The present invention broadly lies in the field of virology and viral vaccines. More particularly,
the present invention relates to expression of SARS-CoV like virus proteins; recombinant
polynucleotides, polypeptides; virus-like particles; immunogenic compositions or vaccines
comprising virus like particles and methods of producing and purifying the same.
BACKGROUND OF THE INVENTION
Virus-like particles (VLPs) resemble their corresponding native viruses with similar overall
structure but lessened original infectious ability due to the absence of viral genome. VLPs are
symmetrically built from hundreds of coat proteins, which can be genetically engineered to
present a regular arrangement of epitope chains on the desired positions of the outer surface.
Currently, VLPs have been widely applied for a variety of applications such as vaccines,
antibody development, delivery systems, bioimaging, and cell targeting. More precisely, VLPs
have proven to be promising candidate vaccines since they: (i) do not comprise a nucleocapsid
and are non-infectious and therefore safe to produce and use, (ii) are more immunogenic than
subunit vaccines because they provide the necessary spatial structure for display of epitopes,
and (iii) elicit humoral, cell-mediated and importantly, mucosal immunity (Krueger et al., Biol.
Chem., 380:275-276, 1998).
WO2017/040387A2 discloses a virus-like particles (VLPs) comprising an antigenic RSV
protein and a composition comprising these VLPs, as well as methods for making and using
these VLPs.
US 8,592,197 B2 discloses a macromolecular protein structure containing (a) a first influenza
virus M1 protein and (b) an additional structural protein, which may include a second or more
influenza virus M1 protein; a first, second or 50 more influenza virus HA protein; a first,
second, or more influenza virus NA protein; and a first, second, or more influenza virus M2
protein.
US 2010/0120092 A1 discloses chimeric or recombinant virus like particles comprising (i) S
polypeptide of an avian hep adenovirus and (ii) a chimeric fusion protein comprising a
polypeptide of interest Covalently attached to a particle-associating portion of L polypeptide
of an avian hepadnavirus.
Of the various biological molecules being developed as potential vaccines or therapeutics,
development of various types of VLPs as next generation vaccines has accelerated over the
past two decades. However, it has been observed that the expression levels of viral proteins in
different platforms vary considerably. In general, the secretory expression of glycoproteins is
difficult. Budding from cell membrane to get envelop is a key step during the eVLP (enveloped
virus like particle) formation process. If eVLP is not efficiently secreted, a cell lysis or other
extraction step might be required, and these steps increase the difficulty for further purification.
A common way to improve expression level of transmembrane glycoproteins is to delete or
replace the transmembrane region which anchors the protein in the membrane (Wang et al,
2018).
Eukaryotic expression systems are more suitable for the production of eVLP. Yeast expression
systems, especially those based on Saccharomyces cerevisiae and Pichia pastoris, have
advantages such as scalable fermentation, low production costs, and PTM process. Yeast has
advantages such as scalable fermentation, low risk of contamination by adventitious agents,
low production costs and the ability to produce VLPs with reliable qualities. Accordingly, yeast
platform has been a preferable choice for productions of both, the nonenveloped viruses but
also of enveloped viruses. It is also understood that the quality and quantity of yeast derived
VLPs is largely influenced by the choice of plasmid and promoter, and the ratio of the structural
proteins produced. VLPs derived from nonenveloped or enveloped viruses can broadly be
classified as the single-layered and multi-layered VLPs (Letters in Applied Microbiology,
2016).
The appropriate formation of complex multi-layered VLPs is restricted to a narrow range of
association energies and protein concentrations. Thus the choice of the host system and design
of expression is critical for a successful assembly. In literature, there are types of
plasmids/vectors used in the transformation of yeast cell like Autonomously Replicating
Plasmids (ARS), Yeast centromere Plasmids (YCps), Yeast Integrating Plasmids (YIp),
Episomal Vectors (YEP plasmids) and Yeast Linear Plasmids (YLp). Episomal expression
vectors (Yep) have been used in literature for the production of VLPs which utilises multiple
episomal vectors. This may cause presence of excess foreign DNA and load in the cells due to
presence of these plasmids as multiple copies.
Two licensed vaccines, the HBV vaccine Engerix-B® and HPV vaccine Gardasil® have been
manufactured using this system. However, in the case of HIV-2, Saccharomyces cerevisiae
cells fail to support the multimerization of the enveloped Gag protein into VLPs and particle
budding from the membrane.
Several other routes for expression of antigens in multi-layered VLPs have also been described
in literature. As one of the strategies used, two antigens VP1 and VP2 for Parvovirus VLP have
been co-expressed under a bicistronic plasmid under the control of the same promoter
(ADH2/GAPDH). In this case, it is seen that expression of VP2 antigen is lower than VP1,
indicating that it is critical to maintain the ratio of both the expression constructs.
In another example, VLP for Coxsackievirus A16 and Enterovirus 71, which are Picornaviridae
and are major causative agents of hand, foot and mouth diseases (HFMD) (Zou et al. 2012),
have been produced. These are composed of VP0, VP1 and VP3 antigens and are each
processed from P1 polypeptide by the 3CD viral protease (Li et al. 2013; Zhao et al. 2013a).
Multilayered VLPs of coxsackievirus A16 and enterovirus 71 have been produced in both S.
cerevisiae and P. pastoris by intracellular expression strategies (Li et al. 2013; Zhao et al.
2013a; Zhang et al. 2015; Wang et al. 2016). There are two different types of expression
strategies for expression plasmids. One is to use two separate expression plasmids, one for
producing the P1 region and the other for the 3CD protease (Zhao et al. 2013a), and the other
strategy is to use a single expression plasmid for both regions (Zhang et al. 2015; Zhou et al.
2016). In both cases, the VLPs produced had potential as vaccines but Zhang et al. obtained
significantly higher levels of expression of enterovirus 71 antigens from cells transformed with
the single expression plasmid than from those transformed with the two separate plasmids.
In another example, Rotavirus VLPs which are composed of VP2, VP6 and VP7 have shown
to be produced intracellularly in S. cerevisiae (Rodriguez-Limas et al. 2011), wherein a single
plasmid harbours the VP2, VP6 and VP7 genes was more effective than using three individual
plasmids, because the introduction of three individual plasmids creates a metabolic burden and
reduced growth rate of the yeast. Moreover, it is important to control the VP7:VP6 expression
ratio during the production of rotavirus structural proteins in order to improve the yield of
authentic multilayered VLPs.
On another aspect, stability of the VLP-based vaccines is one of the most significant and
challenging issues. VLPs being multimeric structures are generally more stable than subunit
vaccines, however, the lack of the viral genome makes them unstable when the conditions
change, especially during downstream processing (DSP) and purification. Generally, it is seen
that eVLPs having a host-derived envelope are more sensitive to the external environment than
the protein-only VLPs. Variations in conditions, e.g., temperature, shear force and chemical
treatment can destroy the integrity and stability of the particles, this structural destruction
further leads to the reduction in immunogenicity of eVLPs, thus, robust purifications processes
are required so as to maintain the VLP structure, conformation and immunogenicity
With the outbreak of severe acute respiratory syndrome (SARS), there is an urgent need for the
development of vaccines for preventing SARS caused by Coronavirus (SARS-CoV).
Coronaviruses commonly cause infections in both humans and animals. Coronavirus virus
particles contain four main structural proteins. These are the spike (S), membrane (M),
envelope (E), and nucleocapsid (N) proteins, all of which are encoded within the 3′ end of the
viral genome.
The S protein (∼150 kDa), homotrimers of the virus encoded S protein make up the distinctive
spike structure on the surface of the virus. S is cleaved by a host cell furin-like protease into
two separate polypeptides noted S1 and S2. S1 makes up the large receptor-binding domain of
the S protein while S2 forms the stalk of the spike molecule. Interactions of the spike protein
from the SARS family of viruses through their receptor binding domain (RBD) -CoV spike
protein receptor-binding domain (RBD) and host receptor angiotensin-converting enzyme 2
(ACE2) has been shown responsible for both cross-species and human-to-human transmissions
of virus. Thus, using S protein or its domains as immunogens may provide protective response
against the spread of the virus and also aid in its clearance.
However, Protein attributes of S protein showed the protein has extremely high number of
cysteine residues along with high hydrophobicity which describes tendency of protein towards
insoluble expression or aggregations if expressed in prokaryote system. Protein is very big in
size and also has high proline residues a structure breaker may increase the instability of the
protein, which makes its expression and purification difficult for vaccine and other purposes.
The M protein is the most abundant structural protein in the virion. It is a small (∼25–30 kDa)
protein with 3 transmembrane domains and is thought to give the virion itsshape. Its interaction
with the S protein and N protein is essential for viral assembly and budding. The N terminal
and the C terminal of the protein have been found to be highly immunogenic and have shown
to induce antibody responses in hosts infected by coronavirus or immunized by attenuated
recombinant virus expressing the M Protein. Moreover, the M proteins of coronaviruses
contain highly conserved glycosylation sequences, and their glycosylation may be related to
the interaction between virus and host. In Alpha coronaviruses, it has been demonstrated that
M protein cooperates with the Spike during the cell attachment and entry. Therefore, mutations
occurring at the N-terminus region, which is exposed to the virus surface, could play a key role
in the host cell interaction. Thus, immunizing with the protein can help generate neutralizing
antibodies which can help clear the virus
The E protein (∼8–12 kDa) transmembrane protein is found in small quantities within the
virion. The E protein facilitates assembly and release of the virus and also has other functions.
E proteins play a part in viral assembly and morphogenesis and blocking its function has been
helpful to contain the virus. The E protein is conserved across β-coronaviruses (Bianchi et al.,
2020; Biomed Research International, 2020) E protein, as a pentameric viroporin-like protein,
is a minor component of the virus membrane though it is deemed to be important for many
stages of virus infection and replication. It is observed that E protein attributes shows that the
protein is high cysteine residues with extremely high hydrophobicity which describes their
propensity towards insoluble expression if expressed in prokaryotic host. The observed
parameter keeps the protein in difficult to express category and suggest the suitability of
eukaryotic host system for expression being membrane protein.
Studies have shown that novel coronaviruses can escape the host immune response either by
exposing non-neutralizing epitopes on their RBDs or due to emerging mutations in the SARSCoV-2 S sequence which mediate escape from neutralizing antibody responses induced by
immunogens designed from the SARS-CoV2. With most of the vaccines targeting the S
protein, emergence of antibody-resistant SARS-CoV-2 variants might limit the therapeutic
usefulness of these vaccines.
One possible route to mitigate this resistance to recognition owing to mutations can be the use
of multiple antigens for generating antibodies and allowing the immune system to recognise
multiple epitopes form multiple antigens. As such, other proteins of SARS-CoV-2 may also
play important roles while developing suitable vaccine candidates. Thus, the presence of three
surface proteins of the SARS-CoV2 “S”, “E” and “M” provide a wider repertoire of antigens
for an effective antibody mediated immune response and vaccines serve as one of the most
important therapeutic mechanisms which help to get acquired immunity against a particular
disease.
Precisely, generation of a humoral immune response is central to development of a vaccine.
The antibodies play a significant role in preventing viral infection by either acting as
neutralizing, enhancing phagocytosis by immune cells and agglutination. However, during
mass vaccination campaigns, it is envisaged that a large number of doses shall be administered
over a short period. There is a high probability of coincidental adverse events.
In such an event, to demonstrate safety of a candidate across multiple dose ranges is of utmost
importance, it is also required to demonstrate stringent safety checks of the vaccine candidate
during pre-licensure stage. Also, efficient expression systems or platforms capable of
expressing SARS-CoV virus like proteins are lacking, which hinders research and development
in this area.
The present invention aims to obviate the problems in prior art and endeavours to provide
efficient VLPs along with their purification and method for expression of SARS-CoVproteins
and methods applicable thereto. The present invention also provides an immunogenic
composition and vaccine comprising the VLPs obtained from the S, E and M proteins of SARSCoV.
OBJECTIVE OF THE INVENTION:
An important objective of the present invention is to provide recombinant VLPs of SARS-CoV
and their utilization as a vaccine candidate.
Another important objective of the present invention is to produce recombinant multi-subunit
VLPs comprising different proteins from SARS-CoV.
Another important objective of the present invention is to provide method of producing the
desired multi-subunit VLPs.
Yet another objective of the present invention is to develop an efficient method for coexpression of multi-subunit and virus like proteins from SARS-CoV, such as, but not limited
to S, M and E proteins.
Another important objective of the present invention is to provide related recombinant
polypeptides and recombinant polynucleotides.
Still another objective is to provide strategies, methods, systems, kits and combinations for
consistent scalable expression and enhanced production of the VLPs of SARS-CoV which
maintains its size range and composition.
Still another objective is to provide a method of producing the scalable amount of VLPs.
Still another objective is to provide a method of purification of VLPs in high yields.
Yet another objective of the present invention is to provide an immunogenic composition
comprising VLPs of SARS-CoV.
Still another objective is to provide safe and efficacious vaccines comprising recombinant
VLPs) of SARS-CoV.
BRIEF DESCRIPTION OF FIGURES AND DRAWINGS
The accompanying drawings illustrate some of the embodiments of the present invention and,
together with the description, explain the invention. These drawings have been provided by
way of illustration and not by way of limitation.
Figure 1: (A) Outline restriction map for S protein expression in episomal vector (B)
Immunoblot analysis of expressed S protein at small scale (C) Immunoblot analysis of
expressed S protein in microsomal fraction as resolved on a 6% gel (loaded as 20µl and 40µl)
(D) Peptide mapping analysis of expressed His tagged S protein. The green highlighted
sequence was identified in high confidence for S protein in peptide mapping analysis.
Figure 2: (A) Restriction map for M protein expression in episomal vector (B) Immunoblot
analysis of expressed M protein at small scale (C) Immunoblot analysis of expressed M- His
tag protein in microsomal fraction (loaded as 10µl, 20µl and 40µl) (D) Peptide mapping
analysis of expressed M His Tag protein in microsomal fraction. The green highlighted
sequences were identified in high confidence for M protein in peptide mapping analysis.
Figure 3: (A) Restriction map for E protein expression in episomal vector (B) Immunoblot
analysis of expressed E_His tag protein at small scale, (C) Immunoblot analysis of expressed
E- His tag protein in microsomal fraction (loaded as: 10µl, 20 µl, 40 µl). (D) Peptide mapping
analysis of expressed E His Tag protein in microsomal fraction. The green highlighted
sequences were identified in high confidence for E protein in peptide mapping analysis.
Figure 4: Immunoblot confirmation of presence of all three antigens S (Fig 4A), E (Fig 4B) and
M (Fig 4C) in the VLP preparation. Samples were prepared post-harvest of the culture as per
the established protocols.
Figure 5: Peptide mapping analysis of the VLP sample was performed using the Mass
spectrometry using a standard protocol. All three antigens have been confirmed for their
presence in the formulation in this analysis.
Figure 6: Antigen specific IgG response to S, E and M proteins as measured by ELISA in sera
samples from mice immunized with eVLP alone or with adjuvant (Alhydrogel + eVLP). Error
bars indicate SEM.
Figure 7: ELISA results showing eVLP specific IgG antibody titers in BALB/c mice
immunized with indicated doses of eVLP either with or without AH.
Figure 8: Neutralization potential (NP) of sera from immunized mice relative to neutralization
potential of convalescent patients’ as reference.
Figure 9: PRNT50 titers of sera samples from different immunized mice groups
Figure 10A: BrDU cell proliferation studies of PBMCs in patient samples subjected to two
doses of VLP formulation (2.5µg-2 Fold; 5µg:1.45 Fold) in comparison to PBMCs from
healthy individuals.
Figure 10B: Cell proliferation studies of PBMCs shows elevated levels of IFN- in the VLP
stimulated patients PBMCs as compared to healthy controls.
Figure 11: Stoichiometric ratio calculation for S, M and E in the produced VLP from co
expression. Standard curve for S1 protein (B) Standard curve for M protein (C) E protein
standard curves.
SUMMARY OF THE INVENTION
The present invention relates to expression of SARS-CoV like virus proteins; recombinant
polynucleotides, polypeptides; constructs, virus-like particles; immunogenic compositions or
vaccines comprising virus like particles. Method of producing the VLPs/expressing the multisubunit virus like proteins. The present invention also provides an efficient method for coexpression of multi-subunit and virus like proteins such as SARS-CoV S, M and E proteins
and related recombinant polypeptides and recombinant polynucleotides.
The present invention also provides immunogenic compositions or vaccines comprising the
VLPs of SARS-CoV. multi-subunit VLPs can be utilized to make.
The present invention also provides strategies, methods, systems, kits and combinations for
scalable expression, purification and enhanced production of the virus like proteins of SARSCoV while maintaining their size range and composition.
DETAILED DESCRIPTION OF THE INVENTION:
The details of one or more embodiments of the invention are set forth in the accompanying
description below including specific details of the best mode contemplated by the inventors for
carrying out the invention, by way of example. It will be apparent to one skilled in the art that
the present invention may be practiced without limitation to these specific details.
Definitions:
The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”,
“include”, “includes”, and “including” are not intended to be limiting. It is to be understood
that both the foregoing general description and this detailed description are exemplary and
explanatory only and are not restrictive.
The term “expression vectors” defines a plasmid or virus designed for gene expression in cells.
The term “host cell” means a host cell used for generation of recombinant proteins.
The term “viral proteins” includes proteins generated by viruses including enzyme proteins as
well as structural proteins such as capsid and viral envelope.
Unless otherwise defined, scientific and technical terms used herein shall have the meanings
that are commonly understood by those of ordinary skill in the art. Further, unless otherwise
required by context, singular terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and
tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and
hybridization described herein are those well-known and commonly used in the art.
Now vis-à-vis the present invention, an important embodiment of the present invention is to
provide recombinant VLPs of SARS-CoV and their utilization as a vaccine candidate.
Another important embodiment of the present invention relates to production of recombinant
multi-subunit VLPs comprising different proteins from SARS-CoV such as but not limited to,
“S” Spike; “M” Membrane protein and “E” protein derive from SARS-CoV-2 virus which have
high importance in vaccine development.
Another embodiment of the present invention is to provide method of producing the desired
multi-subunit VLPs.
Yet another embodiment of the present invention pertains to an efficient method for coexpression of multi-subunit and virus like proteins from SARS-CoV, such as, but not limited
to S, M and E proteins.
Another important embodiment of the present invention is to provide recombinant polypeptides
and recombinant polynucleotides of S, M and E proteins from SARS-CoV.
Still another embodiment of the present invention is to provide strategies, methods, systems,
kits and combinations for scalable expression, purification and enhanced production of the
VLPs of SARS-CoV.
Still another embodiment of the present invention is to provide immunogenic compositions
comprising the VLPs of SARS-CoV.
Still another important embodiment of the present invention is to provide vaccines comprising
recombinant VLPs of SARS-CoV.
In an important aspect of the present invention, codon optimization was done in respective
genes to obtain modified S, M and E proteins with increase in de novo mRNA synthesis rate
and stable mRNA production for efficient expression of proteins and VLPs with high yields
and efficacy. The VLPs of the present invention containing the recombinant proteins able to
generate high immune response and are ideal to be used in an immunogenic composition or
vaccine.
In an embodiment, present invention provides a recombinant polypeptide selected from the
group comprising the SEQ ID Nos 1, 2 and 3 and variants thereof, wherein the recombinant
polypeptide belongs to proteins selected from Spike (S), Membrane (M) and Envelope (E)
proteins, respectively, from SARS-CoV family, and wherein the said proteins are either
individually expressed or co-expressed.
In another embodiment, wherein said variant of the polypeptides comprises a sequence having
at least 70% sequence identity to the polypeptide sequence selected from the group comprising
SEQ ID Nos 1, 2 and 3.
In still embodiment, wherein said polypeptides are obtained after codon optimization.
In yet another embodiment, said S protein is obtained after individual expression of SEQ ID
NO 1 or a variant thereof.
In still another embodiment, said recombinant polypeptide is a full-length S protein which is a
single pass type I membrane glycoprotein.
In yet another embodiment, said M protein is obtained after individual expression of SEQ ID
NO 2 or a variant thereof.
In still another embodiment, wherein said recombinant polypeptide is a full-length M protein
which is a multi-pass membrane glycoprotein.
In another embodiment, said E protein is obtained after individual expression of SEQ ID NO3
or a variant thereof.
In yet another embodiment, said recombinant polypeptide is a full-length E protein which is a
single pass type III membrane glycoprotein.
In another embodiment, the present invention provides a recombinant molecule comprising
recombinant polypeptides wherein said molecule is a complex formed by assembly of
polypeptides defined in SEQ ID NO 1, 2 and 3 or variants thereof and is obtained after coexpression of S, M and E proteins to form an ordered aggregate.
In another embodiment, said recombinant molecule assembles to form VLPs.
In another embodiment, wherein said, the present invention provides a recombinant
polynucleotide selected from the group comprising the SEQ ID Nos 4, 5 and 6 or variants
thereof, wherein the polynucleotides are selected from Spike (S), Membrane (M) and Envelope
(E) genes from SARS-CoV family, and wherein the said genes are cloned individually or in
combination.
In yet another embodiment, wherein said variant of the nucleotides comprises a sequence
having at least 60% sequence identity to the polynucleotide sequence selected from the group
comprising SEQ ID NOs 4, 5 and 6.
In still another embodiment, said polynucleotides are codon biased and optimized.
In yet another embodiment, said S gene has SEQ ID NO 4 and variants thereof.
In another embodiment, said M gene has SEQ ID NO 5 and variants thereof.
In still another embodiment, said E gene has SEQ ID NO 6 and variants thereof.
In another embodiment, said recombinant polynucleotides as claimed in claim 9, wherein the
polypeptides or polynucleotides are selected from, but not limited to, Spike (S), Membrane (M)
and Envelope (E) proteins from SARS-CoV-2.
In another embodiment, the present invention provides a method of expressing SARS-CoV
proteins comprising the steps of:
i. selecting the target genes from the group comprising of S, E and M proteins;
ii. optimizing and codon biasing of target gene sequences;
iii. preparing the construct comprising target genes and yeast-based expression
vectors by cloning of target genes individually or in combination to co-express
the proteins into the expression vector, wherein the expression vector is yeast
based expression vector selected from episomal expression vector and/or
integrative expression vector or a combination of both;
iv. transforming the constructs into Protease deficient yeast host cell to obtain
target proteins.
In another embodiment, the present invention provides a method of expressing S protein from
SARS-CoV, wherein the method comprises the steps of:
i. selecting the target gene S protein;
ii. optimizing and codon biasing of target gene sequence;
iii. preparing the construct comprising S gene and yeast-based expression vector by
cloning of S gene individually to express the said protein into the expression
vector, wherein the expression vector is yeast based episomal or integrative
expression vector;
iv. transforming the construct into Protease deficient yeast host cell to obtain target
protein.
In yet another embodiment, the present invention provides a method of expressing M protein
from SARS-CoV , wherein the method comprises the steps of:
i. selecting the target gene M protein;
ii. optimizing and codon biasing of target gene sequence;
iii. preparing the construct comprising M gene and yeast-based expression vector
by cloning of M gene individually to express the said protein into the expression
vector, wherein the expression vector is yeast based integrative or episomal
expression vector;
iv. transforming the construct into Protease deficient yeast host cell to obtain target
protein.
In another embodiment, the present invention provides a method of expressing E protein from
SARS-CoV, wherein the method comprises the steps of:
i. selecting the target gene E protein;
ii. optimizing and codon biasing of target gene sequence;
iii. preparing the construct comprising E gene and yeast-based expression vector
by cloning of E gene individually to express the said protein into the expression
vector, wherein the expression vector is yeast based episomal or integrative
expression vector;
iv. transforming the construct into Protease deficient yeast host cell to obtain target
protein.
In another embodiment, the present invention provides a method of expressing the SARS-CoV
proteins wherein the method of co-expressing S, E and M proteins comprises the steps of:
i. selecting the S, E and M proteins from SARS-CoV;
ii. cloning “S” and “E” proteins into an episomal expression vector to obtain a
construct having SEQ ID NO 10 and “M” protein into integrative expression
vector to obtain a construct having SEQ ID NO 11;
iii. optimizing and codon biasing of target gene sequences;
iv. preparing the construct comprising the selected genes and yeast vectors;
v. transforming the constructs into yeast host;
vi. over-expressing the target multi-subunit proteins in the cell.
In another embodiment, the present invention provides a method wherein all the three proteins
and variants thereof are either co-expressed individually as episomal or integrative or
combinations thereof.
In still another embodiment, the proteins are selected, but not limited to, the S, M and E Corona
virus proteins.
In yet another embodiment, said yeast host is Saccharomyces cerevisiae.
In another embodiment, the present invention provides a construct comprising the target genes
selected from “S”, “M” and “E” proteins of SARS-CoV having SEQ ID Nos 7, 8 and 9 or
variants thereof, individually or in combination to co-express in yeast-based expression
vectors, wherein the vectors are selected from episomal and/or integrative vectors and wherein
any two proteins are expressed in episomal and one protein is expressed in integrative
expression vector.
In another embodiment, said construct comprises S protein having SEQ ID NO 1 of SARS
CoV virus and a yeast episomal expression vector.
In another embodiment, said construct comprises M protein having SEQ ID NO 2 of SARS
CoV virus and a yeast integrative or episomal expression vector or integrative expression
vector.
In another embodiment, said construct comprises E protein having SEQ ID NO 3 of SARS
CoV virus and a yeast episomal or integrative expression vector.
In another embodiment, said construct is a multiprotein construct comprising “S” and “E”
proteins expressed into an episomal expression vector to obtain a construct having SEQ ID NO
10 and “M” protein expressed into integrative expression vector to obtain a construct having
SEQ ID NO 11.
In another embodiment, the present invention provides a recombinant Corona virus like particle
(VLP) comprising SARS-CoV proteins, wherein the SARS-CoV proteins are selected from;
(i) a SARS-CoV- virus Spike proteins (S);
(ii) a SARS-CoV- virus Membrane proteins (M); and
(iii) a SARS-CoV- virus Envelope proteins (E),
or variants thereof.
In another embodiment, said VLPs comprise one or more SEQ IDs selected from 1, 2 and 3
encoding S, M and E proteins or their variants thereof, in a yeast cell under conditions which
permit the formation of VLPs.
In another embodiment, the present invention provides a method said VLPs maintain a size
range and stoichiometry range for all the co-expressed proteins.
In another embodiment, the present invention provides a method of preparing the target VLPs
comprising the target proteins selected from S, M and E proteins of SARS-CoV, individually
or in combination, comprising the steps of:
i. inserting the target gene selected from S, M and E proteins of SARS-CoV into
protease deficient yeast host cell by either episomal construct or integrative
construct;
ii. transforming the selected genes in protease deficient yeast host strain;
iii. selecting the transformants on selective Yeast Nitrogen Base (YNB) Glucose
medium and without LEU auxotrophic marker in case of integrative construct
and without URA auxotrophic marker in case of episomal construct;
iv. transforming the selected genes in protease deficient yeast host strain;
v. selecting the transformants on YNB with Glucose without URA and LEU
auxotrophic marker plates;
vi. performing transformation;
wherein, transformation is a sequential transformation and performed using
Lithium acetate/SS-DNA/PEG mediated protocol.
In another embodiment, the present invention provides a method of preparing the target VLPs,
comprising the target M protein of SARS-CoV, individually, comprising the steps of:
i. Integrating/episomally inserting the M gene into protease deficient yeast host
cell;
ii. transforming the M gene in Protease deficient yeast host strain;
iii. selecting the transformants on Yeast Nitrogen Base (YNB) Glucose medium
without LEU auxotrophic marker in case of integrative construct and without
URA auxotrophic marker in case of episomal construct;
iv. performing transformation;
wherein, transformation is performed using Lithium acetate/SS-DNA/PEG
mediated protocol.
In another embodiment, the present invention provides a method of preparing the target VLPs
comprising the target E and M protein of SARS-CoV, in combination, comprising the steps of:
i. Integrating/episomally inserting the E or M gene into protease deficient yeast
host cell;
ii. transforming the E or M gene in Protease deficient yeast host strain
iii. selecting the transformants on Yeast Nitrogen Base (YNB) Glucose medium
without LEU auxotrophic marker in case of integrative construct and without
URA auxotrophic marker in case of episomal construct;
iv. transforming the E or M gene in Protease deficient yeast host strain for co
expression;
v. selecting the transformants on YNB Glucose without URA and LEU
auxotrophic marker plates for co-expression using episomal and integration
vector; and selecting the transformants on YNB Glucose without URA for
expression of both using episomal vector or YNB Glucose without LEU for
expression of both using integration vector;
vi. performing transformation;
wherein transformation is performed using Lithium acetate/SS-DNA/PEG mediated protocol.
In another embodiment, the present invention provides a method comprising the target proteins
selected from S, M and E proteins of SARS-CoV, in combination, comprising the steps of:
i. inserting the target gene selected from S, M and E proteins of SARS-CoV into
protease deficient yeast host cells;
ii. selecting the transformants on selective Yeast Nitrogen Base (YNB) Glucose
medium and without LEU auxotrophic marker;
iii. transforming the selected genes in protease deficient yeast host strain;
iv. selecting the transformants on YNB with Glucose without URA and LEU
auxotrophic marker plates;
v. performing transformation;
wherein the transformation is a sequential transformation and is performed using Lithium
acetate/SS-DNA/PEG mediated protocol by incubating the plates at 28ᵒC for 2-4 days for S, E,
and M proteins and cultures were grown in YNB Glucose without URA and LEU media at
28ᵒC for 36 hr and control with URA media.
In another embodiment, the present invention provides a method of producing the scalable
amount of VLPs comprising the steps of:
i. overexpressing recombinant proteins or VLPs in a transformed yeast host cell culture
in a batch or fed batch cultivated system using high density culture, wherein the
media is supplemented with additives, such as glucose, glycerol, either at the time of
inoculation or later at the time when the cells are grown to a high cell density, wherein
high cell density is a cell density where the transformed yeast cells are grown to a
density of up to 60 at OD600 and up to 120-300 g/L, WCW in a time interval of 12-
24 hours at a growth temperature of 28-32 oC;
ii. growing the transformed yeast cell in culture to a stage of log phase growth;
iii. cooling the cultivation media to a temperature in range of 25-28oC;
iv. inducing the cultivation media with induction agents;
v. harvesting the yeast cells containing the VLP by centrifugation or a microfiltration
step, or a mixture of both;
vi. washing the harvested yeast cells with cell lysis buffer consisting of a diafiltration or
buffer exchange step or an ultrafiltration step, or a mixture of both and resuspending
the yeast host cells in the lysis buffer;
vii. disrupting the yeast host cells in lysis buffer by mechanical force or ultrasonic waves,
or a French press or a method of cell lysis, or a combination of the above;
viii. purifying the VLPs obtained in step (vi), using an ion exchange resin or a mix mode
resin or a combination of the two wherein said exchange resin is an anion or cation
or mix mode resin, Capto Core 70;
ix. eluting the VLPs with a buffer containing up to 1-2 M KCl;
x. dialysing the VLPs with a formulation buffer comprising 20-100mM Potassium
phosphate pH 7.2.
xi. supplementing the VLPs with 50 -100 mM KCl, optionally along with 0.0005 –
0.001% Tween 80 and 2-10% Sucrose.
xii. filter-sterilizing the dialyzed VLPs using 0.2 µm filter;
xiii. determining the yield of purified VLP.
In still another embodiment, the high density culture of yeast cells and cultivation medium is
supplemented with an amount of an induction agent such as galactose, glycerol or a mixture of
both wherein the period of induction is from 48-120 hours at 25-28oC.
In another embodiment, the cultivation medium is further supplemented with a boosting
solution containing supplements selected from peptides, amino acids, tryptone and yeast
extract.
In another embodiment, the cultivation media is saturated by air to a level 20-80% saturation.
In another embodiment, the lysis buffer contains 20mM-100 mM Potassium phosphate pH 7.2,
0.0005 % - 0.001% Tween 80 or Tween 20 or a non-ionic detergent, 2mM PMSF and wherein
the lysis buffer is optionally supplemented with a nuclease.
In another embodiment, the yeast cells containing the VLP in lysis buffer is disrupted by
mechanical force, specifically with a high pressure ultrasonic waves, or a method of cell lysis,
where in the yeast cells are lysed or broken to obtain a lysed medium of 60-100% lysed cells
in the lysis buffer.
In another embodiment, said VLPs are harvested by centrifugation step, or a diafiltration or an
ultrafiltration step microfiltration step, or a mixture of both, to produce partially isolated VLPs.
In another embodiment, the eluted VLPs are dialysed by buffer exchange with a formulation
buffer, consisting of 20-100 mM Potassium phosphate pH 7.2.
In another embodiment, the present invention provides a method an immunogenic composition,
comprising the VLPs, along with pharmaceutically acceptable excipients, adjuvants and/or
stabilizers, wherein the VLP formulation is administered with or without an adjuvant.
In another embodiment, said composition is administered intranasal, mucosal, intradermal,
subcutaneous, intramuscular, sublingual or oral.
In another embodiment, the present invention provides a vaccine comprising the VLPs,
wherein said vaccine induces an immune response in a subject.
In another embodiment, said immunogenic composition or the vaccine, wherein said
immunogenic composition or the vaccine induces the immune response in a subject against
multiple serotypes or clades of SARS-CoV virus.
In another embodiment, said SARS-CoV virus is SARS-CoV2 virus.
In another embodiment, the subject is a human and/or animal.
In present invention both integration and episomal expression vector are simultaneously used
for co-expression of full-length SARS CoV-2 structural glycoprotein antigens Spike,
Membrane and Envelope for the VLP production using S. cerevisiae host system. The designed
strategy for the heterologous co-expression of three structural proteins includes the integration
of capsid Membrane (M) protein into the protease deficient yeast host genome while the Spike
(S) and Envelope (E) proteins have been cloned and co expressed in episomal vector to reduce
the excess foreign DNA interference and load in cell for the enhanced efficient expression of
all the proteins in desired stoichiometry ration response.
Electron microscopy of the SARS-CoV-2 virus has, shown it to be pleomorphic with a size
range distribution from 52 to 200nm. Further structural analysis for protein spacing data, basis
the size from coronaviruses have shown a 4 to 5 nm distance between the M dimer molecules.
Basis the size, this leads to ~1100 molecules of M2 in a SARS- CoV virus . Further, the model
predicted presence of ∼90 spikes per particle which exists as a trimer of size 9 to 12 nm and
gives a corona appearance to the virus. Further study from TGEV corona viruses showed that
15 to 30 copies of E protein are present per 100 to 200 molecules of S protein.
Thus, in the current invention, a new approach of co-expression of three proteins is
demonstrated from three independent cassettes which assemble together to form a VLP. While
the expression of the “M” protein was driven from a genomic integration done using pYRI100,
the co-expression of “S” and “E” was driven by an episomal vector pYRE100. This strategy
has led to successful expression of SARS-CoV-2 antigens which lead to formation of VLP at
high concentration in the yeast cytoplasm.
Present invention also shows appropriate assembly of the Corona like structure and ratio of
three recombinantly co-expressed structural proteins S, M and E in produced stable VLPs using
S. cerevisiae as host system and successfully used in eliciting the immune response against all
three proteins in mice and Nabs.
EXAMPLES
The present invention is further described herein below by way of illustration and more
particularly, the following paragraphs are provided in order to describe the best mode of
working the invention and nothing in this section should be taken as a limitation of the claims.
Example 1: Expression of S protein
The Spike protein gene sequence was taken from reference ID strain MN908947.3 (Severe
acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1). The virus is a single stranded
RNA virus with a genome size of 29903 bp. The gene was codon biased and optimized for
expression in yeast host. The glycoprotein was expressed in episomal expression vector
pYRE100 (Fig 1A). The protein was His tagged for ease of analysis of expression and
purification as protein.
The gene was cloned using conventional cloning methodologies and was analysed through
restriction digestion. The construct was transformed into protease deficient S. cerevisiae yeast
host for expression studies and further was taken for scale up and microsomes preparation for
localization and analytics. Expression was characterized by anti His antibody immunoblotting,
and by peptide mapping analysis. The protein can further be purified using standard
purification protocol to get the purified protein.
Example 1.1: Expression analysis and confirmation of expressed proteins using S. Cerevisiae
host
Expression at small scale
The characterized recombinant construct was transformed in yeast host using Lithium
acetate/SS-DNA/PEG mediated protocol known in art and transformants were selected over
YNB Glucose – URA plates along with control transformed with episomal vector backbone.
Few isolated healthy transformed colonies were inoculated in 10ml YNB Glucose - URA media
for episomal expression analysis. All proteins were analysed for expression in 24th hr post
induced (Induction at late log phase with galactose at final concentration of 2% galactose) time
point samples using anti- His antibody by Immuno-blot analysis.
Scale up culture and microsomes preparation:
Scale up culture was performed at shake flask level at 500ml scale was done and analysed for
expression and endoplasmic localization of expressed protein by microsomal preparation. 25
ml of inoculum was prepared for respective proteins in respective media. Late log culture of
the healthy grown pre-culture was inoculated into 450ml of YPD broth in duplicates and was
cultured in shake flasks for 24hrs. The cultures were induced at a final concentration of 2%
galactose for 24hrs. Microsomal preparations were made using methodology known art.
Expression and localization was analysed using His tag antibody immunoblot.
Results
The immunoblot of His tagged S protein showed a band at app. size of 71 kDa molecular weight.
This may be due to the fact that the protein is cleaved into S1 and S2 domains by Kex 2 (native
protease in S.cerevisiae host) mimicking Furin cleavage site and both the fragments are
migrating together at ~71KDa size. Further clone was taken for scale up, microsomes
preparation and localization analysis. Immuno-blot results using anti His antibody from
microsomal preparations also confirmed the expression of the S protein at~71kDa (Fig 1B and
C). The sample were further analysed by peptide mapping analysis to confirm identity of the
protein. for correct and full-length protein expression. The peptide spanning the full-length of
the protein were detected in mass spectrometry analysis (Fig. 1D). This may be due to the fact
that the S1 and the S2 fragments comigrate on the gel (Fig.1B and C). This also further
confirmed the expression of full-length S protein.
Example 2: Expression of M protein
The M protein gene sequence was taken from reference ID strain MN908947.3 (Severe acute
respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1). The gene was codon biased and
optimized for expression in yeast host. The glycoprotein was expressed in episomal expression
vector pYRE100 (Fig. 2A). The protein was His tagged for ease of analysis of expression and
purification as protein.
The gene was cloned, expressed and analysed as described in example 1. The final
characterized construct was transformed into protease deficient S. cerevisiae yeast host for
expression studies and further was taken for scale up to 500ml and microsomes preparation for
localization and analytics as mentioned for example 1. Expression was characterized by anti
His antibody immunoblotting, and by peptide mapping analysis. The protein can further be
purified using standard purification protocol to get the purified protein.
Results
Immuno Blot analysis using anti His antibody showed a specific signal at expected band size
of ~26 kDa of His tagged full length M protein. Also a band at ~31kda was also observed which
may be glycosylated form and also observed in literature. Further, Immuno-blot analysis using
anti His antibody of microsomal preparation confirmed expression of full-length M protein
also confirming the localization to membrane fraction (Fig. 2B and C). The sample was further
analysed using mass spectrometry to confirm protein identity. for correct and full-length
protein expression. Peptide mapping results confirms the sequence identity of the His tagged
M protein (Fig. 2D). In conclusion, present invention has showed and confirmed the expression
of full-length surface transmembrane M structural glycoprotein of SARS CoV2 virus using S.
cerevisiae host system platform.
Example 3: Expression of Full-length SARS CoV2 Envelope (E) capsid glycoprotein
antigen
The E protein gene sequence was taken from reference ID strain MN908947.3 (Severe acute
respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1). The gene was codon biased and
optimized for expression in yeast host. The glycoprotein was expressed in episomal expression
vector pYRE100 (Fig. 3A). The protein was His tagged for ease of analysis of expression and
purification as protein.
The gene was cloned, expressed and analysed as described in example 1 and 2. The final
characterized construct was transformed into protease deficient S. cerevisiae yeast host for
expression studies and further was taken for scale up and microsomes preparation for
localization and analytics. Expression was characterized by anti His antibody immunoblotting,
and by peptide mapping analysis. The protein can further be purified using standard
purification protocol to get the purified protein.
Results
Immuno Blot analysis using anti His antibody showed a signal at a size higher than the expected
size of ~9kDa in case of full length His tagged E protein may be due to the predicted
glycosylation of the envelope protein (Fig. 3B). Further the expression of protein was also
observed in the microsomal preparation prepared from scale to 500ml ((Fig. 3C). The protein
was also confirmed by mass spec analysis (Fig. 3D).
Example 4: Cloning and Expression of multiple antigens S, E and M of an enveloped virus
SARS CoV-2 as co-expression strategy in the yeast expression host system using dual
approach of episomal and integrated expression together
The present examples outline the strategy for co-expression of three viral surface proteins “S”,
“E” and “M”, from SARS CoV-2 virus, using yeast expression platform wherein two antigens
were expressed through episomal expression and the third protein was expressed by integrating
it into the yeast genome. This strategy design further provides an approach of co-expressing
proteins which assemble as an enveloped VLP, maintaining structure and immunogenic
potential similar to the original SARS-COV2 virus.
4.1 Strategy design:
In order to co-express the proteins as VLP maintains, “S” and “E” proteins were cloned into an
episomal expression vector pYRE100. “M” protein was cloned into integrative expression
vector pYRI100. The gene sequences were codon biased and optimized for the cloning and
expression in S. cerevisiae host with respect to certain important key critical parameters like
codon usage with respect to the expression host, rare codon, GC content and AT rich or GC
rich stretches optimization, internal TATA boxes, ribosomal entry sites ,chi sites removal to
increase the mRNA turnover and protein stability that improved the efficiency of gene
expression for expression in yeast host.
Example 4.2: Cloning and construct preparation
The gene for “S” and “E” proteins were cloned using conventional cloning methodologies into
expression plasmid pYRE100 as two separate expression cassettes for co-expression analysis.
The M protein was cloned in pYRI100 integration vector. The cloned gene were analyzed
through restriction digestion. The constructs were transformed into S. cerevisiae host of
recombinant expression platform for expression studies using specific antibodies by
immunoblots analysis.
Example 4.3: Transformation in S. cerevisiae protease deficient host strain:
For co-expression of three proteins, the M protein gene (pYRI100_M) was integrated into
protease deficient S. cerevisiae host genome and transformants were selected over selective
YNB Glucose without LEU auxotrophic marker plates. This was followed by the
transformation of the S and E protein genes (pYRE100_S+E episomal construct) in S.
cerevisiae strain integrated with the M gene construct. Transformants were selected over
selective YNB Glucose without URA and LEU auxotrophic marker plates. Vector pYRE100
plasmid was transformed in protease deficient S. cerevisiae host as control and was selected
over selective YNB Glucose without URA auxotrophic marker plates. Sequential
transformation was performed using Lithium acetate/SS-DNA/PEG mediated protocol by
incubating the plates at 28ᵒC for 2-4 days for S, E, and M protein co-expression analysis.
Example 4.4 Expression analysis of culture:
Cultures were grown for the co expression of three proteins in YNB Glucose without URA and
LEU media at 28ᵒC for 36 hr and control with URA media. At this stage, cultures were harvested
and induced with galactose at a final concentration of 2% in YNB media without URA and
LEU medium for 48 hours. Following induction, the harvested culture was used for expression
analysis.
Example 4.5 Immunoblot analysis:
In order to confirm the presence of the S, E, and M protein, immunoblot analysis was carried
out using the purified VLP. For immunoblot, ~6 µg samples were loaded on SDS-PAGE under
reduced conditions. Immunoblot was developed using 1:1000 dilution of S specific antibody
(Cat#ZHU1076, Sigma-Aldrich) and 1:500 dilution of poly-clonal M (Cat# GTX134866, Gene
Tex) and E (Cat#MBS150849, My Bio-source) specific antibodies. Signals were detected with
ECL solution (Biorad cat#170-5061)..
Results: Immunoblot analysis using commercially available antibodies confirmed the
expression of S, E, and M proteins (Fig. 4 A, B and C). The co-expression of S, E, and M
proteins was further confirmed by mass spectrometry (Fig. 5). The studies confirm that the S,
cerevisiae based expression platform is able to express the three antigens which co-assemble to
form a VLP (Fig. 4).
Example 5: Simultaneous use of the Integrated and Episomal vectors for co-expression
of multiple antigens that assemble to form a VLP using yeast expression platform
The three proteins (S, E and M) were expressed as three separate independent cassettes but
regulated by a common promoter present in the three cassettes. The “S” and “E” proteins were
cloned into an episomal expression vector pYRE100. “M” protein was cloned into integrative
expression vector pYRI100. The expression of three proteins was confirmed independently as
a first step, then as co-expression of S and E protein followed by co expression of all the three
proteins. The constructs were transformed in protease deficient yeast host and expression was
analysed. The co-expression of the three proteins was confirmed by immunoblot analysis as
described previously (Fig. 4).
Example 6: Demonstration of Immune response against all the three antigens in a multiantigen enveloped VLP for SARS CoV-2 virus
In order to assess the immunogenicity and the antibody response against the three antigens
which come together to assemble as an eVLP, specific immune responses against S, E, and M
proteins were investigated by ELISA assay at week five after the initial immunization in mouse
model.
To assess the antigen-specific IgG response, 96-well microtiter plates were coated with 0.25
µg of purified S, E and M proteins were individually at 2-8 °C overnight and blocked with 2%
BSA for 1 h at room temperature. Diluted sera (1:1000) were applied to each well and incubated
for at 37 °C for 2 h, followed by incubation with HRP conjugated goat anti-mouse antibodies
at 37 °C for 1h . The plate was developed using TMB, following 2 M H2SO4 addition to stop
the reaction, and read at 450 nm using an ELISA plate reader.
Results
Evaluation of immune response against the S, E and M proteins showed that mice vaccinated
with or without an adjuvant (Alhydrogel was used as an adjuvant in the studies) could surmount
a immune response against the three eVLP antigens (Fig. 6). Thus, these results highlight the
potential of eVLP in inducing a strong and potent immune response by eliciting effective
antibody responses for all three antigens and, therefore, may serve as an effective vaccine
candidate for novel coronavirus infections. This eVLP based vaccine could be helpful to also
prevent infections from the various variants of the SARS-CoV-2 which may have developed
mutations in the S protein.
Example 7: The multi-antigen enveloped VLP produced in yeast expression system is
highly immunogenic as demonstrated by in vivo studies
This example shows that a triple antigen, eVLP is able to surmount an immune response with
high titers in mice. Also, the antibodies are seen to be neutralizing in nature.
Example 7.1: Humoral immune responses and Immunization in mice
BALB/c mice were immunized with eVLP formulation with and without adjuvant Alhydrogel
(AH). Adjuvant groups included injection with 5 µg eVLP+AH, 10 µg eVLP+AH and 20 µg
eVLP+AH. The eVLP alone group was injected with 10 µg eVLP. Two controls groups were
set with buffer alone injection and the with adjuvant AH only injection. The animals were
injected at 14 days interval (Day 0, 14, 28) with one primary and two booster doses.
Example 7.2: Serum Antibody measurements
IgG mediated antibody response and titer of serum samples collected from immunized animal
sera at 0 and 35th day were determined by enzyme-linked immunosorbent assay (ELISA).
Briefly, 96 well ELISA plate was coated with 0.25 µg of eVLP at 2-8 °C overnight and blocked
with 2% BSA for 1 h at room temperature. Serum dilutions were applied to the well and
incubated for at 37 °C for 2 h, followed by incubation with HRP conjugated goat anti-mouse
antibodies at 37 °C for 1h . The plate was developed using TMB, following 2 M H2SO4 addition
to stop the reaction, and read at 450 nm using an ELISA plate reader (Fig 7).
Example 7.3: SARS-CoV-2 neutralization assays
Neutralization experiments on serum samples of immunized animals and convalescent patients
were performed as per the manufacturer protocol (sVNT Kit, GenScript, USA). Briefly, the
samples were pre-incubated with HRP-RBD to bind the circulating neutralizing antibodies to
HRP-RBD. The mixture was added to an hACE2 protein pre-coated plate and incubated at 37
°C for 15 min. The wells were washed 4 times with PBS and the mixture was incubated with
TMB at 20-25 °C in the dark for 15 min. The stop solution was added, and the plate was read
at 450nm in a microtiter plate reader. Neutralisation potential of mice was plotted relative to
human neutralization potential (reference), taken as 100 percent (Fig 8).
Example 7.4: Plaque reduction neutralization test (PRNT)
For the PRNT assay, Vero E6 cells (1x105
per well) were seeded onto 24-well plates. On the
following day, 30 PFU of infectious wild-type SARS-CoV-2 was incubated with diluted serum
(total volume of 150 µl) at 37 °C for 1 h. The virus-serum mixture was added to the pre-seeded
Vero E6 cells and incubated at 37 °C for 1 h. Following this, 1 ml of 2% Modified Eagle’s
Medium (MEM) containing carboxy methylcellulose (CMC) was added to the infected cells.
After 3 days of incubation, 1 ml 3.7% formaldehyde was added and incubated at room
temperature for 30 min. The formaldehyde solution from each well was discarded, and the cell
monolayer was stained with a crystal violet solution for 60 min. After washing with water,
plaques were counted for PRNT50 calculation. The PRNT assay was performed in a BSL-3
facility.
Results
The total IgG response was evaluated in different mice groups using the sera samples at day 0
and 35. The total IgG binding endpoint titers (EPTs) from all the immunized mice groups were
measured against eVLP. Results showed that all the eVLP vaccinated groups elicited IgGmediated response as compared to controls (Fig. 7). A dose-dependent increase in titer was
observed. Interestingly, the eVLP was seen to be immunogenic even without the adjuvant
suggesting its potential to generate an immune response.
RBD-specific neutralization inhibition was evaluated in eVLP immunized animals and
compared to that with convalescent patients’ sera. Neutralization potential (NP) of
convalescent patients’ sera was used as a reference and considered 100 percent; data from mice
was plotted relative to that. In the neutralization inhibition assay, sera from all the eVLP
vaccinated groups showed significant NP by inhibiting the RBD and ACE2 interaction
compared to control groups (Fig. 8). However, maximum inhibition of RBD and ACE2 cell
surface receptors was observed at the lowest (5µg) dose of adjuvanted eVLP (62.3±0.50%) as
compared to control groups (AH: 6.12±8.06%; Fig. 9).
PRNT is considered as the gold-standard for determining immune protection. To validate the
virus neutralization assay results, a conventional PRNT assay was performed for the vaccinated
groups. On day 3 post-challenge, wild-type virus neutralizing activity capable of reducing
SARS-CoV-2 infectivity by 50% or more (PRNT50) was detected in all eVLP vaccinated
groups (Fig. 6). The serum dilutions yielding 50% virus neutralization (PRNT50 titers) were
higher in eVLP (1:10) and AH plus 5µg eVLP (1:20) groups in comparison to other adjuvanted
groups, where PRNT50 was observed at a dilution of 1:5 (Fig. 9).
The results thus comprehensively show that eVLP was able to surmount an immune response
which has neutralization potential.
Example 8: The eVLP produced using Yeast based expression platform mimics the viral
epitopes and produces a cellular immunity response
To assess if the eVLP mimics the viral epitopes, it was used in cytokine assays from healthy
and convalescent human PBMCs. VLP specific lymphocyte proliferation was evaluated by
ELISA assay using sera from convalescent patients (N=5) and healthy controls sera (N=2). All
the human samples have been taken after adequate ethical committee approval.
Example 8.1: Determination of cell proliferation using BrDU assay
PBMCs were separated from the blood of the convalescent patients and stimulated with two
doses of the VLP (2.5µg and 5µg). The proliferation rate of lymphocytes was measured using
5-Bromo-2-deoxyuridine (BrDU) assay as per the manufacturer instructions (Cell Proliferation
ELISA BrDU, Roche, USA).
Example 8.2: Cytokine profiling of stimulated PBMCs
Healthy control and patient PBMCs (1×105 cells/well) were stimulated with two doses of the
VLP formulation (2.5 µg and 5 µg) at 37 °C in a humified chamber containing 8% CO2 for
120 h. Supernatants were collected, and cytokine staining for IFN-g was performed as per the
manufacturer instructions (Human IFN--g ELISA set (RUO), BD Biosciences, USA).
Results
BrDU cell proliferation studies of PBMCs showed a higher lymphocyte proliferation in patient
samples subjected to two doses of VLP formulation (2.5µg-2 Fold; 5µg:1.45 Fold) in
comparison to PBMCs from healthy individuals (Figure 10A). Further, elevated levels of IFN-
 in the VLP stimulated patients PBMCs (2.16±0.33) was observed as compared to healthy
controls (0.95±0.23), suggesting induction of T helper 1 (Th1)–biased cellular immune
responses (Figure 10B).
The results demonstrate that the VLP formulation containing S, E and M full length proteins
from SARS CoV-2 virus, can mimic the epitopes of the original SARS-CoV2 virus, as it can
stimulate a cell mediated immune response with PBMCs from convalescent patients which
have never been exposed to VLP formulation. In literature, none of the vaccine candidates have
shown a recall of cellular immune response from convalescent patients confirming the
mimicking epitope by a potential vaccine candidate.
Example 9: Safety of the vaccine formulation over large range of dose formulation
Extensive studies were performed to demonstrate that the enveloped VLP for SARS CoV-2
virus, composed of three antigens, S, E and M, and produced using the yeast expression
platform is safe over a large range of doses. Two rodent species have been used to demonstrate
the safety of this formulation: rats and mice.
Example 9.1: Studies in mice (Mus musculus):
Multiple cohorts of Mice (Mus musculus sp.), Strain Balb/c, gender male, age 6-8 weeks, were
taken and under all applicable guidelines, injected intramuscularly with liquid formulation of
the VLP in dose of 5µg, 10 µg, 20 µg, 50 µg, 100 µg and 150 µg. Two vaccine formulations
were studied simultaneously: a) with commercially available adjuvant aluminum hydroxide gel
and b) VLP alone with no adjuvant. In each cohort, five animals were included to maintain
statistical significance of the data observed.
The mice were given three doses, on day 0, day 14 and day 28 and observed for any signs/
symptoms of morbidity or other changes. No mortality was documented through the course of
study. All animals looked healthy post injection, and all through the course of study, there was
no visible changes in their motility, no behavioral changes inside the cage were observed or
fighting wounds or any skin infections were observed, no lack of shininess of hair was
observed. Food habits were observed to be normal for all the groups.
Example 9.2: Studies in mice (Swiss albino): Abnormal Toxicity studies
Abnormal toxicity test with the VLP as vaccine candidate in Swiss albino mice was tested in 5
male mice. The abnormal toxicity test was performed as per Indian Pharmacopoeia IP 2018
and OECD principles on Good Laboratory Practice.
The test was performed with 5 animals. 250 µL of the test candidate, VLP in liquid formulation
was reconstituted to the 250 µL of the adjuvant (Alhydrogel) and mixed / shook well for one
minute. To each mouse, 500 µL of the reconstituted test item was administered immediately
via intraperitoneal route.
The animals were observed for 7 days for any adverse effects or clinical signs and recorded at
30 min, 1, 2, 4, 24 hr and then after once a day till the termination / completion of the
experiment. All animals were normal, and no signs of clinical toxicity were observed
throughout the experiment. All the animals used in this study gained body weight on day 7
when compared to its respective day 0 (dosing start day).
The animals were humanely sacrificed by carbon dioxide asphyxiation at termination of the
experiment.
Based on the results obtained, it was concluded that the formulated eVLP, with the given
Adjuvant, passed the abnormal toxicity test and was classified as “Non-toxic” to the mice under
the conditions of the study conducted (Table 1).
Table 1: Abnormal Toxicity studies in Swiss albino
Example 9.3: Study in Guinea pigs: Abnormal Toxicity studies
Abnormal toxicity test with the VLP formulation as a vaccine candidate was conducted in
another species, using the Guinea Pigs model. The formulation was tested in 2 male guinea
Pigs. The abnormal toxicity test was performed as per Indian Pharmacopoeia IP 2018 and
OECD principles on Good Laboratory Practice (revised 1997, issued January 1998).
The test was performed with 2 animals. 250 µL of the test candidate, VLP in liquid formulation
was reconstituted to the 250 µL of the adjuvant (Alhydrogel) and mixed / shook well for one
minute. To each mouse, 500 µL of the reconstituted test item was administered immediately
via intraperitoneal route.
The animals were observed for 7 days for any adverse effects or clinical signs and recorded at
30 min, 1, 2, 4, 24 hr and then after once a day till the termination / completion of the
experiment. All animals were normal, and no signs of clinical toxicity were observed
throughout the experiment. All the animals used in this study gained body weight on day 7
when compared to its respective day 0 (dosing start day). The animals were humanely
sacrificed by carbon dioxide asphyxiation at termination of the experiment.
A.No
Days of Observation
Acclimatization Phase Treatment Phase
1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7
01 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
02 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
03 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
04 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
05 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Note: 1- Normal.
18.1 Individual body weight and % body weight change
A.No. Group /
Sex
Acclimatizatio
n start day
(g)
Experiment
start day
(g)
Experiment
completion
day
(g)
% Body
weight
change
(Day 0- 7)
01
Initial test /
Male
18.08 19.18 19.97 4.12
02 17.20 18.03 18.75 3.99
03 18.24 18.97 19.76 4.16
04 18.50 19.27 20.04 4.00
05 18.39 19.23 19.98 3.90
Note: A.No.- Animal number; g- Grams; %- Percent.
Results
Based on the results obtained, it was observed that the formulated test item as a vaccine
candidate with the given Adjuvant passed the abnormal toxicity test and was classified as
“Non-toxic” to the guinea pigs under the conditions of study conducted (Table 2).
Table 2: Abnormal Toxicity studies in Guinea pigs
Example 10: Stoichiometric ration calculation for S, M and E in the produced VLP from
co expression
Relative amounts of S, M and E proteins were calculated using quantitative ELISA
methodology. To estimate the individual amount of protein present in VLP, ELISA plate wells
were coated with known amounts of respective purified standard S1, E and M proteins to
generate a standard curve for each protein.
Similarly, purified VLP was coated for the analysis of number of molecules of S, M and E
protein in assembled VLP. Relative quantitative estimation of S: M: E ratio in the VLP was
measured against purified protein standard curve generation.
10.1: ELISA
Purified standard proteins (S1, M and E) and VLP were coated at different amount per well on
ELISA plate for 14hr at 4°C. 1% BSA was used as blocking solution. The proteins were then
detected in ELISA using respective commercially available antibodies along with the VLP.
Clinical signs
A.No
Days of Observation
Acclimatization Phase Treatment Phase
1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7
01 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
02 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Note: 1- Normal.
Individual body weight and % body weight change
A.No. Group /
Sex
Acclimatizatio
n start day
(g)
Experiment
start day
(g)
Experiment
completion
day
(g)
% Body
weight
change
(Day 0- 7)
01 Initial test /
Male
307.02 324.77 341.08 5.02
02 301.60 318.54 339.67 6.63
Note: A.No.- Animal number; g- Grams; %- Percent.
For S1 standard protein, RBD specific antibody (dilution 1:250); for M standard protein, M
specific antibody (1:500); for E standard protein, E specific antibody (1:250) were used along
with VLP coated wells. After washing, respective secondary antibodies were added. Finally,
absorbance measured with TMB solution at 450nm. Graphs were plotted accordingly and
mentioned in Fig. 11 A, B and C.
Results
Calculations for estimation of S1, M and E
A linear plot of OD vs the amount of the protein coated was plotted from which the equation
of straight line was generated (y=m1x+c). Using the derived equation, the amount of protein
(x) in the VLP was calculated using the absorbance value. Further, using the protein amounts
the number of molecules of each protein was determined using online software
(https://www.bioline.com/media/calculator/01_04.html), (Table 3).
Table 3: Calculations for S and M molecules in assembled VLP sample:
Antibody used
for detection
Absorbance Amount of
protein (ng)
Mol wt
(kDa)
Number of
molecules
RBD 0.063 10.9 420 1.56 x 1010
M specific 0.426 6.29 25 1.51 x 1011
E specific 0.0104 0.812 54 0.8 x 1010
Obtained values showed S: M: E stoichiometric value of 1: 9.7 : 0.5 in VLP. The ratios were
calculated for VLPs which showed the expected range of SARS-CoV-2 virion size of 180 to
200nm However, due to pleomorphic nature of the Corona viruses the ratio and size of the VLP
is expected to vary. The M glycoprotein of coronaviruses drives the assembly hence is the
most abundant viral structural protein in the virion and oligomerizes and interacts laterally with
S and E, the other two viral membrane proteins to make VLP while E protein which in literature
is described to be present in low amounts but important for assembly.
Example 11: Production of the scalable amount of VLPs
The VLPs were produced in a transformed yeast cell culture and cultivated in a batch or fedbatch cultivation system using a cultivation medium which is supplemented with at least one
or more additives to support high density culture. Transformed yeast cell was further grown in
a culture to a stage of log phase growth with a media supplemented with additives, such as
glucose, glycerol, either at the time of inoculation or later at the time when the cells are grown
to a high cell density. The transformed yeast cells are grown to a density of up to 60 at OD600
and up to 120-300 g/L, WCW in a time interval of 12-24 hours at a growth temperature of 28-
32 oC after which the temperature is cooled to 25-28oC followed by supplementing the media
with an amount of an induction agent such as galactose, glycerol or a mixture of both and
boosting solution containing peptides, amino acids, tryptone and yeast extract in an amount of
10x concentrated 12 g/L tryptone and 24 g/L yeast extract. The cultivation media is saturated
by air to a level 20-80% saturation and incubated for period induction of high protein
expression, by an additive at 48-120 hours at 25-28oC. The cells were harvested either by
centrifugation step, or a microfiltration step, or a mixture of both resulting into VLPs and
cultivation medium.
The VLPs were washed and buffer was changed to a lysis buffer for cell lysis, by a diafiltration
or buffer exchange or an ultrafiltration step, or a mixture of both to resuspend the yeast cells in
the lysis buffer. The lysis buffer contains 20mM-100 mM Potassium phosphate pH 7.2, 0.0005
% - 0.001% Tween 80 or Tween 20 or a non-ionic detergent, 2mM PMSF and is also
supplemented with a nuclease where the nuclease treatment was given for 2-18 hours at 2-8
oC,
or 37oC, preferably, for 2-3 hours at 37oC or 4-18 at 2-8
oC.
The yeast cells containing the VLP in lysis buffer was disrupted by mechanical force or
ultrasonic waves, or a French press or a method of cell lysis, or a combination of the above and
results into the lysed or broken yeast cells where lysis outcome is up to 60-100% lysed cells in
the lysis buffer containing the nuclease. The yeast cells containing the VLP in lysis buffer was
further disrupted by mechanical force, specifically with a high-pressure mechanical
homogenizer, where the yeast cells were subjected to a mechanical pressure of 200 – 800 Bar,
at 2-8
oC, and the process is repeated 2-8 times or passes to obtain a lysis outcome of 60-100%
lysed cells in the lysis buffer. The method results into the VLPs, from the nuclease treated lysis
buffer and produce partially isolated VLP separating the lysis buffers, nucleases and other
contaminating components.
The exchange buffer solution consists of 20 - 100 mM Potassium phosphate pH 7.2, 0.0005%
- 0.001% Tween 80) and nuclease treated VLP in lysis buffer is buffer exchanged and
diafiltered using 400 - 750 kDa hollow fiber cartridge of I.D of 0.5 – 1.0 mm.
The VLPs were purified using an anion or cation ion exchange resin or a mix mode resin or a
combination of the two. A mix mode resin, Capto Core 700 was used in the present invention.
The VLP material was loaded and eluted with a buffer containing up to 1.5M KCl and
collection of fractions was performed based on A280 and VLP purity. The VLPs were further
taken to dialysis and buffer exchange step with a formulation buffer, consisting of 20-100mM
Potassium phosphate pH 7.2 supplemented with 50 -100 mM KCl, 0.0005 – 0.001% Tween 80
and 2-10% Sucrose. Dialyzed material was sterile filtered using 0.2 µm filter and tested for
yield and the yield was determined to be 60mg-120 mg/L of purified VLP.
The present invention provides VLPs and method of preparing said VLPs for SARS-CoV
which are stable and display appropriate stoichiometric and are capable of inducing
immunogenic response. The present invention also provides the strategy for efficient
expression of the SARS-CoV proteins(S, M and E) individually and in combination. The VLPs
are suitable for incorporated in an efficient vaccine against SARS-CoV.
Advantages of the present invention:
 Recombinant VLPs have significant morphological resemblance to the parent virus,
provide a highly repetitive immunogenic surface structure, and the retention of cell
uptake and immune processing pathways associated with their parent virus. They
themselves are devoid of the parent virus genome and hence are non-pathogenic or
incapable of infection.
 The SARS CoV-2 VLP described in this invention can be bulk expressed in bioreactor
cultures, and using the current expression system and processes, it is capable of millions
of doses in a very short span of time, utilizing advanced in vitro protein expression
systems for producing vaccine-grade VLP suitable for clinical administration.
 Multi-antigen VLP vaccine of the present invention is capable of providing antibody
response against diverse antigens and has ability to recognise the virus even in case of
mutations, which is a very important aspect that is not being addressed by vaccines
available or under development currently.
 The enveloped VLPs have been difficult to express and purify from yeast-based
platform in large quantities; this invention describes the process and method to do so,
which can also be helpful in developing vaccines for other similar viruses and
infections, both in human and animal.
 The potential vaccine candidate in this invention has demonstrated in animal models,
safety, simplicity of production and favourable immunological characteristics to induce
both humoral and cellular immune responses. The SARS cov-2 virus poses a continuous
threat to human health in the pandemic and with multiple variations, which can be
addressed by the current invention.
 Affordability of the dose is an important criterion for any vaccine to be successfully
developed and launched; with the current processes and high yields described in this
application, the vaccine cost per dose can be made highly affordable.

We Claim:

1. A recombinant polypeptide selected from the group comprising the SEQ ID Nos 1, 2
and 3 and variants thereof, wherein the recombinant polypeptide belongs to proteins
selected from Spike (S), Membrane (M) and Envelope (E) proteins, respectively, from
SARS-CoV family, and wherein the said proteins are either individually expressed or
co-expressed.
2. The recombinant polypeptides as claimed in claim 1, wherein the variant of the
polypeptides comprises a sequence having at least 70% sequence identity to the
polypeptide sequence selected from the group comprising SEQ ID NOs 1, 2 and 3.
3. The recombinant polypeptides as claimed in claim 1, wherein the polypeptides are
obtained after codon optimization.
4. The recombinant polypeptide as claimed in claim 1, wherein the S protein is obtained
after individual expression of SEQ ID NO 1 or a variant thereof.
5. The recombinant polypeptide as claimed in claim 4, wherein said recombinant
polypeptide is a full-length S protein which is a single pass type I membrane
glycoprotein
6. The recombinant polypeptide as claimed in claim 1, wherein the M protein is obtained
after individual expression of SEQ ID NO 2 or a variant thereof.
7. The recombinant polypeptide as claimed in claim 6, wherein said recombinant
polypeptide is a full-length M protein which is a multi-pass membrane glycoprotein.
8. The recombinant polypeptide as claimed in claim 1, wherein the E protein is obtained
after individual expression of SEQ ID NO3 or a variant thereof.
9. The recombinant polypeptide as claimed in claim 8, wherein said recombinant
polypeptide is a full-length E protein which is a single pass type III membrane
glycoprotein.
10. A recombinant molecule comprising recombinant polypeptides as claimed in claim 1,
wherein said molecule is a complex formed by assembly of polypeptides defined in
SEQ ID NO 1, 2 and 3 or variants thereof and is obtained after co-expression of S, M
and E proteins to form an ordered aggregate.
11. The recombinant molecule as claimed in claim 10, wherein the recombinant molecule
assembles to form Virus like particles (VLPs).
12. A recombinant polynucleotide selected from the group comprising the SEQ ID Nos 4,
5 and 6 or variants thereof, wherein the polynucleotides are selected from Spike (S),
Membrane (M) and Envelope (E) genes from SARS-CoV family, and wherein the said
genes are cloned individually or in combination.
13. The recombinant polynucleotides as claimed in claim 12, wherein the variant of the
nucleotides comprises a sequence having at least 60% sequence identity to the
polynucleotide sequence selected from the group comprising SEQ ID NOs 4, 5 and 6.
14. The recombinant polynucleotides as claimed in claim 12, wherein the said
polynucleotides are codon biased and optimized.
15. The recombinant polynucleotide as claimed in claim 12, wherein said S gene has SEQ
ID NO 4 and variants thereof.
16. The recombinant polynucleotide as claimed in claim 12, wherein the said M gene has
SEQ ID NO 5 and variants thereof.
17. The recombinant polynucleotide as claimed in claim 12, wherein the said E gene has
SEQ ID NO 6 and variants thereof.
18. The recombinant polypeptides as claimed in claim 1 or the recombinant polynucleotides
as claimed in claim 9, wherein the polypeptides or polynucleotides are selected from,
but not limited to, Spike (S), Membrane (M) and Envelope (E) proteins from SARSCoV-2.
19. A method of expressing SARS-CoV proteins comprising the steps of:
i. selecting the target genes from the group comprising of S, E and M proteins;
ii. optimizing and codon biasing of target gene sequences;
iii. preparing the construct comprising target genes and yeast-based expression
vectors by cloning of target genes individually or in combination to co-express
the proteins into the expression vector, wherein the expression vector is yeast
based expression vector selected from episomal expression vector and/or
integrative expression vector;
iv. transforming the constructs into Protease deficient yeast host cell to obtain
target proteins.
20. A method of expressing S protein from SARS-CoV as claimed in claim 20, wherein the
method comprises the steps of:
i. selecting the target gene S protein;
ii. optimizing and codon biasing of target gene sequence;
iii. preparing the construct comprising S gene and yeast-based expression vector by
cloning of S gene individually to express the said protein into the expression
vector, wherein the expression vector is yeast based episomal or integrative
expression vector;
iv. transforming the construct into Protease deficient yeast host cell to obtain target
protein.
21. A method of expressing M protein from SARS-CoV as claimed in claim 20, wherein
the method comprises the steps of:
i. selecting the target gene M protein;
ii. optimizing and codon biasing of target gene sequence;
iii. preparing the construct comprising M gene and yeast-based expression vector
by cloning of M gene individually to express the said protein into the expression
vector, wherein the expression vector is yeast based integrative or episomal
expression vector;
iv. transforming the construct into Protease deficient yeast host cell to obtain target
protein.
22. A method of expressing E protein from SARS-CoV as claimed in claim 20, wherein
the method comprises the steps of:
i. selecting the target gene E protein;
ii. optimizing and codon biasing of target gene sequence;
iii. preparing the construct comprising E gene and yeast-based expression vector
by cloning of E gene individually to express the said protein into the expression
vector, wherein the expression vector is yeast based episomal or integrative
expression vector;
iv. transforming the construct into Protease deficient yeast host cell to obtain target
protein.
23. A method of expressing the SARS-CoV proteins as claimed in claim 20, wherein the
method of co-expressing S, E and M proteins comprises the steps of:
i. selecting the S, E and M proteins from SARS-CoV;
ii. optimizing and codon biasing of target gene sequences;
iii. cloning “S” and “E” proteins into an episomal expression vector to obtain a
construct having SEQ ID NO 10 and “M” protein into integrative expression
vector to obtain a construct having SEQ ID NO 11;
iv. preparing the construct comprising the selected genes and yeast vectors;
v. transforming the constructs into yeast host;
vi. over-expressing the target multi-subunit proteins in the cell.
24. The method as claimed in claim 20, wherein all the three proteins and variants thereof
are either co-expressed individually as episomal or integrative or combinations thereof.
25. The method as claimed in claim 20, wherein proteins are selected, but not limited to,
the S, M and E Corona virus proteins.
26. The method of expressing SARS-CoV proteins as claimed in claim 20, wherein said
yeast host is Saccharomyces cerevisiae.
27. A construct comprising the target genes selected from “S”, “M” and “E” proteins of
SARS-CoV having SEQ ID Nos 7, 8 and 9 or variants thereof, individually or in
combination to co-express in yeast-based expression vectors, wherein the vectors are
selected from episomal and/or integrative vectors and wherein any two proteins are
expressed in episomal and one protein is expressed in integrative expression vector.
28. The construct as claimed in claim 27 having the SEQ ID 7 wherein the construct
comprises S protein having SEQ ID NO 1 of SARS CoV virus and a yeast episomal
expression vector.
29. The construct as claimed in claim 27 having the SEQ ID 8, wherein the construct
comprises M protein having SEQ ID NO 2 of SARS CoV virus and a yeast integrative
or episomal expression vector.
30. The construct as claimed in claim 27 having the SEQ ID 9, wherein the construct
comprises E protein having SEQ ID NO 3 of SARS CoV virus and a yeast episomal or
integrative expression vector.
31. The construct as claimed in claim 27, wherein the construct is a multiprotein construct
comprising “S” and “E” proteins expressed into an episomal expression vector to obtain
a construct having SEQ ID NO 10 and “M” protein expressed into integrative
expression vector to obtain a construct having SEQ ID NO 10.
32. A recombinant corona virus like particle (VLP) comprising SARS-CoV proteins,
wherein the SARS-CoV proteins are selected from;
(i) a SARS-CoV- virus Spike proteins (S);
(ii) a SARS-CoV- virus Membrane proteins (M); and
(iii) a SARS-CoV- virus Envelope proteins (E),
or variants thereof.
33. The VLP as claimed in claim 32, wherein the VLPs comprise one or more SEQ IDs
selected from 1, 2 and 3 encoding S, M and E proteins or their variants thereof, in a
yeast cell under conditions which permit the formation of VLPs.
34. The VLP as claimed in claim 32, wherein the VLPs maintain a size range and
stoichiometry range for all the co-expressed proteins.
35. A method of preparing the target VLPs as claimed in claim 32 comprising the target
proteins selected from S, M and E proteins of SARS-CoV, individually or in
combination, comprising the steps of:
i. inserting the target gene selected from S, M and E proteins of SARS-CoV into
protease deficient yeast host cell by either episomal construct or integrative
construct;
ii. transforming the selected genes in protease deficient yeast host strain;
iii. selecting the transformants on selective Yeast Nitrogen Base (YNB) Glucose
medium and without LEU auxotrophic marker in case of integrative construct
and without URA auxotrophic marker in case of episomal construct;
iv. transforming the selected genes in protease deficient yeast host strain;
v. selecting the transformants on YNB with Glucose without URA and LEU
auxotrophic marker plates;
vi. performing transformation;
wherein, transformation is a sequential transformation and performed using
Lithium acetate/SS-DNA/PEG mediated protocol.
36. The method of preparing the target VLPs as claimed in claim 35, comprising the target
M protein of SARS-CoV, individually, comprising the steps of:
i. Integrating/episomally inserting the M gene into protease deficient yeast host
cell;
ii. transforming the M gene in Protease deficient yeast host strain;
iii. selecting the transformants on Yeast Nitrogen Base (YNB) Glucose medium
without LEU auxotrophic marker in case of integrative construct and without
URA auxotrophic marker in case of episomal construct;
iv. performing transformation;
wherein, transformation is performed using Lithium acetate/SS-DNA/PEG
mediated protocol.
37. The method of preparing the target VLPs as claimed in claim 35, comprising the target
E and M protein of SARS-CoV, in combination, comprising the steps of:
i. integrating/episomally inserting the E or M gene into protease deficient yeast
host cell;
ii. transforming the E or M gene in Protease deficient yeast host strain
iii. selecting the transformants on Yeast Nitrogen Base (YNB) Glucose medium
without LEU auxotrophic marker in case of integrative construct and without
URA auxotrophic marker in case of episomal construct;
iv. transforming the E or M gene in Protease deficient yeast host strain for co
expression;
v. selecting the transformants on YNB Glucose without URA and LEU
auxotrophic marker plates for co-expression using episomal and integration
vector; and selecting the transformants on YNB Glucose without URA for
expression of both using episomal vector or YNB Glucose without LEU for
expression of both using integration vector;
vi. performing transformation;
wherein transformation is performed using Lithium acetate/SS-DNA/PEG mediated protocol.
38. The method of preparing the target VLPs as claimed in claim 35, comprising the target
proteins selected from S, M and E proteins of SARS-CoV, in combination, comprising
the steps of:
i. inserting the target gene selected from S, M and E proteins of SARS-CoV into
protease deficient yeast host cells;
ii. selecting the transformants on selective Yeast Nitrogen Base (YNB) Glucose
medium and without LEU auxotrophic marker;
iii. transforming the selected genes in protease deficient yeast host strain;
iv. selecting the transformants on YNB with Glucose without URA and LEU
auxotrophic marker plates;
v. performing transformation;
wherein the transformation is a sequential transformation and is performed using Lithium
acetate/SS-DNA/PEG mediated protocol by incubating the plates at 28ᵒC for 2-4 days for S, E,
and M proteins and cultures were grown in YNB Glucose without URA and LEU media at
28ᵒC for 36 hr and control with URA media.
39. A method of producing the scalable amount of VLPs comprising the steps of:
i. overexpressing recombinant proteins or VLPs in a transformed yeast host cell culture
in a batch or fed batch cultivated system using high density culture, wherein the
media is supplemented with additives, such as glucose, glycerol, either at the time of
inoculation or later at the time when the cells are grown to a high cell density, wherein
high cell density is a cell density where the transformed yeast cells are grown to a
density of up to 60 at OD600 and up to 120-300 g/L, WCW in a time interval of 12-
24 hours at a growth temperature of 28-32 oC;
ii. growing the transformed yeast cell in culture to a stage of log phase growth;
iii. cooling the cultivation media to a temperature in range of 25-28oC;
iv. inducing the cultivation media with induction agents;
v. harvesting the yeast cells containing the VLP by centrifugation or a microfiltration
step, or a mixture of both;
vi. washing the harvested yeast cells with cell lysis buffer consisting of a diafiltration or
buffer exchange step or an ultrafiltration step, or a mixture of both and resuspending
the yeast host cells in the lysis buffer;
vii. disrupting the yeast host cells in lysis buffer by mechanical force or ultrasonic waves,
or a French press or a method of cell lysis, or a combination of the above;
viii. purifying the VLPs obtained in step (vi), using an ion exchange resin or a mix mode
resin or a combination of the two wherein said exchange resin is an anion or cation
or mix mode resin, Capto Core 70;
ix. eluting the VLPs with a buffer containing up to 1-2 M KCl;
x. dialysing the VLPs with a formulation buffer comprising 20-100mM Potassium
phosphate pH 7.2.
xi. supplementing the VLPs with 50 -100 mM KCl, optionally along with 0.0005 –
0.001% Tween 80 and 2-10% Sucrose.
xii. filter-sterilizing the dialyzed VLPs using 0.2 µm filter;
xiii. determining the yield of purified VLP.
40. The method as claimed in claim 45, wherein the high-density culture of yeast cells and
cultivation medium as obtained in step (iii) of claim 43, is supplemented with an amount
of an induction agent such as galactose, glycerol or a mixture of both wherein the period
of induction is from 48-120 hours at 25-28oC.
41. The method as claimed in claim 45, wherein the cultivation medium as obtained in step
(iii) of claim 43 is further supplemented with a boosting solution containing
supplements selected from peptides, amino acids, tryptone and yeast extract.
42. The method as claimed in claim 45, wherein the cultivation media is saturated by air to
a level 20-80% saturation.
43. The method as claimed in claim 45, wherein the lysis buffer of step (vi) contains 20mM100 mM Potassium phosphate pH 7.2, 0.0005 % - 0.001% Tween 80 or Tween 20 or a
non-ionic detergent, 2mM PMSF and wherein the lysis buffer is optionally
supplemented with a nuclease.
44. The method as claimed in claim 45, wherein the yeast cells containing the VLP in lysis
buffer is disrupted by mechanical force, specifically with a high pressure ultrasonic
waves, or a method of cell lysis, where in the yeast cells are lysed or broken to obtain
a lysed medium of 60-100% lysed cells in the lysis buffer.
45. The method as claimed in claim 45, wherein VLPs are harvested by centrifugation step,
or a diafiltration or an ultrafiltration step microfiltration step, or a mixture of both, to
produce partially isolated VLPs.
46. The method as claimed in claim 45, wherein the eluted VLPs obtained from step (x),
are dialysed by buffer exchange with a formulation buffer, consisting of 20-100 mM
Potassium phosphate pH 7.2.
47. An immunogenic composition, comprising the VLPs as claimed in claim 32, along with
pharmaceutically acceptable excipients, adjuvants and/or stabilizers, wherein the VLP
formulation is administered with or without an adjuvant.
48. A method of administering the composition as claimed in claim 47, wherein said
composition is administered intranasal, mucosal, intradermally, subcutaneously,
intramuscularly, sublingual or orally.
49. A vaccine comprising the VLPs as claimed in claim 32, wherein said vaccine induces
an immune response in a subject.
50. The immunogenic composition as claimed in claim 47 or the vaccine as claimed in
claim 49, wherein said immunogenic composition or the vaccine induces the immune
response in a subject against multiple serotypes or clades of SARS-CoV virus.
51. The immunogenic composition or the vaccine as claimed in claim 47, wherein the said
SARS-CoV virus is SARS-CoV2 virus.
52. The immunogenic composition as claimed in claim 47 or the vaccine as claimed in
claim 49, wherein the subject is a human and/or animal