DESC:Quadruple Antigen Virus Like Particle (VLP) Vaccine candidate for SARS COV2 and method thereof

FIELD OF THE INVENTION

The present Invention relates to the development of Quadruple Antigen Virus Like Particle (VLP) Vaccine candidate for SARS COV2 in Pichia pastoris expression host.

BACKGROUND OF THE INVENTION

The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Virus-like particle have been studied well in literature. VLP is formed by self- assembly to form a nanostructure including key viral structural proteins. VLP bear a resemblance to molecular and morphological features of authentic viruses but lacks genetic material due to which it is non-infectious and non-replicating. VLP have been successfully applied in vaccinological and virological research. As an accessibly safe and relevant substitute of naturally pathogenic viruses, the construction of SARS-CoV-2 VLPs is much in demand in the ongoing fight against 2019 Coronavirus disease (COVID-19) pandemics. In studies efficient production SARS-CoV-2 VLPs has been shown to in mammalian and plant hosts, where four SARS-CoV-2 structural proteins, expression of membrane protein (M) and small envelope protein (E) are essential for efficient formation and release of SARS-CoV-2 VLPs. Moreover, the corona-like structure presented in SARS-CoV-2 VLPs from Vero E6 cells is more stable and unified, as compared to those from HEK-293T cells and also shown in plant cells. In earlier reports, VLP formation has been shown either by three or four structural protein through the co-transfection as three or four separate plasmids each carrying one structural protein. The data demonstrate that SARS-CoV-2 VLPs possess molecular and morphological properties of native virion particles, which endow such VLPs with a promisingvaccinecandidateandapowerfultoolfortheresearchofSARS-CoV-2.Inthistechnique maintaining the ration of plasmids is a challenge in co-transformation this may affect the formation of VLPs in repeated experiments.

Primary target for SARS-CoV-2 vaccine developers is spike or S protein, which mediates viral engagement with the host cell receptor, the human angiotensin-converting enzyme 2 (ACE2). Many platforms are currently available for vaccine delivery, namely, RNA, DNA, recombinant proteins, live attenuated virus (LAV) and inactivated viruses, viral vectors, or virus-like particles (VLPs). S protein also shown to be the most susceptible for mutations at different geographical regions.

While the production RNA, DNA, and protein-based vaccines are easier to manufacture in comparison to those, which derived from VLPs or LAVs, they are more effective in terms of inducing an immune response, which can sustained over more extended period. This is a striking aspect regarding a potential COVID-19 vaccine development to produce a durable neutralizing antibody (NAb) reaction.

Previous studies indicate that vaccination in humans leads to generate a NAb titres at least comparable to that assayed in convalescent serum following natural COVID-19 infection. While it is easier to produce the RNA, DNA, and protein-based vaccines there are several limitations associated in terms of storage, transport, stability, however VLP based vaccines showed better stability and enhanced immune response.

VLPs are generated by assembling viral proteins (e.g., S, E, M and N proteins) that lack genetic material which mimics viral structure and are therefore not able to replicate, but elicit a high degree of immune response since they are rapidly recognized by antigen- presenting cells. Surfaces VLPs bear a repeating array of viral antigens that leads to the induction of both innate and adaptive immunity, with high neutralizing activity.

Earlier VLPs have been used to deliver vaccine antigens in the case of human papillomavirus (HPV), hepatitis, and influenza viruses. These vaccines can also be lyophilized, making storage and distribution of the vaccine much more straight forward, enabling broader coverage in the current situation where the entire world requires vaccine protection.

The rapid development and urgency of producing a vaccine for the COVID 19 pandemic may increase the risks and failure rate of delivering a safe, effective vaccine. Hence, there is a need of highly effective protein-based vaccine that could be quickly scaled-up in bioreactors.

OBJECTS OF THE INVENTION:

A primary object of the present invention is to overcome the drawback/s associated with the prior art.
An object of the invention is to provide a safe stable and commercially viable vaccine against Covid-19.
Another object of the invention is to provide Quadruple Antigen Virus Like Particle (VLP) Vaccine candidate for SARS COV2 in Pichia pastoris expression host
Another object of the invention is to provide native-like VLPs of SARS-CoV-2, which contain all four structural proteins, and is functional against multiple strains of SARS- CoV-2 and method thereof.
Another object of the invention is to provide novel platform for VLP formation and method thereof.

Summary of the Invention:

In an embodiment, the Invention provides a vaccine for multiple strains of SARS-CoV-2 comprising Quadruple Antigen Virus Like Particle (VLP) of SARS COV2 in Pichia pastoris expression host wherein said VLPs are formed by expression of structural proteins comprising S, E, M and N in Pichia host as secreted in the culture medium.

In another embodiment, the Invention provides an expression cassette comprising 5 different proteins of SARS-COV-2 wherein the proteins comprises are Mpro protease, M, E, N and S wherein the Plasmid 1 comprises sequences for Matrix (M), Envelope (E), Nucleocapsid (N) and with or without the viral Main protease (Mpro); while plasmid 2 comprises sequence for only Spike (S); characterized in that Plasmid 1 is integrated in the genome of Pichia pastoris to develop a modified host which expresses M, E and N)structural proteins, for expression of variable spike protein.

In another embodiment, the Invention provides a process for the production of Quadruple Antigen Virus Like Particle (VLP) Vaccine from culture supernatant for SARS COV2 in Pichia pastoris expression host comprising:
a) cloning the SARS-CoV-2 structural protein sequences in two pPICZ backbone plasmids in Pichia expression host wherein the Plasmid 1 comprises sequences for Matrix (M), Envelope (E), Nucleocapsid (N) and with or without the viral Main protease (Mpro); while plasmid 2 comprises sequence for only Spike (S); characterized in that Plasmid 1 is integrated in the genome of Pichia pastoris to develop a modified host which expresses M, E and N)structural proteins, for expression of variable spike protein.

b) co-transfecting plasmid 1 and plasmid 2 in Pichia expression host to express all 4 structural proteins to form VLPs wherein the plasmid 1 and plasmid 2 were linearized with restriction enzyme SacI and co-transformed in Pichia competent cells followed by selecting the transformants on YPDS-Zeocin plate.

Brief Description of Drawings:

Figure 1: illustrates schematic representation of plasmid 1 (pPICZa-Cassette1) and plasmid 2 (pPICZa-Cassette 2)

Figure 2: illustrates Expression screening to select best expressing colony. a: probed with anti-RBD antibody (1:1000), b. probed with anti-N antibody (1:1000). Secondary antibody 1:20000 (anti-Rabbit-HRP)

Figure 3: illustrates Scale up of the expression at 50ml scale (a.) and 500ml scale (b.)

Figure 4: illustrates purification of VLPs. a: SDS-PAGE analysis, b.: Western blot analysis with anti-Sera. 15% SDS-PAGE and d.: 6% SDS-PAGE respectively, c: probed with anti-N antibody. Fraction 4 NR: fraction 4 non-reduced sample

Figure 5:illustrates analysis of VLP samples after concentration. RD: reduced sample, NR: non reduced sample

Figure 6:illustrates analysis of purified VLPs by specific N (A.) and RBD (B.) antibodies

DETAILED DESCRIPTION OF THE INVENTION

Some embodiments of this invention, illustrating all its features, will now be discussed in detail.

In the present document, the word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment or implementation of the present subject matter described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

While the disclosure is susceptible to various modifications and alternative forms, specific embodiment thereof has been shown by way of example in the drawings and will be described in detail below. It should be understood, however that it is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the disclosure.

The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a setup, device or method that comprises a list of components or steps does not include only those components or steps but may include other components or steps not expressly listed or inherent to such setup or device or method. In other words, one or more elements in a device or system or apparatus proceeded by “comprises… a” does not, without more constraints, preclude the existence of other elements or additional elements in the device or system or apparatus.

It must also be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Although any systems and methods similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred, systems and methods are now described.

The disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms.

The Invention provides a safe stable and commercially viable vaccine against Covid-
19. The Invention develops Quadruple Antigen Virus Like Particle (VLP) Vaccine candidate for SARS COV2 in Pichia pastoris expression host. The strategy or platform technology will be applicable in the area of development of VLPs based vaccines and also to develop multi-subunit protein vaccines to cater any such pandemic situations. This technology will provide an opportunity to develop the vaccines in quick turnaround time.

In an embodiment, the Invention provides native-like VLPs of SARS-CoV-2, which contain all four structural proteins, and is functional against multiple strains of SARS-CoV-2. The VLPs formed by expression of 4 structural proteins (S, E, M and N) in Pichia host as secreted in the culture medium. The strategy for expression of all 4 proteins to form VLPs, expression and maturation of VLPs are drivenby a protease to form individual protein subunits then they come in close contact to form VLPs.

In an embodiment, the Invention provides a process for the production of VLPs from culture supernatant.

In an embodiment, the Invention provides a novel platform for VLP formation which has been designed based on cloning of structural proteins in 2 plasmids.

In Pichia expression host, in an embodiment, the SARS-CoV-2 structural protein sequences were cloned in two pPICZ backbone plasmids. Plasmid 1 contained the sequences for Matrix (M), Envelope (E), Nucleocapsid (N) and with or without the viral Main protease (Mpro); while plasmid 2 contained the sequence for only Spike (S). Plasmid 1 will be integrated in the genome of Pichia pastoris to create a modified host which expresses three (M, E and N) structural proteins. This platform, will help in expression of variable spike protein which can be taken from different geographical region. S protein was shown to be mutation prone; this platform will allow us to express S proteins from different geographical regions to produce VLPs for target specific region.

For Pichia expression host, the SARS-CoV-2 structural protein sequences were cloned in pPICZa and pPICZaA backbone plasmid. Plasmid 1 (pPICZA) contained the sequences for Matrix (M), Envelope (E), Nucleocapsid (N) and with or without the viral Main protease (Mpro); while plasmid 2 (pPICZaA) contained the sequence for only Spike (S). Plasmid 1 and plasmid 2 will be co- transfected in Pichia expression host to express all 4 structural proteins. On transfection all 4 proteins will express to form VLPs. This platform, will help in expression of variable spike protein which can be taken from different geographical region to resolve the issues of multiple mutations in structural protein. S protein was shown to be mutation prone; this platform will allow us to express S proteins from different geographical regions to produce VLPs for target specific region.

In an embodiment, for transformation, Pichia pastoris strain will be grown in YPD at 30°C. After growth cells will be washed and prepared for electroporation 1M Sorbitol. The electroporation of linearized DNA will be done in Pichia pastoris and selected on YPDS plates containing Zeocin at 100 µg/ml and will be incubated from 3–10 days at 30°C or until colonies formed. Double selection will be done to minimize the false positive colonies. This transformed strain will be used to electroporate the plasmid 2 in similar way as done for plasmid 1. Plasmid 2 will be transformed as episomal expression cassette. After transformed of both plasmid 1 and 2 in Pichia pastoris, 20-30 colonies will be selected to isolate best expressing colony.

In an embodiment, for expression analysis single colony will be inoculated in different media eg. BMGY media in a flask and incubate at different temperature 22-30°C in a shaking incubator (250–300 rpm) until culture reaches an OD600 = 2–6. Change the media and induce the culture with various methanol concentration at every 24hr till 120hr. Analysis of protein expression can be done at every 24hr after induction.

VLP Purification: The culture media will be first clarified by centrifugation at 9000 and the particles will be purified on a AKTATM pure 25 FPLC (GE), and eluted using elution buffer. Peak fractions will be collected and dialyzed in formulated buffer and concentrated. Total protein concentration was estimated using a Bradford assay.

In an embodiment, expression analysis of both pellet and supernatant (culture media) will be done by SDS-PAGE, Western blotting, Mass spec analysis.

In an embodiment, morphology and size of VLPs will be characterized by DLS, Electron microscope after purification at small scale of expression.

In an embodiment, after initial characterization, process development will be done for the production of VLPs to perform animal studies for the establishment of immunogenicity, safety, toxicity and production of neutralizing antibodies.

In an embodiment, SARS-CoV-2 structural proteins are been expressed in Pichia expression host system GS115 strain cells. The VLPs are being made and characterized using variety of analytical technique. This gives a confirmation that the constructs are capable of generating VLPs. The binding studies are being done which shows good results. The studies will be submitted in the complete specification.

In an embodiment, the Invention provides constructs comprising two constructs with 5 different proteins of SARS-COV-2. The proteins included are Mpro protease, M, E, N and S. The two cassettes were generated where cassette 1 is with Mpro, M, E and N while cassette 2 carries S protein. This strategy makes it simple to develop a vaccine candidate to different mutant variants in quick turnaround time.

In an embodiment, the Invention comprises following steps:

a) Development of the construct:
Two constructs were prepared with 5 different proteins of SARS-COV-2. The proteins included are Mpro protease, M, E, N and S. The two cassettes were generated where cassette 1 is with Mpro, M, E and N while cassette 2 carries S protein. This strategy makes it simple to develop a vaccine candidate to different mutant variants in quick turnaround time.

b) Transformation in Pichia pastoris:
Both cassettes were linearized with restriction enzyme SacI and co-transformed in Pichia competent cells. The transformants were selected on YPDS-Zeocin plate.

c) Expression screening: Transformants were grown and expression was induced by methanol. The expression was confirmed at 5ml with different colonies.
a. Scale up of expression from 5ml to 50ml to 500ml
i. To check the scalability of the strategy and process, scale up expression experiments were performed. The expression was confirmed at all conditions.
b. Purification at 50ml and 500ml scale
i. Purification optimization were performed at 50 and 500ml scale, both the VLP and spike protein were purified successfully from culture supernatant of expression host.
c. Analysis of purified samples
i. The purified protein was characterized by various analytical techniques which includes SDS-PAGE, Western blotting, SEC-HPLC, Peptide mapping, DLS and Electron microscopy. The VLPs thus produced was found to be stable and immunogenic (data not shown).

Technical advantages/technical benefits being achieved by the present invention as compared to the existing technologies being used earlier:

Presence of all structural proteins of the native virus in a stable VLP can arguably elicit a strong as well as effective immune response against multiple strains of a virus. As the major structural proteins of SARS Coronaviruses – S, M and N - appear to be involved in triggering T-cell mediated immune response, a native-like VLP which contains all structural proteins, is expected to induce a robust response. Although lung immunopathology triggered by N in SARS-CoV vaccine candidates has been reported previously, recent studies on inactivated SARS-CoV-2 formulations which contain all structural proteins have ruled out such concerns.

The claimed invention generates the native-like VLPs by transfection in Pichia expression host, as expression systems are expected to produce the most faithful post-translational modifications (PTM) which is an essential factor for the immunogenicity of glycoproteins. Further, Pichia pastoris cells can be adapted to bioreactor-based methods for large-scale production of VLPs.
The initial studies show the possibility of generating SARS-CoV-2 VLPs that closely resemble the native virus. The particles generated by from Pichia pastoris were purified by an ion-exchange based method, and cross-react to antisera and antibodies, N. The structural similarity with native virus indicates the potential of these native-like VLPs as vaccine candidates.
The technology has advantage of developing a vaccine candidate in quick time by just changing the gene in Cassette 2 which is carrying the spike protein and is shown to be mutation prone and generation of deadly forms of viruses.

The Invention is further described with the help of non-limiting examples:

Example 1:
The construct development part which was design with unique strategy to form virus like particles. The structural proteins have Mpro cleavage site in between of M, E and N in cassette 1. The Mpro protease is responsible for generation of individual structural proteins to form virus structure, our strategy we are mimicking the process in Pichia pastoris and we are able to generate the VLPs as occur in natural process. These VLPs are stable and can itself shown immune response without the adjuvant (not shown here). While cassette 1 carries 3 important structural protein (E, M and N) along with Mpro protease, Cassette 2 carries only spike protein. This allows us to change the most dangerous protease easily and develop a vaccine candidate in quick turnaround time to any such mutations. Therefore, this process is providing a platform to develop vaccine for any mutations by changing the spike protein or any other highly mutated protein.

Expression for VLPs formation was done by co-transformation of linearized cassette 1 and cassette 2 in Pichia pastoris expression host. The transformants were selected in YPD Zeocin plates. Expression was secretory expression in culture supernatant was confirmed at 3ml scale and scale up till 50 to 500ml scale. Purification was done through anion exchange chromatography and purified sample were characterized by various analytical techniques. Since VLPs is expressed and secreted in the culture supernatant this process makes it easy for scale up to generate the material in large amount at reasonable cost.

Example 2:
Construct development
SARS-CoV-2 structural protein sequences were codon optimized and synthesized. The synthesized genes were sub cloned in pPICZa and pPICZaA backbone plasmids between EcoRI and KpnI restriction site. Cassette 1 contained the sequences for Matrix (M), Envelope (E), Nucleocapsid (N) and the viral Main protease (Mpro) forming pPICZa-Cassette 1; while plasmid 2 contained the sequence for Spike (S) and forming pPICZaA-Cassette 2. This strategy provides an opportunity to change the spike protein easily to any mutant variants to develop various vaccine candidates in quick turnaround time. Plasmid 1 is designed for cytoplasmic expression while plasmid 2 is carrying gene for secretory expression. Secretion of cassette 1 proteins will occur only when VLP is formed with spike protein as cassette 1 doesn’t carry any signal peptide.

Example 3:
Transformation in Pichia pastoris
10µg of each plasmid (plasmid 1 and 2) were digested with SacI restriction enzyme to linearize the plasmid. The linearized plasmids were purified by DNA purification kit and concentration was estimated by NanoDrop™ 2000/2000c Spectrophotometers. ~5µg of each linearized plasmids were 80µl of mixed and incubated with the Pichia competent cells for 5mins; the mix was then electroporated using Gene Pulser at defined setting for Pichia pastoris (25µF, 200O, 2 kV). 1ml of ice-cold 1 M sorbitol was immediately added to the electroporation cuvette and transferred the cuvette contents to a sterile 15 ml tube. The tube was incubated at 30°C without shaking for 1 then 50µl suspended cells were platted on YPDS (1%yeast extract, 2% peptone, 2% dextrose, 1 M sorbitol and 2% agar)-agar plates containing the 100µg/ml Zeocin. The 1ml of YPD was added to the remaining cells and were further incubated for 1hr with shaking at 100rpm at 30°C. 200µl of cells were again platted on the YPDS-Zeocin agar plates. The plates were then incubated for 2–3 days at 30°C until colonies form. Pick 10–20 colonies and purify (streak for single colonies) on fresh YPD or YPDS plates containing the appropriate concentration of Zeocin™.

Example 4:
Expression analysis
Expression analysis was screened from the colonies observed on the YPDS-Zeocin agar plates. For expression of extracellular recombinant VLPs, multiples colonies were screened for the production of producing recombinant P. pastoris strain GS115 was inoculated into 2 mL YPD broth as the primary inoculum and incubated at 28°C and 250 rpm for 12-16h till OD reaches to 8-10. For expression studies at 5ml scale, overnight grown cells were pelleted at 1500g for 5min and resuspended in 5mL BMMY medium (with 1.34% YNB, 0.5% methanol and biotin 4x10-1%) to a final OD600nm of ~1. AOX1 induction was sustained by adding methanol (0.5% v/v) at every 24h intervals till 120 h. 1 mL samples were collected every 24h to monitor the cell growth and expression of desired protein. Expression was confirmed in several colonies and was confirmed by specific antibodies against RBD and N in the culture supernatant. Colonies which showed the expression were taken forward for further optimization at different expression scale from 50ml to 500ml scale.

Example 5:
Scale up studies
For scale up studies in 50ml scale selected colony was inoculated in 5ml of YPD media and incubated at 28°C for overnight at 250rpm till OD reaches to 8-10. The culture was then centrifuged at 1500g for 5min and resuspended the cells in 50ml of BMMY media and incubated at 28°C for 72hrs at 250rpm. The methanol concentration was sustained by adding 0.5% methanol at every 24hrs. The expression of VLP was confirmed by anti-Sera and anti-N antibodies. Same has been taken forward for purification optimizations.
To scale up the expression at 500ml scale selected colony was inoculated in 50ml of YPD media and incubated at 28°C for overnight at 250rpm till OD reaches to ~8-10. The culture was then centrifuged at 1500g for 5min and resuspended the cells in 500ml of BMMY media and incubated at 28°C for 72hrs at 250rpm. The methanol concentration was sustained by adding 0.5% methanol at every 24hrs. The expression of VLP was confirmed by anti-Sera and anti-N antibodies. Same has been taken forward for purification optimizations.

Example 6:
Purification
Purification was performed by using Ion exchange (Anion) chromatography. 1ml column was packed with cellufine Q-R, strong anion exchange resin was used. Column was equilibrated with 3.0 column volume (C.V) of 50mM Tris, pH 9.5.
Load: 50 ml of culture supernatant (VLP proteins) pH was adjusted to 9.5 with 1M NaOH and was loaded onto the column and washed with equilibration buffer to remove any unbound protein. Bound protein was eluted by gradient.
Buffer A: Equilibration buffer (50mM Tris-HCl pH 9.5).
Buffer B: 50mM Tris-HCl + 1.0 M NaCl pH 9.5.
Gradient: 0-50% B in 10 C.V & 50-100%B in 5.0 C.V.
Fractions were collected and given for SDS-PAGE and Western blot analysis.
The elution fractions were analyzed by SDS-PAGE and Western blot analysis using anti-Sera at 1:500 dilution. Protein was majorly observed in fraction 4. Fraction 3, 4 and 5 were pooled and concentrated using 5kDa amicon. The final concentration was 1.1mg/ml. The concentrated sample was further used for analytical characterization by different analytical techniques. SDS-PAGE and Western blot analysis were performed by anti-RBD, anti-N and anti-Sera antibodies. The presence of spike and N protein in same elution fraction (fraction 4, Figure 4) confirm the formation of complex with spike and other proteins expressed from cassette 1 of plasmid 1. This confirms the formation of VLPs in culture supernatant of Pichia pastoris. Concentrated fraction 4 sample showed similar profile in both reduced and non-reduced samples, this confirm the correct conformation of the VLPs structures.

Example 7:
Analysis of purified protein
Purified protein was analyzed and characterized by analytical techniques including SDS-PAGE, Western blot (with anti-sera and specific antibodies). Both spike and nucleocapsid proteins were observed in the VLP fraction Figure 6.

CLAIMS:We Claim:

1. A vaccine for multiple strains of SARS-CoV-2 comprising Quadruple Antigen Virus Like Particle (VLP) of SARS COV2 in Pichia pastoris expression host wherein said VLPs are formed by expression of structural proteins comprising S, E, M and N in Pichia host as secreted in the culture medium.

2. An expression cassette comprising 5 different proteins of SARS-COV-2 wherein the proteins comprises are Mpro protease, M, E, N and S wherein the Plasmid 1 comprises sequences for Matrix (M), Envelope (E), Nucleocapsid (N) and with or without the viral Main protease (Mpro); while plasmid 2 comprises sequence for only Spike (S);
characterized in that Plasmid 1 is integrated in the genome of Pichia pastoris to develop a modified host which expresses M, E and N)structural proteins, for expression of variable spike protein.

3. A process for the production of Quadruple Antigen Virus Like Particle (VLP) Vaccine from culture supernatant for SARS COV2 in Pichia pastoris expression host comprising:
c) cloning the SARS-CoV-2 structural protein sequences in two pPICZ backbone plasmids in Pichia expression host wherein the Plasmid 1 comprises sequences for Matrix (M), Envelope (E), Nucleocapsid (N) and with or without the viral Main protease (Mpro); while plasmid 2 comprises sequence for only Spike (S);
characterized in that Plasmid 1 is integrated in the genome of Pichia pastoris to develop a modified host which expresses M, E and N)structural proteins, for expression of variable spike protein.

d) co-transfecting plasmid 1 and plasmid 2 in Pichia expression host to express all 4 structural proteins to form VLPs wherein the plasmid 1 and plasmid 2 were linearized with restriction enzyme SacI and co-transformed in Pichia competent cells followed by selecting the transformants on YPDS-Zeocin plate.

4. The process as claimed in claim 3, wherein the culture media is first clarified by centrifugation at 9000 rpm and the particles are purified on a AKTATM pure 25 FPLC (GE)followed by eluting using elution buffer.
5. The process as claimed in claim 3, wherein the expression host express S proteins from different geographical regions to produce VLPs for target specific region
6. The process as claimed in claim 3, wherein Pichia expression host system comprises GS115 strain cells.
7. The process as claimed in claim 3, wherein the transformation comprises the steps of:
a) Growing Pichia pastoris strain in YPD at 30°C followed by washing and preparing for electroporation in 1M Sorbitol;
b) Performing Electroporation of linearized DNA in Pichia pastoris followed by selection on YPDS plates containing Zeocin at 100 µg/ml
c) Incubation from 3–10 days at 30°C or until colonies formed