FIELD OF THE INVENTION
The present invention relates to Coronavirus 20 19-n Co V spike protein, pol ynucl eoti des
5 encoding said spike protein, antibodies and vaccines for treatment or prevention of2019-nCoV
infection.
BACKGROUND OF THE INVENTION
Since 08 December 2019, several cases of pneumonia ofunknown aetiology have been
10 reported in Wuhan, Hubei province, China. Most patients worked at or lived around the local
Huanan seafood wholesale market, where live animals were also on sale. In the early stages of
this pneumonia, severe acute respiratory infection symptoms occurred, with some patients
rapidly developing acute respiratory distress syndrome (ARDS), acute respiratory failure, and
other serious complications. On 07 January 2020, a novel coronavirus was identified by the
15 Chinese Center for Disease Control and Prevention (CDC) from the throat swab sample of a
patient, and was subsequently named 2019-nCoV by WHO and has now been designated
SARS-Co V -2.
Coronaviruses can cause multiple system infections in various animals and mainly
respiratory tract infections in humans, such as severe acute respiratory syndrome (SARS) and
20 Middle East respiratory syndrome (MERS). Most patients have mild symptoms and good
prognosis.
So far, a few patients with 2019-nCoV have developed severe pneumonia, pulmonary
oedema, ARDS, or multiple organ failure and have died. All costs of2019-nCoV treatment are
covered by medical insurance in China. At present, information regarding the epidemiology
25 and clinical features of pneumonia caused by 20 19-nCo Vis scarce and no vaccine is available.
Therefore, there is an ongoing need for the development of antigens which may be used in
vaccines to prevent and treat 2019-nCoV infection. Further, there is a need to provide antigens
that can be produced at scale inexpensively.
The present invention addresses one or more of the above needs by providing
30 polynucleotides encoding 2019-nCoV antigens, particularly antigens from the spike protein of
2019-nCoV, vectors comprising said polynucleotide, vectors encoding said antigens, and
binding compounds (particularly antibodies and antibody-like molecules including aptamers
and peptides) raised against the antigen, together with the use thereof (either alone or in
combination) in the prevention or treatment of infection with 2019-nCoV. The polynucleotides
wo 2021/165667 2 PCT/GB2021/050383
encoding the antigen are optimised for expression in host cells of interest. Antibodies and
antibody-like molecules raised against the antigen may bind (e.g. specifically bind) to the
antigen.
5 SUMMARY OF THE INVENTION
To-date, no vaccine has been developed for 2019-nCoV. The present inventors have
developed polynucleotides encoding the 2019-nCoV spike protein, said polynucleotides being
optimised for expression in commonly used expression systems. These polynucleotides
provide increased level and duration of expression of the 2019-nCoV spike protein, making
10 them advantageous for large-scale production of this antigen. Furthermore, the polynucleotides
devised by the inventors encode the spike protein amino acid sequence in a form which retains
the conformation of the native spike protein. Thus, the spike protein produced according to
the present invention can give rise to an immunoprotective response, particularly through the
production of neutralising antibodies.
15 Accordingly, the present invention provides an isolated polynucleotide encoding a
spike protein from 2019-nCoV having at least 90% identity with SEQ ID NO: 1, or a fragment
thereof that has a common antigenic cross-reactivity with said spike protein, wherein said
polynucleotide is optimised for recombinant expression.
Said polynucleotide of may be optimised for expression in a host cell selected from:
20 Escherichia coli; yeast, preferably Komagataella or Saccharomyces; and/or mammalian cells,
preferably human cells. Optimisation may occur by omitting one or more cis-acting sequence
motif, said one or more cis-acting sequence motif being independently selected from: an
internal TATA-box; a chi-site; a ribosomal entry site; an AT-rich and/or GC-rich stretch of
sequence; an RNA instability motif; a repeat sequence and/or an RNA secondary structure; a
25 cryptic splice donor site; a cryptic splice acceptance site; and/or any combination of (a) to (i).
Said polynucleotide may integrate into the host cell genome. Said polynucleotide may have a
codon adaptation index (CAl) of at least about 0.80, preferably at least about 0.9, more
preferably at least about 0.93. A polynucleotide of the invention may comprise or consist of a
nucleic acid sequence having at least 90% identity to any one of SEQ ID NO: 2 to 8, 13, 14,
30 26, 27, 28, 30 or 32. The polynucleotide of the invention typically encodes a spike protein, or
fragment thereof which: (i) retains the conformational epitopes present in the native 2019-
nCoV spike protein; (ii) results in the production of neutralising antibodies specific for the
spike protein or fragment thereof when the nucleic acid or the encoded spike protein or
fragment thereof is administered to a subject; and/or (iii) comprises or consists of receptorwo
2021/165667 3 PCT/GB2021/050383
binding domain (RBD) of the 2019-nCoV spike protein, preferably having at least 90% identity
with SEQ ID NO: 15.
The invention further provides an expression construct comprising polynucleotide of
the invention, operably linked to a promoter.
5 The invention further provides a vaccine composition comprising a spike protein from
2019-nCoV having at least 90% identity with SEQ ID NO: 1, or a fragment thereof, that has a
common antigenic cross-reactivity with said spike protein, wherein optionally said fragment
comprises or consists of receptor-binding domain (RBD) of the 2019-nCoV spike protein,
preferably having at least 90% identity with SEQ ID NO: 15. Said vaccine typically results in
1 0 the production of neutrali sing antibodies specific for the spike protein or fragment thereof when
administered to a subject.
The invention also provides a viral vector, RNA vaccine or DNA plasmid that expresses
a spike protein from 2019-nCoV having at least 90% identity with SEQ ID NO: 1, or a fragment
thereof, that has a common antigenic cross-reactivity with said spike protein, wherein
15 optionally said fragment comprises or consists of receptor-binding domain (RBD) of the 2019-
nCoV spike protein, preferably having at least 90% identity with SEQ ID NO: 15. Said viral
vector, RNA vaccine or DNA plasmid may further encode a signal peptide. The signal peptide
may direct secretion from human cells. The viral vector, RNA vaccine or DNA plasmid of the
invention may further express one or more additional antigen or a fragment thereof, preferably
20 one or more additional antigen from 20 19-nCo V, or a fragment thereof The spike protein or
fragment thereof and the one or more additional antigen or fragment thereof may be expressed:
as a fusion protein; or in separate viral vectors, RNA vaccines or DNA plasmids for use in
combination. Said viral vector, RNA vaccine or DNA plasmid may comprise one or more
polynucleotide or expression construct of the invention.
25 The invention also provides a fusion protein comprising a spike protein from 2019-
nCoV having at least 90% identity with SEQ ID NO: 1, or a fragment thereof, that has a
common antigenic cross-reactivity with said spike protein, wherein optionally said fragment
comprises or consists of receptor-binding domain (RBD) of the 2019-nCoV spike protein,
preferably having at least 90% identity with SEQ ID NO: 15. Said VLP or fusion protein may
30 further comprise: the Hepatitis B surface antigen (HBSAg), or a fragment thereof that has a
common antigenic cross-reactivity with said HBSAg; the HPV 18 L1 protein, or a fragment
thereof that has a common antigenic cross-reactivity with said HPV 18 L 1 protein; the Hepatitis
E P239 protein (HEV), or a fragment thereof that has a common antigenic cross-reactivity with
said Hepatitis E P239 protein; and/or the HPV 16 L1 protein, or a fragment thereof that has a
wo 2021/165667 4 PCT/GB2021/050383
common antigenic cross-reactivity with said HPV 16 L1 protein. The fusion protein may be
encoded by a polynucleotide which comprises or consists of a nucleic acid sequence having at
least 90% identity with any one of SEQ ID NO: 3, 5, 6, 8, 26, 27, 29,30, or 32; and/or may the
fusion protein may comprise of consist of an amino acid sequence having at least 90% identity
5 with any one of SEQ ID NO: 9, 10, 11, 12, 28,31, or 33.
The invention also provides a virus-like particle comprising a spike protein from 2019-
nCoV having at least 90% identity with SEQ ID NO: 1, or a fragment thereof, that has a
common antigenic cross-reactivity with said spike protein, wherein optionally said fragment
comprises or consists of receptor-binding domain (RBD) of the 2019-nCoV spike protein,
10 preferably having at least 90% identity with SEQ ID NO: 15. Preferably the VLP comprises
or consists of a fusion protein of the invention.
The invention also provides an antibody, or binding fragment thereof, that specifically
binds to a 2091-nCoV spike protein antigen, or fragment thereof, as herein. Said antibody, or
binding fragment thereof, may be a monoclonal or polyclonal antibody. Said antibody, or
15 binding fragment thereof may be an Fab, F(ab')2, Fv, scFv, Fd or dAb.
20
25
The invention further provides an oligonucleotide aptamer that specifically binds to a
20 19-nCo V spike protein or fragment thereof as defined herein.
The invention provides a vaccine composition comprising the viral vector, and/or RNA
vaccine and/or DNA plasmid of the invention.
The invention also provides a polynucleotide of the invention, and/or the expression
construct of the invention, and/or vaccine composition of the invention, and/or the viral vector
and/or RNA vaccine and/or DNA plasmid of the invention and/or the virus-like particle of the
invention, and/ or the fusion protein of the invention, and/ or the anti body of the invention and/ or
the aptamer of the invention for use in the treatment and/or prevention of 20 19-nCo V infection.
The invention also provides the use of a polynucleotide of the invention, and/or the
expression construct of the invention, and/or vaccine composition of the invention, and/or the
viral vector and/or RNA vaccine and/or DNA plasmid of the invention and/or the virus-like
particle of the invention, and/or the fusion protein of the invention, and/or the antibody of the
invention and/or the aptamer of the invention in the manufacture of a medicament for the
30 prevention and/or treatment of 20 19-nCo V infection.
The invention also provides a method of producing a spike protein from 2019-nCoV
having at least 90% identity with SEQ ID NO: 1, or a fragment thereof, comprising expressing
a polynucleotide of the invention in a host cell, and optionally purifying the spike protein or
5
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fragment. Said method may further comprise formulating said spike protein or fragment thereof
with a pharmaceutically acceptable carrier or diluent.
DESCRIPTION OF FIGURES
Figure 1:
proteins.
Figure 2:
Schematic of the corona virus's structure and the function of the structural
Tabulated results from ELISA reporting antibody titre at dayO and dayl4
following administration of 2019-nCoV spike protein and fusion proteins comprising 2019-
10 nCo V spike protein produced according to the invention.
Figure 3: Western blots showing secreted protein HBSAg(EAAAK)3RBD released into
culture medium after 40h (following concentration by centrifugal filtration). Left blot =
HBSAg antibody. Right blot= 2019-nCoV antibody. For both blots, lane M =marker; lane 1
= HEV-GGGS-RBD #lOB; lane 2 = HBSAg-EAAK-RBD #2A (293F secreted, total protein
15 content by Bradford assay= 750!J.g/ml); lane 3 = HBSAg-EAAAK-RBD #2B (293F secreted,
total protein content by Bradford assay = 500!J.g/ml); lane 4 = CoV-s #17 (293F surface
bounded HisTag)
Figure 4: Graphs illustrating titres at dl4 (A) and d42 (B) in mice inoculated with
HBSAg(EAAAK)3RBD generated in HEK cells, either alone, or using aluminium hydroxide
20 or Addavax™ adjuvants.
Figure 5: Western blots showing secreted protein HEV-(GGGGS)3-RBD produced by E.
coli. Left blot= #lOA Middle blot= #lOB. Right blot= #lOC. For all blots an anti-HEY
mAB was used at a dilution of 1:4000.
Figure 6: Graphs illustrating titres at dl4 (A) and d42 (B) in mice inoculated with HEV-
25 GGGGS-RBD generated in E. coli, either alone, or using aluminium hydroxide or Addavax™
adjuvants.
30
Figure 7: Western blot of recombinant HBSAg-(EAAAK)3-full-length 2019-nCoV spike
protein fusion protein (HBSAg-(EAAAK)3-CoV-s) clone D8-SA01-0l-Ol (4x) and clone D8-
SA01-02-0l (5X) expressed in HEK 293 cells.
DETAILLED DESCRIPTION OF THE INVENTION
Unless otherwise defined herein, scientific and technical terms used in connection with
the present invention shall have the meanings that are commonly understood by those of
wo 2021/165667 6 PCT/GB2021/050383
ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the
event of any latent ambiguity, definitions provided herein take precedent over any dictionary
or extrinsic definition. It should be understood that this invention is not limited to the particular
methodology, protocols, and reagents, etc., described herein and as such can vary. The
5 terminology used herein is for the purpose of describing particular embodiments only, and is
not intended to limit the scope of the present invention, which is defined solely by the claims.
Further, unless otherwise required by context, singular terms shall include pluralities and plural
terms shall include the singular. In this application, the use of "or" means "and/or" unless stated
otherwise. Furthermore, the use of the term "including", as well as other forms, such as
10 "includes" and "included", is not limiting.
The description of embodiments of the disclosure is not intended to be exhaustive or to
limit the disclosure to the precise form disclosed. While specific embodiments of, and examples
for, the disclosure are described herein for illustrative purposes, various equivalent
modifications are possible within the scope of the disclosure, as those skilled in the relevant art
15 will recognize. For example, while method steps or functions are presented in a given order,
alternative embodiments may perform functions in a different order, or functions may be
performed substantially concurrently. The teachings of the disclosure provided herein can be
applied to other procedures or methods as appropriate. The various embodiments described
herein can be combined to provide further embodiments. Aspects of the disclosure can be
20 modified, if necessary, to employ the compositions, functions and concepts of the above
references and application to provide yet further embodiments of the disclosure. Moreover, due
to biological functional equivalency considerations, some changes can be made in protein
structure without affecting the biological or chemical action in kind or amount. These and other
changes can be made to the disclosure in light of the detailed description. All such
25 modifications are intended to be included within the scope of the appended claims.
The publications discussed herein are provided solely for their disclosure prior to the
filing date of the present application. Nothing herein is to be construed as an admission that
such publications constitute prior art to the claims appended hereto.
30 Coronaviruses
Coronaviruses (CoVs) belong to the subfamily Coronavirinae, in the family
Coronaviridae of the order Nidovirales. There are four genera: Alphacoronavirus,
Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Alphacoronaviruses and
wo 2021/165667 7 PCT/GB2021/050383
Betacoronaviruses infect species of mammal, Gammacoronaviruses infect species of bird, and
Deltacoronaviruses infect both species of mammals and birds.
CoVs are large enveloped single positive-sense RNA viruses. Mutation rates of RNA
viruses are greater than DNA viruses, suggesting a more efficient adaptation process for
5 survival.
CoVs have the largest genome among all RNA viruses, typically ranging from 27 to 32
kb. The CoV genome codes for at least four main structural proteins: spike (S), membrane (M),
envelope (E), nucleocapsid (N) proteins and other accessory proteins which aid the replicative
processes and facilitate entry into cells. Figure 1 summarises the coronavirus' s structure and
10 the function of the structural proteins. Briefly, the CoV genome is packed inside a helical capsid
formed by the nucleocapsid and further surrounded by an envelope. Associated with the viral
envelope are at least three structural proteins: the membrane and envelope proteins, which are
involved in virus assembly, and the spike protein, which mediates virus entry into host cells.
Some coronaviruses also encode an envelope-associated hemagglutinin-esterase protein (HE).
15 The spike protein forms large protrusions from the virus surface, giving coronaviruses the
appearance of having crowns, from which the name "Coronavirus" is derived. As well as
mediating virus entry, the spike protein is a critical determinant of viral host range and tissue
tropism and a major inducer of host immune responses.
20 19-nCo V (officially named severe acute respiratory syndrome corona virus 2, SARS-
20 CoV-2, the two terms being used interchangeably herein) is the causative agent of coronavirus
disease 2019 (COVID-19) and is contagious among humans. It is believed that 2019-nCoV
originated in animals, with bats being a likely source given the genetic similarities of 2019-
nCoV to SARS-CoV (79.5%) and bat coronaviruses (96%). Any disclosure herein in relation
to CoVs also applies directly and without restriction to 2019-nCoV.
25 The CoV spike protein comprises three domains: (i) a large ectodomain; (ii) a
transmembrane domain (which passes through the viral envelope in a single pass); and (iii) a
short intracellular tail. The ectodomain consists of three receptor-binding subunits (3 x S 1) and
a trimeric stalk made of three membrane-fusion subunits (3 x S2). During virus entry, S1 binds
to a receptor on the host cell surface for viral attachment, and S2 fuses the host and viral
30 membranes, allowing viral genomes to enter host cells. Receptor binding and membrane fusion
are the initial and critical steps in the coronavirus infection cycle. There is significant
divergence in the receptors targeted by different Co V s.
The 2019-nCoV spike protein or immunogenic fragments thereof have therapeutic
potential as antigens for vaccines against 2019-nCoV infection.
wo 2021/165667 8 PCT/GB2021/050383
Accordingly, as described herein, the invention relates to a 2019-nCoV spike protein
has at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or more identity with SEQ ID NO: 1, or a fragment thereof,
that has a common antigenic cross-reactivity with said spike protein. Preferably the invention
5 relates to a spike protein from 2019-nCoV has at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or more identity with SEQ ID NO: 1, or a fragment thereof,
that has a common antigenic cross-reactivity with said spike protein. More preferably, the
invention relates to a spike protein from 2019-nCoV having least 98%, at least 99% or more
with SEQ ID NO: 1, or a fragment thereof, that has a common antigenic cross-reactivity with
10 said spike protein. The spike protein from 2019-nCoV may comprise or consist of SEQ ID
NO: 1, or a fragment thereof, that has a common antigenic cross-reactivity with said spike
protein.
According to the present invention, the 2019-nCoV spike protein or fragment thereof
encoded by a polynucleotide of the invention maintains one or more conformational epitope
15 present in native 2019-nCoV spike protein. As such, the 2019-nCoV spike protein or fragment
thereof encoded by a polynucleotide of the invention is capable of giving rise to an
immunoprotective effect. Typically said immunoprotective effect comprises the production of
neutralising antibodies (nAb) which specifically bind to the one or more conformational
epitope of the 2019-nCoV spike protein or fragment thereof encoded by a polynucleotide of
20 the invention. A conformational epitope of a Co V spike protein has a specific threedimensional
structure that is found in the tertiary structure of the Co V spike protein. Said one
or more conformational epitope is typically within the ectodomain of the spike protein.
Preferably the 2019-nCoV spike protein or fragment thereof encoded by a polynucleotide of
the invention retains all of the conformational epitopes present in native 2019-nCoV spike
25 protein.
In some preferred embodiments, the invention relates to an immunogenic fragment of
2019-nCoV spike protein which is the receptor-binding domain (RBD) ofthe 2019-nCoV spike
protein. This RBD is responsible for 2019-nCoV binding to a host cell and thus facilitates
entry of 2019-nCoV particles into the host cell. The RBD corresponds to amino acid residues
30 319 to 529 of SEQ ID NO: 1, as described herein is referred to as SEQ ID NO: 15. The RBD
is encoded by bases corresponding to positions 955 to 1597 in the genome ofthe2019-nCoV
virus (Genbank Accession No. MN908947, version 3 of which (MN908947.3) was deposited
17 January 2020). Accordingly, as described herein, the invention relates to an RBD of the
2019-nCoV spike protein has at least 70%, at least 75%, at least 80%, at least 90%, at least
wo 2021/165667 9 PCT/GB2021/050383
95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO:
15. Preferably the invention relates to an RBD of the 2019-nCoV spike protein that has at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with
SEQ ID NO: 15. More preferably, the invention relates to an RBD of the 2019-nCoV spike
5 protein having least 98%, at least 99% or more with SEQ ID NO: 15. The RBD of the 2019-
nCoV spike protein may comprise or consist of SEQ ID NO: 15. Any and all disclosure herein
relating to the 2019-nCoV spike protein (e.g. in relation to polynucleotides, viral vectors, DNA
plasmids, RNA vaccines, virus-like particles (VLPs), fusion proteins, antibodies, compositions
and pharmaceutical compositions, formulations and therapeutic indications) applies equally
10 and without reservation to the RBD of the 2019-nCoV spike protein. References herein to
RBD refer to the RBD of the 2019-nCoV spike protein.
CoVs are large enveloped single positive-sense RNA viruses. Mutation rates of RNA
viruses are greater than DNA viruses, suggesting a more efficient adaptation process for
survival. Thus, there is a risk that antigenic drift will also become a feature of the 2019-nCoV,
15 or if 20 19-nCo V becomes endemic in the population once the pandemic has subsided. Indeed,
research to-date has already identified mutations within the receptor binding domain (RBD) of
the spike protein of 2019-nCoV, particularly G476S and V483A/G, as well as a prevalent
D614G mutation in the vicinity of the S1/S2 site (Saha et al., ChemRxivTM
httrdi_r,i_Qi_QrgL1_Q,_;?._\i:L?JL~h~mi~i_y_j_2_~_2_Q_~_\i:Z,_yJ_), which the evidence suggests can enhance cell
20 entry by the 2019-nCoV virion, and also broaden the host cell tropism. Other mutations
reported in the 2019-nCoV spike protein include S943 (particularly S943P), L5 (particularly
L5F), L8 (particularly L8F), V367 (particularly V367F), H49 (particularly H49Y), Y145
(particularly Y145H/del), Q239 (particularly Q239K), A831 (particularly A831 V), D839
(particularly D839Y/N/E), and P1263 (particularly P1263L), or any combination thereof
25 (Korber et al., BioRxivTM https://doi.org/10.1101/2020.04.29.069054).
Accordingly, the invention advantageously allow 2019-nCoV vaccine antigens to be
modified if required to provide enhanced immunity against strains with mutated spike proteins
as they arise. By way of non-limiting example, any 2019-nCoV spike protein or fragment
thereof according to the invention may be modified (particularly by substitution) at position (i)
30 D614, (ii) V483, (iii) G476, (iv) K417, (v), E484, (vi) N501, (vii) A570, and (viii) P681, or
any combination of (including any two, any three, any four, any five, any six, any seven or all
eight) of (i) to (viii). Alternatively or in addition, the 2019-nCoV spike proteins or fragments
thereof may comprise deletion mutations, including deletions at one or more of amino acid
residues 69, 70 and/or 144. As described herein, the positions of the mutations/modifications
wo 2021/165667 10 PCT/GB2021/050383
typically corresponds to the numbering of amino acids in SEQ ID NO: 1 of the present
invention.
Modification at position D614, particularly the D614G substitution, is preferred. In
particular, any 2019-nCoV spike protein or fragment thereof according to the invention may
5 comprise the following substitutions (i) G476S, (ii) V483A/G, (iii) D614G, (iv) K417N/T, (v),
E484K, (vi) N501Y, (vii) A570D, and (viii) P681H, or any combination of(including any two,
any three, any four, any five, any six, any seven or all eight) of (i) to (viii).
The invention also relates to 2019-nCoV spike proteins or fragments thereof from a
variant 2019-nCoV. In particular, the invention may relate to 2019-nCoV spike proteins or
10 fragments thereof from the B.l.l.7 strain (also known as 201/501Y.Vl, which was first
detected in the UK); the B.l.351 strain (also known as 20H/50l.V2, which was first detected
in South Africa), and/or the PI strain (also known as 20J/501 Y.V3, which was first detected in
Japan and Brazil). The key mutations of the B.l.l.7 stain comprise deletion of residues 69/70
and 144Y, as well as N501Y, A570D, D614G and P681H substitutions. The key mutations of
15 the B.l.351 strain comprise K417N, E484K, N501Y and D614G substitutions. The key
mutations of the P.l strain comprise E484K, K417N.T, N501 Y and D614G.
20
All the disclosure herein in relation to polynucleotides, spike proteins and fragments
thereof, VLPs, fusion proteins and DNA/RNA vaccine applies equally to different variants and
strains of 2019-nCoV unless explicitly stated.
Polynucleotides
The present invention provides a polynucleotide that encodes or expresses (the terms
"encode" and "express" are used interchangeably herein) the protein or immunogenic fragment
of the invention. The term polynucleotide encompasses both DNA and RNA sequences.
25 Herein, the terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" are used
interchangeably.
The invention provides an isolated polynucleotide encoding a spike protein from 2019-
nCoV having at least 90% identity with SEQ ID NO: 1, or a fragment thereof that has a
common antigenic cross-reactivity with said spike protein. For example, the polynucleotide
30 may encode an RBD of the 2019-nCoV spike protein, preferably wherein said RBD has at least
90% identity with SEQ ID NO: 15. Exemplary polynucleotides encoding the RBD are shown
in SEQ ID NO: 13, and the codon-optimised sequence of SEQ ID NO: 14.
The invention also encompasses polynucleotides encoding a variant spike protein from
2019-nCoV, as described above, or fragments thereof that have common antigenic crosswo
2021/165667 11 PCT/GB2021/050383
reactivity with said variant spike protein. Said varint spike proteins typically have at least 90%
identity with SEQ ID NO: 1.
A polynucleotide of the invention may be used for recombinant expression of the
protein or immunogenic fragment of the invention (including in the form of a VLP or fusion
5 protein), or as a DNA/RNA vaccine.
The present inventors are the first to provide improved polynucleotides encoding the
2019-nCoV spike protein or immunogenic fragments thereof In particular, the present
inventors have designed polynucleotides that are optimised for recombinant expression. A
polynucleotide of the invention may be optimised for expression in one or more particular cell
10 type, for example, eukaryotic cells (e.g. mammalian cells, yeast cells, insect cells or plants
cells) or prokaryotic cells (bacterial cells). Typically the polynucleotides are optimised for
expression in bacterial cells, yeast cells or mammalian cells. Preferably said polynucleotides
are optimised for expression in Escherichia coli (for example, BL21(DE3), RV308(DE3),
HMS174(DE3) or K12 strains), Komagataella (formally assigned as Pichia, particularly
15 Komagataella pastoris or Komagataella phaffii), Saccharomyces (particularly Saccharomyces
cerevisiae) or human cells (preferably 293 F cells, HEK 293 cells, HEK 293T cells or HeLa
cells). Other cell types/expression systems of interest include Pichia angusta, Hansenula
polymorpha, Chinese Hamster Ovary (CHO) cells and/or insect cell baculovirus-based
expression systems.
20 The term "optimised" as used herein relates to optimisation for recombinant expression
of the 2019-nCoV spike protein or immunogenic fragment thereof, and includes both codon
optimisation and/or other modifications to the polynucleotide (both in terms of the nucleic acid
sequence and other modifications) which increase the level and/or duration of expression of
the 2019-nCoV spike protein from the polynucleotide within the host cell/organism, or which
25 otherwise provide an advantage when expressing the 2019-nCoV spike protein, or fragment
thereof, from a polynucleotide of the invention.
The term "codon optimised" refers to the replacement of at least one codon within a
base polynucleotide sequence with a codon that is preferentially used by the host organism or
cell in which the polynucleotide is to be expressed. Typically, the most frequently used codons
30 in the host organism are used in the codon-optimised polynucleotide sequence. Methods of
codon optimisation are well known in the art.
By way of non-limiting example, another form of polynucleotide optimisation are
modifications which minimise RNA structure, as structures that involve or otherwise occlude
the RBS and/or start codon in genes expressed in prokaryotes can impair expression.
wo 2021/165667 12 PCT/GB2021/050383
Optimisation also encompasses modifications to the polynucleotide which optimise translation,
either by increasing the rate of translation, or by balancing the rate of translation with the need
to allow for efficient "self' or chaperone-aided protein folding, in which strategically placed
slower codons or codon runs (e.g. at protein domain boundaries) could maximise folding
5 efficiency whilst maintaining a high overall translation rate. Optimisation may also encompass
the removal of deleterious motifs within the nucleic acid sequence of the polynucleotide. By
way of non-limiting example, expressing a gene under control of a T7 promoter in E. coli, it is
preferable to avoid both class I and II transcriptional termination sites. Shine-Dalgarno-like
sequences within the coding sequence may cause incorrect downstream initiation or
10 translational pauses in prokaryotic hosts. For expression in eukaryotic hosts/cells, potential
splice signals, polyadenylation signals and other motifs affecting mRNA processing and
stability may be removed. Other classes of deleterious motifs include sequences that promote
ribosomal frameshifts and pauses. Any combination of modifications may be made to the
polynucleotides of the invention to optimise expression in a host cell of interest.
15 Typically polynucleotides of the invention optimised for expression in bacterial cells,
particularly E. coli, include cloned N-terminal and/or C-terminal deleted amino acids.
Preferably about 1 to 20, more preferably about 1 to 15, most preferably about 5 to 10 cloned
N-terminal and/or C-terminal deleted amino acids are included.
It will be understood by a skilled person that numerous different polynucleotides can
20 encode the same polypeptide as a result of the degeneracy of the genetic code. It is also
understood that skilled persons may, using routine techniques, make nucleotide substitutions
that do not affect the polypeptide sequence encoded by the nucleic acid molecules to reflect
the codon usage of any particular host organism in which the polypeptides are to be expressed.
Therefore, unless otherwise specified, a "polynucleotide that encodes the protein or
25 immunogenic fragment of the invention" includes all polynucleotide sequences that are
degenerate versions of each other and that encode the same amino acid sequence.
30
A polynucleotide of the invention is typically designed so that it is capable of
integrating into the genome of a host cell of interest. Different optimisation strategies may be
used to facilitate integration depending on the desired host cell.
Typically a polynucleotide of the invention is optimised by the removal or omission of
one or more cis-acting sequence motif (also referred to interchangeably as cis-acting elements
or cis-acting regulatory elements). A cis-acting sequence motif is a sequence in the vicinity of
the structural portion of a gene that is required for gene expression. Said one or more cis-acting
sequence motif may be independently selected from: (a) an internal TATA-box; (b) a Chi-site;
wo 2021/165667 13 PCT/GB2021/050383
(c) a ribosomal entry site; (d) an AT-rich and/or GC-rich stretch of sequence; (e) an RNA
instability motif; (f) a repeat sequence and/or an RNA secondary structure; (g) a cryptic splice
donor site; (h) a cryptic splice acceptance site; and/or (i) any combination of (a) to (i). These
cis-acting sequence motifs are known in the art. By way of non-limiting example, regions of
5 high GC content (e.g. above about 70%, preferably above about 80%) and/or low GB content
(e.g. below about 40%, preferably below about 30%) are omitted. Preferably both regions of
high and low GC content are omitted, in combination with the removal or omission of one or
more other cis-acting sequence motif
A polynucleotide of the invention may also be "codon optimised" as described herein.
10 Codon optimisation preferably occurs in addition to the removal or omission of one or more
cis-acting sequence motif as described herein.
The average GC content of a polynucleotide of the invention may also be modified to
optimise expression of said polynucleotide. For example, the average GC content of a
polynucleotide may be in the region of about 40% to about 60%, preferably about 40% to about
15 57%, more preferably about 45% to about 56%.
A polynucleotide of the invention typically has a codon adaptation index (CAl) of at
least about 0.80, preferably at least about 0.9, more preferably at least about 0.91, at least about
0.92, at least about 0.93, at least about 0.94, at least about 0.95, at least about 0.96, at least
about 0.97, at least about 0.98, at least about 0.99, or more up to about 1.0.
20 As a result of the optimising modifications, a polynucleotide of the invention may
increase the expression of the encoded 2019-nCoVspike protein or fragment thereofby at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 100% or more compared with the corresponding non-optimised polynucleotide
sequence. Preferably the expression level is increased by at least 50%, at least 60%, at least
25 70%, at least 80%, at least 90%, at least 100% or more, more preferably at least at least 70%,
at least 80%, at least 90%, at least 100% or more compared with the corresponding nonoptimised
polynucleotide.
A polynucleotide of the invention may be capable of expression in the host cell for at
least one week, at least two weeks, at least three weeks, at least one month, at least two months,
30 at least three months, at least four months or more, preferably at least one month, at least two
months, at least three months, at least four months or more.
The inventors have demonstrated that 2019-nCoV spike protein and fusion proteins
comprising 20 19-nCo V spike protein can be expressed at high levels in a variety of expression
systems/host cells using their rationally designed optimised polynucleotides. Furthermore, the
wo 2021/165667 14 PCT/GB2021/050383
present inventions have surprisingly demonstrated that 2019-nCoV spike protein and fusion
proteins comprising 2019-nCoV spike protein can generate a strong antibody response in mice,
which demonstrates their potential therapeutic utility. The inventors have exemplified their
optimisation methodology by designing and generating optimised polynucleotides and fusion
5 proteins as described in the Examples below.
Accordingly, a polynucleotide of the invention may comprise or consist of a nucleic
acid sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99% or more identity to any one of SEQ ID NOs:
2, 3, 4, 5, 6, 7 or 8. Preferably a polynucleotide ofthe invention may comprise or consist of a
10 nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% or more identity to any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 13, 14, 26,
27, 29,30, or 32. More preferably, a polynucleotide of the invention may comprise or consist
of a nucleic acid sequence having at least 98%, at least 99% or more identity to any one of SEQ
ID NOs: 2, 3, 4, 5, 6, 7, 8, 13, 14, 26, 27, 29,30, or 32. A polynucleotide of the invention may
15 comprise or consist of the nucleic acid sequence of any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8,
13, 14, 26, 27, 29, 30, or 32. In addition, the 5' cloning site, the 3' cloning site, or the 5' and
3' cloning sites identified in any of SEQ ID NOs; 2, 3, 4, 5, 6, 7, 8, 13, 14, 26, 27, 29, 30, or
32, or any variant thereof as described herein, may be deleted. Thus, the invention provides
polynucleotides comprising or consisting of any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 13, 14,
20 26, 27, 29,30, or 32 but lacking the 5' cloning site, the 3' cloning site, or the 5' and 3' cloning
sites identified in any of SEQ ID NOs; 2, 3, 4, 5, 6, 7, 8, 13, 14, 26, 27, 29,30, or 32.
Alternatively, the 5' cloning site, the 3' cloning site, or the 5' and 3' cloning sites identified in
any of SEQ ID NOs; 2, 3, 4, 5, 6, 7, 8, 13, 14, 26, 27, 29, 30, or 32, or any variant thereof as
described herein, may be independently replaced with another appropriate cloning site.
25 Suitable alternative cloning sites are well known in the art.
The invention particularly provides polynucleotides encoding an RBD of the 2019-
nCoV spike protein. Accordingly, a polynucleotide of the invention may comprise or consist
of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to SEQ ID NO: 13,
30 or to the codon-optimised sequence of SEQ ID NO: 14. Preferably a polynucleotide of the
invention may comprise or consist of a nucleic acid sequence having at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or more identity to SEQ ID NO: 13, or to
the codon-optimised sequence of SEQ ID NO: 14. More preferably, a polynucleotide of the
invention may comprise or consist of a nucleic acid sequence having at least 98%, at least 99%
wo 2021/165667 15 PCT/GB2021/050383
or more identity to SEQ ID NO: 13, or to the codon-optimised sequence of SEQ ID NO: 14.
A polynucleotide of the invention may comprise or consist of the nucleic acid sequence of SEQ
ID NO: 13, or the codon-optimised sequence of SEQ ID NO: 14.
A polynucleotide of the invention typically encodes a 2019-nCoV spike protein, or an
5 immunogenic fragment thereof which: (a) retains the conformational epitopes present in the
native 2019-nCoV spike protein; and/or (b) results in the production ofneutralising antibodies
specific for the spike protein or fragment thereof when the nucleic acid or the encoded spike
protein or fragment thereof is administered to a subject.
The polynucleotide of the invention typically expresses a spike protein from 2019-
10 nCoV having at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 1, or a fragment
thereof, that has a common antigenic cross-reactivity with said spike protein. Preferably a
polynucleotide of the invention expresses a spike protein from 20 19-nCo V having at least 90%,
at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ
15 ID NO: 1, or a fragment thereof, that has a common antigenic cross-reactivity with said spike
protein. More preferably, a polynucleotide of the invention expresses a spike protein from
2019-nCoV having least 98%, at least 99% or more with SEQ ID NO: 1, or a fragment thereof,
that has a common antigenic cross-reactivity with said spike protein. A polynucleotide of the
invention may express a spike protein from 2019-nCoV comprising or consisting of SEQ ID
20 NO: 1, or a fragment thereof, that has a common antigenic cross-reactivity with said spike
protein.
A polynucleotide of the invention may be comprised in an expression construct to
facilitate expression of the 2019-nCoV spike protein or fragment thereof Accordingly, the
invention further provides an expression construct comprising polynucleotide the invention.
25 Typically, in such an expression construct a polynucleotide of the invention is operably linked
to a suitable promoter. The polynucleotide may be linked to a suitable terminator sequence.
The polynucleotide may be linked to both a promoter and terminator. Suitable promoter and
terminator sequences are well known in the art.
The choice of promoter will depend on where the ultimate expressiOn of the
30 polynucleotide will take place. In general, constitutive promoters are preferred, but inducible
promoters may likewise be used. The construct produced in this manner includes at least one
part of a vector, in particular regulatory elements. The vector is preferably capable of
expressing the nucleic acid in a given host cell. Any appropriate host cell may be used, such
wo 2021/165667 16 PCT/GB2021/050383
as mammalian, bacterial, insect, yeast, and/or plant host cells. In addition, cell-free expression
systems may be used. Such expression systems and host cells are standard in the art.
The 2019-nCoV spike protein or immunogenic fragment thereof encoded or expressed
(the two terms are used interchangeably herein) by a polynucleotide of the invention typically
5 retain the same binding affinity for its receptor as the native 2019-nCoV spike protein. In the
context of the present invention, this may mean having a binding affinity for the 20 19-nCo V
spike protein receptor of at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or
more of that of the native 2019-nCoV spike protein. Preferably the 2019-nCoV spike protein
or immunogenic fragment thereof expressed by a polynucleotide of the invention have a
10 binding affinity for the 20 19-nCo V spike protein of at least 90%, at least 95%, at least 99% or
more of that of the native 2019-nCoV spike protein.
In some embodiments, the 2019-nCoV spike protein or immunogenic fragment thereof
expressed by a polynucleotide of the invention have a binding affinity for the 20 19-nCo V spike
protein receptor greater than that of the full-length protein. For example, the 20 19-nCo V spike
15 protein or immunogenic fragment thereof expressed by a polynucleotide of the invention of the
invention may have a binding affinity of at least 100%, at least 110%, at least 120%, or at least
150% or more of that of the native 20 19-nCo V spike protein.
In other embodiments, the 2019-nCoV spike protein or immunogenic fragment thereof
expressed by a polynucleotide ofthe invention may have a binding affinity for the 2019-nCoV
20 spike protein receptor less than that of the native 2019-nCoV spike protein. For example, the
2019-nCoV spike protein or immunogenic fragment thereof expressed by a polynucleotide of
the invention may have a binding affinity of less than 80%, less than 70%, less than 60%, less
than 50% or less of that of the native 2019-nCoV spike protein.
The binding affinity of a 2019-nCoV spike protein or immunogenic fragment thereof
25 expressed by a polynucleotide of the invention for its receptor may be quantified in terms of
dissociation constant (Kct). Kct may be determined using any appropriate technique, but SPR is
generally preferred in the context of the present invention.
An immunogenic fragment of the 2019-nCoV spike protein expressed by a
polynucleotide of the invention are typically greater than 200 amino acids in length. 2019-
30 nCo V spike protein fragments of the present invention may comprise or consist of at least 200,
at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least
1000, at least 1100, or more amino acid residues in length. The fragments of the invention
have a common antigenic cross-reactivity with the 20 19-nCo V spike protein. In some
preferred embodiments, the immunogenic fragment of the 2019-nCoV spike protein expressed
wo 2021/165667 17 PCT/GB2021/050383
by a polynucleotide of the invention is an RBD of the 2019-nCoV spike protein as defined
herein, preferably wherein said RBD has at least 90% identity with SEQ ID NO: 15.
The 2019-nCoV spike protein or immunogenic fragment thereof expressed by a
polynucleotide of the invention may additionally comprise a leader sequence, for example to
5 assist in the recombinant production and/or secretion of the 2019-nCoV spike protein or
immunogenic fragment thereof Any suitable leader sequence may be used, including
conventional leader sequences known in the art. Suitable leader sequences include Bip leader
sequences, which are commonly used in the art to aid secretion from insect cells and human
tissue plasminogen activator leader sequence (tPA), which is routinely used in viral and DNA
10 based vaccines and for protein vaccines to aid secretion from mammalian cell expression
platforms.
The 2019-nCoV spike protein or immunogenic fragment thereof expressed by a
polynucleotide of the invention may additionally comprise anN- or C-terminal tag, for example
to assist in the recombinant production and/or purification of the 2019-nCoV spike protein or
15 immunogenic fragment thereof Any N-or C-terminal tag may be used, including conventional
tags known in the art. Suitable tags sequences include C-terminal hexa-histidine tags and the
"C-tag" (the four amino acids EPEA at the C-terminus), which are commonly used in the art
to aid purification from heterologous expression systems, e.g. insect cells, mammalian cells,
bacteria, or yeast. In other embodiments, the 2019-nCoV spike protein or immunogenic
20 fragment thereof expressed by a polynucleotide of the invention are purified from heterologous
expression systems without the need to use a purification tag.
The 2019-nCoV spike protein or immunogenic fragment thereof expressed by a
polynucleotide of the invention may comprise a leader sequence and/or a tag as defined herein.
25 Viral Vectors, DNA Plasmids and RNA Vaccines
The present invention also provides a vector: (a) comprising a polynucleotide of the
invention; and/or (b) encoding a 2019-nCoV spike protein or immunogenic fragment thereof
of the invention. The vector(s) may be present in the form of a vaccine composition or
formulation.
30 The vector of the invention typically expresses a spike protein from 2019-nCoV having
at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or more identity with SEQ ID NO: 1, or a fragment thereof, that has
a common antigenic cross-reactivity with said spike protein. Preferably a vector of the
invention expresses a spike protein from 2019-nCoV having at least 90%, at least 95%, at least
wo 2021/165667 18 PCT/GB2021/050383
96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 1, or a fragment
thereof, that has a common antigenic cross-reactivity with said spike protein. More preferably,
a vector of the invention expresses a spike protein from 2019-nCoV having least 98%, at least
99% or more with SEQ ID NO: 1, or a fragment thereof, that has a common antigenic cross-
5 reactivity with said spike protein. A vector of the invention may express a spike protein from
2019-nCoV comprising or consisting of SEQ ID NO: 1, or a fragment thereof, that has a
common antigenic cross-reactivity with said spike protein. In some preferred embodiments, the
immunogenic fragment of the 2019-nCoV spike protein expressed by a vector ofthe invention
is an RBD of the 2019-nCoV spike protein as defined herein, preferably wherein said RBD has
10 at least 90% identity with SEQ ID NO: 15.
15
The vector of the invention may express a spike protein or immunogenic fragment
thereof as defined herein which further comprises a signal peptide. Typically said signal
peptide directs secretion of the 20 19-nCo V spike protein or fragment thereof from a host cell
of interest, such as a human cell, an E. coli cell or a yeast cell.
The vector of the invention may further express one or more additional antigen or a
fragment thereof The spike protein or fragment thereof and the one or more additional antigen
or fragment thereof may be expressed as a fusion protein. Alternatively, separate vectors
expressing the 2019-nCoV spike protein or fragment thereof and the one or more additional
antigen or fragment thereof may be used. In such instances, said separate vectors may be used
20 in combination, either sequentially or simultaneously. The one or more additional antigen may
be the same antigen or a different antigen from 2019-nCoV, or a fragment thereof More
preferably, said one or more additional antigen is a different antigen from 2019-nCoV, such as
an antigen from the 2019-CoV membrane protein or envelope protein.
The vector(s) of the invention may comprise any polynucleotide or expression construct
25 as defined herein, or any combination thereof
The vector(s) may be a viral vector. Such a viral vector may be an adenovirus (of a
human serotype such as AdHu5, a simian serotype such as ChAd63, ChAdOX1 or ChAdOX2,
or another form), an adena-associated virus (AA V), or a poxvirus vector (such as a modified
vaccinia Ankara (MV A)), or an adeno associated virus (AA V). ChAdOX1 and ChAdOX2 are
30 disclosed in W02012/172277 (herein incorporated by reference in its entirety). ChAdOX2 is
a BAC-derived and E4 modified AdC68-based viral vector. Preferably said viral vector is an
AA V vector adenovirus.
Viral vectors are usually non-replicating or replication impaired vectors, which means
that the viral vector cannot replicate to any significant extent in normal cells (e.g. normal
wo 2021/165667 19 PCT/GB2021/050383
human cells), as measured by conventional means- e.g. via measuring DNA synthesis and/or
viral titre. Non-replicating or replication impaired vectors may have become so naturally (i.e.
they have been isolated as such from nature) or artificially (e.g. by breeding in vitro or by
genetic manipulation). There will generally be at least one cell-type in which the replication-
S impaired viral vector can be grown - for example, modified vaccinia Ankara (MV A) can be
grown in CEF cells. By way of non-limiting example, the vector may be selected from a human
or simian adenovirus or a poxvirus vector.
Typically, the viral vector is incapable of causing a significant infection in an animal
subject, typically in a mammalian subject such as a human or other primate.
10 The vector(s) may be a DNA vector, such as a DNA plasmid. The vector(s) may be an
RNA vector, such as a mRNA vector or a self-amplifying RNA vector. The DNA and/or RNA
vector(s) of the invention may be capable of expression in eukaryotic and/or prokaryotic cells,
particularly any host cell type described herein, or in a subject to be treated.
Typically the DNA and/or RNA vector(s) are capable of expression in a human, E. coli
15 or yeast cell.
The present invention may be a phage vector, such as an AA V /phage hybrid vector as
described in Hajitou et al., Cell 2006; 125(2) pp. 385-398; herein incorporated by reference.
The nucleic acid molecules of the invention may be made using any suitable process
known in the art. Thus, the nucleic acid molecules may be made using chemical synthesis
20 techniques. Alternatively, the nucleic acid molecules of the invention may be made using
molecular biology techniques.
Vector(s) ofthe present invention may be designed in silica, and then synthesised by
conventional polynucleotide synthesis techniques.
25 Virus-Like Particles
Virus-like particles (VLPs) are particles which resemble viruses but do not contain viral
nucleic acid and are therefore non-infectious. They commonly contain one or more virus
capsid or envelope proteins which are capable of self-assembly to form the VLP. VLPs have
been produced from components of a wide variety of virus families (Noad and Roy (2003),
30 Trends in Microbiology, 11 :438-444; Grgacic et al., (2006), Methods, 40:60-65). Some VLPs
have been approved as therapeutic vaccines, for example Engerix-B (for hepatitis B), Cervarix
and Gardasil (for human papilloma viruses).
Accordingly, the invention provides a VLP comprising a 2019-nCoV spike protein or
immunogenic fragment thereof of the invention. A VLP of the invention typically comprises
wo 2021/165667 20 PCT/GB2021/050383
a spike protein from 2019-nCoV having at least 70%, at least 75%, at least 80%, at least 90%,
at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ
ID NO: 1, or a fragment thereof, that has a common antigenic cross-reactivity with said spike
protein. Preferably a VLP of the invention comprises a spike protein from 2019-nCoV having
5 at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity
with SEQ ID NO: 1, or a fragment thereof, that has a common antigenic cross-reactivity with
said spike protein. More preferably, a VLP of the invention comprises a spike protein from
2019-nCoV having least 98%, at least 99% or more with SEQ ID NO: 1, or a fragment thereof,
that has a common antigenic cross-reactivity with said spike protein. A VLP of the invention
10 may comprise a spike protein from 2019-nCoV comprising or consisting of SEQ ID NO: 1, or
a fragment thereof, that has a common antigenic cross-reactivity with said spike protein. In
some preferred embodiments, the immunogenic fragment of the 2019-nCoV spike protein
comprised in a VLP of the invention is an RBD of the 2019-nCoV spike protein as defined
herein, preferably wherein said RBD has at least 90% identity with SEQ ID NO: 15.
15 The skilled person will understand that VLPs can be synthesized through the individual
expression of viral structural proteins, which can then self-assemble into the virus-like
structure. Combinations of structural capsid proteins from different viruses can be used to
create recombinant VLPs. In additions, antigens or immunogenic fragments thereof can be
fused to the surface of VLPs. By way of non-limiting example, antigens or immunogenic
20 fragments thereof of the invention may be coupled to a VLP using the SpyCatcher-SpyTag
system (as described by Brune, Biswas, Howarth).
A VLP of the invention may comprise one or more additional protein antigen. The one
or more additional antigen may be the same antigen or a different antigen from 2019-nCoV, or
a fragment thereof More preferably, said one or more additional antigen is a different antigen
25 from 2019-nCoV, such as an antigen from the 2019-CoV membrane protein or envelope
protein.
A VLP of the invention may comprise a fusion protein as described herein. A VLP of
the invention may comprise a fusion protein of the 2019-nCoV spike protein or immunogenic
fragment thereof with Hepatitis B surface antigen (HBSAg), human papillomavirus (HPV) 18
30 L1 protein, HPV 16 L1 protein and/or Hepatitis E P239, preferably Hepatitis B surface antigen.
Although these other viral proteins have been described in fusion proteins previous, to-date,
there are no reports of fusion proteins being successfully generated comprising proteins of the
size of 2019-nCoV spike protein. Furthermore, there are known limitations regarding the
choice of expression systems for such fusion proteins. The present inventors have surprisingly
wo 2021/165667 21 PCT/GB2021/050383
demonstrated that VLPs/fusion proteins comprising 2019-nCoV spike protein can be produced
recombinantly in E. coli, yeast and human cells, and that these VLPs/fusion proteins can elicit
an (immunoprotective) antibody response in animal models.
Thus, a VLP of the invention may be encoded by a polynucleotide which comprises or
5 consists of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to any
one of SEQ ID NOs: 3, 5, 6 or 8. Preferably a VLP of the invention may be encoded by a
polynucleotide which comprises or consists of a nucleic acid sequence having at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to any one of
10 SEQ ID NOs: 3, 5, 6 or 8. More preferably, a VLP of the invention may be encoded by a
polynucleotide which comprises or consists of a nucleic acid sequence having at least 98%, at
least 99% or more identity to any one of SEQ ID NOs: 3, 5, 6 or 8. A VLP of the invention
may be encoded by a polynucleotide which comprises or consists of a nucleic acid sequence of
any one of SEQ ID NOs: 3, 5, 6 or 8.
15 A VLP of the invention may be encoded by a polynucleotide which comprises or
consists of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to any
one of SEQ ID NOs: 26, 27, 29,30, or 32. Preferably a VLP of the invention may be encoded
by a polynucleotide which comprises or consists of a nucleic acid sequence having at least
20 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to any
one of SEQ ID NOs: 26, 27, 29, 30, or 32. More preferably, a VLP of the invention may be
encoded by a polynucleotide which comprises or consists of a nucleic acid sequence having at
least 98%, at least 99% or more identity to any one of SEQ ID NOs: 26, 27, 29, 30, or 32. A
VLP of the invention may be encoded by a polynucleotide which comprises or consists of the
25 nucleic acid sequence of any one of SEQ ID NOs: 26, 27, 29, 30, or 32.
A VLP of the invention may comprise or consist of an amino acid sequence having at
least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% or more identity to any one of SEQ ID NO: 9, 10, 11 or 12. Preferably
a VLP of the invention may comprise or consist of an amino acid sequence having at least 90%,
30 at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to any one
of SEQ ID NOs: 9, 10, 11 or 12. More preferably, a VLP of the invention may comprises or
consists of an amino acid sequence having at least 98%, at least 99% or more identity to any
one of SEQ ID NOs: 9, 10, 11 or 12. A VLP of the invention may comprise or consist of an
amino acid sequence of any one of SEQ ID NOs: 9, 10, 11 or 12.
wo 2021/165667 22 PCT/GB2021/050383
A VLP of the invention may comprise or consist of an amino acid sequence having at
least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% or more identity to any one of SEQ ID NOs: 28,31, or 33. Preferably
a VLP of the invention may comprise or consist of an amino acid sequence having at least 90%,
5 at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to any one
of SEQ ID NOs: 28, 31, or 33. More preferably, a VLP of the invention may comprises or
consists of an amino acid sequence having at least 98%, at least 99% or more identity to any
one of SEQ ID NOs: 28, 31, or 33. A VLP of the invention may comprise or consist of an
amino acid sequence of any one of SEQ ID NOs: 28, 31, or 33.
10 The use of VLP may increase the efficacy of the immunoprotective response induced
by the 2019-nCoV spike protein or immunogenic fragment and/or may increase the duration
of the immunoprotective response as defined herein.
Fusion Proteins
15 The invention further provides a fusion protein comprising a 2019-nCoV spike protein
or immunogenic fragment thereof of the invention. A fusion protein of the invention typically
comprises a spike protein from 2019-nCoV having at least 70%, at least 75%, at least 80%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity
with SEQ ID NO: 1, or a fragment thereof, that has a common antigenic cross-reactivity with
20 said spike protein. Preferably a fusion protein of the invention comprises a spike protein from
2019-nCoV having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or more identity with SEQ ID NO: 1, or a fragment thereof, that has a common antigenic
cross-reactivity with said spike protein. More preferably, a fusion protein of the invention
comprises a spike protein from 2019-nCoV having least 98%, at least 99% or more with SEQ
25 ID NO: 1, or a fragment thereof, that has a common antigenic cross-reactivity with said spike
protein. A fusion protein of the invention may comprise a spike protein from 2019-nCoV
comprising or consisting of SEQ ID NO: 1, or a fragment thereof, that has a common antigenic
cross-reactivity with said spike protein. In some preferred embodiments, the immunogenic
fragment of the 2019-nCoV spike protein comprised in a fusion protein of the invention is an
30 RBD of the 2019-nCoV spike protein as defined herein, preferably wherein said RBD has at
least 90% identity with SEQ ID NO: 15.
A fusion protein of the invention typically also comprises a non-2019-nCoV domain or
element, typically a non-2019-nCoV protein, polypeptide or peptide domain or element. A
fusion protein of the invention may comprise the 2019-nCoV spike protein or immunogenic
wo 2021/165667 23 PCT/GB2021/050383
fragment thereof and one or more of: Hepatitis B surface antigen (HBSAg); human
papillomavirus (HPV) 18 L1 protein; HPV 16 L1 protein; and/or Hepatitis E P239 (HEV),
preferably Hepatitis B surface antigen. As described above in the context of VLPs, the present
inventors have surprisingly demonstrated that fusion proteins comprising 2019-nCoV spike
5 protein can be produced recombinantly in E. coli, yeast and human cells, and that these fusion
proteins can elicit an (immunoprotective) antibody response in animal models.
A fusion protein of the invention may be encoded by a polynucleotide which comprises
or consists of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to any
10 one of SEQ ID NO: 3, 5, 6 or 8. Preferably a fusion protein of the invention may be encoded
by a polynucleotide which comprises or consists of a nucleic acid sequence having at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to any
one of SEQ ID NOs: 3, 5, 6 or 8. More preferably, a fusion protein of the invention may be
encoded by a polynucleotide which comprises or consists of a nucleic acid sequence having at
15 least 98%, at least 99% or more identity to any one of SEQ ID NOs: 3, 5, 6 or 8. A VLP of the
invention may be encoded by a polynucleotide which comprises or consists of a nucleic acid
sequence of any one of SEQ ID NOs: 3, 5, 6 or 8.
A fusion protein of the invention may be encoded by a polynucleotide which comprises
or consists of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least
20 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to any
one of SEQ ID NOs: 26, 27, 29,30, or 32. Preferably a fusion protein of the invention may be
encoded by a polynucleotide which comprises or consists of a nucleic acid sequence having at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity
to any one of SEQ ID NOs: 26, 27, 29, 30, or 32. More preferably, a fusion protein of the
25 invention may be encoded by a polynucleotide which comprises or consists of a nucleic acid
sequence having at least 98%, at least 99% or more identity to any one of SEQ ID NOs: 26,
27, 29, 30, or 32. A fusion protein of the invention may be encoded by a polynucleotide which
comprises or consists of the nucleic acid sequence of any one of SEQ ID NOs: 26, 27, 29, 30,
or 32.
30 A fusion protein of the invention may comprise or consist of an amino acid sequence
having at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or more identity to any one of SEQ ID NO: 9, 10, 11 or 12.
Preferably a fusion protein of the invention may comprise or consist of an amino acid sequence
having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more
wo 2021/165667 24 PCT/GB2021/050383
identity to any one of SEQ ID NOs: 9, 10, 11 or 12. More preferably, a fusion protein of the
invention may comprises or consists of an amino acid sequence having at least 98%, at least
99% or more identity to any one of SEQ ID NOs: 9, 10, 11 or 12. A fusion protein of the
invention may comprise or consist of an amino acid sequence of any one of SEQ ID NOs: 9,
5 10

CLAIMS
1. An isolated polynucleotide encoding a spike protein from 2019-nCoV having at least
90% identity with SEQ ID NO: 1, or a fragment thereof that has a common antigenic
5 cross-reactivity with said spike protein, wherein said polynucleotide is optimised for
recombinant expression.
10
15
2. The polynucleotide of claim 1, which is optimised for expression in a host cell selected
from:
3.
(a) Escherichia coli;
(b) yeast, preferably Komagataella or Saccharomyces; and/or
(c) mammalian cells, preferably human cells.
The polynucleotide of claim 1 or 2, wherein one or more cis-acting sequence motif is
omitted, said one or more cis-acting sequence motif being independently selected from:
(a) an internal TAT A-box;
(b) a chi-site;
20 (c) a ribosomal entry site;
25
30
4.
(d) an AT -rich and/or GC-rich stretch of sequence;
(e) an RNA instability motif;
(f) a repeat sequence and/or an RNA secondary structure;
(g) a cryptic splice donor site;
(h) a cryptic splice acceptance site; and/or
(i) any combination of (a) to (i).
The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide integrates
into the host cell genome.
5. The polynucleotide of any one of claims 1 to 4, which has a codon adaptation index (CAl)
of at least about 0.80, preferably at least about 0.9, more preferably at least about 0.93.
wo 2021/165667 78 PCT/GB2021/050383
6. The polynucleotide of any one of claims 1 to 5, which comprises or consists of a nucleic
acid sequence having:
(a) at least 90% identity to SEQ ID NO: 2;
5 (b) at least 90% identity to SEQ ID NO: 3;
(c) at least 90% identity to SEQ ID NO: 4;
(d) at least 90% identity to SEQ ID NO: 5;
(e) at least 90% identity to SEQ ID NO: 6;
(f) at least 90% identity to SEQ ID NO: 7;
10 (g) at least 90% identity to SEQ ID NO: 8
(h) at least 90% identity to SEQ ID NO: 13
(i) at least 90% sequence identity to SEQ ID NO: 14
G) at least 90% identity to SEQ ID NO: 26
(k) at least 90% identity to SEQ ID NO: 27
15 (1) at least 90% identity to SEQ ID NO: 29;
(m)at least 90% identity to SEQ ID NO: 30; or
(n) at least 90% identity to SEQ ID NO: 32.
7.
20
The polynucleotide of any one of claims 1 to 6, wherein the encoded spike protein, or
fragment thereof:
(a) retains the conformational epitopes present in the native 2019-nCoV spike protein;
(b) results in the production of neutralising antibodies specific for the spike protein or
fragment thereof when the nucleic acid or the encoded spike protein or fragment
25 thereof is administered to a subject; and/or
30
8.
(c) comprises or consists of receptor-binding domain (RBD) of the 2019-nCoV spike
protein, preferably having at least 90% identity with SEQ ID NO: 15.
An expression construct comprising polynucleotide of any one of claims 1 to 7, operably
linked to a promoter.
9. A vaccine composition comprising a spike protein from 2019-nCoV having at least 90%
identity with SEQ ID NO: 1, or a fragment thereof, that has a common antigenic crossreactivity
with said spike protein, wherein optionally said fragment comprises or consists
wo 2021/165667 79 PCT/GB2021/050383
of receptor-binding domain (RBD) of the 2019-nCoV spike protein, preferably having at
least 90% identity with SEQ ID NO: 15.
10. The composition of claim 9, which results in the production of neutralising antibodies
5 specific for the spike protein or fragment thereof when administered to a subject.
11. A viral vector, RNA vaccine or DNA plasmid that expresses a spike protein from 2019-
nCoV having at least 90% identity with SEQ ID NO: 1, or a fragment thereof, that has a
common antigenic cross-reactivity with said spike protein, wherein optionally said
10 fragment comprises or consists of receptor-binding domain (RBD) of the 2019-nCoV
spike protein, preferably having at least 90% identity with SEQ ID NO: 15.
15
12. The viral vector, RNA vaccine or DNA plasmid of claim 11, which expresses the spike
protein or fragment thereof, further comprising a signal peptide.
13. The viral vector, RNA vaccine or DNA plasmid of claim 12, wherein the signal peptide
directs secretion from human cells.
14. The viral vector, RNA vaccine or DNA plasmid of any one of claims 11 to 13, wherein
20 the viral vector, RNA vaccine or DNA plasmid further expresses one or more additional
antigen or a fragment thereof, preferably one or more additional antigen from 2019-
nCo V, or a fragment thereof
15. The viral vector, RNA vaccine or DNA plasmid of claim 14, wherein the spike protein
25 or fragment thereof and the one or more additional antigen or fragment thereof are
expressed:
30
(a)
(b)
as a fusion protein; or
in separate viral vectors, RNA vaccmes or DNA plasmids for use m
combination.
16. The viral vector, RNA vaccine or DNA plasmid of any one of claims 11 to 15, which
comprises one or more polynucleotide as defined in any one of claims 1 to 7 or an
expression construct of claim 8.
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17. A fusion protein comprising a spike protein from 20 19-nCo V having at least 90% identity
with SEQ ID NO: 1, or a fragment thereof, that has a common antigenic cross-reactivity
with said spike protein, wherein optionally said fragment comprises or consists of
5 receptor-binding domain (RBD) of the 2019-nCoV spike protein, preferably having at
least 90% identity with SEQ ID NO: 15.
10
15
18. The fusion protein of claim 17, which further comprises:
(a)
(b)
(c)
(d)
the Hepatitis B surface antigen, or a fragment thereof that has a common
antigenic cross-reactivity with said Hepatitis B surface antigen;
the HPV 18 L 1 protein, or a fragment thereof that has a common antigenic crossreactivity
with said HPV 18 L 1 protein;
the Hepatitis E P239 protein, or a fragment thereof that has a common antigenic
cross-reactivity with said Hepatitis E P239 protein; and/or
the HPV 16 L 1 protein, or a fragment thereof that has a common antigenic crossreactivity
with said HPV 16 L 1 protein;
wherein optionally:
20 (i) the fusion protein is encoded by a polynucleotide which comprises or consists
of a nucleic acid sequence having at least 90% identity with any one of SEQ ID NO: 3,
5, 6, 8, 26, 27, 29,30, or 32; and/or
25
(ii) the fusion protein comprises of consists of an amino acid sequence having at
least 90% identity with any one of SEQ ID NO: 9, 10, 11, 12, 28,31, or 33.
19. A virus-like particle (VLP) comprising a spike protein from 2019-nCoV having at least
90% identity with SEQ ID NO: 1, or a fragment thereof, that has a common antigenic
cross-reactivity with said spike protein, wherein optionally said fragment comprises or
consists of receptor-binding domain (RBD) of the 20 19-nCo V spike protein, preferably
30 having at least 90% identity with SEQ ID NO: 15;
wherein optionally said VLP comprises or consists of a fusion protein as defined in claim
17 or 18.
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20. An antibody, or binding fragment thereof, that specifically binds to a 2091-nCoV spike
protein antigen, or fragment thereof, as defined in claim 1.
21. The antibody, or binding fragment thereof, of claim 20, wherein the antibody IS a
5 monoclonal or polyclonal antibody.
22. The antibody, or binding fragment thereof, of claim 20 or 21, wherein the antibody is an
Fab, F(ab')2, Fv, scFv, Fd or dAb.
10 23. An oligonucleotide aptamer that specifically binds to a 2019-nCoV spike protein or
15
fragment thereof as defined in any claim 1.
24. A vaccine composition comprising the viral vector, and/or RNA vaccine and/or DNA
plasmid of any one of claims 11 to 16.
25. The polynucleotide of any one of claims 1 to 7, and/or the expression construct of claim
8, and/or vaccine composition of any one of claims 9, 10 and/or 24, and/or the viral vector
and/or RNA vaccine and/or DNA plasmid of any one of claims 11 to 16, and/or the viruslike
particle of claim 19, and/or the fusion protein of claim 17 or 18, and/or the antibody
20 of any one of claims 20 to 22 and/or the aptamer of claim 23 for use in the treatment
and/or prevention of 2019-nCoV infection.
26. Use of the polynucleotide of any one of claims 1 to 7, and/or the expression construct of
claim 8, and/or vaccine composition of any one of claims 9, 10 and/or 24, and/or the viral
25 vector and/or RNA vaccine and/or DNA plasmid of any one of claims 11 to 16, and/or
the virus-like particle of claim 19, and/or the fusion protein of claim 17 or 18, and/or the
antibody of any one of claims 20 to 22 and/or the aptamer of claim 23 in the manufacture
of a medicament for the prevention and/or treatment of2019-nCoV infection.
30 27. A method of producing a spike protein from 2019-nCoV having at least 90% identity
with SEQ ID NO: 1, or a fragment thereof, comprising expressing a polynucleotide as
defined in any one of claims 1 to 7 in a host cell, and optionally purifying the spike
protein or fragment.