transcript. The poly-A tail protects mRNA from degradation, aids in exporting the mature mRNA to the cytoplasm, and is also involved in binding proteins to initiate translation.

[0067] Vaccine

[0068] The term “vaccine” or “immunological preparation” refers to a biological composition comprising an immunogen or antigenic molecule that stimulates immune response in body to generate prophylactic or therapeutic effect against disease causing antigen.

[0069] The invention provides a mRNA vaccine composition comprising a. a mRNA comprises a gene of interest encoding at least one spike protein and one or more structural protein selected from envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants; b. suitable pharmaceutical acceptable excipient.

[0070] The suitable pharmaceutical acceptable excipient is Lipid nanoparticle (LNP). Lipid nanoparticles typically comprise ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles of the disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551 ;

[0071] PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016000129; PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491 all of which are incorporated by reference herein in their entirety.

[0072] In an embodiment the vaccines of the present disclosure are typically formulated in lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises at least one ionizable cationic lipid, at least one non-cationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG) -modified lipid.

[0073] In an embodiment, the nucleic acid encapsulated in lipid nano particle comprises cationic lipid, neutral lipid, one polymer conjugated lipid or PEG-lipid and steroid or cholesterol.

[0074] In some embodiments, the lipid nanoparticle comprises 40-50 mol% ionizable lipid, optionally 45- 50 mol%, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol , 48.5 mol%, 49 mol%, or 49.5 mol%.

[0075] In some embodiments, the lipid nanoparticle comprises 30-45 mol% sterol, optionally 35- 40 mol%, for example, 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 35-35 mol%, 35- 36 mol%, 36-37 mol%, 38-38 mol%, 38-39 mol%, or 39-40 mol%.

[0076] In some embodiments, the lipid nanoparticle comprises 5-15 mol% helper lipid, optionally 10-12 mol%, for example, 5-6 mol%, 6-7 mol%, 7-8 mol%, 8-9 mol%, 9-10 mol%, 10-11 mol%, 11-12 mol%, 12-13 mol%, 13-14 mol%, or 14-15 mol%.

[0077] In some embodiments, the lipid nanoparticle comprises 1-5% PEG lipid, optionally 1-3 mol%, for example 1.5 to 2.5 mol%, 1-2 mol%, 2-3 mol%, 3-4 mol%, or 4-5 mol%.

[0078] In some embodiments, the lipid nanoparticle comprises 20-60 mol% ionizable cationic lipid. For example, the lipid nanoparticle may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable cationic lipid. In some embodiments, the lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable cationic lipid. In some embodiments, the lipid nanoparticle comprises 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, or 55 mol% ionizable cationic lipid.

[0079] In an embodiment, the encapsulation efficiency of lipid nanoparticle is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95%, at least 97.5%, at least 98%, at least 99%, at least 99.5% or more.

[0080] In preferred embodiment, the encapsulation efficiency of lipid nanoparticle is not less than 40%,

[0081] In an embodiment, the lipid nanoparticle average size selected from about lOOOnm or less, about 900 nm or less, about 800 nm or less, about 700 nm or less, about 600 nm or less, about 500 nm or less, about 400 nm or less, about 300 nm or less, about 200 nm or less, about 175 nm or less, about 150 nm or less, about 145 nm or less, about 140 nm or less, about 135 nm or less, about 130 nm or less, about 125 nm or less, about 120 nm or less, about 115 nm or less, about 100 nm or less, about 90 nm or less, about 80 nm or less, about 75 nm or less, about 50 nm or less.

[0082] GOI

[0083] The term “GOI” or “gene of interest” refers to nucleic acid sequence encoding structural proteins which are selected from spike protein (S), envelope protein ™, membrane protein (M), nucleocapsid protein (N or NC) of SARS-CoV-2 ; for example, the N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. The GOI can be obtained from a variety of sources, including source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. mRNA

[0084] The term “mRNA” or “messenger RNA” refers to nucleic acid sequence / single-stranded RNA with at least one open reading frame that can be translated by a cell or organism comprising the mRNA. The mRNA is made from a DNA template during the process of transcription. The role of mRNA is to carry protein information from the DNA. The mRNA delivered into cell or organism it reaches to cell’s cytoplasm the protein-making machinery reads the mRNA sequence and translates each three-base codon into its corresponding amino acid in a growing protein chain.

[0085] In an embodiment, the mRNA size is about more than 4kb. In certain embodiment, the mRNA size is about 8kb, about lOkb, about l lkb, and about 12kb.

[0086] In an embodiment, the mRNA comprises at least one spike protein and one or more structural protein selected from envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants. In another embodiment, the mRNA comprises at least more than one spike protein preferably two spike protein and one or more structural protein selected from envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0087] In another embodiment, mRNA comprises at least one spike protein and one or more structural protein selected from envelope (E) and membrane (M) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0088] In another embodiment, mRNA comprises at least one spike protein and one or more structural protein selected from envelope (E) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0089] In another embodiment, the mRNA comprises at least one spike protein and one or more structural protein selected from membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0090] Omicron Variant

[0091] The term “Omicron variant” or “B.1.1.529” refers to SARS-CoV-2 variant where large number of mutations observed in the spike protein which is most supposed to increase the transmissibility rate. It is designated as “variant of concern” on 26 November 2021 by WHO. The variant is characterised by more than 30 amino acid changes, three small deletions and one small insertion in the spike protein compared with the original virus, of which 15 are located in the receptor binding domain (residues 319-541). The SARS-CoV-2 omicron has 1270 aa in spike protein, 222 aa in membrane protein and 416 aa in nucleocapsid protein. Other genomic regions also carry several mutations and deletions. The variant has three mutations at the furin cleavage site. The furin cleavage site increases SARS-CoV-2 infectivity.

[0092] Delta Variant

[0093] The term “Delta variant” or “B.1.617.2” refers to SARS-CoV-2 variant, a globally dominant strain was first detected in India in late 2020 and named on 31 May 2021. The substitutions T478K, P681R and L452R are known to affect transmissibility of the virus. The SARS-CoV-2 delta spike protein has 1271 aa, and envelope protein has 75 aa. The B.1.617.2 spike has 10 mutations which produce alterations in the amino-acid sequences of the proteins it encodes. The delta variant spike protein with 1271 aa and envelope with 75 aa. The Delta with K417N corresponded to lineages AY.l, AY.2 and AY.3 has been nicknamed “Delta plus” or “Nepal variant”.

[0094] Spike Protein The term “Spike protein” or “S protein” refers to structural protein of SARS-CoV-2 with a size of 180-200 kDa, consists of an extracellular N-terminus, a transmembrane ™ domain anchored in the viral membrane, and a short intracellular C-terminal segment. The total length of SARS-CoV- 2 S is 1273 aa and consists of a signal peptide (amino acids 1-13) located at the N-terminus, the SI subunit (14-685 residues), and the S2 subunit (686-1273 residues); the last two regions are responsible for receptor binding and membrane fusion, respectively. In the SI subunit, there is an N-terminal domain (14-305 residues) and a receptor-binding domain (RBD, 319-541 residues); the fusion peptide (FP) (788-806 residues), heptapeptide repeat sequence 1 (HR1) (912-984 residues), HR2 (1163-1213 residues), TM domain (1213-1237 residues), and cytoplasm domain (1237-1273 residues) comprise the S2 subunit. The S protein normally exists in a metastable, prefusion conformation, S protein trimers visually form a characteristic bulbous, crown-like halo surrounding the viral particle based on the structure of coronavirus S protein monomers, the SI and S2 subunits form the bulbous head and stalk region. The NTD also plays a regulatory role in the conformational changes of the S protein. The RBD structure with neighbouring NTD helps to enhance the binding affinities. Once the virus interacts with the host cell, extensive structural rearrangement of the S protein occurs, allowing the virus to fuse with the host cell membrane. The spikes are coated with polysaccharide molecules to camouflage them, evading surveillance of the host immune system during entry.

[0095] A spike protein that is less than the full-length spike protein is one or more domains and / or subunits of the spike protein having at least one amino acid less than of the full-length spike protein or a fusion protein and multi component having one or more domains linked together in an non-natural order or sequence.

[0096] The RBD (receptor-binding domain) fragment short immunogenic fragment in SARS-CoV-2 S protein allows virus to gain entry into cells and cause infection by its strong affinity to ACE2 receptor. The region of SARS-CoV-2 RBD at residues 319 to 541 of S protein. The similarity in sequence of RBD between SARS-CoV and SARS-CoV-2 is close to 73.5%

[0097] Membrane Protein

[0098] The term “Membrane protein” or “M protein” refers to SARS-CoV-2 structural protein is the most abundant structural protein it spans the membrane bilayer, leaving a short NH2-terminal domain outside the virus and a long COOH terminus (cytoplasmic domain) inside the virion. The M protein can bind to all other structural proteins, binding to M protein helps to stabilize N proteins and promotes completion of viral assembly by stabilizing the N protein-RNA complex, inside the internal virion. It has a triple-helix bundle, and forms a single 3 -transmembrane domain. Envelope Protein

[0099] The term “Envelope protein” or “E protein” refers to SARS-CoV-2 structural protein with a size of 8-12 kDa involved in a wide spectrum of functional repertoire. E protein have 75-109 amino acid integral membrane protein with a short 7-12 residue hydrophilic N-terminal domain followed by a hydrophobic transmembrane domain of ~25 residues and a long hydrophilic C-terminal domain. The E-protein of SARS-CoV-2 shares sequence similarity with that of SARS-CoV-1.

[0100] Nucleocapsid Protein

[0101] The term “Nucleocapsid protein” or “N or NC protein” refers to SARS-CoV-2 structural protein that is more conserved and stable, with 90% amino acid homology and fewer mutations over time. It have five domains N-terminal domain (NTD), an RNA-binding domain (RBD), central linker (LINK), a dimerization domain, and a predicted disordered C-terminal domain (CTD). N proteins of many coronaviruses are highly immunogenic and are expressed abundantly during infection. N protein is a representative antigen for the T-cell response in a vaccine setting, inducing SARS- specific T-cell proliferation and cytotoxic activity. The middle or C-terminal region of the SARS- CoV N protein is important for eliciting antibodies against SARS-CoV during the immune response.

[0102] In-vitro Transcription

[0103] The term “IVT” or “ In-vitro transcription” refers to the use of DNA as a template to simulate the transcription process to generate RNA in vitro cell-free system. In-vitro transcription: preparative RNA yields in analytical scale reactions (ID Pokrovskaya, V V Gurevich, Analytical Biochemistry, Volume 220, Issue 2, 1 August 1994, Pages 420-423)

[0104] Chromatography

[0105] The term “Chromatography” used herein for the separation of biomolecule such as plasmid DNA and mRNA from mixture based on size, surface charge and / or interaction the purification method is selected from the group consisting of cation exchange chromatography, anion exchange chromatography, membrane absorbers, reversed phase chromatography, normal phase chromatography, size exclusion chromatography, hydrophobic interaction chromatography, mixed mode chromatography, affinity chromatography, hydroxylapatite (HA) chromatography, HPLC, core bead chromatography or combinations thereof. The Ion exchange chromatography or IEX refers to the separation of molecules on the basis of differences in their net surface charge. The Affinity chromatography, the separation based on interactions between two molecules, purification by placing one of the interacting molecules, referred to as affinity ligand, onto a solid matrix to create a stationary phase while the target molecule is in the mobile phase. The Mixed / Multi-mode chromatography or “MMC” utilize more than one form of interaction between the stationary phase and analytes in order to achieve the separation, with multiple retention modes on a single MMC column provides additional dimension to a separation method by its unique selectivity and retention of a variety of compounds, especially polar and charged molecules.

[0106] In an embodiment, the gene of interest encodes at least two structural proteins.

[0107] In an embodiment, the gene of interest encodes at least three structural proteins.

[0108] In an embodiment, the gene of interest encodes at least four structural proteins.

[0109] In an embodiment, the gene of interest encodes at least five structural proteins.

[0110] In an embodiment, the invention related to the preparation of expression vector construct comprises; a) promoter sequence; b) untranslated regions (3’ UTR and 5’ UTR); c) more than one Internal Ribosome Entry Site (IRES) element; d) gene of interest encoding more than one structural protein of SARS-CoV-2; e) polyadenylation poly(A) tail.

[0111] In an embodiment, wherein the gene of interest comprises structural proteins of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants wherein the variants share 60% identity to wuhan coronavirus.

[0112] In certain embodiment, variants share at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to wuhan coronavirus.

[0113] In preferred embodiment, the coronavirus variants are selected from Omicron and Delta virus.

[0114] In an embodiment, wherein the gene of interest comprises spike (S) protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0115] In an embodiment, wherein the gene of interest comprises nucleocapsid (N or NC) protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0116] In an embodiment, wherein the gene of interest comprises envelope (E) protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0117] In an embodiment, wherein the gene of interest comprises membrane (M) protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants. In an embodiment the novel expression vector construct comprising at least one spike protein of Omicron.

[0118] In an embodiment the novel expression vector construct comprising at least one spike protein of Omicron and one spike protein of Delta variant.

[0119] In an embodiment, the novel expression multicistronic vector construct to produce mRNA vaccine encoding spike (S), envelope (E), membrane (M), and nucleocapsid (N or NC) proteins of SARS- CoV-2.

[0120] In an embodiment, the novel expression multicistronic vector construct to produce mRNA vaccine encoding full or partial spike (S) protein of Omicron and Delta variant of coronavirus.

[0121] In an embodiment, an expression vector encoding more than one structural protein of Severe Acute Respiratory Syndrome Coronavirus- 2 (SARS-CoV-2) comprising: a) suitable promoter; b) untranslated region; c) structural protein of SARS-CoV-2 comprising; i. spike protein and one or more structural protein selected from; ii. membrane protein; iii. envelope protein; and iv. nucleocapsid protein; d) more than one Internal ribosomal entry sites (IRES) element; e) untranslated region; f) and polyadenylation (Poly A) tail.

[0122] In an embodiment, the expression vector is not mono-cistronic and essentially encoding more than one structural protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0123] In an embodiment, the expression vector is capable to encode mRNA having size more than 4 kb.

[0124] In an embodiment, the expression vector is capable to encode mRNA size more than 4kb, 8kb, and 12kb.

[0125] In an embodiment, the expression vector comprises spike protein of SARS-CoV-2, first spike protein and second spike protein both derived from different SARS-CoV-2. In an embodiment, the expression vector comprises spike protein of SARS-CoV-2, first spike protein derived from alpha, beta, gamma, delta, eta, Iota, kappa, lambda, Mu, Omicron and emerging variants thereof.

[0126] In an embodiment, the expression vector comprises spike protein of SARS-CoV-2, second spike protein derived from alpha, beta, gamma, delta, eta, Iota, kappa, lambda, Mu, Omicron and emerging variants thereof.

[0127] In an embodiment, the expression vector comprises of SARS-CoV-2 envelope (E), membrane (M), and nucleocapsid (N) derived from alpha, beta, gamma, delta, eta, Iota, kappa, lambda, Mu, Omicron and emerging variants thereof.

[0128] In an embodiment, the expression vector encoding more than one structural protein of SARS-CoV- 2 comprises; a) T7 Promoter; b) 5’ untranslated region (5’ UTR); c) spike protein of SARS-CoV-2 delta variant; d) internal Ribosome Entry Site (IRES) element; e) spike protein of SARS-CoV-2 omicron variant; f) internal Ribosome Entry Site (IRES) element; g) membrane protein of SARS-CoV-2 omicron variant; h) internal Ribosome Entry Site (IRES) element; i) envelope protein of SARS-CoV-2 delta variant; j) internal Ribosome Entry Site (IRES) element; k) nucleocapsid protein of SARS-CoV-2 omicron variant; l) 3’ untranslated region (3’ UTR); m) polyadenylation Poly(A) tail.

[0129] In an embodiment, the expression vector comprises nucleotide sequence of SEQ ID No: 1.

[0130] In an embodiment, the expression vector comprises nucleotide sequence is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SARS- CoV-2 sequence of SEQ ID No: 1.

[0131] In an embodiment, the expression vector comprises the accession number MTCC 25576.

[0132] In an embodiment, the expression vector capable to encode mRNA having size about 12kb. In an embodiment, the expression vector encoding at least one domain of spike protein and other structural proteins of SARS-CoV-2 comprises; a) T7 promoter; b) 5’ untranslated regions (5’ UTR); c) SI domain of spike protein of SARS-CoV-2 delta variant; d) internal Ribosome Entry Site (IRES) element; e) SI domain of spike protein of SARS-CoV-2 omicron variant; f) internal Ribosome Entry Site (IRES) element; g) membrane protein of SARS-CoV-2 omicron variant; h) internal Ribosome Entry Site (IRES) element; i) envelope protein of SARS-CoV-2 delta variant; j) internal Ribosome Entry Site (IRES) element; k) nucleocapsid protein of SARS-CoV-2 omicron variant; l) 3’ untranslated region (3’ UTR); m) polyadenylation poly(A) tail.

[0133] In an embodiment, the expression vector comprises nucleotide sequence SEQ ID No: 2.

[0134] In an embodiment, the expression vector comprises nucleotide sequence is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SARS- CoV-2 sequence of SEQ ID No: 1.

[0135] In an embodiment, the expression vector encoding more than one structural protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) comprising: a) T7 promoter; b) 5’ untranslated region (5’ UTR); c) spike protein of SARS-CoV-2 delta variant; d) internal Ribosome Entry Site (IRES) element; e) envelope protein of SARS-CoV-2 delta variant; f) internal Ribosome Entry Site (IRES) element; g) membrane protein of SARS-CoV-2 delta variant; h) internal Ribosome Entry Site (IRES) element; i) nucleocapsid protein of SARS-CoV-2 delta variant; j) 3’ untranslated region (3’ UTR); and k) polyadenylation (Poly A) tail. In an embodiment, the expression vector comprises nucleotide sequence SEQ ID No: 3.

[0136] In an embodiment, the expression vector comprises nucleotide sequence is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SARS- CoV-2 sequence of SEQ ID No: 1.

[0137] In an embodiment, the expression vector expressed mRNA size is more than 4kb.

[0138] In an embodiment, the preparation of expression vector construct encoding structural protein of SARS-CoV-2 omicron variant comprises; a) promoter sequence; b) 5’ untranslated regions (5’ UTR); c) spike protein of SARS-CoV-2 omicron variant; d) 3’ untranslated region (3’ UTR); e) polyadenylation poly(A) tail.

[0139] In an embodiment, the invention related to the preparation of expression vector construct comprises; a) promoter sequence; b) untranslated regions; c) gene of interest more than one structural protein selected from spike (S), envelope (E), membrane (M), and nucleocapsid (N or NC); d) more than one Internal Ribosome Entry Site (IRES) element; e) polyadenylation poly(A) tail.

[0140] In the present invention the capability of expression vector encoding structural proteins of SARS- COV- 2 to transcribe large fragments of DNA into mRNA selected from about 8kb, about 10 kb, and about 12 kb.

[0141] In an embodiment, the expression vector construct comprises SARS-CoV-2 spike (S) protein, the length of spike (S) protein from about 1250 to about 1290 amino acid residues.

[0142] In an embodiment, the expression vector construct comprises SARS-CoV-2 envelope (E) protein, the length of envelope (E) protein from about 50 to about 90 amino acid residues.

[0143] In an embodiment, the expression vector construct comprises SARS-CoV-2 membrane (M) protein, the length of membrane (M) protein from about 200 to about 250 amino acid residues. In an embodiment, the expression vector construct comprises SARS-CoV-2 nucleocapsid (N or NC), the length of nucleocapsid (N or NC) protein from about 390 to about 446 amino acid residues.

[0144] In an embodiment, the expression vector construct comprises polyadenylation (Poly A) tail contains about 80 to about 120 adenosine nucleotides.

[0145] In an embodiment, the expression vector construct comprises polyadenylation (Poly A) tail contains 110 adenosine nucleotides.

[0146] In an embodiment, the expression vector construct comprises polyadenylation (Poly A) tail contains more than 100 adenosine nucleotides.

[0147] In an embodiment, the SARS-CoV-2 variant selected from alpha, beta, gamma, delta, eta, Iota, kappa, lambda, Mu, Omicron and emerging variants thereof.

[0148] In preferred embodiment, the SARS-CoV-2 variant is Omicron.

[0149] In another preferred embodiment, the SARS-CoV-2 variant is Delta.

[0150] In an embodiment, the expression construct comprises as gene of interest is SARS-CoV-2 protein.

[0151] In another embodiment, the expression construct comprises as gene of interest selected from spike protein, nucleocapsid protein, membrane protein, envelope protein.

[0152] In an embodiment, the expression construct comprises as gene of interest is spike protein.

[0153] In certain embodiment, the expression construct comprises as gene of interest is SI domain of spike protein.

[0154] In certain embodiment, the expression construct comprises as gene of interest is membrane protein.

[0155] In certain embodiment, the expression construct comprises as gene of interest is envelope protein.

[0156] In certain embodiment, the expression construct comprises as gene of interest is nucleocapsid protein.

[0157] In an embodiment, the expression construct comprises as gene of interest is Receptor binding domain of spike (S) protein.

[0158] In an embodiment, the nucleotide sequence encodes structural proteins is at least 80% identical to SARS-CoV-2 immunogenic variant.

[0159] In certain embodiment, the expression construct comprises nucleotide sequence encodes structural proteins is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SARS-CoV-2 immunogenic variant.

[0160] In certain embodiment, the expression construct comprises nucleotide sequence encodes structural proteins is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to Omicron SARS-CoV-2.

[0161] In certain embodiment, the expression construct comprises nucleotide sequence encodes structural proteins is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to Delta SARS-CoV-2.

[0162] In an embodiment, the invention related to the preparation of expression vector construct comprises; a) promoter sequence; b) untranslated regions (3’ UTR and 5’ UTR); c) gene of interest encoding more than one structural protein selected from spike (S), envelope (E), membrane (M), and nucleocapsid (N); d) more than one Internal Ribosome Entry Site (IRES) element; e) polyadenylation poly(A) tail.

[0163] In an embodiment, the invention related to a process for the preparation of vaccine vector construct encoding more than one antigenic polypeptide comprises; a) promoter sequence to initiate the reaction for expression of polypeptide; b) 5’ UTR untranslated region to serve as entry point for the ribosome during translation; c) internal Ribosome Entry Site (IRES) element initiates translation in a cap-dependent and independent manner; d) 3’ UTR untranslated region regulates mRNA-based processes; e) polyadenylation poly(A) tail provides stability and allows mature messenger RNA molecule to export.

[0164] In an embodiment, the invention provides a process for the preparation of mRNA from vector construct comprises; a) selection and identification of gene of interest for various antigen; b) cloning the selected gene of interest in a vector; c) isolation of suitable plasmid; d) linearization of isolated plasmid; e) performing the in-vitro reaction for the preparation mRNA copies; f) purification of mRNA by chromatography methods.

[0165] In an embodiment, a process for the purification of mRNA comprises; a) transfect the expression vector into E. Coli host cells; b) harvesting of cells followed by lysis, neutralization and precipitation; c) isolation and purification of plasmid; d) linearization of purified plasmid; e) performing the in-vitro reaction for the preparation mRNA; f) purification of mRNA by chromatography methods.

[0166] In an embodiment, the linearization of isolated plasmid using restriction endonuclease selected from EcoRI, EcoRII, BamHI, Hindlll, TaqI, Notl, HinFI, Sau3AI, PvuII*, Smal*, Haelll*, Hgal, Alul*, EcoRV*, EcoP15I, Rsal, Kpnl, Pstl, BstBI , Mfel, BciVI, Xhol, PacI, BstEII, SacI, Sall, Seal, SacI, Tthllll, EagI, Sadi, Spel, SphI, Stul*, Bglll, Xbal, Nrul.

[0167] In an embodiment, the expression vector comprises kozak sequence before initiation codon.

[0168] In an embodiment, the in-vitro transcription uses cell-free system for the generation of RNA transcripts.

[0169] In-vitro transcription: preparative RNA yields in analytical scale reactions (ID Pokrovskaya, V V Gurevich, Analytical Biochemistry, Volume 220, Issue 2, 1 August 1994, Pages 420-423)

[0170] In an embodiment, the promoter for controlling RNA in-vitro transcription based on DNA dependent RNA polymerases selected from T7, T3, and SP6 RNA polymerases.

[0171] In an embodiment, the regulatory promoter sequence is T7 promoter.

[0172] In another embodiment, the promoter sequence selected from simian vacuolating virus 40 (SV40), cytomegalovirus (CMV), elongation factor (EF)-l, lactase LAC4, pPolh, trp, ZPL, A0X1, GALI, GAL10, nmtl, nmt42, nmt81 and glyceraldehyde-3-phosphate dehydrogenase (GAP).

[0173] In an embodiment, the purification of mRNA using chromatography method selected from high performance liquid chromatography (HPLC), low normal pressure liquid chromatography methods, gas chromatography, reversed phase HPLC (RP-HPLC), affinity chromatography, ion exchange chromatography, hydroxyapatite chromatography, core bead flow-through chromatography, Oligo dT chromatography.

[0174] In an embodiment, mRNA is purified using affinity chromatography.

[0175] In an embodiment, mRNA is purified using ion exchange chromatography.

[0176] In an embodiment, mRNA is purified using Oligo dT chromatography.

[0177] In an embodiment, mRNA is purified using high performance liquid chromatography (HPLC).

[0178] In an embodiment, mRNA is purified using mixed mode chromatography.

[0179] In an embodiment, the analysis of purified mRNA using methods selected from agarose gel electrophoresis and in-vitro cell-based expression.

[0180] In another embodiment, the structural protein of SARS-CoV-2 expressed on HEK 293 cell line.

[0181] In another embodiment, the method for expression of structural protein of SARS-CoV-2 selected from flow cytometry, dot blot, and western blotting,

[0182] In an embodiment, the expressed mRNA has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% similarity to SARS-CoV-2 sequence of SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3.

[0183] In an embodiment, a mRNA vaccine composition comprising; a. a mRNA comprises a gene of interest encoding at least one spike protein and one or more structural protein selected from envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants; b. suitable pharmaceutical acceptable excipient; wherein the suitable pharmaceutical acceptable excipient is lipid nanoparticle (LNP).

[0184] In an embodiment, the lipid nanoparticles typically comprise ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest.

[0185] In an embodiment, the vaccines of the present disclosure are typically formulated in lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises at least one ionizable cationic lipid, at least one non-cationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG) -modified lipid. In an embodiment, the nucleic acid encapsulated in lipid nano particle comprises cationic lipid, neutral lipid, one polymer conjugated lipid or PEG-lipid and steroid or cholesterol.

[0186] In some embodiments, the lipid nanoparticle comprises 40-50 mol% ionizable lipid, optionally 45- 50 mol%, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol , 48.5 mol%, 49 mol%, or 49.5 mol%.

[0187] In some embodiments, the lipid nanoparticle comprises 30-45 mol% sterol, optionally 35- 40 mol%, for example, 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 35-35 mol%, 35- 36 mol%, 36-37 mol%, 38-38 mol%, 38-39 mol%, or 39-40 mol%.

[0188] In some embodiments, the lipid nanoparticle comprises 5-15 mol% helper lipid, optionally 10-12 mol%, for example, 5-6 mol%, 6-7 mol%, 7-8 mol%, 8-9 mol%, 9-10 mol%, 10-11 mol%, 11-12 mol%, 12-13 mol%, 13-14 mol%, or 14-15 mol%.

[0189] In some embodiments, the lipid nanoparticle comprises 1-5% PEG lipid, optionally 1-3 mol%, for example 1.5 to 2.5 mol%, 1-2 mol%, 2-3 mol%, 3-4 mol%, or 4-5 mol%.

[0190] In some embodiments, the lipid nanoparticle comprises 20-60 mol% ionizable cationic lipid. For example, the lipid nanoparticle may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable cationic lipid. In some embodiments, the lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable cationic lipid. In some embodiments, the lipid nanoparticle comprises 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, or 55 mol% ionizable cationic lipid.

[0191] In an embodiment, the nucleic acid encapsulated in lipid nano particle comprises cationic lipid, neutral lipid, one polymer conjugated lipid or PEG-lipid and steroid or cholesterol.

[0192] In an embodiment, the encapsulation efficiency of lipid nanoparticle is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95%, at least 97.5%, at least 98%, at least 99%, at least 99.5% or more.

[0193] In preferred embodiment, the encapsulation efficiency of lipid nanoparticle is not less than 40%.

[0194] In an embodiment, the lipid nanoparticle average size selected from about lOOOnm or less, about 900 nm or less, about 800 nm or less, about 700 nm or less, about 600 nm or less, about 500 nm or less, about 400 nm or less, about 300 nm or less, about 200 nm or less, about 175 nm or less, about 150 nm or less, about 145 nm or less, about 140 nm or less, about 135 nm or less, about 130 nm or less, about 125 nm or less, about 120 nm or less, about 115 nm or less, about 100 nm or less, about 90 nm or less, about 80 nm or less, about 75 nm or less, about 50 nm or less.

[0195] In an embodiment, the mRNA vaccine comprising; a. a mRNA encoded by the expression construct as claimed in claim 1 or claim 8 or claim 14 or claim 17 comprises a gene of interest encoding at least one spike protein and one or more structural protein selected from envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants; b. suitable pharmaceutical acceptable excipient selected from cationic lipid, neutral lipid, one polymer conjugated lipid or PEG-lipid and steroid or cholesterol.

[0196] In an embodiment, the mRNA vaccine provides immune response to at least more than one structural protein selected from spike (S), envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.

[0197] In an embodiment, the expression vector expressed mRNA capable to encode more than one structural protein to provide immune response against SARS-CoV-2 virus.

[0198] The present invention provides an example for illustrative purpose and scope should not be considered limiting to them.

[0199] Example 1: Preparation of mRNA vaccine construct

[0200] The multicistron construct is designed to have better coverage and efficiency against SARS-COV- 2. The structural sequences of virus from the database were analyzed, it is observed that other structural proteins of Severe Acute Respiratory Syndrome Coronavirus 2 as membrane, enveloped and nucleocapsid apart from the spike are more conserved and less susceptible to mutation. The vaccine construct was designed to increase the breadth of immune defense against coronavirus infection as an improved mRNA vaccine. The vector construct was prepared by following the steps below: a) The amino acid sequences of SARS-CoV-2 and emerging variants were retrieved from http s : / / co vid 19dashboard .re eneron . com / ?tab=Home , https: / / marks.hms.harvard.edu / sars-cov-2 / , https: / / www.gisaid.org / and analyzed for lineage, mutation identification for different variants. b) Based on the amino acid sequences in the emerging mutants were reverse translated to DNA sequence which were codon optimized as per human amino acid codon usage. c) The pKashiv_multicistronic_3 DNA vector construct (-15255 bp) comprises the elements as shown in Figure 1 & 3 (SEQ ID NO: 1) and respective sequence mentioned as “pKashiv_Multicistron_3 Vector DNA (-15255 bp)” and cloned into pHSG298 plasmid. d) Preparation of clone of SARS-CoV-2 delta and omicron variant sequences in the pHSG298 vector using restriction enzyme cloning. e) Transforming the E. coli host with the recombinant plasmid and further selection on Kanamycin containing media. f) Screening the transformed colonies for positive clone and confirmation by sequencing and other identification methods known in the art. g) Culturing the recombinant E. coli cells in the desired media following by purification of plasmid DNA. h) Linearization of plasmid DNA by restriction enzyme to use further for in-vitro transcription reaction using T7 RNA polymerase. i) Preparation of mRNA through in-vitro transcription (IVT) and purification using suitable chromatography column.

[0201] Example 2: Preparation of mRNA vaccine construct

[0202] The multicistron construct is designed to have better coverage and efficiency against SARS-COV- 2. The structural sequences of virus from the database were analyzed, it is observed that other structural proteins of Severe Acute Respiratory Syndrome Coronavirus 2 as membrane, enveloped and nucleocapsid apart from the spike are more conserved and less susceptible to mutation. The vaccine construct was designed to increase the breadth of immune defence against coronavirus infection as an improved mRNA vaccine. The vector construct was prepared by following the steps below: a) The amino acid sequences of SARS-CoV-2 and emerging variants were retrieved from http s : / / co vid 19dashboard .re eneron . com / ?tab=Home , https: / / marks.hms.harvard.edu / sars-cov-2 / , https: / / www. isaid.or / and analyzed for lineage, mutation identification for different variants. b) Based on the amino acid sequences in the emerging mutants were reverse translated to DNA sequence which were codon optimized as per human amino acid codon usage. c) The pKashiv_multicistron_4 DNA vector construct (-11718bp) comprises the elements as shown in Figure 2 & 4 (SEQ ID NO: 2) and respective sequence mentioned as “pKashiv Multicistron 4 Vector DNA (-11718 bp)” and clone into pHSG298 plasmid. d) Preparation of clone of SARS-CoV-2 delta and omicron variant sequences in the pHSG298 vector using restriction enzyme cloning. e) Transforming the E. coli host with the recombinant plasmid and further selection on Kanamycin containing media. f) Screening the transformed colonies for positive clone and confirmation by sequencing and other identification methods known in the art. g) Culturing the recombinant E. coli cells in the desired media following by purification of plasmid DNA. h) Linearization of plasmid DNA by restriction enzyme to use further for in-vitro transcription reaction using T7 RNA polymerase. i) Preparation of mRNA through in-vitro transcription (IVT) and purification using suitable chromatography column.

[0203] Example 3: Preparation of pDNA (~10876bp), clone generation, selection and to use pDNA as template for mRNA preparation

[0204] The multicistron construct is designed to have better coverage and efficiency against emerging Severe Acute Respiratory Syndrome Coronavirus 2 (SAR-CoV-2). The structural sequences of virus from the database were analyzed, it is observed that other structural proteins of Severe Acute Respiratory Syndrome Coronavirus 2 as membrane, envelope and nucleocapsid apart from the spike are more conserved and less susceptible to mutation. The vaccine construct is designed to increase the breadth of immune defense against coronavirus infection as an improved mRNA vaccine. The vector construct was prepared by following the steps below: a) The amino acid sequences of SARS-CoV-2 and emerging variants were retrieved from http s : / / co vid 19dashboard .regeneron . com / ?tab=Home , https: / / marks.hms.harvard.edu / sars-cov-2 / , https: / / www.gisaid.org / and analyzed for lineage, mutation identification for different variants. b) Based on the amino acid sequences in the emerging mutants were reverse translated to DNA sequence which were codon optimized as per human amino acid codon usage. c) The pKashiv_multi-DNA vector construct (~10876bp) comprises the elements as shown in Figure 5 & 6 (SEQ ID NO: 3) and respective sequence mentioned as “pKashiv multi- DNA vector (-10876 bp)” and to clone into pHSG298 plasmid. d) The clone was prepared using SARS-CoV-2 delta variant sequences in the pHSG298 vector using restriction enzyme cloning. e) The E. coli host was transformed with the recombinant plasmid and further selection on Kanamycin containing media. f) The transformed colonies were screened for positive clone and confirmed by sequencing and other identification methods. g) The recombinant E. coli cells were grown in the desired media in shake flask and allowed to grow for 12- 16 hours at 37°C at suitable RPM. h) Cells were harvested by centrifugation followed by cell lysis, neutralization and precipitation for plasmid DNA purification. i) Purification of plasmid DNA carried out using Ion exchange chromatography. j) Plasmid DNA (~11 kb) was observed on 1% agarose gel electrophoresis in figure 7. k) Linearization of plasmid DNA was done by by restriction enzyme followed by initiation of in-vitro transcription reaction using T7 RNA polymerase and co-transcriptional capping reagent in addition to available IVT reagents as shown in Table 1. The reaction was further treated by DNAsel. l) The mRNA through in-vitro transcription (IVT) was purified using mixed-mode and affinity chromatography. m) Purified mRNA (~8kb) was subjected to physicochemical testing such as agarose gel electrophoresis shown in Figure 11, quantification, purity in addition to in-vitro cell-based expression as shown in Figure 8-9.

[0205] Example 4: Preparation of pDNA (~15255bp), clone generation and selection a) The gene sequence of Covid- 19 was identified from literature and data base. b) Designing of gene of interest (pKashiv_multicistronic_3 DNA) as in Figure 1 and Figure 3 (SEQ ID NO: 1) and codon optimization as per E.coli host was done. c) The codon optimized gene of interest (GOI) was chemically synthesized and used for cloning in pHSG298 vector construct. d) Both vector (pHSG298) and insert (pKashiv_multicistronic_3 DNA) were digested with restriction enzyme EcoRI & BamHI and gel purified for ligation reaction. e) Ligated product was transformed in E.coli DH5 host and inoculated the selected colonies in chemically define media containing antibiotics as a selection marker and incubated at 37°C with 160 rpm for overnight for growth. f) The grown culture was taken out from shaker incubator and performed plasmid isolation using Qiagen Kit. g) The eluted DNA was digested with EcoRI and Bam Hl to check the presence of gene of insert (GOI). h) The release of insert ~12kb DNA band along with ~2.5kb vector analysis was observed on 1% agarose gel electrophoresis in Figure 10 and shortlisted the positive clones. i) Further confirmation of positive clones was done using various enzymes, cut at specific sites and finalized the positive clone for cell bank preparation j) The cell bank of final clone was prepared in CD supreme media using 15% glycerol. k) Cell bank stored at -80°C for long term storage. l) The bank was inoculated in desire media for plasmid DNA isolation followed by in-vitro transcription to prepare mRNA with gene of interest (GO I).

[0206] Example 5: plasmid DNA (~15255bp) to use as template for mRNA preparation a) The selected clone was resuscitated and grown in the chemically defind media in shake flasks for 12- 16 hours at 37°C at 140-160 RPM, cells were harvested by centrifugation. b) Cell pellet was resuspended and further subjected to alkaline cell lysis along with SDS. The suspension was neutralized, and salt precipitation was carried-out. c) Purification of plasmid DNA was carried out using Ion exchange chromatography. d) Purified pDNA was linearized by restriction enzyme Xba I at defined process conditions. e) Linearized DNA was used as template at a template concentration of 10-150 microgram / ml of in-vitro transcription (IVT) reaction using T7 RNA polymerase and co-transcriptional capping reagent in addition to required IVT reagents for 2-3 hrs. as shown in Table 1. f) DNAsel treatment was carried-out to remove residual DNA. g) Quantification of mRNA at this stage was done by Ribogreen based quantification method and an agarose gel electrophoresis was done to identify the size of the mRNA. h) Purification of mRNA carried out using mixed-mode and affinity chromatography. i) Purified mRNA (~12kb) was subjected to physicochemical testing such as agarose gel electrophoresis shown in Figure 11, quantification, purity in addition to in-vitro cell-based expression. The expressed spike protein, membrane protein, envelope protein and nucleocapsid protein of SARS-CoV-2 as shown in Figure 12-15.

[0207] Table 1: Reaction mixture for in-vitro transcription

Claims

Claims1. An expression vector encoding more than one structural protein of Severe Acute Respiratory Syndrome Coronavirus- 2 (SARS-CoV-2) comprising: a) suitable promoter; b) untranslated region; c) structural protein of SARS-CoV-2 comprising; i. spike protein and one or more structural protein selected from; ii. membrane protein; iii. envelope protein; and iv. nucleocapsid protein; d) more than one Internal ribosomal entry sites (IRES) element; e) untranslated region; f) and Polyadenylation (Poly A) tail.

2. The expression vector as claimed in claim 1, wherein spike protein of SARS-CoV-2 comprises first spike protein and second spike protein are derived from different SARS- CoV-2.

3. The expression vector as claimed in claim 1, wherein the expression vector is capable to encode mRNA having size more than 4kb.

4. The expression vector as claimed in claim 3, wherein the expression vector is capable to encode mRNA having size more than 4kb, 8kb, and 12kb.

5. The expression vector as claimed in claim 2, wherein the first spike protein derived from alpha, beta, gamma, delta, eta, Iota, kappa, lambda, Mu, Omicron and emerging variants thereof.

6. The expression vector as claimed in claim 2, wherein the second spike protein, wherein derived from alpha, beta, gamma, delta, eta, Iota, kappa, lambda, Mu, Omicron and emerging variants thereof.

7. The expression vector as claimed in claim 1, wherein the envelope (E), membrane (M), and nucleocapsid (N) derived from alpha, beta, gamma, delta, eta, Iota, kappa, lambda, Mu, Omicron and emerging variants thereof.

8. The expression vector in claim 1, encoding more than one structural protein of SARS-CoV- 2 comprises; a) T7 promoter; b) 5’ untranslated region (5’ UTR); c) spike protein of SARS-CoV-2 delta variant;32d) internal Ribosome Entry Site (IRES) element; e) spike protein of SARS-CoV-2 omicron variant; f) internal Ribosome Entry Site (IRES) element; g) membrane protein of SARS-CoV-2 omicron variant; h) internal Ribosome Entry Site (IRES) element; i) envelope protein of SARS-CoV-2 delta variant; j) internal Ribosome Entry Site (IRES) element; k) nucleocapsid protein of SARS-CoV-2 omicron variant; l) 3’ untranslated region (3’ UTR); m) polyadenylation poly (A) tail.

9. The expression vector as claimed in claim 8, wherein expression vector comprises nucleotide sequence of SEQ ID No: 1.

10. The expression vector as claimed in claim 9, wherein comprises nucleotide sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SARS-CoV-2 sequence of SEQ ID No: 1.

11. The expression vector as claimed in claim 8, comprises the accession number MTCC 25576.

12. The expression vector as claimed in claim 8, capable to encode mRNA having size more than 4 kb.

13. The expression vector as claimed in claim 12, capable to encode mRNA having size about 12kb.

14. The expression vector in claim 1, encoding at least one domain of spike protein and other structural proteins of SARS-CoV-2 comprises; a) T7 promoter; b) 5’ untranslated regions (5’ UTR); c) SI domain of spike protein of SARS-CoV-2 delta variant; d) internal Ribosome Entry Site (IRES) element; e) SI domain of spike protein of SARS-CoV-2 omicron variant; f) internal Ribosome Entry Site (IRES) element; g) membrane protein of SARS-CoV-2 omicron variant; h) internal Ribosome Entry Site (IRES) element; i) envelope protein of SARS-CoV-2 delta variant; j) internal Ribosome Entry Site (IRES) element; k) nucleocapsid protein of SARS-CoV-2 omicron variant;33l) 3’ untranslated region (3’ UTR); m) polyadenylation poly(A) tail.

15. The expression vector in claim 14, wherein expression vector comprises nucleotide sequence SEQ ID No: 2.

16. The expression vector as claimed in claim 15, wherein comprises nucleotide sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SARS-CoV-2 sequence of SEQ ID No: 2.

17. The expression vector in claim 1, encoding more than one structural protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) comprising: a) T7 promoter; b) 5’ untranslated region (5’ UTR); c) spike protein of SARS-CoV-2 delta variant; d) internal Ribosome Entry Site (IRES) element; e) envelope protein of SARS-CoV-2 delta variant; f) internal Ribosome Entry Site (IRES) element; g) membrane protein of SARS-CoV-2 delta variant; h) internal Ribosome Entry Site (IRES) element; i) nucleocapsid protein of SARS-CoV-2 delta variant; j) 3’ untranslated region (3’ UTR); and k) polyadenylation (Poly A) tail.

18. The expression vector in claim 17, wherein expression vector comprises nucleotide sequence SEQ ID No: 3.

19. The expression vector as claimed in claim 18, wherein comprises nucleotide sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SARS-CoV-2 sequence of ID No: 3.

20. The expression vector construct as claimed in claim 1 or claim 8 or claim 14 or claim 17, wherein the Poly A tail contains about 80 to about 120 adenosine nucleotides.

21. The expression vector construct as claimed in claim 20, wherein the Poly A tail contains 110 adenosine nucleotides.

22. The expression vector construct as claimed in claim 1 or claim 8 or claim 14 or claim 17, wherein the length of spike (S) protein from about 1250 to about 1290 amino acid residues.

23. The expression vector construct as claimed in claim 1 or claim 8 or claim 14 or claim 17, wherein the length of envelope (E) protein from about 50 to about 90 amino acid residues.

24. The expression vector construct as claimed in claim 1 or claim 8 or claim 14 or claim 17, wherein the length of membrane (M) protein from about 200 to about 250 amino acid residues.

25. The expression vector construct as claimed in claim 1 or claim 8 or claim 14 or claim 17, wherein the length of nucleocapsid (N) protein from about 390 to about 446 amino acid residues.

26. The expression vector construct as claimed in claim 1 or claim 8 or claim 14 or claim 17, wherein the expressed mRNA has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% similarity to SARS-CoV-2 sequence of SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3.

27. The expression vector construct as claimed in claim 1 or claim 8 or claim 14 or claim 17, wherein the expressed mRNA capable to encode more than one structural protein to provide immune response against SARS-CoV-2 virus.

28. A process for the purification of mRNA comprises; a) transfect the expression vector as claimed in claim 1 or claim 8 or claim 14 or claim 17 into E. Coli host cells; b) harvesting of cells followed by lysis, neutralization, and precipitation; c) isolation and purification of plasmid; d) linearization of purified plasmid; e) performing the in-vitro transcription reaction for the preparation mRNA; f) purification of mRNA by chromatography methods.

29. The purification of mRNA as claimed in claim 28, purified using suitable chromatography selected from high performance liquid chromatography (HPLC), low normal pressure liquid chromatography method, gas chromatography, reversed phase HPLC (RP-HPLC), affinity chromatography, mixed-mode chromatography, ion exchange chromatography, hydroxyapatite chromatography, core bead flow-through chromatography, Oligo dT chromatography .

30. The purified mRNA as claimed in claim 29, analysed using agarose gel electrophoresis and in-vitro cell-based expression.The purified mRNA as claimed in claim 30, encapsulated in lipid nano particle comprises cationic lipid, neutral lipid, one polymer conjugated lipid or PEG-lipid and steroid or cholesterol. A mRNA vaccine comprising; c. a mRNA encoded by the expression construct as claimed in claim 1 or claim 8 or claim 14 or claim 17 comprises a gene of interest encoding at least one spike protein and one or more structural protein selected from envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants; d. suitable pharmaceutical acceptable excipient selected from cationic lipid, neutral lipid, one polymer conjugated lipid or PEG-lipid and steroid or cholesterol. The mRNA vaccine as claimed in claim 32, provides immune response to at least more than one structural protein selected from spike (S), envelope (E), membrane (M) and nucleocapsid (N) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its variants.36

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