# Patent

## Title
Chimeric protein for coronavirus vaccine

## Abstract

The disclosure relates to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9 or a sequence having at least 97%-100% sequence identity to one of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9 for use as an immunogen for the purpose of eliciting an immune response in a subject susceptible to infection with a coronavirus. The disclosed polypeptide is further useful in reducing the severity of symptoms associated with a coronavirus infection. In addition to use in a protein-based vaccine, the polypeptide of the disclosure can be encoded by a nucleic acid/ribonucleic acid and used in a nucleic acid vaccine or viral vector vaccine.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the priority of U.S. Provisional application No. 63/056,377 filed Jul. 24, 2020, the contents of which are hereby incorporated by reference in their entirety into the instant disclosure.


FIELD OF THE DISCLOSURE
The disclosure relates to an immunogenic polypeptide and compositions for treatment of or protection from infection by coronavirus. In particular, the disclosure relates to a chimeric polypeptide for immunizing subjects against coronavirus, such as SARS CoV-2.
BACKGROUND
The desperate need for a COVID-19 vaccine has given rise to over 160 vaccine candidates that are currently under study around the world. These utilize four main platforms: RNA vaccines, DNA vaccines, recombinant protein vaccines, and vectored vaccines, each of which has specific advantages and disadvantages (reviewed in Corey et al. A strategic approach to COVID-19 vaccine R & D. Science 368:948-950, 2020, which is herein incorporated by reference in its entirety into the instant disclosure).
Nucleic acid vaccines can be generated rapidly once a viral target sequence is identified. However, while there had been a fair amount of data on the use of nucleic acid vaccines in early-phase clinical tests, prior to development and emergency use of covid-2 vaccines by Pfizer/BioNTech and Moderna, none had been approved for widespread use.
Replication-defective adenoviral vectors are generally safe and immunogenic but pre-existing immunity to the vector can hamper immunogenicity.
Recombinant protein vaccines (influenza, papillomavirus, hepatitis B, varicella-zoster) are in widespread use in the human population but require more time to manufacture than nucleic acid vaccines.
The majority of COVID-19 vaccine candidates target the Spike (S) glycoprotein of SARS-CoV-2. The S glycoprotein is a favored target because it is generally accepted that neutralizing antibodies against it play a predominant role in protection from infection. SARS-CoV-2 has, however, developed multiple strategies to evade or by-pass the immune system.
The S glycoprotein has at least two such strategies. First, a dense glycan shield covers the region in the S glycoprotein that makes contact with the cell's receptor for entry (Wrapp et al. 2020). Second, the S protein contains immunodominant sequences that induce antibodies that are not neutralizing (He et al. 2004; 2006). An effective vaccine will have to circumvent the immune evasion strategies of this virus.
To overcome problems with the prior art approaches, the instant disclosure provides an alternative strategy, in particular with respect to designing a suitable immunogen that overcomes the immune avoidance strategies of the virus, and induces a robust and protective immune response.
SUMMARY OF THE DISCLOSURE
In one aspect, the disclosure relates to a recombinant, chimeric, non-naturally occurring polypeptide consisting of or comprising the amino acid sequence of SEQ ID NO: 1 (see Table 1 below) or a sequence having at least 97% sequence identity to SEQ ID NO: 1. The polypeptide comprises a modified Spike receptor-binding domain (RBD) and an HA2 sequence lacking the transmembrane domain. Using methods known to those of skill in the art, the polypeptide can be expressed in a variety of vaccine platforms.
In another aspect, the disclosure relates to a polypeptide comprising consisting of the sequence of SEQ ID NO: 5 (see Table 1) or a sequence having at least 97% sequence identity to SEQ ID NO: 5. The polypeptide comprises the modified S RBD, the HA2 sequence lacking the transmembrane domain plus an HA signal sequence, which enables the polypeptide to be expressed as a secreted molecule.
In yet another aspect, the disclosure relates to a polypeptide comprising or consisting of the sequence of SEQ ID NO: 6 (see Table 1) or a sequence having at least 97% sequence identity to SEQ ID NO: 6. This polypeptide comprises the modified S RBD sequence, an HA signal sequence, the HA2 sequence including the transmembrane domain.
In another aspect, the disclosure relates to a polypeptide comprising or consisting of the sequence of SEQ ID NO: 9 (see Table 1) or a sequence having at least 97% sequence identity to SEQ ID NO: 9. This polypeptide comprises the modified S RBD sequence, an S protein signal sequence, and HA2 sequence including the transmembrane domain.
In another aspect, the disclosure relates to a nucleic acid that encodes a polypeptide of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 9 or an amino acid sequence having at least 97% sequence identity to one of SEQ ID NOS: 1, 5, 6, or 9.
In yet another aspect, the disclosure relates to a composition comprising an amino acid with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 9 or an amino acid sequence having at least 97% sequence identity to one of SEQ ID NOS: 1, 5, 6, or 9.
In yet another aspect, the disclosure relates to a vector comprising a nucleic acid that encodes an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 9 or an amino acid sequence having at least 97% sequence identity to one of SEQ ID NOS: 1, 5, 6, or 9.
In one aspect, the disclosure relates to a composition comprising a viral vector, said viral vector comprising a nucleic acid encoding a polypeptide of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 9 or an amino acid sequence having at least 95% sequence identity to one of SEQ ID NOS: 1, 5, 6, or 9.
In one aspect, the invention relates to a composition as described above, wherein said composition is a pharmaceutical composition.
In one aspect, the invention relates to a composition as described above, wherein said composition is a vaccine composition.
In one aspect, the invention relates to a composition as described above, wherein said composition is capable of inducing a protective immune response against SARS-Cov-2 in a human.









TABLE 1





SEQ




ID




NO:

Description

















1
NITNLCPFGE
An embodiment of the



VFNATRFASV
polypeptide of the



YAWNRKRISN
disclosure comprising



CVADYSVLYN
modified Spike



SASFSTFKCY
RBD and HA2 (without



GVSPTKLNDL
transmembrane domain of



CFTNVYADSF
HA2)



VIRGDEVRQI




APGQTGKIAD




YNYKLPDDFT




GCVIAWNSNN




LDSKVGGNYN




YLYRLFRKSN




LKPFERDIST




EIYQAGSTPC




NGVEGFNCYF




PLQSYGFQPT




NGVGYQPYRW




VLSFELLHAP




ATVCGPKKSG




FRHQNSEGTG




QAADLKSTQA




AIDQINGKLN




RVIEKTNEKF




HQIEKEFSEV




EGRIQDLEKY




VEDTKIDLWS




YNAELLVALE




NQHTIDLTDS




EMNKLFEKTR




RQLRENAEEM




GNGCFKIYHK




CDNACIESIR




NGTYDHDVYR




DEALNNRFQI




KGVELKSGYK




DW



 


2
NITNLGPFGE
Spike (S) RBD



VFNATRFASV




YAWNRKRISM




CVADYSVLYN




SASFSTFKCY




GVSPTKLNDL




CFTNWADSFV




IRGDEVRQIA




PGQTGKIADY




NYKLPDDFTG




CVIAWNSNNL




DSKVGGNYNY




LYRLFRKSNL




KPFERDISTE




IYQAGSTPCN




GVEGFNCYFP




LQSYGFQPTM




GVGYQPYRWV




LSFELUHAPA




TVCGPKKS



 


3
GFRHQNSEGT
HA2 minus transmembrane



GQAADLKSTG
domain residues 23-185



AAIDQINGKL




NRVIEKTNEK




FHQIEKEFSE




VEGRIQDLEK




YVEDTKIDLW




SYNAELLVAL




ENQHTIDLTD




SEMNKLFEKT




RRQLRENAEE




MGNGCFKIYH




KCDNACIESI




RNGTYDHDVY




RDEALNNRFQ




IKGVELKSGY




KDW



 


4
MKTIIALSYI
HA signal sequence



FCLALG



 


5
MKTIIALSYI
Embodiment of polypeptide



FCLALGNITN
to be expressed as secreted



LCPFGEVF
molecule



NATRFASVYA




WNRKRISNCV




ADYSVLYMSA




SFSTFKCYGV




SPTKLNDLCF




TNVYADSFVI




RGDEVRQIAP




GQTGKIADYN




YKLPDDFTGC




VIAWNSNNLD




SKVGGNYNYL




YRLFRKSNLK




PFERDISTEI




YQAGSTPCNG




VEGFNGYFPL




QSYGFQPTKG




VGYQPYRWVL




SFELLHAPAT




VCGPKKSGFR




HQNSEGTGQA




ADLKSTQAAD




QINGKLNRVI




EKTNEKFHQI




EKEFSEVEGR




IQDLEKWEDT




KIDLWSYNAE




LLVALENGHT




IDLTDSEMNK




LFEKTRRQLR




ENAEEMGNGC




FKIYHKCDNA




CIESIRNGTY




DHDVYRDEAL




NNRFQIKGVE




LKSGYKDW



 


6
MKTIIALSYI
Embodiment of polypeptide



FCLALGNITN
to be expressed as plasma



LCPFGEVFNA
membrane-bound molecule



TRFASWAWNR




KRISNCVADY




SVLYNSASFS




TFKCYGVSPT




KLNOLCFTNV




YADSFVIRGD




EVRQIAPGQT




GKIADYNYKL




PDDFTGCVIA




WNSNNLDSKV




GGNYNYLYRL




FRKQNLKPFE




RDISTEIYQA




GSTPCNGVEG




FNCYFPIQSY




GFQPTNGVGY




QPYRWVLSFE




LLHAPATVCG




PKKSGFRHQN




SEGTGQMDLK




STQAAIDQIN




GKLNIRVIEK




TNEKFHQIEK




EFSEVEGRIQ




DLEKYVEDTK




IDLWSYNAEL




LVALENQHTI




DLTDSEMNKL




FEKTRRQLRE




NAEEMGNGCF




KIYHKCDNAC




IESIRNGTYD




HDVYRDEALN




NRFQIKGVEL




KSGYKDWILW




ISFAISCFLL




CWLLGFIMWA




CQRGNIRCNI




CI



 


7
MFVFLVLLPL
Spike signal sequence



VSS



 


8
GFRHQNSEGT
HA2 sequence including



GQAADLKSTQ
transmembrane sequence



AAIDQFNGKL




NRVIEKTNEK




FHQIEKEFSE




VEGRIQDLEK




YVEDTKIDLW




SYNAELLVAL




ENQHTIDLTD




SEMNKLFEKT




RRQLRENAEE




MGNGCFKIYH




KCDNACIESI




RNGTYDHDVY




RDEALNNRFQ




IKGVELKSGY




KDWILWISFA




ISCFLLCWLL




GFIMWACQRG




NIRCNICI



 


9
MFVFLVUPLV
Polypeptide to be expressed



SSNITNLCPF
as an ER/Golgi membrane-



GEVFNATRFA
bound molecule comprising



SVYAWNRKRI
Covid-19 S protein signal



SNCVADYSVL
sequence, RBD and HA2



YNSASFSTFK
including transmembrane



CYGVSPTKLN
sequence



DLCFTNVYAD




SFVIRGDEVR




QFAPGQTGKI




ADYNYKLPDD




FTGCVIAWNS




NNLDSKVGGN




YNYLYRLFRK




SNLKPFERDI




STEIYQAGST




PCNGVEGFNC




YFPLQSYGFQ




PTNGVGYQPY




RWVLSFELLH




APATVCGPKK




SGFRHQNSEG




TGQAADLKST




QAAIDQINGK




LNRVIEKTNE




KFHQIEKEFS




EVEGRIQDLE




KYVEDTKIDL




WSYNAELLVA




LENQHTIDLT




DSEMNKLFEK




TRRQLRENAE




EMGNGCFKIY




HKCDNACIES




IRNGTYDHDV




YRDEALNNRF




QIKGVELKSG




YKDWILWISF




AISCFLLCWL




LGFIMWACQR




GNIRCNICI











DETAILED DESCRIPTION
All patents, published applications and other publications and references are hereby incorporated by reference in their entirety into the present disclosure.
As discussed above, the majority of COVID-19 vaccine candidates target the S glycoprotein of SARS-CoV-2 because it is generally accepted that neutralizing antibodies against it play a predominant role in protection from infection. SARS-CoV-2 has, however, developed multiple strategies to evade or by-pass the immune system.
The S glycoprotein has at least two such strategies. First, a dense glycan shield covers the region in the S glycoprotein that makes contact with the cell's receptor for entry (Wrapp et al. Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation. Science 367: 1260-1263, 2020, which is hereby incorporated by reference in its entirety into the instant disclosure).
Second, the S protein contains immunodominant sequences that induce antibodies that are not neutralizing (He et al. Identification of Immunodominant Sites on the Spike Protein of Severe Acute Respiratory Syndrome (SARS) Coronavirus: implication for Developing SARS Diagnostics and Vaccines. J. Immunol. 173:4050-4057, 2004; and He et al. Antigenic and immunogenic Characterization of Recombinant Baculovirus-Expressed Severe Acute Respiratory Syndrome Coronavirus Spike Protein: Implication for Vaccine Design. J. Virol. 80(12): 5757-5767, 2006; both are hereby incorporated by reference in their entirety into the instant disclosure). An effective vaccine will have to circumvent the immune evasion strategies of this virus.
The present disclosure describes a recombinant polypeptide useful for eliciting an immune response in a subject who is susceptible to infection with coronavirus. The polypeptide is a chimera comprising elements of the S protein of SARS-Cov-2 as well as a hemagglutinin (HA) moiety of influenza virus, a chimera, which can be expressed in any of four known vaccine platforms.
In designing a new vaccine candidate, three main hurdles were addressed: (1) the inherent immune evasion strategies of the S glycoprotein, (2) the necessity of manufacturing hundreds of millions of vaccine doses, and (3) protection from future coronavirus pandemics. The vaccine candidate of the disclosure is a chimeric molecule that overcomes these obstacles.
Immunogen Design
To counter the two immune evasion strategies of the native S glycoprotein, the vaccine target had to be modified. This was done by removing the amino-terminal sequence of Spike, which is heavily glycosylated, along with the carboxy-terminal sequence that induces non-neutralizing antibodies. The resulting vaccine target is a modified RBD (receptor-binding domain) containing multiple neutralization antigenic sites and just two glycosylation sites. (RBD portion of Spike glycoprotein of Covid-19: Genbank:QHU79173.2 surface glycoprotein [Severe acute respiratory syndrome coronavirus 2])
The next step involved choosing a protein stalk for the modified RBD that is capable of trimerization since trimers are generally more immunogenic than monomers. The protein stalk was also chosen with scale-up in mind. The HA2 moiety of influenza virus (HA2 of influenza A can be found at the following url: uniprot.org/uniprot/P03437:>sp|P03437|HEMA_I68A0 Hemagglutinin OS=Influenza A virus (strain A/Aichi/2/1968 H3N2) OX=387139 GN=HA PE=1 SV=1) was chosen as the stalk because HA2 is also immunologically quiescent, allowing for focusing of the immune response to the neutralization antigenic sites in the RBD. In one embodiment, the resulting chimeric protein consists of the RBD of SARS-CoV-2 tethered to the HA2 moiety of influenza virus. Importantly, since about half of the chimeric protein is hemagglutinin, scale-up for a protein vaccine could utilize existing technologies that produce massive doses of flu vaccines comprising the hemagglutinin molecule (Flublok, Flucelvax).
In one embodiment, the chimeric protein molecule was subsequently refined using a molecular modeling approach. Recent work by Wrapp et al. (Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation. Science 367: 1260-1263, 2020) resolved the structure of the S trimer in its pre-fusion state. The predominant state of the trimer has one of the three RBDs rotated up which allows the display of neutralization sensitive epitopes. The chimeric molecule described herein retains the pre-fusion state of RBD for display of epitopes and the post-fusion state of HA2 for trimeric structure.
An initial study to evaluate the immunogenicity of the disclosed polypeptide on a self-amplifying RNA vaccine platform was performed. Mice were immunized intramuscularly with the RNA vaccine version of the disclosed polypeptide and blood samples were collected two weeks later. Serum samples were assayed by ELISA for RBD-specific IgG antibodies. Mice immunized with the chimeric protein vaccine induced a robust RBD-specific Ab response (data not shown.)
Four weeks after immunization, serum samples were assayed for neutralization activity against SARS-CoV-2 using a plaque reduction assay. The chimeric protein vaccine induced a strong neutralizing antibody response.
Lastly, the design of the disclosed chimeric protein lends itself to a pan-coronavirus vaccine strategy. For example, the RBD sequence of SARS-CoV-2 could easily be swapped out of the polypeptide of the disclosure and replaced with that of other coronaviruses, SARS-CoV-3, -4, -5 etc., to generate vaccines for subsequent pandemics.

## Claims

1. A recombinant polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

2. An isolated nucleic acid that encodes the recombinant polypeptide of claim 1.

3. The isolated nucleic acid of claim 2, wherein the isolated nucleic acid is DNA.

4. The isolated nucleic acid of claim 2, wherein the isolated nucleic acid is RNA.

5. A composition comprising the recombinant polypeptide of claim 1 and a pharmaceutically acceptable carrier.

6. A method of reducing the likelihood of infection of a subject with a coronavirus comprising administering to said subject a therapeutically effective amount of a composition of claim 5.