WO2023062651A1

WO2023062651A1 - Virus-like particles for respiratory syncytial virus and method of preparation thereof

Info

Publication number: WO2023062651A1
Application number: PCT/IN2022/050914
Authority: WO
Inventors: Harshad PADMANABH PATIL; Ahmedali Shabbir MANDVIWALA
Original assignee: Padmanabh Patil Harshad
Priority date: 2021-10-13
Filing date: 2022-10-13
Publication date: 2023-04-20

Abstract

The present invention relates to virus-like particles (VLP) for respiratory syncytial virus (RSV) comprising of prefusogenic F (preFG) associated with G and M proteins using baculovirus system wherein the VLP is having lipid bilayer. Also, the present invention provides a method for preparation of respiratory syncytial virus-virus like particles (RSV-VLP). Further, the present invention provides an immunogenic composition wherein the immunogenic composition induces a highly effective immune response in a subject. Also, the present invention provides a VLP candidate vaccine for RSV wherein said vaccine is embedded with or without adjuvants in the lipid bilayer of VLP.

Description

Title of invention

VIRUS-LIKE PARTICLES FOR RESPIRATORY SYNCYTIAL VIRUS AND METHOD OF PREPARATION THEREOF

Field of Invention

The present invention relates to virus-like particles (VLP) for respiratory syncytial virus (RSV) and its method of use. More particularly, the present invention relates to a VLP for RSV and its method of preparation, an immunogenic composition, a vaccine comprising of said VLP and a kit comprising of said VLP for diagnosis using baculovirus system.

Background of Invention

Human respiratory syncytial virus (RSV) is a major pathogen that leads to severe lower and upper respiratory tract infection (LRTI and URTI) for which considerable efforts are ongoing worldwide for the treatment and for the development of a vaccine for the prevention of RSV infection.

RSV, belongs to family Pneumovirdae, is an enveloped virus with a single-stranded, negativesense, RNA genome containing 10 genes encoding a total of 11 proteins including four nucleocapsid proteins (nucleocapsid N protein, phosphoprotein P, large polymerase subunit L and transcription elongation factor M2-1), three transmembrane envelope glycoprotein (fusion (F) protein, attachment glycoprotein (G) protein and small hydrophobic (SH) protein), two non-structural proteins (NS1 and NS2), the matrix (M) protein, and M2-2, and an RNA regulatory factor. The G and F membrane proteins are major protective antigens, inducing host-protective RSV-neutralizing antibodies.

A humanized monoclonal antibody against the viral surface F protein is the only prophylactic product in the market which is recommended for infants who are at high risk including preterm infants and infants with chronic lung disease.

Reportedly, RSV-VLP made using F0 are metastable or are fused together with prefusion-F (preF) with or without G or M proteins are known. In 2019, Smith et al., has reported RSV prefusogenic F which is significantly more immunogenic than prefusion F, and produced higher levels of anti-F IgG, PCA, and neutralizing antibodies than prefusion F.

Similarly, in 2019, Patel etal., reported prefusogenic F proteins to be more immunogenic than prefusion and post fusion F.

US20130122032A1 relates to modified or mutated RSV fusion (Fj proteins and methods for making and using them including as an immunogenic composition such as vaccine for the treatment and/or prevention of RSV infection.

EP3109258B1 relates to compositions and methods for eliciting an immune response specific for RSV.

W02014160463A1 relates to RSV antigens including a recombinant RSV F protein stabilized in a prefusion conformation and methods for generating an immune response in a subject.

However, inventions heretofore known suffer from disadvantages including that they do not induce qualitatively superior antibody response against F, or do not produce antibody response against G or M proteins. Further, none of the cited prior arts reported the RSV-VLP comprising of preFG protein associated with G and M proteins using baculovirus expression vector system (BEVS).

Accordingly, there is a need for an approach that helps to resolve problems of the state of the art by providing a VLP comprising of preFG protein associated with G and M proteins using a BEVS. Further, there is a need for the presence of a lipid bilayer in the VLP that allows the incorporation of traditional adjuvants as well as adjuvants having lipophilic tails into the lipid bilayer of the RSV-VLP.

Definitions

Virus-like-particles: Virus-like particles (VLPs) are structures made up of one or more single or different proteins with the ability to self-assemble. The VLP lack the genetic material and are not capable of infecting the host cell. The VLPs can non-enveloped or enveloped. VLP are called enveloped if they contain a cell derived lipid membrane. VLP formation can be conferment directly by electron microscopy and indirectly by ultracentrifugation, immunological and biochemical methods.

RSV: Respiratory syncytial virus (RSV) is one of the most common respiratory viruses that usually cause mild, cold-like symptoms. Most people recover in a week or two, but RSV can be serious, especially for infants and older adults. RSV is the most common cause of bronchiolitis (inflammation of the small airways in the lung) and pneumonia (infection of the lungs) in children younger than 5 year of age.

RSV G protein: The RSV large glycoprotein G is involved in the attachment of the virus to the cells. The RSV G glycoproteins along with RSV F protein are the only viral protective antigens. G protein in VLP will be responsible for generation of antibodies against G protein of RSV.

RSV F protein: The RSV fusion (F) glycoprotein is responsible for the fusion of the virus with the cell. F causes the virion membrane to fuse with a target cell membrane. The functional F protein trimer in the virion membrane is in a metastable prefusion (preF) form which quickly gets transformed into stable postfusion F (postF). PreFG is the more immunogenic form of RSV preF constructed from the near full-length RSV/A2 F protein in which furin cleavage site II was mutated to retain the native p27. PreFG will be responsible for generation of antibodies against F protein of RSV.

RSV M protein: The RSV M protein is a nonglycosylated internal virus component. RSV M appears to play two roles typical i.e. it helps organize virus components at the plasma membrane for budding, and it may silence viral RNA synthesis in preparation for packaging into the virus particle. In VLP formation, it the main function of M protein is to interact with preFG and G to form a VLP structure that is secreted out of the cell.

Lipid bilayer: It is a thin polar membrane made of two layers of lipid molecules with individual lipid molecules able to diffuse rapidly within their own monolayer. During VLP formation, it is derived from the cell during VLP release from the cells. Antigen: It is a foreign substance which upon that induces an immune response in the body. In current document, VLP is an antigen against which antibody and T cell response will be induced.

Codon optimized: Also called as codon optimization. It refers to experimental approaches designed to improve the codon composition of a recombinant gene based on various criteria without altering the amino acid sequence. In this document, the codon optimization was done based on the ExpiSF9 cells.

Baculovirus expression system: The baculovirus vector system is widely used for the expression of recombinant proteins in cultured insect cells. Baculovirus is a double -stranded DNA virus that commonly infects insect cells. The cloning vector in our case is pFastBad which is optimized for use with bacmid. The bacmid technology is based on the incorporation of the components of the Tn7 integration signal. The gene of interest is first cloned into the pFastBad vector having gentamicin resistance gene which is flanked by the Tn7 transposon terminal elements. pFastBad vector is then transformed into E. coli carrying the bacmid shuttle vector and a helper plasmid. The pFastBad with gene of interest is then transformed into E. coli carrying the bacmid shuttle vector and a helper plasmid. Colonies containing recombinant bacmids are identified by blue /white screening (non-recombinant colonies are blue whereas recombinant colonies are white) by transposon insertion. White colonies having insert are identified, propagated and purified bacmid with inserted gene is then used to transfect SF9 or ExpiFF9 cells to generate live baculovirus, which after further propagated and used to generate VLP. In this document, three different bacmids specific for preFG, G and M are developed. Transfection of these bacmids individually in SF9 or ExpiSF9 generated baculoviruses specific for preFG, G and M that were propagated and used for making VLP.

Suspension culture: A cell suspension culture is a type of cell culture in which single cells or small aggregates of cells are allowed to function and multiply in an agitated growth medium. In this document the cells are ExpiSF9™ and the medium is ExpiSf™ CD Medium. Inducer: Also termed as enhancer. It is a chemically defined animal origin-free formulation developed to be used along with ExpiSf™ CD Medium to enhance protein production in ExpiSf9™ cells, resulting in maximal protein yields.

Multiplicity of infection (MOI): MOI is the ratio of infectious virus particles to cells in a culture.

Objectives of the Invention

The main objective of the present invention is to provide virus-like particles (VLP) for respiratory syncytial virus (RSV) comprising of preFG and G proteins associated M protein using a BEVS wherein the VLP is having lipid bilayer.

Another objective of the present invention is to provide an antigen comprising of an effective amount of preFG, G and M proteins of RSV wherein the immunogenic composition induces an immune response in a subject.

Another objective of the present invention is to provide a method for preparation of respiratory syncytial virus-virus like particles (RSV-VLP) based on preFG, G and M proteins of RSV.

Yet another objective of the present invention is to provide RSV-VLP for developing diagnostics for RSV.

Still another object of the present invention is to provide VLP candidate vaccine with lipid bilayer for RSV comprising of preFG, G and M proteins of RSV.

Summary of the invention

The present invention relates to VLP for RSV comprising of preFG protein associated with G and M proteins using a baculovirus expression system wherein the VLP is having lipid bilayer. Also, the present invention provides a method for preparation of RSV-VLPs based on preFG, G and M proteins of RSV.

The present invention further provides an immunogenic composition comprising of an effective amount of preFG, G and M proteins of RSV wherein the immunogenic composition induces a highly effective immune response.

In an embodiment, the present invention provides an antigen for developing a diagnostic kit comprising of a RSV-VLPs comprising of preFG, G and M proteins of RSV. In yet another embodiment, the present invention provides a VLP candidate vaccine for RSV comprising of a VLP comprising of preFG, G and M proteins of RSV wherein said VLP is associated with or without adjuvants in the lipid bilayer of VLP.

Detailed description of invention

The present invention now will be described hereinafter with reference to the detailed description, in which some, but not all embodiments of the invention are indicated. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout. The present invention is described fully herein with non-limiting embodiments and exemplary experimentation.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "a VLP" includes a mixture of two or more such VLPs.

The present invention relates to VLP for RSV comprising of preFG protein associated with G and M proteins using a BEVS wherein the VLP is having lipid bilayer. Also, the present invention provides a method for preparation of respiratory syncytial virus-virus like particles (RSV-VLP) based on preFG, G and M proteins of RSV.

In a preferred embodiment, the present invention provides an immunogenic composition comprising of an effective amount of preFG, G and M proteins of RSV wherein the immunogenic composition induces a highly effective immune response in a subject.

In another embodiment, the present invention provides a diagnostic kit comprising of a RSV- VLP comprising of preFG, G and M proteins of RSV.

In yet another embodiment, the present invention provides a VLP candidate vaccine for RSV comprising of a VLP comprising of preFG, G and M proteins of RSV wherein said vaccine is embedded with or without adjuvants in the lipid bilayer of VLP.

The invention is further ascribed below by reference of the following, non-limiting examples: EXAMPLE 1

METHODOLOGY

A) Generation of pFastBac vectors specific for RSV Prefusogenic F (PreFG), Glycoprotein (G) and Matrix (M) (Fig.1)

Codon optimized RSV preFG sequence is synthetically generated and cloned into pFastBac vector. To insert RSV G and M, into pFastBacl, RNA is first extracted from RSV virus propagated on Hep2 cells. Complementary DNA (cDNA) is prepared using gene specific reverse primers for M and G (Table 1). G and M genes are amplified using gene specific forward (Table 2) and reverse primer. Amplified G and M PCR products and pFastBacl vector are digested separately with BamHI and Xhol restriction enzymes at 37°C for 60 min. After digestion, the PCR products and vector are ligated using a T4 DNA ligase and products are transformed DH5a E. coli. Positive colonies are screened for transformation of recombinant vectors by colony PCR using gene specific primer pairs (Table 1 and 2). Plasmids are extracted from positive colonies and confirmed for gene insertion into vector by digestion with BamHI and Xhol restriction enzymes.

Table 1 discloses reverse primer sequences.

* Bold and underlined letters indicate site for restriction enzyme Xhol

Table 2 discloses forward primer sequences.

*Bold and underlined letters indicate site for restriction enzyme BamHl

B) Generation of RSV M, G and F genes specific recombinant bacmids

Bacmids for the individual genes preFG, G and M are generated by transforming the pFastBacl plasmids into DHIOBac E. coli. Transformed cells are selected for recombinant bacmids on LB agar plates containing kanamycin, gentamycin, tetracycline, IPTG and X-gal. White colonies are selected, streaked onto fresh selection plates and grown. Isolated colonies are picked and confirmed for gene insertion by PCR with overlapping primers across the insertion sites (Table 3). Bacmids are extracted from positive colonies by alkaline lysis.

Table 3 discloses sequences ofM13F and M13R primers

C) Production of recombinant baculovirus expressing RSV-PreFG, G and M proteins

For production of recombinant baculoviruses, ExpiSf9 cells are transfected with bacmids containing the gene of interest. Cells are incubated at 27°C in a non-humidified non-CCh atmosphere incubator on an orbital shaker platform. After 96hrs of infection, transfected cells are centrifuged at 300g for 5 minutes. Supernatant is collected, aliquoted and stored at -80°C. Further stocks are prepared by infecting 100 pl ExpiSf enhancer (Gibco, USA) treated 5 x 10⁶ viable cells/mL in 25 mL media with 500 pL baculovirus stock. Virus supernatants harvested at 5 days post infection, are clarified by centrifugation, aliquoted and stored at - 80°C for further use.

D) Production and purification of RSV virus like particles (VLP)

For production of RSV-VLPs, ExpiSF9 cells were treated with inducer and were co-infected with different MOI of recombinant baculoviruses specific for RSV-PreFG, G and M. Cells were incubated at 27°C in a non-humidified non-C02 atmosphere under agitation for 96 hrs. Following incubation, cells were removed, RSV-VLP was concentrated and buffer exchanged with phosphate buffer saline using tangential filtration flow. The solution with RSV-VLP was layered over 20-60% sucrose density gradient in PBS and ultracentrifuged for 3hrs at 4°C.

The RSV-VLP herein produced will consist of following protein sequence,

PreFG protein [SEQ ID 1]:

E) Confirmation of RSV-VLPs

RSV-VLPs produced with PreFG, G and M proteins were confirmed by western-blot and transmission electron microscopy (TEM), protein estimation and ELISA.

F) Immunization and collection of samples

Female Balb/cmice (6-8 weeks) were used for immunization studies. All procedures on mice were executed under ketamine (100 mg/kg) /xylazine (10 mg/kg) anesthesia. Mice, n=6/experimental group, were used for immunization. Immunization was done twice on day 1 and 21 i.e. with an interval of 3 weeks, via intramuscular route. For i.m. delivery, 50 pl solution was administered in hind limb. Blood was collected 7 days post second dose and serum was obtained for antibody response evaluation.

RESULTS

Recombinant pFastBac vectors for RSV-M and G

RSV-M and G genes corresponding to 770bp and 908bp were amplified (Fig 2A), and purified for generation of M and G specific pFastBac!. Purified PCR products were inserted into pFastBac vector and cloned into DH5a E. coli. Extracted plasmid was confirmed for gene insertion by digestion with BamHI and Xhol restriction enzymes. Release of inserted M (770bp) and G (908bp) products from pFastBac vector (4.7kb) (Fig 2B and 2C) confirm construction of recombinant pFastBac vectors specific to RSV-M and G.

Recombinant pFastBac vectors for RSV-preFG

Recombinant plasmids containing PreFG were confirmed for insertion of the genes by digestion with restriction enzymes BamHI and Hindlll, the sites of which were incorporated into the 5’ and 3’ end of the gene sequence respectively. Digestion with these enzymes released inserted PreFG (1695bp) from the pFastBac vector (4.7kb) as shown in Fig 3.

Recombinant bacmid for RSV M, G and F Recombinant bacmids specific for M, G and PreFG was extracted from DHIOBac E. coli by alkaline lysis method (Fig. 4A-C). PCR analysis was done to verify the presence of gene of interest in the recombinant bacmids. Recombinant bacmids of M, G and PreFG gave products of sizes 3.0kb, 3.2kb, 4.0kb respectively.

Characterization of RSV-VLP

Purified RSV-VLP were obtained using equilibrium density sucrose gradient (Fig. 5A). Protein content and OD of ELISA coincided with each other indicating presence of RSV- reactive structure (Fig. 5B). Protein content gradually dropped but binding of antibodies persists till 50-60% sucrose interface indicating formation of particles with various mass (Fig. 5B). TEM image (Fig. 5C) offractions at35% sucrose confirmed formation ofround VLP with size between 70-100 nm having lipid bilayer structure. Western blot analysis (Fig 5D) confirmed presence of proteins preFG, G and M at 30-35% sucrose interface, while 50-60% sucrose interface lacked G protein. Presence of lipid was confirmed by phosphate determination (Fig. 5E) which indicated that the adjuvants having lipophilic tail might get associated with VLP. The purified VLP were also found to be binding to immune serum from mice infected with RSV (Fig. 5F) indicating VLP formed are indeed RSV specific.

Standardization of RSV-VLP production

To make RSV-VLP, ExpiSF9 cells were infected with three rBV specific to M, G and prefusogenic F, with a hypothesis that all three rBV will infect single cell and the resultant proteins produced within the cell will interact with each other to for the VLP. Thus, the amount of rBV for each protein should not be a limiting factor. In addition, manufacture of the ExpiSF9 system recommended treatment of cells with an inducer for increased yield. Therefore, in this experiment, the cells were treated with inducer a day before infection and infected with either theoretical 10 or 1 MOI of rBV.

Addition of inducer enhanced the yield of the VLP (Fig 6A-D). Protein content and ELISA OD coincide with each other in presence of inducer (Fig 6A-B). On the contrary, the protein yield and the OD values were much lower in absence of inducer (>10 fold) (Fig 6C-D). Infection with 1 MOI resulted in high yield of the VLP in presence of inducer. Immunogenicity of RSV-VLP

The preFG, G and M protein-based RSV-VLP were highly immunogenic (Fig. 7A-D) and induced significantly higher RSV-F protein, RSV-M protein and RSV-VLP IgG antibodies compared to B PL-inactivated RSV. Antibodies against RSV-G protein were also induced but the levels were not significantly different than induced by BPL-inactivated RSV. A dose dependent increase in IgG OD values was seen with increase in RSV-VLP immunization.

Detailed Description of Drawings

A complete understanding of the virus-like particles (VLP) and method for the preparation of VLP of the present invention may be obtained by reference to the following drawings:

Figure 1 depicts systematic representation of constructs of different RSV proteins and antigenic composition of single VLP. (A) Diagrammatic representation of the preFG protein construct. (B) The preFG is prepared by removing amino acids from position 137 to 146 and replacing amino acids atpositions in F2 region, furine cleavage site and Flregion ofwildtype RSV F protein. (C) Diagrammatic representation of the G protein constructs. (D) Diagrammatic representation of M protein constructs. (E) Diagrammatic representation single VLP antigenic composition.

Figure 2 depicts a polymerase chain reaction (PCR) image of (A) Amplified PCR of RSV M and G are shown in lanes 1 and 2 respectively. Lane M consists of a broad range DNA marker. (B) Restriction digestion confirmation for RSV-M clone, which shows release of insert (770bp) from the vector (4.7kb) in lane 1. Lanes Ml and M2 are loaded with lOObp and Ikb DNA markers respectively. (C) Restriction digestion confirmation for RSV-G clone, which shows release of insert (908bp) from the vector (4.7kb) in lane 1. Lane 2 consists of undigested clone. Lane M consists of Ikb DNA marker.

Figure 3 depicts a confirmation image of RSV-preFG clones wherein Lane 1 shows restriction digestion of PreFGto release insert of 1695bp from pFastBac vector (4.7kb). Lane M consists Ikb DNA marker. Figure 4 depicts analysis of recombinant bacmids by PCR wherein (A) PCR confirmation image of RSV-M bacmid, which shows PCR product of 3.0 kb in Lane 1. Lane 2 shows the PCR product generated by PCR of non-recombinant bacmid. (B) PCR confirmation image of RSV- G bacmid, which shows PCR product 3.2kb in Lane 1. Lane 2 shows the PCR product generated by PCR of non-recombinant bacmid. (C) PCR confirmation image of RSV-PreFG bacmid, which shows PCR product of 4.0kb in Lane 1. Lane M is loaded with a Ikb DNA marker.

Figure 5 depicts analysis of RSV-VLPs wherein (A) supernatant of infected ExpiSF9 cells collected after ultracentrifugation was purified using 20-60% sucrose density gradient. A cloud was notice in 35% sucrose. (B) Fractions were collected to determine protein content and binding to anti-RSV-IgG positive serum. (C) VLP formation was confirmed by TEM while

(D) presence of proteins M, G and prefusogenic F in VLP were confirmed by Western-blot.

(E) Phosphate determination confirmed presence of lipid bilayer that was observed in TEM images. (F) Purified VLP were also reactive to the serum from mice infected with live RSV.

Figure 6. depicts yield of RSV-VLP wherein ExpiSF9 cells were infected with rBV specific to preFG, G and M at (A) 10 or (B) 1 MOI in presence of ExpiSF9 inducer. Purified cell supernatant collected 5 days post infection was subjected to 20-60% density gradient ultracentrifugation and collected fraction were analyzed for protein content and RSV antibody reactivity.

Figure 7. depicts antibody response after immunization with RSV-VLP. BALB/c (n=6) mice were used to evaluate antibody response induced after immunization with p-propiolactone inactivated RSV and RSV-VLP. Mice were immunized twice by intramuscular route on day 0 and 21. Blood was collected 7 days after the second immunization and serum obtained was used to determine IgG antibody presence against (A) RSV-F, (B) RSV-G, (C) RSV-M and (D) RSV-VLP. Mann-Whitney U test was used to perform statistics. Levels of significance are presented as * p < 0.05, ** p < 0.01. Advantages of the present invention The immunogenic composition of the present invention induces a highly effective immune response. The present invention provides a VLP candidate vaccine for RSV wherein the VLP is having a lipid bilayer that allows the use of traditional adjuvants as well as incorporation of adjuvants having lipophilic tails into the lipid bilayer of the RSV- VLP and a diagnostic kit. The use of combination of preFG together with G and M proteins for the preparation of VLP is not reported anywhere in the prior art.

Claims

I/We Claim, 1. A virus-like particle (VLP) for respiratory syncytial virus (RSV) comprising at least one respiratory syncytial virus (RSV) Prefusogenic F protein, at least one respiratory syncytial virus (RSV) G protein and; at least one respiratory syncytial virus (RSV) surface matrix (M) protein and; lipid bilayer; wherein, the prefusogenic F protein is characterized to fuse virus with cells, G protein is characterized to bind the said RSV VLP to cells and M protein is characterized to provide structural stability to cells, wherein the immunogenic composition of said RSV VLP induces an effective immune response. 2. A VLP for RSV as claimed in claim 1, wherein the RSV PreFG protein is codon optimized or non-codon optimized. 3. A VLP for RSV as claimed in claim 1, wherein the RSV G and M protein is non-codon optimized or codon optimized. 4. A VLP for RSV as claimed in claim 1, wherein, surface protein comprises modifications to one or more amino acid residues. 5. A VLP for RSV as claimed in claim 1, wherein said M protein is derived from human strain of RSV or other paramyxoviruses or influenza virus preferably from human strain of RSV. 6. The VLP for RSV as claimed in claim 1, wherein the lipid bilayer is derived from the cells. 7. The VLP for RSV as claimed in claim 1 is embedded with one or more adjuvants. 8. The VLP for RSV as claimed in claim 1 is immunogenic in humans or animals. 9. The VLP for RSV as claimed in claim 1 is administered by intramuscular, intradermal, mucosal routes such as pulmonary, nasal, sublingual, buccal, oral routes of immunization in humans or animals. 10. Method of making the VLP for RSV in adherent or suspension culture with RSV prefusogenic F protein, glycoprotein (G) and matrix protein (m), comprises the steps of: a. Generation of vectors specific for RSV Prefusogenic F(PreFG) of SEQ ID 1 flanked by BamHI and HindIII restriction enzymes. b. Generation of vectors specific for Glycoprotein (G) of SEQ ID 2 and Matrix (M) of SEQ ID 3 flanked by BamHI and XhoI restriction enzymes. c. Generation of RSV preFG, G and M specific genes specific recombinant bacmids. d. Production of recombinant baculovirus expressing RSV PreFG, G and M proteins. e. Production and purification of RSV virus like particles (VLP) using baculovirus specific for RSV PreFG, G and M proteins as obtained in step d. Method of making the VLP for respiratory syncytial virus (RSV) in suspension culture with RSV prefusogenic F protein with matrix protein (m) and glycoprotein (G) as claimed in claim 10, wherein, production and purification of RSV virus like particles (VLP) comprises the steps of: a. Treating suspension cells with inducer and co-infecting with different MOI of said recombinant baculoviruses specific for RSV-PreFG, G and M. b. Incubating cells at 22-37°C in a humidified or non-humidified non-C02 atmosphere under agitation for 3-5 days. c. Removing cells by appropriate method such as centrifugation or filtration, concentrating RSV-VLP and performing buffer exchange with any buffer, preferably phosphate buffer saline (PBS) using tangential filtration flow. d. Purifying the RSV-VLP using appropriate methods such as fast protein liquid chromatography techniques or 20-60% sucrose density gradient ultracentrifugation using any buffer, preferably phosphate buffer saline (PBS) for 1-3hrs at 2-8°C.