DESC:FIELD OF INVENTION
[001] The present disclosure broadly relates to the field of immunobiology, and
particularly discloses immunogenic peptides, and vaccine compositions for
5 eliciting immune response against Sarbecoviruses. The present disclosure also
relates to a method for producing the vaccine composition as well as the method
of eliciting an immune response against diverse Sarbecoviruses, in a subject, by
administering the subject with an effective amount of said vaccine composition.
BACKGROUND OF THE INVENTION
10 [002] Coronaviruses (CoVs) have consistently posed a significant risk of
dissemination. Zoonotic viruses such as betacoronaviruses continue to represent a
global hazard through spillover events into human population. This has been
witnessed multiple times in the last 30 years by severe acute respiratory
syndrome coronavirus 1 (SARS CoV-1), Middle East respiratory syndrome
15 coronavirus (MERS CoV) outbreaks, and the latest severe acute respiratory
syndrome coronavirus 2 (SARS CoV-2) pandemic. The unprecedented
appearance and spread of coronavirus disease of 2019 (COVID-19) at the global
level accelerated the development of various vaccines utilizing multiple platforms,
out of which some were approved by the regulatory agencies and rolled out for
20 public usage. However, the rapid evolution of SARS CoV-2 resulted in emergence
of variants which exhibited immune escape and rendered the vaccines less
effective (Li et al., 2024; Liu et al., 2024; Lyke et al., 2022).
[003] Sarbecoviruses are a subgenus of betacoronaviruses that includes the SARS-
CoV, MERS-CoV, and the more recently identified SARS-CoV-2, which causes
25 COVID-19. In Sarbecoviruses such as SARS CoV-1 and SARS CoV-2, the viral
spike protein (S) is the principal target of neutralising antibodies that prevent
infection. Within spike, the immunodominant receptor-binding domain (RBD) is
the primary target of neutralising antibodies as observed in COVID-19
convalescent sera and vaccine recipients (Premkumar et al., 2020; Yang et al.,
30 2020).
2
[004] The attachment and entry of the viral particles into host cells is facilitated by
the S-proteins through their binding to ACE2 (angiotensin-converting enzyme-2).
Protein S is comprised of two distinct subunits, namely S1 and S2. The S1 subunit,
positioned in the N-terminal region, encompasses RBD responsible for
5 recognizing the host cell receptor (Huang et al., 2020; Lan et al., 2020). The RBDs
of SARS-CoV-2, SARS CoV-1 as well as MERS CoV have been demonstrated to
be highly suitable targets for the development of vaccines across various platforms
owing to their exceptional level of antigenicity and capability to effectively
stimulate robust immune responses (Ahmed et al., 2021; Malladi, Patel, et al.,
10 2021; Tai et al., 2020, 2023).
[005] Human infection with viruses against which pre-existing immunity is not
prevalent in the population can result in epidemics and even pandemics with
substantial morbidity and mortality. These outbreaks can have a negative influence
on public health and the economy worldwide. Considering the number of viruses
15 which have various zoonotic reservoirs and the potential to infect humans, future
pandemic preparedness is of utmost importance to mitigate these risks.
[006] Therefore, there remains a dire need for the development of a comprehensive
thermostable, safe and effective pan-sarbecovirus vaccine formulation, which can
elicit a targeted immune response and is also capable of eliciting a broadly
20 neutralizing cross reactive response against diverse Sarbecoviruses.
SUMMARY OF INVENTION
[007] In an aspect of the present disclosure, there is provided an immunogenic
polypeptide comprising a polypeptide selected from the group consisting of: a
25 polypeptide having an amino acid sequence of at least 95% sequence identity to
the sequence selected from SEQ ID NO: 1 or SEQ ID NO: 59; a polypeptide
having an amino acid sequence of at least 95% sequence identity to the sequence
selected from SEQ ID NO: 3 or SEQ ID NO: 61; a polypeptide having an amino
acid sequence of at least 95% sequence identity to the sequence selected from SEQ
30 ID NO: 5 or SEQ ID NO: 63; a polypeptide having an amino acid sequence of at
least 95% sequence identity to the sequence selected from SEQ ID NO: 7 or SEQ
3
ID NO: 65; a polypeptide having an amino acid sequence of at least 95% sequence
identity to the sequence selected from SEQ ID NO: 9 or SEQ ID NO: 67, and a
polypeptide having an amino acid sequence of at least 95% sequence identity to
the sequence selected from SEQ ID NO: 85 or SEQ ID NO: 69, wherein the
5 polypeptide comprises substitution mutations A17P, Y34W, and P196L.
[008] In an aspect of the present disclosure, there is provided a polynucleotide
encoding the immunogenic polypeptide as described herein.
[009] In another aspect of the present disclosure, there is provided a recombinant
vector containing the polynucleotide as disclosed herein operably linked to a
10 promoter.
[0010] In another aspect of the present disclosure, there is provided a
recombinant host cell comprising the recombinant vector as described herein.
[0011] In another aspect of the present disclosure, there is provided a vaccine
composition comprising the immunogenic polypeptide as disclosed herein, and a
15 pharmaceutically acceptable carrier.
[0012] In an aspect of the present disclosure, there is provided a method for
producing the vaccine composition comprising: (a) culturing the recombinant host
cell as described herein, under suitable conditions to obtain the immunogenic
polypeptide; (b) subjecting the immunogenic polypeptide to purification; and (c)
20 contacting the immunogenic polypeptide of step (b) with a pharmaceutically
acceptable carrier, to obtain the vaccine composition.
[0013] In an aspect of the present disclosure, there is provided a method of
eliciting an immune response against diverse Sarbecoviruses, in a subject,
comprising administering the subject with an effective amount of the vaccine
25 composition as disclosed herein.
[0014] In an aspect of the present disclosure, there is provided a kit
comprising the immunogenic polypeptide or the vaccine composition as described
herein, and an instruction leaflet.
[0015] These and other features, aspects, and advantages of the present
30 subject matter will be better understood with reference to the following description
and appended claims. This summary is provided to introduce a selection of
4
concepts in a simplified form. This summary is not intended to identify key
features or essential features of the claimed subject matter, nor is it intended to be
used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
5
[0016] The following drawings form a part of the present specification and
are included to further illustrate aspects of the present disclosure. The disclosure
may be better understood by reference to the drawings in combination with the
detailed description of the specific embodiments presented herein.
10 [0017] Figure 1 depicts the results related to expression and purification of
Sarbecoviruses RBD derivatives. Coomassie stained profiles of Sarbecoviruses
RBDs purified from Expi293F cells are shown, in accordance with the
embodiments herein.
[0018] Figure 2 depicts relative thermal stability of Sarbecoviruses RBD
15 derivatives: Nano-DSF profiles showing thermal melting temperature for
Sarbecoviruses RBD derivatives: wild type (WT) and stabilized (St), in
accordance with the embodiments herein.
[0019] Figure 3 depicts relative thermal tolerance of Sarbecoviruses RBD
derivatives: Purified proteins were incubated at temperatures ranging from 4-70?C
20 for 2 hours followed by Nano-DSF profiles of (A) wild type and (B) stabilized
RBD derivatives, in accordance with the embodiments herein.
[0020] Figure 4 depicts immunogenicity studies in mice: Endpoint ELISA
IgG titers against different Sarbecoviruses RBDs obtained after two
immunizations with (A) individual stabilized RBD oligomers and (B) stabilized
25 RBD derivative cocktail formulated with adjuvant, in accordance with the
embodiments herein.
[0021] Figure 5 depicts immunogenicity studies in mice: Pseudovirus
neutralization titers against (A) SARS CoV-1, (B) SARS CoV-2, (C) BtKY72 and
(D) heterologous pseudoviruses including LYRa3, SHC014 and Khosta-2 from
30 sera obtained after two immunizations, in accordance with the embodiments
herein.
5
[0022] Figure 6 illustrates efficacy of Stabilized RBD trimer and RBD-S2
monomer cocktail formulation, wherein A) is a schematic representation of the
immunization protocol, and (B) depicts body weight change and lung viral titres
in mice post SARS-CoV-1 challenge and (C) depicts body weight change and
5 lung viral titres in mice post SARS-CoV-2 BA.5 challenge, in accordance with the
embodiments herein.
[0023] Figure 6 illustrates long-term thermal stability of the RBD cocktail
immunogens wherein (A) depicts thermal denaturation profile and (B) depicts
binding affinity of the RBD cocktail immunogens from day0- day 30 for proteins
10 incubated at 4°C and 37°C determined by Nano-DSF and BLI experiments
respectively. Lyophilized proteins were incubated at the indicated temperatures
and reconstituted in PBS prior to measurements, in accordance with the
embodiments herein.
15 DETAILED DESCRIPTION OF THE INVENTION
[0024] Those skilled in the art will be aware that the present disclosure is
subject to variations and modifications other than those specifically described. It
is to be understood that the scope of the present disclosure includes all such
20 variations and modifications that may be apparent to a person skilled in the art in
light of the present disclosure. The disclosure also includes all such steps, features,
compositions, and compounds referred to or indicated in this specification,
individually or collectively, and any and all combinations of any or more of such
steps or features.
25 Definitions
[0025] For convenience, before further description of the present disclosure,
certain terms employed in the specification, and examples are delineated here.
These definitions should be read in the light of the remainder of the disclosure and
understood as by a person of skill in the art. The terms used herein have the
30 meanings recognized and known to those of skill in the art, however, for
6
convenience and completeness, particular terms and their meanings are set forth
below.
[0026] The articles “a”, “an” and “the” are used to refer to one or to more
than one (i.e., to at least one) of the grammatical object of the article.
5 [0027] The terms “comprise” and “comprising” are used in the inclusive,
open sense, meaning that additional elements may be included. It is not intended
to be construed as “consists of only”.
[0028] Throughout this specification, unless the context requires otherwise
the word “comprise”, and variations such as “comprises” and “comprising”, will
10 be understood to imply the inclusion of a stated element or step or group of element
or steps but not the exclusion of any other element or step or group of element or
steps.
[0029] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably.
15 [0030] The term “pharmaceutically acceptable carrier” refers to any known
carriers, excipients, adjuvants known to a person skilled in the art, which can be
used in therapeutic and vaccine formulations. The term “pharmaceutically
effective amount” or “effective amount”, as used herein, refers to an amount that
is effective in eliciting an immune response, in a subject.
20 [0031] The term “vaccine composition” refers to a composition that elicits a
prophylactic or therapeutic immune response in a subject. Typically, a vaccine
composition elicits an antigen-specific immune response to an antigen of a
pathogen, for example a viral pathogen, or to a cellular constituent correlated with
a pathological condition.
25 [0032] The term “subject” refers to any animal, including a mammal, e.g.,
human and non-human mammals. Examples of non-human animals include non-
human primates, dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, mice, rats,
hamsters, guinea pigs and etc. Unless otherwise noted, the terms “patient” or
“subject” are used herein interchangeably. Preferably, the subject is human.
30 [0033] Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary skill
7
in the art to which this disclosure belongs. Although any methods and materials
similar or equivalent to those described herein can be used in the practice or testing
of the disclosure, the preferred methods, and materials are now described. All
publications mentioned herein are incorporated herein by reference.
5 [0034] Embodiments herein provide immunogenic polypeptides having
Sarcebovirus derived stabilized receptor binding domain (RBD) fragments. The
wildtype RBD fragments obtained from various Sarbecoviruses, particularly
SARS-CoV-1 (Clade 1a), WIV-1 (Clade 1a), RaTG13 (Clade 1b), RmYN02
(Clade 2), BtKY72 (Clade 3), and SARS CoV-2 XBB1.5 are stabilized by
10 introducing specific stabilizing mutations at various positions on the amino acid
sequence. The present inventors observed that the introduction of these mutations
substantially improved thermal stability by about ~7°C, and enhanced purified
yield by about 3-23-fold, relative to corresponding wildtype (WT) RBDs, without
affecting their binding to conformation specific ligands.
15 [0035] Further, in some embodiments, the immunogenic polypeptides
comprise Sarbecovirus derived stabilized receptor binding domain (RBD)
fragment, and a fragment of S2 subunit of SARS-CoV-2 spike protein . The
fragment of S2 subunit may be attached N-terminally or C-terminally to the
stabilized RBD, preferably via a linker. In an embodiment, the fragment of S2
20 subunit is attached C-terminally to the stabilized RBD. The fragment of S2 subunit
of SARS-CoV-2 spike protein may be engineered to include substitution mutations
at positions 817, 892, 899, 942, 969 and 973, positions in respect of the wildtype
S2 subunit of SARS-CoV-2 spike protein (Accession Id: YP_009724390.1). In an
embodiment, the amino acid sequence of the fragment of S2 subunit of SARS-
25 CoV-2 spike protein is engineered to substitute amino acid residue at positions
817, 892, 899, and 942 with proline (i.e. F817P, A892P, A899P, and A942P
respectively) and substitute amino acid residue at positions 969 and 973 with
aspartic acid (i.e. N969D and I973D respectively). In an embodiment, the
fragment of S2 subunit of SARS-CoV-2 spike protein has an amino acid sequence
30 as depicted in SEQ ID NO: 83 (corresponding nucleotide sequence is depicted in
SEQ ID NO: 84).
8
[0036] In an embodiment, the stabilized RBD fragments obtained from
Sarbecoviruses: SARS-CoV-1 (Clade 1a), WIV-1 (Clade 1a), RaTG13 (Clade 1b),
RmYN02 (Clade 2), BtKY72 (Clade 3), and SARS CoV-2 XBB1.5 are depicted
in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
5 and SEQ ID NO: 85, respectively.
[0037] In an embodiment, the polypeptides having a combination of
sarbecovirus derived stabilized receptor binding domain (RBD) fragment, and a
fragment of S2 subunit of SARS-CoV-2 spike protein: Stabilized SARS CoV-1
RBD-S2, Stabilized WIV-1 RBD-S2, Stabilized RaTG13 RBD-S2, Stabilized
10 RmYN02 RBD-S2, Stabilized BtKY72 RBD-S2, and Stabilized SARS CoV-2
XBB1.5 RBD-S2 are depicted in SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:
63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69, respectively.
[0038] Substitution mutations and stabilizing mutations, according to
embodiments herein, are also described herein by use of the following notation:
15 amino acid residue substituted: amino acid position in the sequence: amino acid
residue substitute. Accordingly, an amino acid substitution with, for instance, if
the amino acid substitution is of phenylalanine (F) with proline (P), at position 111
in a sequence, it may be indicated as F111P. Further, alternative amino acid
residues substitutes are represented by the notation: amino acid substitute/ amino
20 acid substitute/ amino acid substitute. For instance, if amino acid substitution of
phenylalanine (F) is with proline (P) or serine (S), at position 111 in a sequence, it
may be indicated as F111P/S. Similarly, when more than one alternatives are
possible, it may be indicated as F111P/S/A, and so on. In general, when more than
one alternatives amino acid residues are possible at a position, it may be indicated
25 as P/S which is intended to mean that any of proline or serine may be present at
that position Similarly, N/I may indicate that any of asparagine or isoleucine may
be present at that position, and so on. Such notations are generally known to a
person skilled in the art and generally used in representing amino acid
residues/substitutions in a given amino acid sequence. Also, amino acids are
30 generally represented by single letter and three letter abbreviations, for example
“Alanine” is represented by single letter code “A” and three letter code “ala” or
9
“Ala”. Similarly, the single letter code include R (Arginine), N (Asparagine), D
(Aspartic acid), C (Cysteine), E (Glutamic acid), Q (Glutamine), G (Glycine), H
(Histidine), I (Isoleucine), L (Leucine), K (Lysine), M (Methionine), F
(Phenylalanine), P (Proline), S (Serine), T (Threonine), W (Tryptophan), Y
5 (Tyrosine), and V (Valine). Such representations are generally used and well
understood by a person skilled in the art. The present disclosure in describing the
present invention employs such representations or phrases which is intended to
mean the generally acceptable meaning in the art.
[0039] Embodiments herein also provide vaccine compositions comprising
10 one or more of the immunogenic polypeptides as described herein. It has been
observed by the present inventors that a cocktail formulation comprising two or
more of the immunogenic polypeptides as described herein significantly augments
the yield, thermostability, and immunogenicity of the RBD fragments. The present
disclosure provides stabilization of RBD fragments obtained from different clades
15 of Sarbecoviruses, which is combined to achieve a pan-Sarbecoviruses vaccine
formulation.
[0040] The immunogenic polypeptides, according to embodiments herein,
are resistant to 2-hour incubation at temperatures of up to 60? in PBS, in contrast
to corresponding WT RBDs. Further, lyophilized cocktail formulations, according
20 to embodiments herein, retained antigenicity even after storage at 37°C for over
15 days. In an embodiment, the vaccine composition is a lyophilized composition.
Immunogenic polypeptide
[0041] Embodiments herein provide immunogenic polypeptides. In an
embodiment, the immunogenic polypeptide comprises a polypeptide having an
25 amino acid sequence of at least 95% sequence identity to the sequence selected
from SEQ ID NO: 1 or SEQ ID NO: 59, wherein the polypeptide comprises
substitution mutations Y34W and P195L. In an embodiment of the present
disclosure, the identity is at least 95%, 96%, 97%, 98%, 99%, or 99.5% to the
amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 59. In another
30 embodiment of the present disclosure, the immunogenic polypeptide comprising a
10
polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ
ID NO: 59.
[0042] In an embodiment of the present disclosure, there is provided an
immunogenic polypeptide comprising a polypeptide having an amino acid
5 sequence of at least 95% sequence identity to the sequence selected from SEQ ID
NO: 3 or SEQ ID NO: 61, wherein the polypeptide comprises substitution
mutations Y34W and P195L. In another embodiment of the present disclosure, the
identity is at least 95%, 96%, 97%, 98%, 99%, 99.5% to the amino acid sequence
as set forth in SEQ ID NO: 3or SEQ ID NO: 61. In an embodiment of the present
10 disclosure, there is provided an immunogenic polypeptide comprising a
polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ
ID NO: 61.
[0043] In an embodiment of the present disclosure, there is provided an
immunogenic polypeptide comprising a polypeptide having an amino acid
15 sequence of at least 95% sequence identity to the sequence selected from SEQ ID
NO: 5 or SEQ ID NO: 63, wherein the polypeptide comprises substitution
mutations A17P, Y34W, and P196L. In another embodiment of the present
disclosure, the identity is at least 95%, 97%, 98%, 99%, 99.5% to the amino acid
sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 63. In an embodiment of
20 the present disclosure, there is provided an immunogenic polypeptide comprising
a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 5 or SEQ
ID NO: 63.
[0044] In an embodiment of the present disclosure, there is provided an
immunogenic polypeptide comprising a polypeptide having an amino acid
25 sequence of at least 95% sequence identity to the sequence selected from SEQ ID
NO: 7 or SEQ ID NO: 65, wherein the polypeptide comprises substitution
mutations Y34W and P177L. In another embodiment of the present disclosure, the
identity is at least 95%, 96%, 97%, 98%, 99%, 99.5% to the amino acid sequence
selected from SEQ ID NO: 7 or SEQ ID NO: 65. In an embodiment of the present
30 disclosure, there is provided an immunogenic polypeptide comprising a
11
polypeptide having an amino acid sequence as set forth in SEQ ID NO: 7 or SEQ
ID NO: 65.
[0045] In an embodiment of the present disclosure, there is provided an
immunogenic polypeptide comprising a polypeptide having an amino acid
5 sequence of at least 95% sequence identity to the sequence selected from SEQ ID
NO: 9 or SEQ ID NO: 67, wherein the polypeptide comprises substitution
mutations Y34W and P195L. In another embodiment of the present disclosure, the
identity is at least 95%, 96%, 97%, 98%, 99%, 99.5% to the amino acid sequence
selected from SEQ ID NO: 9 or SEQ ID NO: 67. In an embodiment of the present
10 disclosure, there is provided an immunogenic polypeptide comprising a
polypeptide having an amino acid sequence selected from SEQ ID NO: 9 or SEQ
ID NO: 67.
[0046] In an embodiment of the present disclosure, there is provided an
immunogenic polypeptide comprising a polypeptide having an amino acid
15 sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID
NO: 85 or SEQ ID NO: 69, wherein the polypeptide comprises substitution
mutations A17P, Y34W, and P196L. In another embodiment of the present
disclosure, the identity is at least 95%, 96%, 97%, 98%, 99%, 99.5% to the amino
acid sequence selected from SEQ ID NO: 85 or SEQ ID NO: 69. In an embodiment
20 of the present disclosure, there is provided an immunogenic polypeptide
comprising a polypeptide having an amino acid sequence selected from SEQ ID
NO: 85 or SEQ ID NO: 69.
[0047] In an embodiment of the present disclosure, there is provided an
immunogenic polypeptide comprising a polypeptide having an amino acid
25 sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 59, SEQ ID NO: 61,
SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69.
Full length Immunogenic polypeptide
[0048] The immunogenic polypeptide, according to embodiments herein,
30 may further comprise one or more peptides such as TPA signal sequence,
oligomerization domain, His-tag, etc. In an embodiment, there is provided an
12
immunogenic polypeptide comprising a polypeptide having at least 95% sequence
identity to an amino acid sequence selected from the group consisting of SEQ ID
NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID
NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and
5 SEQ ID NO: 69, wherein the immunogenic polypeptide further comprises a
peptide selected from TPA signal peptide; oligomerization domain; HRV3C
protease cleavage site or a portion thereof; His tag; one or more linkers; or
combinations thereof.
[0049] In an embodiment, there is provided an immunogenic polypeptide
10 comprising a polypeptide having at least 95% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 59, SEQ ID NO: 61,
SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69, wherein
the polypeptide is further attached to a oligomerization domain, preferably via a
15 linker.
[0050] Various oligomerization domains are known and may be used in
embodiments herein. Examples of oligomerization domains includes human
cartilage matrix protein (hCMP), chicken CMP (cCMP), fish cartilage matrix
protein (F1CMP), fish isoform 2 cartilage matrix protein (F2-CMP), leucine
20 Zipper with double cysteine (CCIZ), Synthetic oligomerization domain (cCMP-
IZm), foldon, or glycosylated leucine zipper sequence (Gly IZ). In an embodiment,
the oligomerization domain is a synthetic oligomerization domain (cCMP-IZm)
having an amino acid sequence as set forth in SEQ ID NO. 38.
[0051] In an embodiment, there is provided an immunogenic polypeptide
25 comprising a polypeptide having at least 95% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 59, SEQ ID NO: 61,
SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69, wherein
the polypeptide is further attached, at the N-terminal end, to a TPA signal peptide
30 having an amino acid sequence as set forth in SEQ ID NO. 36.
13
[0052] In an embodiment, there is provided an immunogenic polypeptide
comprising a polypeptide having at least 95% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 59, SEQ ID NO: 61,
5 SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69, wherein
the polypeptide is further attached at the C-terminal end to a HRV3C protease
cleavage site having an amino acid sequence as set forth in SEQ ID NO. 40.
[0053] The TPA signal peptide; oligomerization domain; HRV3C protease
cleavage site or a portion thereof; and/or the His tag, may be attached to the
10 polypeptide having at least 95% sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:
5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:
63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69 by one or more linkers.
[0054] Various linkers are known and may be used in embodiments herein.
15 In an embodiment, the linker is having an amino acid sequence selected from
“AAS”, “S”, “AS”, “GT”, "GSAGS" (SEQ ID
NO.52), "ASSEGTMMRGELKN"(SEQ ID NO.53), and/or "GS".
[0055] In an embodiment, the immunogenic polypeptide further comprises
a peptide “EIS” attached at the N-terminal end. The peptide “EIS” is a vector
20 derived peptide and is attached to the N-terminal end of the immunogenic
polypeptide. In an embodiment, the immunogenic polypeptide comprises TPA
signal peptide at the N-terminal end of the immunogenic polypeptide attached by
the peptide “EIS”.
[0056] In an embodiment, the immunogenic polypeptide comprises
25 a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1
attached to a oligomerization domain at C-terminal end of the polypeptide by a
linker “GS”, wherein the oligomerization domain is further attached to a HRV3C
protease cleavage site by a linker “AAS” followed by a His-tag using a linker “S”,
wherein the polypeptide is attached to a peptide of sequence “EIS” at the N-
30 terminal end which is attached to TPA signal peptide, wherein the oligomerization
domain has an amino acid sequence as set forth in SEQ ID NO. 38. In an
14
embodiment of the present disclosure, there is provided an immunogenic peptide
comprising a polypeptide having an amino acid sequence of at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity
to a sequence as set forth in SEQ ID NO: 13, wherein the polypeptide comprises
5 substitution mutations Y60W and P221L. In an embodiment of the present
disclosure, there is provided an immunogenic peptide comprising a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID NO:
13.
[0057] In an embodiment, the immunogenic polypeptide comprises
10 a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1
attached to a oligomerization domain at C-terminal end of the polypeptide by a
linker “GS”, wherein the oligomerization domain is further attached to a HRV3C
protease cleavage site by a linker “AAS”, wherein the polypeptide is attached to a
peptide of sequence “EIS” at the N-terminal end, wherein the oligomerization
15 domain has an amino acid sequence as set forth in SEQ ID NO. 38. In an
embodiment of the present disclosure, there is provided an immunogenic peptide
comprising a polypeptide having an amino acid sequence of at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity
to a sequence as set forth in SEQ ID NO: 15, wherein the polypeptide comprises
20 substitution mutations Y37W and P198L. In an embodiment of the present
disclosure, there is provided an immunogenic peptide comprising a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID NO:
15.
[0058] In an embodiment, the immunogenic polypeptide comprises
25 a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3
attached to a oligomerization domain at C-terminal end of the polypeptide by a
linker “AS”, wherein the oligomerization domain is further attached to a HRV3C
protease cleavage site by a linker “GT” followed by a His-tag using a linker “S”,
wherein the polypeptide is attached to a peptide of sequence “EIS” at the N-
30 terminal end which is attached to TPA signal peptide, wherein the oligomerization
domain has an amino acid sequence as set forth in SEQ ID NO. 38. In an
15
embodiment of the present disclosure, there is provided an immunogenic peptide
comprising a polypeptide having an amino acid sequence of at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity
to a sequence as set forth in SEQ ID NO: 18, wherein the polypeptide comprises
5 substitution mutations Y60W and P221L. In an embodiment of the present
disclosure, there is provided an immunogenic peptide comprising a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID NO:
18.
[0059] In an embodiment, the immunogenic polypeptide comprises
10 a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3
attached to a oligomerization domain at C-terminal end of the polypeptide by a
linker “AS”, wherein the oligomerization domain is further attached to a HRV3C
protease cleavage site by a linker “GT”, wherein the polypeptide is attached to a
peptide of sequence “EIS” at the N-terminal end which is attached to TPA signal
15 peptide, wherein the oligomerization domain has an amino acid sequence as set
forth in SEQ ID NO. 38. In an embodiment of the present disclosure, there is
provided an immunogenic peptide comprising a polypeptide having an amino acid
sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
at least 99.5% sequence identity to a sequence as set forth in SEQ ID NO: 20,
20 wherein the polypeptide comprises substitution mutations Y37W and P198L. In
an embodiment of the present disclosure, there is provided an immunogenic
peptide comprising a polypeptide having an amino acid sequence selected from
the group consisting of SEQ ID NO: 20.
[0060] In an embodiment, the immunogenic polypeptide comprises
25 a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 5
attached to a oligomerization domain at C-terminal end of the polypeptide by a
linker “AS”, wherein the oligomerization domain is further attached to a HRV3C
protease cleavage site by a linker “GT” followed by a His-tag using a linker “S”,
wherein the polypeptide is attached to a peptide of sequence “EIS” at the N-
30 terminal end which is attached to TPA signal peptide, wherein the oligomerization
domain has an amino acid sequence as set forth in SEQ ID NO. 38. In an
16
embodiment of the present disclosure, there is provided an immunogenic peptide
comprising a polypeptide having an amino acid sequence of at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity
to a sequence as set forth in SEQ ID NO: 23, wherein the polypeptide comprises
5 substitution mutations A43P, Y60W, and P222L. In an embodiment of the present
disclosure, there is provided an immunogenic peptide comprising a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID NO:
23.
[0061] In an embodiment, the immunogenic polypeptide comprises
10 a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 5
attached to a oligomerization domain at C-terminal end of the polypeptide by a
linker “AS”, wherein the oligomerization domain is further attached to a HRV3C
protease cleavage site by a linker “GT”, wherein the polypeptide is attached to a
peptide of sequence “EIS” at the N-terminal end which is attached to TPA signal
15 peptide, wherein the oligomerization domain has an amino acid sequence as set
forth in SEQ ID NO. 38. In an embodiment of the present disclosure, there is
provided an immunogenic peptide comprising a polypeptide having an amino acid
sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
at least 99.5% sequence identity to a sequence as set forth in SEQ ID NO: 25,
20 wherein the polypeptide comprises substitution mutations A20P, Y37W and
P199L. In an embodiment of the present disclosure, there is
provided an immunogenic peptide comprising a polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO: 25.
[0062] In an embodiment, the immunogenic polypeptide comprises
25 a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 7
attached to a oligomerization domain at C-terminal end of the polypeptide by a
linker “AS”, wherein the oligomerization domain is further attached to a HRV3C
protease cleavage site by a linker “GT” followed by a His-tag using a linker “S”,
wherein the polypeptide is attached to a peptide of sequence “EIS” at the N-
30 terminal end which is attached to TPA signal peptide, wherein the oligomerization
domain has an amino acid sequence as set forth in SEQ ID NO. 38. In an
17
embodiment of the present disclosure, there is provided an immunogenic peptide
comprising a polypeptide having an amino acid sequence of at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity
to a sequence as set forth in SEQ ID NO: 28, wherein the polypeptide comprises
5 substitution mutations Y60W and P203L. In an embodiment of the present
disclosure, there is provided an immunogenic peptide comprising a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID NO:
28.
[0063] In an embodiment, the immunogenic polypeptide comprises
10 a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 7
attached to a oligomerization domain at C-terminal end of the polypeptide by a
linker “AS”, wherein the oligomerization domain is further attached to a HRV3C
protease cleavage site by a linker “GT”, wherein the polypeptide is attached to a
peptide of sequence “EIS” at the N-terminal end which is attached to TPA signal
15 peptide, wherein the oligomerization domain has an amino acid sequence as set
forth in SEQ ID NO. 38. In an embodiment of the present disclosure, there is
provided an immunogenic peptide comprising a polypeptide having an amino acid
sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
at least 99.5% sequence identity to a sequence as set forth in SEQ ID NO: 30,
20 wherein the polypeptide comprises substitution mutations Y37W and P180L. In
an embodiment of the present disclosure, there is provided an immunogenic
peptide comprising a polypeptide having an amino acid sequence selected from
the group consisting of SEQ ID NO: 30.
[0064] In an embodiment, the immunogenic polypeptide comprises
25 a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 9
attached to a oligomerization domain at C-terminal end of the polypeptide by a
linker “AS”, wherein the oligomerization domain is further attached to a HRV3C
protease cleavage site by a linker “GT” followed by a His-tag using a linker “S”,
wherein the polypeptide is attached to a peptide of sequence “EIS” at the N-
30 terminal end which is attached to TPA signal peptide, wherein the oligomerization
domain has an amino acid sequence as set forth in SEQID NO. 38. In an
18
embodiment of the present disclosure, there is provided an immunogenic peptide
comprising a polypeptide having an amino acid sequence of at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity
to a sequence as set forth in SEQ ID NO: 33, wherein the polypeptide comprises
5 substitution mutations Y60W, and P221L. In an embodiment of the present
disclosure, there is provided an immunogenic peptide comprising a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID NO:
33.
[0065] In an embodiment, the immunogenic polypeptide comprises
10 a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 9
attached to a oligomerization domain at C-terminal end of the polypeptide by a
linker “AS”, wherein the oligomerization domain is further attached to a HRV3C
protease cleavage site by a linker “GT”, wherein the polypeptide is attached to a
peptide of sequence “EIS” at the N-terminal end which is attached to TPA signal
15 peptide, wherein the oligomerization domain has an amino acid sequence as set
forth in SEQID NO. 38. In an embodiment of the present disclosure, there is
provided an immunogenic peptide comprising a polypeptide having an amino acid
sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
at least 99.5% sequence identity to a sequence as set forth in SEQ ID NO: 35,
20 wherein the polypeptide comprises substitution mutations Y37W and P198L. In
an embodiment of the present disclosure, there is provided an immunogenic
peptide comprising a polypeptide having an amino acid sequence selected from
the group consisting of SEQ ID NO: 35.
[0066] In an embodiment of the present disclosure, there is provided an
25 immunogenic peptide comprising a polypeptide having an amino acid sequence
selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 28, SEQ
ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 71, SEQ ID NO: 73,
SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 81.
30 [0067] In an embodiment, there is provided a polynucleotide encoding the
immunogenic polypeptide as described herein.
19
[0068] In another embodiment, the polynucleotide has a nucleotide sequence
selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:
6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO:
24, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID
5 NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ
ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, and SEQ ID NO:
82. The polynucleotide may be deoxy ribose nucleic acid (DNA) or ribose nucleic
acid (RNA). In an embodiment, the polynucleotide is selected from DNA, RNA,
or messenger RNA (mRNA).
10 [0069] In an embodiment, there is provided a recombinant vector containing
the polynucleotide as disclosed herein, operably linked to a promoter.
[0070] In an embodiment, there is provided a recombinant host cell
comprising the recombinant vector as described herein. The host cell is a bacterial
cell, yeast cell, insect cell, or mammalian cell. The bacterial cell is Escherichia
15 coli, and the yeast cell is selected from the group consisting of Pichia X33, Pichia
GlycoSwitch® , DSMZ 70382, GS115, KM71, KM71H, BG09, GS190, GS200,
JC220, JC254, JC227, JC300-JC308, YJN165, and CBS7435, and wherein the
insect cell is selected from the group consisting of Expi-Sf9®, Sf9, High Five® ,
Sf21, and S2, and wherein the mammalian cell is selected from the group consisting
20 of Expi293F®Expi-CHO-S ® , CHO-K1, CHO-S, HEK293F® , CHOBC™,
SLIM™ , SPOT™ , SP2/0 , Sp2/0- Ag14, CHO DG44, HEK 293S, HEK 293 Gnt1-
/- ,HEK293-EBNA1, CHOL-NSO, and NSO. Further, the host cell may be selected
from OPENPichia (NCYC 2543 hoc1tr), OPENPichia his4, NCYC 2543 type
strain, OPENPichia pep4, OPENPichia yps1, OPENPichia pep4 yps1, and
25 OPENPichia mutS strains.
[0071] Embodiments herein provide vaccine compositions. In an
embodiment, there is provided a vaccine composition comprising the
immunogenic polypeptide as described herein, and a pharmaceutically acceptable
carrier. The pharmaceutically acceptable carrier is an adjuvant selected from an
30 oil-in-water adjuvant, a polymer and water adjuvant, a water-in-oil adjuvant, an
aluminum hydroxide adjuvant, and combinations thereof.
20
[0072] In an exemplary embodiment of the present disclosure, the
pharmaceutically acceptable carrier is selected from the group consisting of
alhydrogel (aluminium hydroxide adjuvant), Alhydrogel CpG, Addavax (oil-in-
water adjuvant), SWE (squalene-in-water emulsion adjuvant), and MF59.
5 [0073] In an embodiment, the vaccine composition comprises an
immunogenic polypeptide of amino acid sequence of at least 95% sequence
identity to amino acid sequence selected from the group consisting of SEQ ID NO:
1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:
13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID
10 NO: 25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ
ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,
SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:
77, SEQ ID NO: 79, and SEQ ID NO: 81. In an embodiment, the vaccine
composition comprises an immunogenic polypeptide of amino acid sequence of
15 amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ
ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ
ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:
59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID
20 NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ
ID NO: 79, and SEQ ID NO: 81.
[0074] In an embodiment, the vaccine composition comprises an
immunogenic polypeptide of amino acid sequence of at least 95% sequence
identity to amino acid sequence selected from the group consisting of SEQ ID NO:
25 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID
NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ
ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,
SEQ ID NO: 79, and SEQ ID NO: 81. In an embodiment, the vaccine composition
comprises an immunogenic polypeptide of amino acid sequence of amino acid
30 sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 20,
SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID NO:
21
61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID
NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and
SEQ ID NO: 81.
[0075] In another embodiment, the vaccine composition is a cocktail
5 formulation comprising a combination of at least 2, at least 3, at least 4 or 5
immunogenic polypeptides of amino acid sequence having at least 95% identity to
a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9. In another embodiment, the vaccine
composition is a cocktail formulation comprising a combination of the
10 immunogenic polypeptides of amino acid sequence having at least 95% identity to
a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:
28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID
15 NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ
ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79,
and SEQ ID NO: 81. In another embodiment, the vaccine composition is a cocktail
formulation comprising a combination of the immunogenic polypeptides of amino
acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:
20 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO:
15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID
NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 59, SEQ
ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69,
SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO:
25 79, and SEQ ID NO: 81.
[0076] In yet another embodiment, the vaccine composition is a cocktail
formulation comprising a combination of at least 2, at least 3, at least 4 or 5
immunogenic polypeptides of amino acid sequence having at least 95% identity to
a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO:
30 20, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID
NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ
22
ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79,
and SEQ ID NO: 81. In yet another embodiment, the vaccine composition is a
cocktail formulation comprising a combination immunogenic polypeptides of
amino acid sequence having at least 95% identity to a sequence selected from the
5 group consisting of SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID
NO: 30, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ
ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,
SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 81. In yet
another embodiment, the vaccine composition is a cocktail formulation comprising
10 a combination immunogenic polypeptides of amino acid sequence selected from
the group consisting of SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ
ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,
SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO:
73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 81.
15 [0077] In an embodiment of the present disclosure, there is provided method
for producing the vaccine composition as disclosed herein, said method
comprising: (a) culturing the recombinant host cell as described herein, to express
the immunogenic polypeptide as described herein; (b) subjecting the immunogenic
polypeptide to purification; and (c) contacting the immunogenic polypeptide of
20 step (b) with a pharmaceutically acceptable carrier, to obtain the vaccine
composition. The vector, according to embodiments herein, comprising the
nucleotide encoding the immunogenic polypeptide may be introduced into the host
cell by transfection, to obtain the recombinant host cell which may then be cultured
to express the immunogenic polypeptide as described herein. Various transfection
25 methods are known for eg: lipofection, electroporation, etc which may be used to
achieve the recombinant host cell as described herein. The expressed
immunogenic polypeptide may then be purified by using protein purification
methods generally known in field (for eg: Ni-NTA affinity chromatography) to
obtain the purified immunogenic polypeptide. The immunogenic polypeptide may
30 then be mixed with a suitable pharmaceutically acceptable carrier to obtain the
23
vaccine composition. One or more immunogenic polypeptides may be mixed with
the pharmaceutically acceptable carrier.
[0078] In an embodiment of the present disclosure, there is provided method
for producing the vaccine composition as disclosed herein, said method
5 comprising: (a) culturing the recombinant host cell as described herein, under
suitable conditions to express the immunogenic polypeptide, wherein the
immunogenic polypeptide has an amino acid sequence of at least 95% sequence
identity to amino acid sequence selected from the group consisting of SEQ ID NO:
1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:
10 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID
NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ
ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,
SEQ ID NO: 79, and SEQ ID NO: 81; (b) subjecting the immunogenic polypeptide
to purification; and (c) contacting the immunogenic polypeptide of step (b) with a
15 pharmaceutically acceptable carrier, to obtain the vaccine composition.
[0079] In another embodiment, there is provided a method of eliciting an
immune response against diverse Sarbecovirus in a subject, the method comprising
administering the subject with an effective amount of the vaccine composition as
disclosed herein. In another embodiment, the Sarbecovirus is selected from clade
20 1a, clade 1b, clade 2, or clade 3 virus. In another embodiment, the Sarbecoviruses
is selected from the group consisting of SARS-CoV-1 (clade 1a), WIV-1 (clade
1a), SARS-CoV-2(clade 1b), RaTG13 (clade 1b), RmYN02 (clade 2), and
BtKY72 (clade 3).
[0080] In another embodiment, the Sarbecovirus is selected from the group
25 consisting of SARS-CoV-1 (clade 1a), WIV-1 (clade 1a), SARS-CoV-2(clade 1b),
RaTG13 (clade 1b), RmYN02 (clade 2), BtKY72 (clade 3), Pangolin_GD,
RaTG13 (MN996532), Pangolin_GX-P2V (EPI_ISL_410542), SARS-CoV-
1_Urbani_HP03 (AY278741), SARS-CoV-1_BJ02_HP03M (AY278487), SARS-
CoV-1_HGZ8L1-A_HP03E (AY394981), SARS-CoV-1_GD01_HP03L
30 (AY278489), SARS-CoV-1_GZ-C_HP03L (AY394979), SARS-CoV-1_Sino1-
11_HP03L (AY485277), SARS-CoV-1_Sin852_HP03L (AY559082), SARS-
24
CoV-1_SZ3_PC03 (AY304486), SARS-CoV-1_SZ13_PC03 (AY304487),
SARS-CoV-1_SZ1_PC03 (AY304489), SARS-CoV-1_GD03T0013_HP04
(AY525636), SARS-CoV-1_GZ0402_HP04 (AY613947), SARS-CoV-1_PC4-
127_PC04 (AY613951), SARS-CoV-1_PC4-137_PC04 (AY627045), SARS-
5 CoV-1_PC4-13_PC04 (AY613948), WIV16 (KT444582), WIV1 (KF367457),
Rs7327 (KY417151), LYRa11 (KF569996), Rs4231 (KY417146), RsSHC014
(KC881005), Rs4084 (KY417144), BM48-31 (NC014470), BtKY72
(KY352407), ZXC21 (MG772934), ZC45 (MG772933), JL2012 (KJ473811), Rf1
(DQ412042), HeB2013 (KJ473812), 273-2005 (DQ648856), Rf4092
10 (KY417145), YN2013 (KJ473816), RmYN02 (EPI_ISL_412977), As6526
(KY417142), Rs4237 (KY417147), Rs4081 (KY417143), Rp3 (DQ071615), 279-
2005 (DQ648857), Shaanxi2011 (JX993987), Yunnan2011 (JX993988), Rs4247
(KY417148), HKU3-13 (GQ153548), HKU3-1 (DQ022305), GX2013
(KJ473815), Longquan-140 (KF294457), HKU3-8 (GQ153543), HuB2013
15 (KJ473814), Hp-BCoV_Zhejiang_2013 (KF636752), HKU1 (KF686346), OC43
(KX344031), MERS-CoV (NC_019843), Bat-CoV-GCCDC1 (MT350598),
Rousettus-CoV_HKU9 (MG762674), Rc-o319 (LC556375), RacCS203
(MW251308), RshSTT182 (EPI_ISL_852604), PDF-2370, PRD-0038, RsYN04
(EPI_ISL_1699444), BB9904 (KR559017), Khosta-1 (MZ190137), Khosta-2
20 (MZ190138), and RhGB01 (MW719567).
[0081] In an embodiment, the vaccine composition is administered by a
mode selected from the group consisting of intranasal, subcutaneous, intravenous,
intra-arterial, intra-peritoneal, intramuscular, intradermal, oral, dermal, and
buccal.
25 [0082] In another embodiment, there is provided a kit comprising the
immunogenic polypeptide as disclosed herein; or the vaccine composition as
described herein, and an instruction leaflet.
[0083] Although the subject matter has been described with reference to
specific embodiments, this description is not meant to be construed in a limiting
30 sense. Various modifications of the disclosed embodiments, as well as alternate
embodiments of the subject matter, will become apparent to persons skilled in the
25
art upon reference to the description of the subject matter. It is therefore
contemplated that such modifications can be made without departing from the
spirit or scope of the present subject matter as defined.
5 EXAMPLES
[0084] The disclosure will now be illustrated with working examples, which
is intended to illustrate the working of disclosure and not intended to take
restrictively to imply any limitations on the scope of the present disclosure. Unless
10 defined otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood to one of ordinary skill in the art to which this
disclosure belongs. Although methods and materials similar or equivalent to those
described herein can be used in the practice of the disclosed methods and
compositions, the exemplary methods, devices and materials are described herein.
15 It is to be understood that this disclosure is not limited to particular methods, and
experimental conditions described, as such methods and conditions may vary.
[0085] Substitution mutations, in accordance with the embodiments
herein are depicted in Table 1.
20 Table 1: depicts the substitution mutations, according to the present disclosure.
SEQ ID
NO.
Virus
name
Mutations:
positions in
respect of RBD
Mutations: positions
in respect of the
immunogenic
polypeptide
SEQ ID
NO: 1
SARS
CoV-1
Y352W, P513L Y34W, P195L
SEQ ID
NO: 3
WIV-1 Y365W, P526L Y34W, P195L
26
SEQ ID
NO: 5
RaTG13 A348P, Y365W,
P527L
A17P, Y34W, P196L
SEQ ID
NO: 7
RmYN02 Y344W, P487L Y34W, P177L
SEQ ID
NO: 9
BtKY72 Y355W, P516L Y34W, P195L
SEQ ID
NO: 15
Mature
stabilized
SARS
CoV-1
Y352W, P513L Y37W, P198L
SEQ ID
NO: 20
Mature
stabilized
WIV-1
Y365W, P526L Y37W, P198L
SEQ ID
NO: 25
Mature
stabilized
RaTG13
A348P, Y365W,
P527L
A20P, Y37W, P199L
SEQ ID
NO: 30
Mature
stabilized
RmYN02
Y344W, P487L Y37W, P180L
SEQ ID
NO: 35
Mature
stabilized
BtKY72
Y355W, P516L Y37W, P198L
SEQ ID
NO: 13
TPA-
SARS
CoV-1
Y352W, P513L Y60W, P221L
SEQ ID
NO: 18
TPA-
WIV-1
Y365W, P526L Y60W, P221L
SEQ ID
NO: 23
TPA-
RaTG13
A348P, Y365W,
P527L
A43P, Y60W, P222L
27
SEQ ID
NO: 28
TPA-
RmYN02
Y344W, P487L Y60W, P203L
SEQ ID
NO: 33
TPA-
BtKY72
Y355W, P516L Y60W, P221L
SEQ ID
NO: 59
SARS
CoV-1
RBD-S2
Y352W, P513L Y34W, P195L
SEQ ID
NO: 61
WIV-1
RBD-S2
Y365W, P526L Y34W, P195L
SEQ ID
NO: 63
RaTG13
RBD-S2
A348P, Y365W,
P527L
A17P, Y34W, P196L
SEQ ID
NO: 65
RmYN02
RBD-S2
Y344W, P487L Y34W, P177L
SEQ ID
NO: 67
BtKY72
RBD-S2
Y355W, P516L Y34W, P195L
SEQ ID
NO: 69
SARS
CoV-2
XBB1.5
A348P, Y365W,
P527L
A17P, Y34W, P196L
[0086] Nucleic acid and amino acid sequences as disclosed in the present
disclosure
Table 2 depicts sequences used in the present disclosure
SEQ ID NO. Sequence
Type
Description
SEQ ID NO: 1 Protein Stabilized SARS CoV-1
SEQ ID NO: 2 Nucleotide Stabilized SARS CoV-1
SEQ ID NO: 3 Protein Stabilized WIV-1
SEQ ID NO: 4 Nucleotide Stabilized WIV-1
SEQ ID NO: 5 Protein Stabilized RaTG13
SEQ ID NO: 6 Nucleotide Stabilized RaTG13
SEQ ID NO: 7 Protein Stabilized RmYN02
28
SEQ ID NO: 8 Nucleotide Stabilized RmYN02
SEQ ID NO: 9 Protein Stabilized BtKY72
SEQ ID NO: 10 Nucleotide Stabilized BtKY72
SEQ ID NO: 11 Protein WT SARS CoV-1 (Accession Id : 5XLR)
SEQ ID NO: 12 Nucleotide WT SARS CoV-1 (Accession Id:
MK062184.1)
SEQ ID NO: 13 Protein tPA signal-linker-Stabilized SARS CoV-
1 RBD sequence-linker-cCMP IzM V2
oligomer motif-linker-HRV3C site-
10xHis tag-linker-stop codon
SEQ ID NO: 14 Nucleotide tPA signal-linker-Stabilized SARS CoV-
1 RBD sequence-linker-cCMP IzM V2
oligomer motif-linker-HRV3C site-
10xHis tag-linker-stop codon
SEQ ID NO: 15 Protein Mature stabilized SARS CoV-1 sequence
SEQ ID NO: 16 Protein WT WIV-1 (Accession Id: 7TTY)
SEQ ID NO: 17 Nucleotide WT WIV-1 (Accession Id: KC881007.1)
SEQ ID NO: 18 Protein tPA signal-linker-Stabilized WIV-1 RBD
sequence-linker-cCMP IzM V2 oligomer
motif-linker-HRV3C site-10xHis tag-
linker-stop codon
SEQ ID NO: 19 Nucleotide tPA signal-linker-Stabilized WIV-1 RBD
sequence-linker-cCMP IzM V2 oligomer
motif-linker-HRV3C site-10xHis tag-
linker-stop codon
SEQ ID NO: 20 Protein Mature stabilized WIV-1 sequence
SEQ ID NO: 21 Protein WT RaTG13 (Accession Id: 7DRV)
SEQ ID NO: 22 Nucleotide WT RaTG13 (Accession Id:
MN996532.2)
29
SEQ ID NO: 23 Protein tPA signal-linker-Stabilized RaTG13
RBD sequence-linker-cCMP IzM V2
oligomer motif-linker-HRV3C site-
10xHis tag-linker-stop codon
SEQ ID NO: 24 Nucleotide tPA signal-linker-Stabilized RaTG13
RBD sequence-linker-cCMP IzM V2
oligomer motif-linker-HRV3C site-
10xHis tag-linker-stop codon
SEQ ID NO: 25 Protein Mature stabilized RaTG13 sequence
SEQ ID NO: 26 Protein WT RmYN02
SEQ ID NO: 27 Nucleotide WT RmYN02 (Accession Id:
EPI_ISL_412977)
SEQ ID NO: 28 Protein tPA signal-linker-Stabilized RmYN02
RBD sequence-linker-cCMP IzM V2
oligomer motif-linker-HRV3C site-
10xHis tag-linker-stop codon
SEQ ID NO: 29 Nucleotide tPA signal-linker-Stabilized RmYN02
RBD sequence-linker-cCMP IzM V2
oligomer motif-linker-HRV3C site-
10xHis tag-linker-stop codon
SEQ ID NO: 30 Protein Mature stabilized RmYN02 sequence
SEQ ID NO: 31 Protein WT BtKY72 (Accession Id: 8K4U)
SEQ ID NO: 32 Nucleotide WT BtKY72 (Accession Id:
KY352407.1)
SEQ ID NO: 33 Protein tPA signal-linker-Stabilized BtKY72
RBD sequence-linker-cCMP IzM V2
oligomer motif-linker-HRV3C site-
10xHis tag-linker-stop codon
30
SEQ ID NO: 34 Nucleotide tPA signal-linker-Stabilized BtKY72
RBD sequence-linker-cCMP IzM V2
oligomer motif-linker-HRV3C site-
10xHis tag-linker-stop codon
SEQ ID NO: 35 Protein Mature stabilized BtKY72 sequence
SEQ ID NO: 36 Protein tPA signal
SEQ ID NO: 37 Nucleotide tPA signal (Nucleotide sequence for SEQ
ID 31)
SEQ ID NO: 38 Protein cCMP IzM V2 oligomer motif
SEQ ID NO: 39 Nucleotide cCMP IzM V2 oligomer motif
(Nucleotide sequence for SEQ ID 33)
SEQ ID NO: 40 Protein HRV3C site
SEQ ID NO: 41 Nucleotide HRV3C site (Nucleotide sequence for
SEQ ID 35)
SEQ ID NO: 42 Protein His tag
SEQ ID NO: 43 Nucleotide His tag (Nucleotide sequence for SEQ ID
37)
SEQ ID NO: 44 Protein linker 1
SEQ ID NO: 45 Nucleotide linker 1 (Nucleotide sequence for SEQ
ID 33)
SEQ ID NO: 46 Protein linker 2
SEQ ID NO: 47 Nucleotide linker 2 (Nucleotide sequence for SEQ
ID 35)
SEQ ID NO: 48 Protein linker 3
SEQ ID NO: 49 Nucleotide linker 3 (Nucleotide sequence for SEQ
ID 37)
SEQ ID NO: 50 Protein Stop Codon
SEQ ID NO: 51 Nucleotide Stop codon (Nucleotide sequence for
SEQ ID 50)
31
SEQ ID NO: 52 Protein linker 4
SEQ ID NO: 53 Nucleotide linker 4
SEQ ID NO: 54 Protein Stabilized SARS CoV-1 RBD sequence-
linker-cCMP IzM V2 oligomer motif-
linker-HRV3C site-10xHis tag-linker-
stop codon (without TPA)
SEQ ID NO: 55 Protein tPA signal-linker-Stabilized WIV-1 RBD
sequence-linker-cCMP IzM V2 oligomer
motif-linker-HRV3C site-10xHis tag-
linker-stop codon (without TPA)
SEQ ID NO: 56 Protein Stabilized RaTG13 RBD sequence-
linker-cCMP IzM V2 oligomer motif-
linker-HRV3C site-10xHis tag-linker-
stop codon
SEQ ID NO: 57 Protein Stabilized RmYN02 RBD sequence-
linker-cCMP IzM V2 oligomer motif-
linker-HRV3C site-10xHis tag-linker-
stop codon
SEQ ID NO: 58 Protein Stabilized BtKY72 RBD sequence-
linker-cCMP IzM V2 oligomer motif-
linker-HRV3C site-10xHis tag-linker-
stop codon
SEQ ID NO: 59 Protein Stabilized SARS CoV-1 RBD-S2
SEQ ID NO: 60 nucleotide Stabilized SARS CoV-1 RBD-S2
SEQ ID NO: 61 Protein Stabilized WIV-1 RBD-S2
SEQ ID NO: 62 nucleotide Stabilized WIV-1 RBD-S2
SEQ ID NO: 63 Protein Stabilized RaTG13 RBD-S2
SEQ ID NO: 64 nucleotide Stabilized RaTG13 RBD-S2
SEQ ID NO: 65 Protein Stabilized RmYN02 RBD-S2
SEQ ID NO: 66 nucleotide Stabilized RmYN02 RBD-S2
SEQ ID NO: 67 Protein Stabilized BtKY72 RBD-S2
32
SEQ ID NO: 68 nucleotide Stabilized BtKY72 RBD-S2
SEQ ID NO: 69 Protein Stabilized SARS CoV-2 XBB1.5 RBD-
S2
SEQ ID NO: 70 nucleotide Stabilized SARS CoV-2 XBB1.5 RBD-
S2
SEQ ID NO: 71 Protein tPA signal-linker- Stabilized SARS CoV-
1 RBD-linker-S2 sequence-linker-
HRV3C site-His tag-stop codon
SEQ ID NO: 72 nucleotide tPA signal-linker-Stabilized SARS CoV-
1 RBD-linker-S2 sequence-linker-
HRV3C site-His tag-stop codon
SEQ ID NO: 73 Protein tPA signal-linker- Stabilized WIV-1
RBD-linker-S2 sequence-linker-HRV3C
site-His tag-stop codon
SEQ ID NO: 74 nucleotide tPA signal-linker- Stabilized WIV-1
RBD-linker-S2 sequence-linker-HRV3C
site-His tag-stop codon
SEQ ID NO: 75 Protein tPA signal-linker-Stabilized RaTG13
RBD-S2-linker-S2 sequence-linker-
HRV3C site-His tag-stop codon
SEQ ID NO: 76 nucleotide tPA signal-linker- Stabilized RaTG13
RBD-S2-linker-S2 sequence-linker-
HRV3C site-His tag-stop codon
SEQ ID NO: 77 Protein tPA signal-linker- Stabilized RmYN02
RBD-linker-S2 sequence-linker-HRV3C
site-His tag-stop codon
SEQ ID NO: 78 nucleotide tPA signal-linker-Stabilized RmYN02
RBD-linker-S2 sequence-linker-HRV3C
site-His tag-stop codon
SEQ ID NO: 79 Protein tPA signal-linker- Stabilized BtKY72
RBD-linker-S2 sequence-linker-HRV3C
site-His tag-stop codon
SEQ ID NO: 80 nucleotide tPA signal-linker-Stabilized BtKY72
RBD-linker-S2 sequence-linker-HRV3C
site-His tag-stop codon
SEQ ID NO: 81 Protein tPA signal-linker- Stabilized SARS CoV-
2 XBB1.5 RBD-linker-S2 sequence-
linker-HRV3C site-His tag-stop codon
SEQ ID NO: 82 nucleotide tPA signal-linker- Stabilized SARS CoV-
2 XBB1.5 RBD-linker-S2 sequence-
linker-HRV3C site-His tag-stop codon
SEQ ID NO: 83 Protein
S2 subunit of SARS-CoV-2
33
SEQ ID NO: 84 nucleotide
S2 subunit of SARS-CoV-2
SEQ ID NO: 85 Protein SARS CoV-2 XBB1.5 RBD
SEQ ID NO: 86 nucleotide SARS CoV-2 XBB1.5 RBD
Example 1: Stabilization of diverse Sarbecoviruses RBDs
[0087] The percentage amino acid identity of the specific RBDs was
compared with SARS-CoV2 RBD and with other members of the respective
5 clades. Considering the conservation of RBD sequence within the clade, SARS-
CoV-1 and WIV-1 (clade 1a); SARS-CoV-2 B.1, SARS-CoV-2 XBB1.5 and
RaTG13 (clade 1b); RmYN02 (clade 2) and BtKY72 (clade 3) were selected as
the representatives for their respective clades. The sequence and structural
conservation of the target residues was analysed. While residues 365 and 527 were
10 fully conserved in all the selected Sarbecoviruses, it was interesting to note that
one of the SARS CoV-2 stabilizing mutations A348P was naturally present in
clade 1a, 2 and 3 viruses. A348P, Y365W and P527L mutations were introduced
in the selected Sarbecoviruses in order to study the transferability of these
mutations in diverse sequence backgrounds.
15
Methods
[0088] The polypeptide fragments were prepared using the immunogenic
polypeptide sequences depicted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,
SEQ ID NO: 7, and SEQ ID NO: 9, and the oligomerization domain (SEQ IN NO:
20 38). The polypeptide fragments were expressed and tested for stability and
immunogenicity. The polypeptide fragments had the sequences as set forth in SEQ
ID NO: 13 without TPA signal, SEQ ID NO: 18 without TPA signal, SEQ ID NO:
23 without TPA signal, SEQ ID NO: 28 without TPA signal, and SEQ ID NO: 33
without TPA signal. The sequences as set forth in SEQ ID NO: 13 without TPA
25 signal, SEQ ID NO: 18 without TPA signal, SEQ ID NO: 23 without TPA signal,
SEQ ID NO: 28 without TPA signal, and SEQ ID NO: 33 without TPA signal are
34
as depicted in SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57,
and SEQ ID NO: 58, respectively.
[0089] Each of the polypeptides having sequences depicted in SEQ ID NO:
1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, were
5 independently fused with a fragment of S2 subunit (SEQ ID NO: 83) at the C-
terminal end using a linker “AS” to obtain stabilized SARS CoV-1 RBD-S2 (SEQ
ID NO: 59), Stabilized WIV-1 RBD-S2 (SEQ ID NO: 61); Stabilized RaTG13
RBD-S2 (SEQ ID NO: 63); Stabilized RmYN02 RBD-S2 (SEQ ID NO: 65); and
Stabilized BtKY72 RBD-S2 (SEQ ID NO: 67), respectively. The polypeptide
10 having a sequence depicted in SEQ ID NO: 85 was fused with a fragment of S2
subunit (SEQ ID NO: 83) at the C-terminal end using a linker “AS” to obtain
stabilized SARS CoV-2 XBB1.5 RBD-S2 (SEQ ID NO: 69).
[0090] The cocktail formulations were prepared using the polypeptide
fragments as depicted in SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18
15 without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 without
TPA signal, and SEQ ID NO: 33 without TPA signal, combined with SWE
adjuvant, at 1:1 v/v polypeptide fragments: adjuvant ratio, and neutralization titers.
The 1:1 v/v polypeptide fragments: adjuvant ratio. The cocktail formulations may
also be prepared using the polypeptide fragments selected from SEQ ID NO: 1,
20 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 15,
SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO:
71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ
ID NO: 81.
25 Protein Expression and Purification
[0091] The genes encoding Sarbecoviruses RBD proteins and monoclonal
antibodies (mAbs) were synthesized at GenScript (USA) and TWIST Biosciences
(USA). All the RBD derivatives [ i.e. SEQ ID NO: 13 without TPA signal, SEQ
ID NO: 18 without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO:
30 28 without TPA signal, and SEQ ID NO: 33 without TPA signal] were purified
from transiently transfected Expi293F cells following the manufacturer’s
35
guidelines (Gibco, Thermo Fisher, Waltham, MA, USA). Briefly, Expi293F cells
were diluted to a density of 3 × 106 cells/mL. For transfection, the desired plasmid
(pCDNA5FRTTO mammalian expression vector) was complexed with
ExpiFectamine293 according to the manufacturer’s protocol and transiently
5 transfected into Expi293F cells. Enhancer 1 and Enhancer 2 were added 16 hours
post transfection. The culture supernatant was collected after 5 days, and protein
was purified through Ni-NTA affinity chromatography using Ni Sepharose 6 fast-
flow resin (GE Healthcare, Chicago, IL, USA) for RBD derivatives. The
supernatant was added to a pre-equilibrated Ni-NTA column. Following a 2-
10 column wash with 1× PBS (pH 7.4) supplemented with 20 mM imidazole, the
protein was eluted in 1X PBS with 300mM imidazole (pH 7.4). Pooled eluted
fractions were dialyzed thrice against 1× PBS (pH 7.4). For expression and
purification of mAbs, Expi293F cells were co-transfected by Heavy and Light
chain plasmids in 1:1 ratio by using polyethylenimine, and supernatants were
15 harvested after 5 days post-transfection. The antibodies were purified from
supernatants by using Protein A/G beads, dialyzed and stored in phosphate
buffered saline (PBS) for further use. Purified protein samples were analyzed on
12% SDS-PAGE gel and quantified using NanoDrop spectrophotometer (Figure
1, Table 3). Table 3 depicts summary of purified yields of Sarbecoviruses RBD
20 derivatives. Similarly, the immunogenic polypeptide selected from SEQ ID NO:
1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:
15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID
NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and
SEQ ID NO: 81 may also be expressed and purified.
25 Table 3:
RBD Clade Yield(mg/L)
WT CoV -1 Trimer 1a 72
Stabilized CoV-1 Trimer 1a 84
WT WIV -1 Trimer 1a 54
36
Stabilized WIV-1 Trimer 1a 144
WT RaTG13 Trimer -RBD 1b 5.6
Stabilized RaTG13 Trimer- RBD 1b 85
WT RmYN02 Trimer 2 8.09
Stabilized RmYN02 Trimer 2 189
WT BtKY72 Trimer -RBD 3 12.4
Stabilized BtKY72 Trimer -RBD 3 48.6
Thermal Unfolding experiments
[0092] Thermal melting studies of the RBD derivatives [ i.e. SEQ ID NO:
13 without TPA signal, SEQ ID NO: 18 without TPA signal, SEQ ID NO: 23
without TPA signal, SEQ ID NO: 28 without TPA signal, and SEQ ID NO: 33
5 without TPA signal] were performed using nanoDSF (Prometheus NT.48), as
described in Chattopadhyay, G. et.al, 2019, at a temperature range of 20 °C to 95
°C. Experimental measurements were conducted at 100% LED intensity with an
initial discovery scan, and the scan counts (350 nm) ranged between 2000 and
3000. For lyophilized protein, reconstitution was done using 1X PBS buffer before
10 the DSF experiment. The results have been summarized in Figure 2.
SPR binding studies
[0093] Kinetics titrations were performed using a CM5 sensor chip (Cytiva)
at 25°C. The activation of the carboxymethylated-dextran gold surface was
achieved by injecting EDC/s-NHS (2/4 mM) solution in autoclaved water pH 7.0
15 injected at 5 µL/min for 400 seconds. Following the activation step, a 10 µg/mL
solution of Protein-G in sodium acetate (NaOAc) pH 5.0 was injected over the
activated surface at 5 µL/min. After covalent modification of the sensor surface, a
quenching solution of ethanolamine pH 8.5 (Cytiva) was injected over the surface
for 600 seconds to cap any residual active NHS esters. PBS (1X) pH 7.4 was used
20 for the running buffer during titration. During the kinetics assay, one flow cell
channel with only Protein G served as a reference channel to monitor and subtract
37
binding responses due to non-specific interactions. 200-300 RU of monoclonal
antibodies at 1 µg/mL were captured onto the chip surface for each cycle at 5
µL/min for 60 seconds, followed by injection of RBD derivatives for 200 seconds.
Then a dissociation step was performed using an injection of running buffer for
5 200 seconds. Following the dissociation step, regeneration of the Protein G surface
was performed using 1 injection of 0.1M glycine-HCl, pH 2.0 at 30 µL/min for 40
seconds. The flow rate for association and dissociation was 30 µL/min. The
kinetics traces were reference subtracted using the responses of the reference
channel in each cycle and blank subtracted using a zero-concentration cycle. Then
10 the kinetics constants ka, kd and KD values were determined using Biacore T200
evaluation software.
Mice Immunizations
[0094] Female C57BL/6 mice (6–8 weeks old, n = 5/group) were immunized
intramuscularly with RBD derivatives [ i.e. SEQ ID NO: 13 without TPA signal,
15 SEQ ID NO: 18 without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ
ID NO: 28 without TPA signal, and SEQ ID NO: 33 without TPA signal],
(5µg/animal in 100 µL of 1× PBS, pH 7.4) adjuvanted with SWE (1:1 v/v antigen:
adjuvant ratio) (Sepivac SWE Batch No. 200915012131, Cat. No. 80748J,
SEPPIC SA) on days 0 (prime), and 21 (boost). RBD cocktail group was
20 immunized with 5µg/antigen having RBD derivatives from different clades as
mentioned. Sera were isolated from blood drawn on days prior to prime (day -1),
post-prime (day 14), and post-boost (day 35) through retro-orbital puncture.
[0095] For the assessment of RBD cocktail efficacy in pre-immunized mice,
female BALB/c mice (6–8 weeks old, n = 5/group) were first immunized with
25 stabilized monomeric SARS CoV-2 RBD (1µg/animal in 100 µL of 1× PBS, pH
7.4) adjuvanted with SWE (1:1 v/v antigen: adjuvant ratio) (Sepivac SWE Batch
No. 200915012131, Cat. No. 80748J, SEPPIC SA) on days 0 (prime), and then
boosted with RBD cocktail formulation (having SEQ ID NO: 13 without TPA
signal, SEQ ID NO: 18 without TPA signal, SEQ ID NO: 23 without TPA signal,
30 SEQ ID NO: 28 without TPA signal, and SEQ ID NO: 33 without TPA signal)
(5µg/antigen, total 30µg) on day 30 and day 51. Sera obtained before prime and
38
post boost immunizations was used to conduct ELISA and pseudoviral
neutralization assays. These studies were performed at Central Animal Facility,
Indian Institute of Science. The Institutional Animal Ethics committee approved
all animal studies (IAEC no. CAF/ETHICS/002/2023). Similarly, studies may also
5 be performed using cocktail formulations having SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 20,
SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 71, SEQ ID NO:
73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and/or SEQ ID NO: 81.
Enzyme-linked immunosorbent assay (ELISA)
10 [0096] ELISA was used to quantify the Sarbecoviruses RBD-specific IgG
titers in serum as described in Ahmed, S. et al. 2021. Briefly, ELISA plates were
coated with 4 µg/mL His tagged RBD derivatives at 25 °C, 1h. The coated plates
were blocked using blocking buffer (PBS containing 3% skimmed milk) for 1 hour
at 25 °C. The sera (maximum concentration, 1:80-1:1280) from mice, were serially
15 diluted 1:4 in PBST (PBS containing 3% skimmed milk and 0.05% Tween-20) for
8 dilutions, followed by incubation with the coated ELISA plates for 60 min at
25 °C. After washing with PBST three times, HRP-conjugated secondary antibody
was added to the plates and incubated for 60 min at 25 °C. After incubation with
the secondary antibody, the plates were washed with PBST three times, followed
20 by adding 3,3’,5,5’-tetramethylbenzidine (TMB) to visualize the reaction. Finally,
6N HCl was used to stop the reaction. The chromogenic signal was measured at
405 nm using an ELISA plate reader (Maxome Labsciences Cat # P3-5x10NO).
The serum dilution with a signal observed two-fold above the negative control
(empty blocked wells) was considered the endpoint titer for ELISA.
25
Sarbecoviruses Pseudovirus Preparation and Neutralization Assay
[0097] HIV-1-based pseudotyped viruses were employed in pseudoviral
neutralization assays, following a method previously described [Malladi SK, et al.
Immunogenicity and Protective Efficacy of a Highly Thermotolerant, Trimeric
30 SARS-CoV-2 Receptor Binding Domain Derivative. ACS Infect Dis. 2021 Aug
13;7(8):2546-2564. doi: 10.1021/acsinfecdis.1c00276. Epub 2021 Jul 14. PMID:
39
34260218; PMCID: PMC8996237.]. Briefly, adherent HEK293T cells were
transiently transfected with plasmid DNA pHIV-1 NL4-3?env-Luc and
Sarbecoviruses spike plasmids, using the ProFection mammalian transfection kit
(Cat# E1200, Promega Inc., Singapore) for pseudovirus production. The genes
5 encoding Spike proteins from SARS CoV-2 VOCs were synthesized at GenScript
(USA) while spike plasmids encoding Sarbecoviruses spikes were kindly gifted by
Dr. Pamela J. Bjorkman (Caltech, USA). The culture supernatant was harvested
48 h post-transfection, filtered through a 0.22 µm filter, and stored at -80 °C.
Adherent HEK293 cells expressing hACE-2 and TMPRSS2 receptors (BEI
10 resources, NIH, Catalog No. NR-55293) were cultured in a growth medium
consisting of DMEM with 5% Fetal Bovine Serum (Thermo Fisher) and
penicillin–streptomycin (100 U/mL). Mice serum samples were heat-inactivated
and then serially diluted in the growth medium, starting from 1:20 dilutions. In the
next step, the pseudotyped virus was incubated with the serially diluted sera in a
15 total volume of 100 µL for 1 h at 37 °C. The adherent cells were then trypsinized,
and 1 × 104 cells/well were added to achieve a final volume of 200 µL/well. The
plates were further incubated for 48 h in a humidified CO2 incubator at 37 °C.
After incubation, neutralization was measured as an indicator of luciferase activity
in the cells (relative luminescence units) using Nano-Glo luciferase substrate (Cat
20 # N1110, Promega). Luminescence was measured using a Cytation-5 multimode
reader (Bio-Tech Inc., Oklahoma City, OK, USA). The luciferase activity,
measured as relative luminescence units (RLU), in the absence of sera was
considered as 100% infection. The serum dilution resulting in half-maximal
neutralization of the pseudovirus (ID50) relative to the no-serum control was
25 determined from neutralization curves.
Biolayer Interferometry experiment
[0098] The long-term stability of lyophilized RBD cocktail the RBD cocktail
(having SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18 without TPA signal,
SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 without TPA signal, and
30 SEQ ID NO: 33 without TPA signal) was assessed through DSF and BLI binding
studies. BLI measurements were made using ForteBio biosensors (Fortebio -
40
Sartorius). All data collection were performed at 25°C using settings of Standard
Kinetics Acquisition rate at a sample plate shake speed of 1000 rpm. mAbs were
loaded onto Protein G sensors, subsequently they were dipped into 1x PBS buffer
for 60 seconds to obtain baseline and then dipped into wells containing RBD
5 derivatives at different concentrations in 1X PBS to monitor antibody association.
The dissociation step was monitored for 200 seconds by dipping Ab-bound sensors
into buffer. Antigen specific binding responses were obtained by subtracting
responses of blank sensors tested in parallel with 1X kinetics buffer. The specific
binding responses were fitted using ForteBio Data Analysis 12.0 software tool.
10 Statistical Analysis
[0099] The p values for ELISA binding and neutralization titres were
analyzed with a two-tailed Mann–Whitney test using the GraphPad Prism software
9.0.0 (* indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001). VOC
pseudoviral neutralization titre data were analyzed with non-parametric Kruskal–
15 Wallis with Dunn’s multiple-comparison tests using the GraphPad Prism software
9.0.0 (* indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001).
Example 2: Thermal stabilization and yield enhancement by transfer of
stabilizing mutations to diverse Sarbecoviruses RBDs without affecting
20 conformational integrity
[00100] SARS-CoV-1 shares 74% amino acid identity with SARS CoV-2
RBD and is closely related to another clade 1a member, WIV-1 (96% identical)
(Table 4). Table 4 depicts percentage amino acid identity of the polypeptide of
receptor binding domain (RBD) of Sarbecoviruses from different clades with that
25 of SARS CoV-2.
41
Table 4:
Virus Clade Host Species
% aa identity
with SARS-
CoV2(RBD)
SARS CoV-2 ½ or 1b Homo Sapiens 100
RaTG13 ½ or 1b Rhinolophus affinis 90
SARS Cov-1 1 or 1a Homo Sapiens 74
WIV-1 1 or 1a Rhinolophus sinicus 77
RsSHC014 1 or 1a Rhinolophus sinicus 77
RfI 2 Rhinolophus ferrumequium 70
RmYN02 2 Rhinolophus malayanus 68
BtKY72 3 Rhinolophus sp. 74
[00101] WIV-1 is found in horseshoe bats (Rhinolophus sinicus) in China; its
usage of ACE2 as the receptor highlights the potential for zoonotic transmission
5 to humans. RaTG13 exhibited high genetic similarity with SARS-CoV-2, it was
also identified in horseshoe bats (Rhinolophus affinis) in Yunnan Province, China.
Similar to SARS-CoV-2, RaTG13 is capable of utilizing the angiotensin-
converting enzyme 2 (ACE2) receptor for cell entry. RmYN02 is a recently
discovered clade-2 virus which contains two deletions in the RBD that prevents it
10 from using ACE2. Another SARS-related CoV that was included by the inventors,
is BtKY72 (clade-3) which was identified in Kenyan Rhinolophus bats and shows
human ACE2-dependent entry. As oligomerization of antigen results in enhanced
immunogenicity, the inventors expressed the wild type (WT) and stabilized (St)
derivatives of these Sarbecoviruses RBDs in Expi-293F cells (Recombinant host
15 cell) as oligomers through genetic fusion of a disulfide linked oligomerization
motif at the respective C termini. For comparison, monomeric WT and Stabilized
derivatives of SARS-CoV-1 RBD were also expressed and purified.
42
[00102] Purification of these mammalian cell expressed proteins was
performed via Ni-affinity chromatography. A significant enhancement in the yield
(~4-23-fold increase) of the RBD derivatives (Immunogenic polypeptides) was
observed upon introduction of the stabilizing mutations. The effect of these
5 mutations on protein thermal stability was probed by assessing the apparent
thermal melting temperature (Tm) of the WT and stabilized derivatives through
nano-DSF. A notable increase (~7°C) in the Tm of the stabilized derivatives
relative to the wild type RBDs was observed. To verify proper folding of these
RBD derivatives, the binding was examined with a selected panel of broadly
10 neutralizing antibodies (bNAbs) that bind to different epitopes on RBD by using
SPR. Due to the lack of conservation in class 1 and class 2 RBD epitopes, it is
improbable for antibodies within these categories to exhibit significant cross-
reactivity towards Sarbecoviruses RBDs. However, class 3 and class 4 RBD-
binding antibodies present more promising opportunities for neutralization across
15 various clades, thereby offering potential protection against emerging
Sarbecoviruses. The epitope of bnAb10-40 is similar to the previously defined
‘class 4’ antibody epitope, it makes polar contacts and hydrophobic interactions
with RBD residues (377-385). ADG-20 binds to a class ¼ epitope that overlaps
with the ACE2 binding site. This bnAb binds to RBDs from clade 1a, 1b and 3;
20 however, there was no binding observed for the clade 2 RmYN02 RBD which also
does not bind ACE2. S2X259 targets the conserved antigenic site II within the
RBD. It interacts with amino acid residues 369-386, 404-411 and 499-50836. All
the RBD derivatives (i.e. SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18
without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 without
25 TPA signal, and SEQ ID NO: 33 without TPA signal) except the stabilized
RmYN02 oligomer bound to this bnAb, this could be due to loss of conservation
of residue D405 and G504 in RmYN02 RBD (i.e. SEQ ID NO: 28 without TPA
signal) which are crucial for interaction with S2X259. All the stabilized RBD
derivatives also bound to class 4 bnAb CR3022.
30 [00103] To probe the thermal tolerance conferred by these mutations, the WT
and St RBD derivatives were incubated at different temperatures ranging from 4°C
43
to 70°C for 2 hours. The proteins were cooled to room temperature and subjected
to DSF. The thermal melt curves clearly demonstrated the extended thermal
tolerance of the stabilized derivatives relative to the corresponding WT RBDs
(Figure 3). Collectively, these results illustrate that mutations are stabilizing in all
5 the Sarbecoviruses included in this study; they not only result in enhanced thermal
stability and thermal tolerance but also increase the yield of the purified proteins
significantly. Binding experiments confirm that the stabilized RBD derivatives are
properly folded.
10 Example 3: Eliciting neutralizing antibody responses in mice against diverse
Sarbecoviruses using stabilized RBD derivatives and a corresponding
cocktail formulation.
[00104] To assess the immunogenicity of the stabilized Sarbecoviruses RBD
derivatives, female C57BL/6 mice (n=5/group) were immunized intramuscularly
15 with individual RBD derivatives (5mg of stabilized RBDs from SARS-CoV-1 and
WIV-1 (clade 1a); SARS CoV-2 and RaTG13 (clade 1b); RmYN02 (clade 2) and
BtKY72 (clade 3), individually, (i.e. SEQ ID NO: 13 without TPA signal, SEQ ID
NO: 18 without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28
without TPA signal, and SEQ ID NO: 33 without TPA signal) adjuvanted with
20 SWE (pharmaceutically acceptable carrier) in a prime-boost regimen.
[00105] The immunogenicity of a Sarbecoviruses RBD cocktail (Vaccine
composition) having 5mg of each of the stabilized RBDs from SARS-CoV-1 and
WIV-1 (clade 1a); SARS CoV-2 and RaTG13 (clade 1b); RmYN02 (clade 2) and
BtKY72 (clade 3) (i.e. SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18
25 without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 without
TPA signal, and SEQ ID NO: 33 without TPA signal) formulated with SWE as the
adjuvant was evaluated.
[00106] Post boost immunization, ELISA against respective RBDs was done
to determine the serum IgG titres (Figure 4). High antibody titres were observed
30 upon immunization with individual RBD derivatives, as well as with the RBD
cocktail. In the context of SARS CoV-1 immunogens, enhancement in the
44
immunogenicity of the monomeric RBD derivative upon stabilization; however,
in trimeric format this difference was insignificant. The stabilized monomeric
SARS CoV-1 RBD derivative elicited higher neutralizing antibody (NAb) titres
against homologous (SARS-CoV-1, WIV-1) and heterologous (LYRa3, SHC014)
5 clade1a pseudoviruses related to WT RBD. In contrast, the WT and mutant
trimeric derivatives demonstrated comparable neutralizing titres. The RBD
cocktail elicited good neutralization against homologous SARS CoV-1, WIV-1
(clade 1a); SARS CoV-2 B.1 (clade 1b) and BtKY72 (clade 3) and heterologous
pseudoviruses (clade1a: LYRa3, SHC014; clade3: Khosta-2) (Figure 5).
10 Importantly with a single RBD immunization, clade matched pseudoviruses were
effectively neutralized but no cross neutralization of mismatched pseudoviruses
was observed. This emphasizes the need for incorporating RBDs from all the
clades to achieve a broadly neutralizing pan-sarbecoviruses response.
Unfortunately, the neutralization titres against clade 2 viruses could not be
15 assessed as they do not bind ACE2 and thus a suitable neutralization assay was not
available. Overall, the RBD cocktail formulation elicited good neutralizing titres
against all the RBDs incorporated in the formulation confirming the
immunogenicity of the RBD derivatives in individual as well as cocktail format.
It also elicited neutralization against all the heterologous pseudoviruses tested,
20 indicating elicitation of a broad neutralization response in the immunized animals.
Example 4: Induction of an immunogenic response in pre-immunized mice
using stabilized RBD cocktail (vaccine composition).
[00107] The inventors aimed to deduce whether the RBD cocktail formulation
25 (vaccine composition) could induce comparable responses in pre-immunized
animals and naïve mice. Female BALB/c mice (n=5/group) were immunized with
SARS CoV-2 B.1 RBD (5mg) followed by two boost immunizations with the
stabilized RBD cocktail formulation (30mg). Serum ELISA demonstrated the
presence of IgG titres against Sarbecoviruses RBDs after boost 1, boost 2 did not
30 result in further increase in antibody titres. However, with sera from the first boost,
only weak and sporadic neutralization of homologous as well as heterologous
45
pseudovirus was observed. Consistent, broad neutralization was observed only
after the second boost of the RBD cocktail. Consistent with the previous results in
naïve mice; the pan-Sarbecoviruses formulation was able to elicit broad
neutralizing responses against Sarbecoviruses after two immunizations in pre-
5 immunized mice, confirming its relevance in the current scenario.
Example 5: Analysing antigenicity retention of the lyophilized RBD cocktail
(vaccine composition).
[00108] To assess the long-term stability of the RBD cocktail formulation
10 (having SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18 without TPA signal,
SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 without TPA signal, and
SEQ ID NO: 33 without TPA signal), it was subjected to lyophilization and
incubated at either 4 or 37°C for a duration of one month. The stability of the
formulations were confirmed through DSF and BLI experiments. As depicted in
15 Figure 7, the lyophilized formulation stored at both 4 and 37°C exhibited similar
melting profiles up until day 15. However, a slight reduction in stability was
observed on day 30. Additionally, the conformational integrity of the RBD
cocktail was assessed by examining binding with the previously described bNAbs
(CR3022, 10-40, ADG-20, S2X259). Consistent with the DSF analysis, the
20 lyophilized formulation demonstrated appropriate binding curves with all the
antibodies until day 15, with a slight decrease in binding signal observed on day
30. These findings illustrate the long-term stability of the cocktail formulation
when in a lyophilized state, an important factor to consider when evaluating
potential vaccine candidate formulation.
25 Results and Conclusion
[00109] The results demonstrate that the stabilizing mutations are effective
for clade members: SARS CoV-1, WIV-1 (clade 1a); SARS CoV-2 B.1 (clade 1b)
and BtKY72 (clade 3) and heterologous pseudoviruses (clade1a: LYRa3, SHC014;
clade3: Khosta-2). The inventors have expressed the stabilized trimeric RBD
30 derivatives (as depicted in SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18
without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 without
46
TPA signal, and SEQ ID NO: 33 without TPA signal), in mammalian cells
(recombinant host cell) and reported good purification yields. The stabilized RBD
derivatives (as depicted in SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18
without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 without
5 TPA signal, and SEQ ID NO: 33 without TPA signal) showed significant
enhancement in the apparent melting temperature and short-term thermal tolerance
than WT RBDs. All the stabilized RBDs resulted in high IgG titres in mice in
individual and cocktail format and thus proved to be highly immunogenic.
Stabilized trimeric RBD derivatives as depicted in SEQ ID NO: 15, SEQ ID NO:
10 20, SEQ ID NO: 25, SEQ ID NO: 30, and SEQ ID NO: 35 would also achieve
similar results. The RBD cocktail formulation (having immunogenic polypeptides
as depicted in SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18 without TPA
signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 without TPA signal,
and SEQ ID NO: 33 without TPA signal) performed comparable to the individual
15 clade-matched immunogens and better in case of heterologous pseudoviral
neutralization. Overall, the absence of cross-reactivity within clades appears to be
evident based on the findings from pseudoviral neutralization assays following
vaccination with individual RBD derivatives, underscoring the significance of
exposure to a variety of antigens in order to develop a broad response, such as the
20 cocktail formulation that the present inventors have employed. It was observed
that in addition to naive mice, the RBD cocktail formulation (vaccine composition)
of the present disclosure demonstrates immunogenicity in SARS CoV-2 RBD pre-
immunized mice as well, mirroring the present situation after COVID-19
vaccination drive. Further, the presently disclosed RBD cocktail (vaccine
25 composition) exhibited remarkable thermal stability following lyophilization for a
period of upto two weeks when stored at 37°C. In summary, the results show that
stabilization and production of diverse Sarbecoviruses RBD derivatives to be used
as vaccine antigens is a technically feasible and scalable process. The significant
improvement in yield and thermostability of immunogens by transfer of identified
30 stabilizing mutations as described, would enhance the manufacturing efficiency
47
and distribution of RBD-based vaccines for Sarbecoviruses on a global scale,
addressing the pressing need for quick, widespread and equitable vaccination.
Example 6: Protective efficacy of Stabilized RBD trimer and RBD-S2
5 monomer cocktail formulation (vaccine composition).
[00110] K18-hACE2 mice were immunized with SWE adjuvanted Stabilized
RBD oligomer cocktail (1:1 v/v), and SWE adjuvanted RBD-S2 monomer
cocktail.
[00111] The stabilized RBD oligomer cocktail included WIV-1 (SEQ ID
10 NO. 55), RaTG13 (SEQ ID NO. 56), RmYN02 (SEQ ID NO. 57), BtKY72 (SEQ
ID NO. 58); 2µg/antigen; total=8µg/mouse. Stabilized RBD-S2 monomer cocktail
included WIV-1 (SEQ ID NO. 61), RaTG13 (SEQ ID NO. 63), RmYN02 (SEQ
ID NO. 65), BtKY72 (SEQ ID NO. 67); 2µg/antigen; total=8µg/mouse).
[00112] [00102] To compare the protective efficacy of the stabilized
15 RBD oligomer cocktail and Stabilized RBD-S2 monomer cocktail; K18-hACE2
mice (n=5/group) were immunized intramuscularly with the cocktail formulations
(8µg/mouse) adjuvanted with SWE in a prime-boost regimen. These mice were
challenged with heterologous SARS-CoV-1 and SARS-CoV-2 BA.5 viruses post
boost immunization.
20 [00113] Figure 6 illustrates efficacy of stabilized RBD oligomer cocktail and
Stabilized RBD-S2 monomer cocktail, wherein A) is a schematic representation
of the immunization protocol, and (B) depicts body weight change and lung viral
titres in mice post SARS-CoV-1 challenge and (C) depicts body weight change
and lung viral titres in mice post SARS-CoV-2 BA.5 challenge. Stabilized RBD-
25 S2 monomer cocktail showed enhanced protective efficacy against heterologous
clade 1a and clade1b Sarbecoviruses.
48
I/We Claim:
1. An immunogenic polypeptide comprising a polypeptide selected from the
group consisting of:
5 a. a polypeptide having an amino acid sequence of at least 95%
sequence identity to the sequence selected from SEQ ID NO: 1 or SEQ ID NO:
59, wherein the polypeptide comprises substitution mutations Y34W and
P195L;
b. a polypeptide having an amino acid sequence of at least 95%
10 sequence identity to the sequence as selected from SEQ ID NO: 3 or SEQ ID
NO: 61, wherein the polypeptide comprises substitution mutations Y34W and
P195L;
c. a polypeptide having an amino acid sequence of at least 95%
sequence identity to the sequence selected from SEQ ID NO: 5 or SEQ ID NO:
15 63, wherein the polypeptide comprises substitution mutations A17P, Y34W,
and P196L;
d. a polypeptide having an amino acid sequence of at least 95%
sequence identity to the sequence selected from SEQ ID NO: 7 or SEQ ID NO:
65, wherein the polypeptide comprises substitution mutations Y34W and
20 P177L;
e. a polypeptide having an amino acid sequence of at least 95%
sequence identity to the sequence selected from SEQ ID NO: 9 or SEQ ID NO:
67, wherein the polypeptide comprises substitution mutations Y34W and
P195L; and
25 f. a polypeptide having an amino acid sequence of at least 95%
sequence identity to the sequence selected from SEQ ID NO: 85 or SEQ ID
NO: 69, wherein the polypeptide comprises substitution mutations A17P,
Y34W, and P196L.
2. The immunogenic polypeptide as claimed in claim 1, wherein the
30 polypeptide is having an amino acid sequence selected from the group consisting
of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
49
SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO:
67, and SEQ ID NO: 69.
3. The immunogenic polypeptide as claimed in claim 1, wherein the
polypeptide further comprises a peptide selected from TPA signal peptide;
5 oligomerization domain; HRV3C protease cleavage site or a portion thereof; His
tag; one or more linkers; or combinations thereof.
4. The immunogenic polypeptide as claimed in claim 3, wherein the
oligomerization domain has an amino acid sequence as set forth in SEQ ID NO.
38.
10 5. The immunogenic polypeptide as claimed in claim 3, wherein the TPA signal
peptide is having an amino acid sequence as set forth in SEQ ID NO. 36, and
wherein the HRV3C protease cleavage site is having an amino acid sequence as
set forth in SEQ ID NO. 40.
6. The immunogenic polypeptide as claimed in claim 3, wherein the linker has
15 an amino acid sequence selected from “AAS”, “S”, “AS”, “GT”,"GSAGS", and/or
"GS".
7. The immunogenic polypeptide as claimed in claim 3, wherein the
polypeptide has an amino acid sequence selected from the group consisting of SEQ
ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23,
20 SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO:
35, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID
NO: 79, and SEQ ID NO: 81.
8. A polynucleotide encoding the immunogenic polypeptide as claimed in any
one of claims 1 to 7.
25 9. The polynucleotide as claimed in claim 8, wherein the polynucleotide has a
nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID
NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 60, SEQ
ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70,
30 SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:
80, and SEQ ID NO: 82.
50
10. The polynucleotide as claimed in claim 8, wherein the polynucleotide is
selected from DNA, RNA, or mRNA.
11. A recombinant vector containing the polynucleotide as claimed in any one
of claims 8 to 10 operably linked to a promoter.
5 12. A recombinant host cell comprising the recombinant vector as claimed in
claim 11.
13. The recombinant host cell as claimed in claim 12, wherein the host cell is a
bacterial cell, yeast cell, insect cell, and mammalian cell.
14. The recombinant host cell as claimed in claim 12, wherein the bacterial cell
10 is Escherichia coli, and wherein the yeast cell is selected from the group consisting
of Pichia X33, Pichia GlycoSwitch® , DSMZ 70382, GS115, KM71, KM71H,
BG09, GS190, GS200, JC220, JC254, JC227, JC300-JC308, YJN165, and
CBS7435, and wherein the insect cell is selected from the group consisting of Expi-
Sf9®, Sf9, High Five® , Sf21, and S2, and wherein the mammalian cell is selected
15 from the group consisting of Expi293F®Expi-CHO-S®, CHO-K1, CHO-S,
HEK293F® , CHOBC™, SLIM™ , SPOT™ , SP2/0 , Sp2/0- Ag14, CHO DG44,
HEK 293S, HEK 293 Gnt1-/- ,HEK293-EBNA1, CHOL-NSO, and NSO.
15. A vaccine composition comprising the immunogenic polypeptide as claimed
in anyone of the claims 1-7, and a pharmaceutically acceptable carrier.
20 16. The vaccine composition as claimed in claim 15, wherein the vaccine
composition is a cocktail formulation comprising a combination of at least 2, at
least 3, at least 4, or 5 immunogenic polypeptides of amino acid sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ
ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18,
25 SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO:
30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID
NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ
ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO:
81.
30 17. The vaccine composition as claimed in claim 15, wherein the
pharmaceutically acceptable carrier is an adjuvant selected from an oil-in-water
51
adjuvant, a polymer and water adjuvant, a water-in-oil adjuvant, an aluminum
hydroxide adjuvant, and combinations thereof.
18. A method for producing the vaccine composition as claimed in claim 15, said
method comprising: (a) culturing the recombinant host cell as claimed in any one
5 of claims 12 to 14 to express the immunogenic polypeptide as claimed in any one
of the claims 1-7; (b) subjecting the immunogenic polypeptide to purification; and
(c) contacting the immunogenic polypeptide of step (b) with a pharmaceutically
acceptable carrier, to obtain the vaccine composition.
19. A method of eliciting an immune response against Sarbecoviruses in a
10 subject, the method comprising administering the subject with an effective amount
of the vaccine composition as claimed in any one of claims 15-18.
20. The method as claimed in claim 19, wherein the Sarbecovirus is selected
from clade 1a, clade 1b, clade 2, or clade 3 virus.
21. The method as claimed in claim 19, wherein the Sarbecovirus is selected
15 from the group consisting of SARS-CoV-1 (clade 1a), WIV-1 (clade 1a), SARS-
CoV-2(clade 1b), RaTG13 (clade 1b), RmYN02 (clade 2), and BtKY72 (clade 3).
22. The method as claimed in claim 19, wherein the vaccine composition is
administered by a mode selected from the group consisting of intranasal,
subcutaneous, intravenous, intra-arterial, intra-peritoneal, intramuscular,
20 intradermal, oral, dermal, and buccal.
23. A kit comprising the immunogenic polypeptide as claimed in any one of the
claims 1-7; or the vaccine composition as claimed in anyone of the claims 15-18,
and an instruction leaflet.
24. The method as claimed in claim 19, wherein the Sarbecovirus is selected
25 from the group consisting of SARS-CoV-1 (clade 1a), WIV-1 (clade 1a), SARS-
CoV-2(clade 1b), RaTG13 (clade 1b), RmYN02 (clade 2), BtKY72 (clade
3), Pangolin_GD, RaTG13 (MN996532), Pangolin_GX-P2V (EPI_ISL_410542),
SARS-CoV-1_Urbani_HP03 (AY278741), SARS-CoV-1_BJ02_HP03M
(AY278487), SARS-CoV-1_HGZ8L1-A_HP03E (AY394981), SARS-CoV-
30 1_GD01_HP03L (AY278489), SARS-CoV-1_GZ-C_HP03L (AY394979),
SARS-CoV-1_Sino1-11_HP03L (AY485277), SARS-CoV-1_Sin852_HP03L
52
(AY559082), SARS-CoV-1_SZ3_PC03 (AY304486), SARS-CoV-
1_SZ13_PC03 (AY304487), SARS-CoV-1_SZ1_PC03 (AY304489), SARS-
CoV-1_GD03T0013_HP04 (AY525636), SARS-CoV-1_GZ0402_HP04
(AY613947), SARS-CoV-1_PC4-127_PC04 (AY613951), SARS-CoV-1_PC4-
5 137_PC04 (AY627045), SARS-CoV-1_PC4-13_PC04 (AY613948), WIV16
(KT444582), WIV1 (KF367457), Rs7327 (KY417151), LYRa11 (KF569996),
Rs4231 (KY417146), RsSHC014 (KC881005), Rs4084 (KY417144), BM48-31
(NC014470), BtKY72 (KY352407), ZXC21 (MG772934), ZC45 (MG772933),
JL2012 (KJ473811), Rf1 (DQ412042), HeB2013 (KJ473812), 273-2005
10 (DQ648856), Rf4092 (KY417145), YN2013 (KJ473816), RmYN02
(EPI_ISL_412977), As6526 (KY417142), Rs4237 (KY417147), Rs4081
(KY417143), Rp3 (DQ071615), 279-2005 (DQ648857), Shaanxi2011
(JX993987), Yunnan2011 (JX993988), Rs4247 (KY417148), HKU3-13
(GQ153548), HKU3-1 (DQ022305), GX2013 (KJ473815), Longquan-140
15 (KF294457), HKU3-8 (GQ153543), HuB2013 (KJ473814), Hp-
BCoV_Zhejiang_2013 (KF636752), HKU1 (KF686346), OC43 (KX344031),
MERS-CoV (NC_019843), Bat-CoV-GCCDC1 (MT350598), Rousettus-
CoV_HKU9 (MG762674), Rc-o319 (LC556375), RacCS203 (MW251308),
RshSTT182 (EPI_ISL_852604), PDF-2370, PRD-0038, RsYN04
20 (EPI_ISL_1699444), BB9904 (KR559017), Khosta-1 (MZ190137), Khosta-2
(MZ190138), and RhGB01 (MW719567).
25. The recombinant host cell as claimed in claim 12, wherein the host cell is
selected from the group consisting of OPENPichia (NCYC 2543 hoc1tr),
OPENPichia his4, NCYC 2543 type strain, OPENPichia pep4, OPENPichia yps1,
25 OPENPichia pep4 yps1, and OPENPichia mutS strains.
26. The vaccine composition as claimed in claim 15, wherein the vaccine
composition is a lyophilized composition.
53
Date 19 October 2024
MALATHI LAKSHMIKUMARAN
IN/PA-1433
Agent for the Applicant
To,
The Controller of Patents
The Patent Office at Chennai
ABSTRACT
IMMUNOGENIC POLYPEPTIDES AND VACCINE COMPOSITIONS
AGAINST DIVERSE SARBECOVIRUSES
The present disclosure relates to immunogenic polypeptides having Sarcebovirus
5 derived stabilized receptor binding domain (RBD) fragments. The immunogenic
polypeptides comprise specific stabilizing mutations at various positions on the
amino acid sequence. Further, the present invention also provides vaccine
compositions comprising one or more of the immunogenic polypeptides, methods
and uses thereof.
10
54
,CLAIMS:I/We Claim:
1. An immunogenic polypeptide comprising a polypeptide selected from the
group consisting of:
5 a. a polypeptide having an amino acid sequence of at least 95%
sequence identity to the sequence selected from SEQ ID NO: 1 or SEQ ID NO:
59, wherein the polypeptide comprises substitution mutations Y34W and
P195L;
b. a polypeptide having an amino acid sequence of at least 95%
10 sequence identity to the sequence as selected from SEQ ID NO: 3 or SEQ ID
NO: 61, wherein the polypeptide comprises substitution mutations Y34W and
P195L;
c. a polypeptide having an amino acid sequence of at least 95%
sequence identity to the sequence selected from SEQ ID NO: 5 or SEQ ID NO:
15 63, wherein the polypeptide comprises substitution mutations A17P, Y34W,
and P196L;
d. a polypeptide having an amino acid sequence of at least 95%
sequence identity to the sequence selected from SEQ ID NO: 7 or SEQ ID NO:
65, wherein the polypeptide comprises substitution mutations Y34W and
20 P177L;
e. a polypeptide having an amino acid sequence of at least 95%
sequence identity to the sequence selected from SEQ ID NO: 9 or SEQ ID NO:
67, wherein the polypeptide comprises substitution mutations Y34W and
P195L; and
25 f. a polypeptide having an amino acid sequence of at least 95%
sequence identity to the sequence selected from SEQ ID NO: 85 or SEQ ID
NO: 69, wherein the polypeptide comprises substitution mutations A17P,
Y34W, and P196L.
2. The immunogenic polypeptide as claimed in claim 1, wherein the
30 polypeptide is having an amino acid sequence selected from the group consisting
of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO:
67, and SEQ ID NO: 69.
3. The immunogenic polypeptide as claimed in claim 1, wherein the
polypeptide further comprises a peptide selected from TPA signal peptide;
5 oligomerization domain; HRV3C protease cleavage site or a portion thereof; His
tag; one or more linkers; or combinations thereof.
4. The immunogenic polypeptide as claimed in claim 3, wherein the
oligomerization domain has an amino acid sequence as set forth in SEQ ID NO.
38.
10 5. The immunogenic polypeptide as claimed in claim 3, wherein the TPA signal
peptide is having an amino acid sequence as set forth in SEQ ID NO. 36, and
wherein the HRV3C protease cleavage site is having an amino acid sequence as
set forth in SEQ ID NO. 40.
6. The immunogenic polypeptide as claimed in claim 3, wherein the linker has
15 an amino acid sequence selected from “AAS”, “S”, “AS”, “GT”,"GSAGS", and/or
"GS".
7. The immunogenic polypeptide as claimed in claim 3, wherein the
polypeptide has an amino acid sequence selected from the group consisting of SEQ
ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23,
20 SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO:
35, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID
NO: 79, and SEQ ID NO: 81.
8. A polynucleotide encoding the immunogenic polypeptide as claimed in any
one of claims 1 to 7.
25 9. The polynucleotide as claimed in claim 8, wherein the polynucleotide has a
nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID
NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 60, SEQ
ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70,
30 SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:
80, and SEQ ID NO: 82.
10. The polynucleotide as claimed in claim 8, wherein the polynucleotide is
selected from DNA, RNA, or mRNA.
11. A recombinant vector containing the polynucleotide as claimed in any one
of claims 8 to 10 operably linked to a promoter.
5 12. A recombinant host cell comprising the recombinant vector as claimed in
claim 11.
13. The recombinant host cell as claimed in claim 12, wherein the host cell is a
bacterial cell, yeast cell, insect cell, and mammalian cell.
14. The recombinant host cell as claimed in claim 12, wherein the bacterial cell
10 is Escherichia coli, and wherein the yeast cell is selected from the group consisting
of Pichia X33, Pichia GlycoSwitch® , DSMZ 70382, GS115, KM71, KM71H,
BG09, GS190, GS200, JC220, JC254, JC227, JC300-JC308, YJN165, and
CBS7435, and wherein the insect cell is selected from the group consisting of Expi-
Sf9®, Sf9, High Five® , Sf21, and S2, and wherein the mammalian cell is selected
15 from the group consisting of Expi293F®Expi-CHO-S®, CHO-K1, CHO-S,
HEK293F® , CHOBC™, SLIM™ , SPOT™ , SP2/0 , Sp2/0- Ag14, CHO DG44,
HEK 293S, HEK 293 Gnt1-/- ,HEK293-EBNA1, CHOL-NSO, and NSO.
15. A vaccine composition comprising the immunogenic polypeptide as claimed
in anyone of the claims 1-7, and a pharmaceutically acceptable carrier.
20 16. The vaccine composition as claimed in claim 15, wherein the vaccine
composition is a cocktail formulation comprising a combination of at least 2, at
least 3, at least 4, or 5 immunogenic polypeptides of amino acid sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ
ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18,
25 SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO:
30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID
NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ
ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO:
81.
30 17. The vaccine composition as claimed in claim 15, wherein the
pharmaceutically acceptable carrier is an adjuvant selected from an oil-in-water
adjuvant, a polymer and water adjuvant, a water-in-oil adjuvant, an aluminum
hydroxide adjuvant, and combinations thereof.
18. A method for producing the vaccine composition as claimed in claim 15, said
method comprising: (a) culturing the recombinant host cell as claimed in any one
5 of claims 12 to 14 to express the immunogenic polypeptide as claimed in any one
of the claims 1-7; (b) subjecting the immunogenic polypeptide to purification; and
(c) contacting the immunogenic polypeptide of step (b) with a pharmaceutically
acceptable carrier, to obtain the vaccine composition.
19. A method of eliciting an immune response against Sarbecoviruses in a
10 subject, the method comprising administering the subject with an effective amount
of the vaccine composition as claimed in any one of claims 15-18.
20. The method as claimed in claim 19, wherein the Sarbecovirus is selected
from clade 1a, clade 1b, clade 2, or clade 3 virus.
21. The method as claimed in claim 19, wherein the Sarbecovirus is selected
15 from the group consisting of SARS-CoV-1 (clade 1a), WIV-1 (clade 1a), SARS-
CoV-2(clade 1b), RaTG13 (clade 1b), RmYN02 (clade 2), and BtKY72 (clade 3).
22. The method as claimed in claim 19, wherein the vaccine composition is
administered by a mode selected from the group consisting of intranasal,
subcutaneous, intravenous, intra-arterial, intra-peritoneal, intramuscular,
20 intradermal, oral, dermal, and buccal.
23. A kit comprising the immunogenic polypeptide as claimed in any one of the
claims 1-7; or the vaccine composition as claimed in anyone of the claims 15-18,
and an instruction leaflet.
24. The method as claimed in claim 19, wherein the Sarbecovirus is selected
25 from the group consisting of SARS-CoV-1 (clade 1a), WIV-1 (clade 1a), SARS-
CoV-2(clade 1b), RaTG13 (clade 1b), RmYN02 (clade 2), BtKY72 (clade
3), Pangolin_GD, RaTG13 (MN996532), Pangolin_GX-P2V (EPI_ISL_410542),
SARS-CoV-1_Urbani_HP03 (AY278741), SARS-CoV-1_BJ02_HP03M
(AY278487), SARS-CoV-1_HGZ8L1-A_HP03E (AY394981), SARS-CoV-
30 1_GD01_HP03L (AY278489), SARS-CoV-1_GZ-C_HP03L (AY394979),
SARS-CoV-1_Sino1-11_HP03L (AY485277), SARS-CoV-1_Sin852_HP03L
(AY559082), SARS-CoV-1_SZ3_PC03 (AY304486), SARS-CoV-
1_SZ13_PC03 (AY304487), SARS-CoV-1_SZ1_PC03 (AY304489), SARS-
CoV-1_GD03T0013_HP04 (AY525636), SARS-CoV-1_GZ0402_HP04
(AY613947), SARS-CoV-1_PC4-127_PC04 (AY613951), SARS-CoV-1_PC4-
5 137_PC04 (AY627045), SARS-CoV-1_PC4-13_PC04 (AY613948), WIV16
(KT444582), WIV1 (KF367457), Rs7327 (KY417151), LYRa11 (KF569996),
Rs4231 (KY417146), RsSHC014 (KC881005), Rs4084 (KY417144), BM48-31
(NC014470), BtKY72 (KY352407), ZXC21 (MG772934), ZC45 (MG772933),
JL2012 (KJ473811), Rf1 (DQ412042), HeB2013 (KJ473812), 273-2005
10 (DQ648856), Rf4092 (KY417145), YN2013 (KJ473816), RmYN02
(EPI_ISL_412977), As6526 (KY417142), Rs4237 (KY417147), Rs4081
(KY417143), Rp3 (DQ071615), 279-2005 (DQ648857), Shaanxi2011
(JX993987), Yunnan2011 (JX993988), Rs4247 (KY417148), HKU3-13
(GQ153548), HKU3-1 (DQ022305), GX2013 (KJ473815), Longquan-140
15 (KF294457), HKU3-8 (GQ153543), HuB2013 (KJ473814), Hp-
BCoV_Zhejiang_2013 (KF636752), HKU1 (KF686346), OC43 (KX344031),
MERS-CoV (NC_019843), Bat-CoV-GCCDC1 (MT350598), Rousettus-
CoV_HKU9 (MG762674), Rc-o319 (LC556375), RacCS203 (MW251308),
RshSTT182 (EPI_ISL_852604), PDF-2370, PRD-0038, RsYN04
20 (EPI_ISL_1699444), BB9904 (KR559017), Khosta-1 (MZ190137), Khosta-2
(MZ190138), and RhGB01 (MW719567).
25. The recombinant host cell as claimed in claim 12, wherein the host cell is
selected from the group consisting of OPENPichia (NCYC 2543 hoc1tr),
OPENPichia his4, NCYC 2543 type strain, OPENPichia pep4, OPENPichia yps1,
25 OPENPichia pep4 yps1, and OPENPichia mutS strains.
26. The vaccine composition as claimed in claim 15, wherein the vaccine
composition is a lyophilized composition.