WO2021229448A1

WO2021229448A1 - Rna replicon encoding a stabilized corona virus spike protein

Info

Publication number: WO2021229448A1
Application number: PCT/IB2021/054022
Authority: WO
Inventors: Jason DEHART; Christian MAINE; Brett Steven MARRO; Johannes Petrus Maria Langedijk; Lucy RUTTEN; Ronald Vogels; Marijn Van Der Neut Kolfschoten; Jaroslaw JURASZEK; Aneesh VIJAYAN
Original assignee: Janssen Pharmaceuticals, Inc.
Priority date: 2020-05-11
Filing date: 2021-05-11
Publication date: 2021-11-18
Also published as: MX2022014167A; US20210347828A1; AU2021271300A1; JP2023525785A; BR112022022942A2; KR20230009489A; CA3183498A1; CN116096409A; EP4149537A1

Abstract

RNA replicons encoding stabilized recombinant pre-fusion SARS CoV-2 S proteins are described. Also described are pharmaceutical compositions and uses of the RNA replicons.

Description

RNA Replicon Encoding a Stabilized Corona Virus Spike Protein Cross-Reference to Related Application

This application claims priority to U.S. Provisional Application No. 63/023,150, filed on May 11, 2020, the disclosure of which is incorporated herein by reference in its entirety.

Reference to Sequence Listing Submitted Electronically

This application contains a sequence, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “JPI6050WOPCTl_Sequence_Listing” and a creation date of April 26, 2021 and having a size of 2.09 MB. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

Field of Invention

The present invention relates to the field of medicine. The invention, in particular, relates to a self-replicating RNA encoding a stabilized recombinant pre-fusion Corona virus spike (S) protein, in particular a SARS CoV-2 S protein, and uses thereof, e.g., in vaccines.

Background of the Invention

RNA replicons are replicons derived from RNA viruses, from which at least one gene encoding an essential structural protein has been deleted. See, e.g., Zimmer, Viruses, 2010, 2(2): 413-434. They are unable to produce infectious progeny but still retain the ability to replicate the viral RNA and transcribe the viral RNA polymerase. Genetic information encoded by the RNA replicon can be amplified many times, resulting in high levels of antigen expression. Additionally, replication/transcription of replicon RNA is strictly confined to the cytosol, and does not require any cDNA intermediates, nor is any recombination with or integration into the chromosomal DNA of the host required.

Corona viruses (CoVs) are enveloped viruses responsible for mild respiratory tract infections and atypical pneumonia in humans. CoVs are a large family of enveloped, single- stranded positive-sense RNA viruses belonging to the order Nidovirales, which can infect a broad range of mammalian and avian species, causing respiratory or enteric diseases. Corona viruses possess large, trimeric spike glycoproteins (S) that mediate binding to host cell receptors as well as fusion of viral and host cell membranes. SARS-CoV-2 is a corona virus that emerged in humans from an animal reservoir in 2019 and rapidly spread globally. SARS-CoV-2 is a beta-coronavirus, like MERS-CoV and SARS- CoV, all of which have their origin in bats. The name of the disease caused by the virus is corona virus disease 2019, abbreviated as COVID-19. Symptoms of COVID-19 range from mild symptoms to severe illness and death for confirmed COVID-19 cases. In the case of SARS-CoV- 2, the S protein is the major surface protein. The S protein forms homotrimers and is composed of an N-terminal SI subunit and a C-terminal S2 subunit, responsible for receptor binding and membrane fusion, respectively. Recent cryogenic electron microscopy (cryoEM) reconstructions of the CoV trimeric S structures of alpha-, beta-, and delta-coronaviruses revealed that the SI subunit comprises two distinct domains: an N-terminal domain (SI NTD) and a receptor-binding domain (S 1 RBD). SARS-CoV-2 makes use of its S 1 RBD to bind to human angiotensin converting enzyme 2 (ACE2) (Hoffmann et. al. (2020); Wrapp et. al. (2020)).

Corona viridae S proteins are classified as class I fusion proteins and are responsible for fusion. The S protein fuses the viral and host cell membranes by irreversible protein refolding from the labile pre-fusion conformation to the stable post-fusion conformation. Like many other class I fusion proteins, Corona virus S protein requires receptor binding and cleavage for the induction of conformational change that is needed for fusion and entry (Belouzard et al. (2009); Follis et al. (2006); Bosch et al. (2008), Madu et al. (2009); Walls et al. (2016)). Priming of SARS-CoV2 involves cleavage of the S protein by ftirin at a ftirin cleavage site at the boundary between the SI and S2 subunits (S1/S2), and by TMPRSS2 at a conserved site upstream of the fusion peptide (S2’) (Bestle et al. (2020); Hoffmann et. al. (2020)).

In order to refold from the pre-fusion to the post-fusion conformation, there are two regions that need to refold, which are referred to as the refolding region 1 (RRl) and refolding region 2 (RR2) (FIG. 1). For all class I fusion proteins, the RRl includes the fusion protein (FP) and heptad repeat 1 (HR1). After cleavage and receptor binding the stretch of helices, loops and strands of all three protomers in the trimer transform to a long continuous trimeric helical coiled coil. The FP, located at the N-terminal segment of RRl, is then able to extend away from the viral membrane and inserts in the proximal membrane of the target cell. Next, the refolding region 2 (RR2), which is located C-terminal to RRl, and closer to the transmembrane region (TM) and which includes the heptad repeat 2 (HR2), relocates to the other side of the fusion protein and binds the HR1 coiled-coil trimer with the HR2 domain to form the six-helix bundle (6HB).

When viral fusion proteins, like the SARS CoV-2 S protein, are used as vaccine components, the fusogenic function of the proteins is not important. In fact, only the mimicry of the vaccine component to the virus is important to induce reactive antibodies that can bind the virus. Therefore, for development of robust efficacious vaccine components it is desirable that the meta-stable fusion proteins are maintained in their pre-fusion conformation. It is believed that a stabilized fusion protein, such as a SARS CoV-2 S protein, in the pre-fusion conformation can induce an efficacious immune response.

In recent years, several attempts have been made to stabilize various class I fusion proteins, including Corona virus S proteins. A particularly successful approach was shown to be the stabilization of the so-called hinge loop at the end of RRl preceding the base helix (WO2017/037196, Krarup et al. (2015); Rutten et al. (2020), Hastie et al. (2017)). This approach has also proved successful for Corona virus S proteins, as shown for SARS-CoV, MERS-CoV and SARS-CoV2 (Pallesen et al. (2016); Wrapp et al. (2020)). Although the proline mutations in the hinge loop indeed increase the expression of the Corona virus S protein, the S protein may still suffer from instability. Thus, for improved vaccine design of S proteins which can for example be used as tools, e.g. as a bait for monoclonal antibody isolation, further stabilization is desired.

Since the novel SARS-CoV-2 virus was first observed in humans in late 2019, over 150 million people have been infected and more than 3 million have died as a result of COVID-19, in particular because SARS-CoV-2, and corona viruses more generally, lack effective treatment. In addition, there is currently no vaccine available to prevent coronavirus induced disease (COVID- 19), leading to a large unmet medical need. Since emerging infectious diseases, such as COVID- 19, present a major threat to public health and economic systems, there is an urgent need for novel components that can be used, e.g., in vaccines to prevent coronavirus induced respiratory disease.

Summary of the Invention

The present invention provides an RNA replicon, also referred to as a self-replicating RNA molecule, encoding a stabilized pre-fusion SARS CoV-2 S protein, e.g., SARS CoV-2 S protein that is stabilized in the pre-fusion conformation, or a fragment or variant thereof.

In certain embodiments, the pre-fusion SARS CoV-2 S proteins encoded by the RNA replicon are soluble proteins, preferably trimeric soluble proteins.

In certain embodiments, an RNA replicon of the application comprises, ordered from the 5’- to 3’-end:

(1) a 5’ untranslated region (5’-UTR) required for nonstructural protein-mediated amplification of an RNA virus; (2) a polynucleotide sequence encoding at least one, preferably all, of non-structural proteins of the RNA virus;

(3) a subgenomic promoter of the RNA virus;

(4) a polynucleotide sequence encoding the recombinant pre-fusion SARS CoV-2 S protein or the fragment or variant thereof; and

(5) a 3’ untranslated region (3’-UTR) required for nonstructural protein-mediated amplification of the RNA virus.

In certain embodiments, the self-replicating RNA molecule is an alphavirus-derived RNA replicon. In certain embodiments, the RNA replicon comprises one or more alphavirus non structural protein genes. In certain embodiments, the RNA replicon comprises genetic elements required for RNA replication and lacks those genetic elements encoding gene products necessary for viral particle assembly, and the RNA replicon is delivered to a subject in a composition containing no viral protein, such as in a lipid composition (e.g., a lipid nanoparticle) or another suitable composition. In other embodiments, the RNA replicon comprises genetic elements required for RNA replication and those genetic elements encoding gene products necessary for viral particle assembly, and the RNA replicon is delivered to a subject in a composition containing one or more viral proteins, such as a viral like particle. In further embodiments, the RNA replicon comprises one or more modifications that enhance gene expression and/or confer a resistance to the innate immune system, such as stem-loops or downstream loops (a DLP motif) that enhance the translation of RNA under the control of a subgenomic promoter (Fovlov et ak, J Virol. 1996, 70: 1182-90).

In certain embodiments, examples of self-replicating RNA molecules, compositions and methods to create and use such molecules that are useful for the present invention are described in U.S. Patent Application Publication US2018/0104359, US2013/0177639, US2013/0149375,

US 2014/0242152, International Patent Application Publication WO2018/075235 or U.S. Patent US 10,022,435, the contents of which are incorporated herein by references in their entirety.

For example, the RNA replicons can include one or more components such as a 5’ UTR, a viral capsid enhancer Downstream Uoop (DUP), and an Old World alphavirus nsP3 hypervariable domain or a chimeric nsP3 hypervariable domain containing a portion of a New World alphavirus nsP3 hypervariable domain and another portion derived from an Old World alphavirus nsP3 hypervariable domain, as described in U.S. Patent Application Publications US2018/0104359, US2018/0171340, and US2020/0109178 respectively, each of which is incorporated herein by reference in its entirety.

Preferably, an RNA replicon of the application comprises, ordered from the 5’ - to 3 ’-end, (1) an alphavirus 5’ untranslated region (5’-UTR),

(2) a 5’ replication sequence of an alphavirus non-structural gene nspl,

(3) a downstream loop (DLP) motif of a virus species,

(4) a polynucleotide sequence encoding an autoprotease peptide,

(5) a polynucleotide sequence encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4,

(6) an alphavirus subgenomic promoter,

(7) the polynucleotide sequence encoding the recombinant pre-fusion SARS CoV-2 S protein or the fragment or variant thereof,

(8) an alphavirus 3' untranslated region (3' UTR), and

(9) optionally, a poly adenosine sequence.

The invention further provides compositions, preferably immunogenic compositions, comprising an RNA replicon encoding a stabilized pre-fusion SARS CoV-2 S protein or a fragment or variant thereof of the application.

The invention also provides compositions for use in inducing an immune response against SARS CoV-2 S protein, and in particular to the use of an RNA replicon of the application as a vaccine against SARS-CoV-2 associated disease, such as COVID-19.

In an embodiment, the self-replicating RNA molecule is encapsulated in, bound to or adsorbed on a liposome, a lipoplex, a lipid nanoparticle, or combinations thereof, preferably the self-replicating RNA molecule is encapsulated in a lipid nanoparticle. Preferably, the self- replicating RNA molecule is encapsulated in a lipid nanoparticle.

The invention also relates to methods for inducing an immune response against SARS CoV-2 in a subject, comprising administering to the subject an effective amount of an RNA replicon encoding a pre-fusion SARS CoV-2 S protein or a fragment or variant thereof of the application. Preferably, the induced immune response is characterized by the induction of neutralizing antibodies to the SARS CoV-2 virus and/or protective immunity against the SARS CoV-2 virus.

In particular aspects, the invention relates to methods for inducing anti-SARS CoV-2 S protein antibodies in a subject, comprising administering to the subject an effective amount of an immunogenic composition comprising an RNA replicon encoding a pre-fusion SARS CoV-2 S protein, or a fragment or variant thereof, of the application.

In certain embodiments, the composition or vaccine is administered in a prime-boost administration of a first and a second dose, wherein the first dose primes the immune response, and the second dose boosts the immune response. The prime-boost administration can, for example, be a homologous prime-boost, wherein the first and second dose comprise the same antigen or a fragment or variant thereof (e.g., the SARS-CoV-2 spike protein) expressed from the same vector (e.g., an RNA replicon). The prime-boost administration can, for example, be a heterologous prime-boost, wherein the first and second dose comprise the same antigen or a fragment or variant thereof (e.g., the SARS-CoV-2 spike protein) expressed from the same or different vector (e.g., an RNA replicon, an adenovirus, an mR A, or a plasmid). In some embodiments of a heterologous prime-boost administration, the first dose comprises an adenovirus vector comprising the SARS-CoV-2 spike protein or a fragment or variant thereof and a second dose comprising an RNA replicon vector comprising the SARS-CoV-2 spike protein or a fragment or variant thereof. In some embodiments of a heterologous prime-boost administration, the first dose comprises an RNA replicon vector comprising the SARS-CoV-2 spike protein or a fragment or variant thereof and a second dose comprising an adenovirus vector comprising the SARS-CoV-2 spike protein or a fragment or variant thereof. In certain aspects, the RNA replicon vaccine used in a homologous prime-boost or a heterologous prime-boost administration comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 5-194, or a fragment or variant thereof.

Brief Description of the Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

FIG.l: Schematic representation of the conserved elements of the fusion domain of a SARS CoV-2 S protein. The head domain contains an N-terminal (NTD) domain, the receptor binding domain (RBD) and domains SD1 and SD2. The fusion domain contains the fusion peptide (FP), refolding region 1 (RRl), refolding region 2 (RR2), transmembrane region (TM) and cytoplasmic tail. Cleavage site between SI and S2 and the S2’ cleavage sites are indicated with arrow

FIGs. 2 A and 2B: Analytical SEC samples of semi-stable SARS-CoV-2 S trimer proteins after freeze thaw cycles. S trimer protein according to SEQ ID NO: 3 (A) and the same protein in which the tag was replaced by a C-tag (B) after flash freezing in liquid Nitrogen and thawing 1 time (dark solid line) and 5 times (light solid line), compared with unfrozen S protein (dashed line). The peak at 5 minutes corresponds to the S trimer.

FIG. 3: Percentage of S trimer expression for S proteins with indicated mutations as measured by ACE2-Fc binding in AlphaFISA assay compared with control unstable uncleaved SARS-CoV-2 S (with ftirin site mutation) (SEQ ID NO: 2). The recombinant S proteins tested contain a single amino acid substitution, as indicated in the figure, introduced into the backbone of unstable uncleaved SARS-CoV-2 S ectodomain (SEQ ID NO: 2) (Furin KO, left panel) and into the backbone of the semi-stable uncleaved SARS-CoV-2 S with the double proline mutations in the hinge loop at position 986 and 987 (SEQ ID NO: 3) (Furin KO + PP, right panel). Analysis was performed on crude cell culture supernatants.

FIGs. 4A-4G: Analytical SEC profile of semi-stabilized uncleaved SARS-CoV-2 S with two stabilizing mutations to Proline in the hinge loop (+PP) (SEQ ID NO: 3) (A-C) and unstable uncleaved SARS-CoV-2 S protein (SEQ ID NO: 2) (D-F) (dashed lines), compared to variants with indicated point mutations (A, D) A892P, (B, E) A942P, (C, F) D614N in black, D614M in dark grey and D614L in light grey (solid line). Analysis was performed on crude cell culture supernatants. The peak at 5 minutes corresponds to the S trimer. G) SEC-MALS with purified stabilized S protein with A942P mutation (SEQ ID NO: 5). SEC signal is shown in grey thick line and corresponding to the left axis. The black thin line shows the molar mass traces (right y axis). The dn/dc value used is 0.185.

FIG. 5: Percentage of S trimer expression for S proteins with indicated mutations as measured by ACE2-Fc binding in AlphaLISA assay compared with control unstable uncleaved SARS-CoV-2 S (with furin site mutation) (SEQ ID NO: 2). The recombinant S proteins tested contain single amino acid substitution or a disulfide bridge, as indicated in the figure, introduced into the backbone of unstable uncleaved SARS-CoV2 S ectodomain (SEQ ID NO: 2) (Furin KO, left panel) and into the backbone of semi-stable uncleaved SARS-CoV-2 S with the double proline in the hinge loop at position 986 and 987 (SEQ ID NO: 3) (Furin KO + PP, right panel). Analysis was performed on crude cell culture supernatants.

FIGs. 6A-6H: Analytical SEC profile of semi-stabilized uncleaved SARS-CoV2 S + PP (SEQ ID NO: 3) (A-D) and unstable uncleaved SARS-CoV2 S protein (SEQ ID NO: 2) (E-H) (dashed lines), compared to variants with indicated point mutation or disulfide bridge (solid line). Analysis was performed on crude cell culture supernatants. The peak at 5 minutes corresponds to the S trimer.

FIG.7 is a schematic illustration of a self-amplifying RNA derived from an alphavirus that contains a 5'cap, nonstructural genes (NSP1-4), 26S subgenomic promoter (arrow), the SARS-CoV2 S protein (SARS-CoV2), and a 3' polyadenylated tail.

FIGs. 8A-8E: ELISA assay results of spike protein specific antibodies elicited after homologous prime-boost administration of RNA replicon constructs (SMAART-1159 and SMAART-1158). FIG. 8A shows a schematic of the prime-boost administration. FIG. 8B shows a graph of the results of an ELISA assay for spike protein specific antibodies at day 14. FIG. 8C shows a graph of the results of an ELISA assay for spike protein specific antibodies at day 27. FIG. 8D shows a graph of the results of an ELISA assay for spike protein specific antibodies at day 42. FIG. 8E shows a graph of the results of an ELISA assay for spike protein specific antibodies at day 54.

FIG. 9: Shows a graph of the results of neutralizing antibody production elicited at day 27 of the homologous prime-boost administration of the RNA replication constructs (SMAART- 1159 and SMAART-1158).

FIGs. 10A-10B: ELISpot assay results of spike protein specific IFNy secreting T cells in the spleens of immunized animals. FIG. 10A shows a graph of the results of the assay to measure spike protein specific IFNy secreting T cells in the spleen at day 14. FIG. 10B shows a graph of the results of the assay to measure spike protein specific IFNy secreting T cells in the spleen at day 54.

FIGs. 11A-11E: ELISA assay results of spike protein specific antibodies elicited after heterologous prime-boost administration of an adenoviral construct and an RNA replicon construct (Ad26NCOV030 and SMARRT-1159). FIG. 11A shows a schematic of the prime- boost administration. FIG. 1 IB shows a graph of the results of an ELISA assay for spike protein specific antibodies at day 14. FIG. 11C shows a graph of the results of an ELISA assay for spike protein specific antibodies at day 27. FIG. 1 ID shows a graph of the results of an ELISA assay for spike protein specific IgG titers at day 42. FIG. 1 IE shows a graph of the results of an ELISA assay for spike protein specific IgG titers at day 54.

FIGs. 12A-12B: ELISA assay results of IgGl (FIG. 12A) and IgG2 (FIG. 12B) isotype levels in the serum.

FIG. 13: Shows a graph of the results of neutralizing antibody production elicited at day 56 of the heterologous prime-boost administration.

FIGs. 14A-14B: ELISpot results of spike protein specific IFNy secreting T cells in the spleens of immunized animals. FIG. 14A shows a graph of the results of the assay for peptide pool 1 to measure spike protein specific IFNy secreting T cells in the spleen. FIG. 14B shows a graph of the results of the assay for peptide pool 2 to measure spike protein specific IFNy secreting T cells in the spleen.

Detailed Description of the Invention As explained above, the spike protein (S) of SARS-CoV-2 and of other Corona viruses is involved in fusion of the viral membrane with a host cell membrane, which is required for infection. SARS-CoV-2 S RNA is translated into a 1273 amino acid precursor protein, which contains a signal peptide sequence at the N-terminus (e.g., amino acid residues 1-13 of SEQ ID NO: 1) which is removed by a signal peptidase in the endoplasmic reticulum. Priming of the S protein typically involves cleavage by host proteases at the boundary between the S 1 and S2 subunits (S1/S2) in a subset of coronaviruses (including SARS CoV-2), and at a conserved site upstream of the fusion peptide (S2’) in all known corona viruses. For SARS-CoV-2, ftirin cleaves at S1/S2 between residues 685 and 686, and subsequently within S2 at the S2’ site between residues at position 815 and 816 by TMPRSS2. C-terminal to the S2’ site the proposed fusion peptide is located at the N-terminus of the refolding region 1 (FIG. 1).

A vaccine against SARS-CoV-2 infection is currently not yet available. Several vaccine modalities are possible, such as genetically based or vector-based vaccines or, e.g., subunit vaccines based on purified S protein. Since class I proteins are metastable proteins, increasing the stability of the pre-fusion conformation of fusion proteins increases the expression level of the protein because less protein will be misfolded, and more protein will successfully transport through the secretory pathway. Therefore, if the stability of the pre-fusion conformation of the class I fusion protein, like SARS CoV-2 S protein is increased, the immunogenic properties of a vector-based vaccine will be improved since the expression of the S protein is higher and the conformation of the immunogen resembles the pre-fusion conformation that is recognized by potent neutralizing and protective antibodies. For subunit-based vaccines, stabilizing the pre fusion S conformation is even more important. Besides the importance of high expression, which is needed to manufacture a vaccine successfully, maintenance of the pre-fusion conformation during the manufacturing process and during storage over time is critical for protein-based vaccines. In addition, for a soluble, subunit-based vaccine, the SARS CoV-2 S protein needs to be truncated by deletion of the transmembrane (TM) and the cytoplasmic region to create a soluble secreted S protein (sS). Because the TM region is responsible for membrane anchoring and increases stability, the anchorless soluble S protein is considerably more labile than the full- length protein and will even more readily refold into the post-fusion end-state. In order to obtain soluble S protein in the stable pre-fusion conformation that shows high expression levels and high stability, the pre-fusion conformation thus needs to be stabilized. Because also the full length (membrane-bound) SARS CoV-2 S protein is metastable, the stabilization of the pre fusion conformation is also desirable for the full-length SARS CoV-2 S protein, i.e., including the TM and cytoplasmic region, e.g., for any DNA, RNA, live attenuated, or vector-based vaccine approach.

The present invention thus provides stabilized, recombinant pre-fusion SARS CoV-2 S proteins or fragments or variants thereof, comprising an S 1 and an S2 domain, and comprising at least one mutation selected from the group consisting of a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 into a proline (P), a mutation of the amino acid at position 892, a mutation of the amino acid at position 614, a mutation of the amino acid at position 572, a mutation of the amino acid at position 532, a disulfide bridge between residues 880 and 888, and a disulfide bridge between residues 884 and 893, wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO: 1, and fragments thereof. According to the invention it has been demonstrated that the presence of specific amino acids and/or a disulfide bridge at the indicated positions increases the stability of the proteins in the pre-fusion conformation. According to the invention, the specific amino acids or disulfide bridges are introduced by substitution (mutation) of the amino acid at that position into a specific amino acid according to the invention. According to the invention, the proteins thus comprise one or more mutations in their amino acid sequence, i.e., the naturally occurring amino acid at these positions has been substituted with another amino acid. In certain embodiments, the proteins or fragments or variants thereof comprise an amino acid sequence, wherein the amino acid at position 892 is not alanine (A), the amino acid at position 614 is not aspartic acid (D) or glycine (G), the amino acid at position 532 is not asparagine (N) and/or amino acid at position 572 is not threonine (T).

In certain embodiments, the proteins or fragments or variants thereof comprise at least two mutations comprising a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 into proline (P), and a mutation selected from the group consisting of a mutation of the amino acid at position 892, a mutation of the amino acid at position 614, a mutation of the amino acid at position 572, a mutation of the amino acid at position 532, a disulfide bridge between residues 880 and 888 and a disulfide bridge between residues 884 and 893.

In certain embodiments, the proteins or fragments or variants thereof comprise at least two mutations comprising a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 into proline (P), and a mutation selected from the group consisting of a mutation of the amino acid at position 892, a mutation of the amino acid at position 614, a mutation of the amino acid at position 572 and a mutation of the amino acid at position 532, a disulfide bridge between residues 880 and 888 and a disulfide bridge between residues 884 and 893, provided that the proteins do not comprise both the disulfide bridge between residues 880 and 888 and the disulfide bridge between residues 884 and 893.

In certain embodiments, the proteins or fragments or variants thereof thus comprise a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941- 945 into proline (P), a mutation of the amino acid at position 892, a mutation of the amino acid at position 614, a mutation of the amino acid at position 572, and/or a mutation of the amino acid at position 532, and/or either a disulfide bridge between residues 880 and 888 or a disulfide bridge between residues 884 and 893.

In a preferred embodiment, the disulfide bridge is a disulfide bridge between residues 880 and 888. According to the invention it is to be understood that “a disulfide bridge between residues 880 and 880” means that the amino acids at the positions 880 and 888 have been mutated into cysteine (C). Similarly, it is to be understood that “a disulfide bridge between residues 884 and 893” means that the amino acids at the positions 884 and 893 have been mutated into cysteine (C).

In certain embodiments, the at least one mutation in the loop region corresponding to amino acid residues 941-945 is a mutation of the amino acid at position 942 into proline (P).

In certain embodiments, the mutation at position 892 is a mutation into proline (P).

In certain embodiments, the mutation at position 614 is a mutation into asparagine (N).

In certain embodiments, the mutation at position 532 is a mutation into proline (P).

In certain embodiments, the mutation at position 572 is a mutation into isoleucine (I).

In certain preferred embodiments, the proteins or fragments or variants thereof comprise a mutation of the amino acid at position 942 into P, a disulfide bridge between the amino acid residues at positions 880 and 888, and a mutation of the amino acid at position 614 into N.

An amino acid according to the invention can be any of the twenty naturally occurring (or ‘standard’ amino acids) or variants thereof, such as, e.g., D-amino acids (the D-enantiomers of amino acids with a chiral center), or any variants that are not naturally found in proteins, such as, e.g., norleucine. The standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties such as cysteine, that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that induces turns of the polypeptide backbone, and glycine that is more flexible than other amino acids. Table 1 shows the abbreviations and properties of the standard amino acids. It will be appreciated by a skilled person that the mutations can be made to the protein or fragment or variant thereof by routine molecular biology procedures.

In certain embodiments, the present invention provides recombinant SARS-CoV-2 S proteins, and fragments or variants thereof, wherein the amino acid at position 942 is P, the amino acid at position 892 is P, the amino acid at position 614 is N, the amino acid at position 532 is P and/or the amino acid at position 572 is I, and/or which comprise a disulfide bridge between residues 880 and 888 or a disulfide bridge between residues 884 and 893, wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO: 1.

In certain embodiments, the SARS CoV-2 S proteins or fragments or variants thereof further comprise a deletion of the ftirin cleavage site. A deletion of the ftirin cleavage, e.g., by mutation of one or more amino acids in the ftirin cleavage site (such that the protein is not cleaved by ftirin), renders the protein uncleaved, which further increases its stability. Deleting the furin cleavage site can be achieved in any suitable way that is known to the skilled person. In certain embodiments, the deletion of the ftirin cleavage site comprises a mutation of the amino acid at position 682 into serine (S) and/or a mutation of the amino acid at position 685 into glycine (G).

In certain embodiments, the proteins or fragments or variants thereof further comprise a mutation of the amino acids at position 986 and 987 into proline (P).

In certain embodiments, the invention provides SARS-CoV 2 proteins or fragments or variants thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5-194 or fragments or variants thereof.

The term “fragment” as used herein refers to a peptide that has an amino-terminal and/or carboxy-terminal and/or internal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence of a SARS CoV-2 S protein, for example, the full-length sequence of a SARS CoV-2 S protein. It will be appreciated that for inducing an immune response and in general for vaccination purposes, a protein needs not to be full length nor have all its wild type functions, and fragments of the protein are equally useful. A fragment according to the invention is an immunologically active fragment, and typically comprises at least 15 amino acids, or at least 30 amino acids, of the SARS CoV-2 S protein. In certain embodiments, it comprises at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 amino acids, of the SARS CoV-2 S protein.

The term “variant” as used herein refers to a SARS CoV-2 S protein that comprises a substitution or deletion of at least one amino acid from the wild type SARS CoV-2 S protein sequence (SEQ ID NO: 1). A variant can be naturally or non-naturally occurring. A variant can comprise at least one, at least two, at least three, at least four, at least five, or at least ten substitution or deletions as compared to the wild type SARS CoV-2 S protein sequence (SEQ ID NO: 1). In certain embodiments, a variant can, for example, be greater than 95% identical with the wild type SARS CoV-2 S protein sequence (SEQ ID NO: 1). Examples of SARS CoV-2 protein variants can include, but are not limited to, the B.1.1.7, B.1.351, P.1, B.1.427, and B.1.429, B.1.526, B.l.526.1, B.1.525, B.1.617, B.1.617.1, B.1.617.2, B.1.617.3, and P.2 variants, as described on cdc.gov/coronavirus/2019-ncov/cases-updates/variant- surveillance/variant-info.html accessed on May 10, 2021.

In certain embodiments, the proteins according to the invention are soluble proteins, e.g., S protein ectodomains, and comprise a truncated S2 domain. As used herein a “truncated” S2 domain refers to a S2 domain that is not a full length S2 domain, i.e., wherein either N- terminally or C-terminally one or more amino acid residues have been deleted. According to the invention, at least the transmembrane domain and cytoplasmic domain are deleted to permit expression as a soluble ectodomain. For the stabilization of such soluble SARS CoV-2 S protein in the pre -fusion conformation, a heterologous trimerization domain, such as a fibritin - based trimerization domain, may be fused to the C-terminus of the Corona virus S protein ectodomain. This fibritin domain or ‘Foldon’ is derived from T4 fibritin and was described earlier as an artificial natural trimerization domain (Letarov et al., (1993); S-Guthe et al., (2004)). Thus, in certain embodiments, the transmembrane region has been replaced by a heterologous trimerization domain. In a preferred embodiment, the heterologous trimerization domain is a foldon domain comprising the amino acid sequence of SEQ ID NO:4. However, it is to be understood that according to the invention other trimerization domains are also possible.

The pre-fusion SARS CoV-2 S proteins or fragments or variants thereof according to the invention are stable, i.e., do not readily change into the post-fusion conformation upon processing of the proteins, such as, e.g., upon purification, freeze-thaw cycles, and/or storage, etc. In certain embodiments, the pre-fusion SARS CoV-2 S proteins or fragments or variants have an increased stability as compared to SARS CoV-2 S proteins or fragments or variants without the mutations of the invention, e.g., as indicated by an increased melting temperature (measured by, e.g., differential scanning fluorimetry).

The proteins according to the invention may comprise a signal peptide, also referred to as signal sequence or leader peptide, corresponding to amino acids 1-13 of SEQ ID NO: 1. Signal peptides are short (typically 5-30 amino acids long) peptides present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. In certain embodiments, the proteins according to the invention do not comprise a signal peptide.

In certain embodiments, the proteins comprise a tag sequence, such as a HIS-Tag or C- Tag. A His-Tag (or polyhistidine-tag) is an amino acid motif in proteins that consists of at least five histidine (H) residues, preferably placed at the N- or C-terminus of the protein, which is generally used for purification purposes. In certain embodiments, the proteins according to the invention do not comprise a tag sequence. Alternatively, other tags like a C-tag can be used for these purposes.

The invention also provides methods for stabilizing a SARS CoV-2 S protein, said method comprising introducing in the amino acid sequence of a SARS CoV-2 S protein at least one mutation selected from the group consisting of a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 into proline (P), a mutation of the amino acid at position 892, a mutation of the amino acid at position 614, a mutation of the amino acid at position 572, a mutation of the amino acid at position 532, a disulfide bridge between residues 880 and 888, and a disulfide bridge between residues 884 and 893, wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO: 1.

In certain embodiments, the methods comprise introducing at least two mutations comprising a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 into proline (P), and a mutation selected from the group consisting of a mutation of the amino acid at position 892, a mutation of the amino acid at position 614, a mutation of the amino acid at position 572, a mutation of the amino acid at position 532, a disulfide bridge between residues 880 and 888 and a disulfide bridge between residues 884 and 893.

In certain embodiments, the methods comprise introducing at least two mutations comprising a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 into proline (P), and a mutation selected from the group consisting of a mutation of the amino acid at position 892, a mutation of the amino acid at position 614, a mutation of the amino acid at position 572, a mutation of the amino acid at position 532, a disulfide bridge between residues 880 and 888 and a disulfide bridge between residues 884 and 893, provided that the proteins do not comprise both the disulfide bridge between residues 880 and 888 and the disulfide bridge between residues 884 and 893.

In certain embodiments, the at least one mutation in the loop region corresponding to amino acid residues 941-945 is a mutation of the amino acid at position 942 into proline (P). In certain embodiments, the mutation at position 892 is a mutation into proline (P).

In certain embodiments, the methods further comprise deleting the furin cleavage site. Deleting the furin cleavage site may be achieved in any way known in the art. In certain embodiments, the deletion of the furin cleavage site comprises introducing a mutation of the amino acid at position 682 into serine (S) and/or a mutation of the amino acid at position 685 into glycine (G).

In certain embodiments, the methods further comprise introducing a mutation of the amino acids at position 986 and 987 into proline (P).

The invention also provided SARS CoV-2 proteins obtainable by the methods described herein.

The present invention further provides nucleic acid molecules encoding the SARS CoV-2 S proteins or fragments or variants thereof according to the invention. The term “nucleic acid molecule” as used in the present invention refers to a polymeric form of nucleotides (i.e., polynucleotides) and includes both DNA (e.g., cDNA, genomic DNA) and RNA, and synthetic forms and mixed polymers of the above.

In preferred embodiments, the nucleic acid molecules encoding the proteins or fragments or variants thereof according to the invention are codon-optimized for expression in mammalian cells, preferably human cells, or insect cells. Methods of codon-optimization are known and have been described previously (e.g., WO 96/09378 for mammalian cells). A sequence is considered codon-optimized if at least one non-preferred codon as compared to a wild type sequence is replaced by a codon that is more preferred. Herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage for a specific organism can be found in codon frequency tables, such as in world wide web site: kazusa.or.jp/codon. Preferably, more than one non preferred codon, preferably most or all non-preferred codons, are replaced by codons that are more preferred. Preferably, the most frequently used codons in an organism are used in a codon- optimized sequence. Replacement by preferred codons generally leads to higher expression.

It will be understood by a skilled person that numerous different polynucleotides and nucleic acid molecules can encode the same protein or fragment or variant thereof 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 protein sequence encoded by the nucleic acid molecules to reflect the codon usage of any particular host organism in which the proteins are to be expressed. Therefore, unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may or may not include introns.

Nucleic acid sequences can be cloned using routine molecular biology techniques, or generated c/e novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g., GeneArt, GenScript, Invitrogen, Eurofins).

The invention also provides vectors comprising a nucleic acid molecule as described above. In certain embodiments, a nucleic acid molecule according to the invention thus is part of a vector. Such vectors can easily be manipulated by methods well known to the person skilled in the art and can for instance be designed for being capable of replication in prokaryotic and/or eukaryotic cells. In addition, many vectors can be used for transformation of eukaryotic cells and will integrate in whole or in part into the genome of such cells, resulting in stable host cells comprising the desired nucleic acid in their genome. The vector used can be any vector that is suitable for cloning DNA and that can be used for transcription of a nucleic acid of interest.

Preferably, the vector is a self-replicating RNA replicon.

As used herein, “self-replicating RNA molecule,” which is used interchangeably with “self-amplifying RNA molecule” or “RNA replicon” or “replicon RNA” or “saRNA,” refers to an RNA molecule engineered from genomes of plus-strand RNA viruses that contains all of the genetic information required for directing its own amplification or self-replication within a permissive cell. A self-replicating RNA molecule resembles mRNA. It is single-stranded, 5'- capped, and 3'-poly-adenylated and is of positive orientation. To direct its own replication, the RNA molecule 1) encodes polymerase, replicase, or other proteins which can interact with viral or host cell -derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process; and 2) contain cis-acting RNA sequences required for replication and transcription of the subgenomic replicon-encoded RNA. Thus, the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, can be translated themselves to provide in situ expression of a gene of interest, or can be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the gene of interest. The overall result of this sequence of transcriptions is a huge amplification in the number of the introduced replicon RNAs and so the encoded gene of interest becomes a major polypeptide product of the cells.

In certain embodiment, an RNA replicon of the application comprises, ordered from the 5’- to 3 ’-end: (1) a 5’ untranslated region (5’-UTR) required for nonstructural protein-mediated amplification of an RNA virus; (2) a polynucleotide sequence encoding at least one, preferably all, of non-structural proteins of the RNA virus; (3) a subgenomic promoter of the RNA virus; (4) a polynucleotide sequence encoding the recombinant pre-fusion SARS CoV-2 S protein or the fragment or variant thereof; and (5) a 3’ untranslated region (3’-UTR) required for nonstructural protein-mediated amplification of the RNA virus.

In certain embodiments, a self-replicating RNA molecule encodes an enzyme complex for self-amplification (replicase polyprotein) comprising an RNA-dependent RNA-polymerase function, helicase, capping, and poly-adenylating activity. The viral structural genes downstream of the replicase, which are under control of a subgenomic promoter, can be replaced by a pre fusion SARS CoV-2 S protein or the fragment or variant thereof described herein. Upon transfection, the replicase is translated immediately, interacts with the 5' and 3' termini of the genomic RNA, and synthesizes complementary genomic RNA copies. Those act as templates for the synthesis of novel positive-stranded, capped, and poly-adenylated genomic copies, and subgenomic transcripts. Amplification eventually leads to very high RNA copy numbers of up to 2 x 10⁵ copies per cell. Thus, much lower amounts of saRNA compared to conventional mRNA suffice to achieve effective gene transfer and protective vaccination (Beissert et al., Hum Gene Ther. 2017, 28(12): 1138-1146).

Subgenomic RNA is an RNA molecule of a length or size which is smaller than the genomic RNA from which it was derived. The viral subgenomic RNA can be transcribed from an internal promoter, whose sequences reside within the genomic RNA or its complement. Transcription of a subgenomic RNA can be mediated by viral-encoded polymerase(s) associated with host cell -encoded proteins, ribonucleoprotein(s), or a combination thereof. Numerous RNA viruses generate subgenomic mRNAs (sgRNAs) for expression of their 3'-proximal genes.

In some embodiments of the present disclosure, a pre-fusion SARS CoV-2 S protein or a fragment or variant thereof thereof described herein is expressed under the control of a subgenomic promoter. In certain embodiments, instead of the native subgenomic promoter, the subgenomic RNA can be placed under control of internal ribosome entry site (IRES) derived from encephalomyocarditis viruses (EMCV), Bovine Viral Diarrhea Viruses (BVDV), polioviruses, Foot-and-mouth disease viruses (FMD), enterovirus 71, or hepatitis C viruses. Subgenomic promoters range from 24 nucleotide (Sindbis virus) to over 100 nucleotides (Beet necrotic yellow vein virus) and are usually found upstream of the transcription start.

In some embodiments, the RNA replicon includes the coding sequence for at least one, at least two, at least three, or at least four nonstructural viral proteins (e.g., nsPl, nsP2, nsP3, nsP4). Alphavirus genomes encode non-structural proteins nsPl, nsP2, nsP3, and nsP4, which are produced as a single polyprotein precursor, sometimes designated P1234 (ornsPl-4 or nsP1234), and which is cleaved into the mature proteins through proteolytic processing. nsPl can be about 60 kDa in size and may have methyltransferase activity and be involved in the viral capping reaction. nsP2 has a size of about 90 kDa and may have helicase and protease activity while nsP3 is about 60 kDa and contains three domains: a macrodomain, a central (or alphavirus unique) domain, and a hypervariable domain (HVD). nsP4 is about 70 kDa in size and contains the core RNA-dependent RNA polymerase (RdRp) catalytic domain. After infection the alphavirus genomic RNA is translated to yield a P 1234 polyprotein, which is cleaved into the individual proteins. In disclosing the nucleic acid or polypeptide sequences herein, for example sequences of nsPl, nsP2, nsP3, nsP4, also disclosed are sequences considered to be based on or derived from the original sequence.

In some embodiments, RNA replicon includes the coding sequence for a portion of the at least one nonstructural viral protein. For example, the RNA replicon can include about 10%,

20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or a range between any two of these values, of the encoding sequence for the at least one nonstructural viral protein. In some embodiments, the RNA replicon can include the coding sequence for a substantial portion of the at least one nonstructural viral protein. As used herein, a “substantial portion” of a nucleic acid sequence encoding a nonstructural viral protein comprises enough of the nucleic acid sequence encoding the nonstructural viral protein to afford putative identification of that protein, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (see, for example, in “Basic Local Alignment Search Tool”; Altschul S F et ak, J. Mol. Biol. 215:403-410, 1993). In some embodiments, the RNA replicon can include the entire coding sequence for the at least one nonstructural protein. In some embodiments, the RNA replicon comprises substantially all the coding sequence for the native viral nonstructural proteins. In certain embodiments, the one or more nonstructural viral proteins are derived from the same virus. In other embodiments, the one or more nonstructural proteins are derived from different viruses.

The RNA replicon can be derived from any suitable plus-strand RNA viruses, such as alphaviruses or flaviviruses. Preferably, the RNA replicon is derived from alphaviruses. The term “alphavirus” describes enveloped single-stranded positive sense RNA viruses of the family Togaviridae. The genus alphavirus contains approximately 30 members, which can infect humans as well as other animals. Alphavirus particles typically have a 70 nm diameter, tend to be spherical or slightly pleomorphic, and have a 40 nm isometric nucleocapsid. The total genome length of alphaviruses ranges between 11,000 and 12,000 nucleotides and has a 5'cap and 3' poly-A tail. There are two open reading frames (ORFs) in the genome, non-structural (ns) and structural. The ns ORF encodes proteins (nsPl-nsP4) necessary for transcription and replication of viral RNA. The structural ORF encodes three structural proteins: the core nucleocapsid protein C, and the envelope proteins P62 and El that associate as a heterodimer. The viral membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells through membrane fusion. The four ns protein genes are encoded by genes in the 5' two-thirds of the genome, while the three structural proteins are translated from a subgenomic mRNA colinear with the 3' one-third of the genome.

In some embodiments, the self-replicating RNA useful for the invention is an RNA replicon derived from an alphavirus virus species. In some embodiments, the alphavirus RNA replicon is of an alphavirus belonging to the VEEV/EEEV group, or the SF group, or the SIN group. Non-limiting examples of SF group alphaviruses include Semliki Forest virus, O'Nyong- Nyong virus, Ross River virus, Middelburg virus, Chikungunya virus, Barmah Forest virus, Getah virus, Mayaro virus, Sagiyama virus, Bebaru virus, and Una virus. Non-limiting examples of SIN group alphaviruses include Sindbis virus, Girdwood S. A. virus, South African Arbovirus No. 86, Ockelbo virus, Aura virus, Babanki virus, Whataroa virus, and Kyzylagach virus. Non- limiting examples of VEEV/EEEV group alphaviruses include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), and Una virus (UNAV).

Non-limiting examples of alphavirus species include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), and Buggy Creek virus. Virulent and avirulent alphavirus strains are both suitable. In some embodiments, the alphavirus RNA replicon is of a Sindbis virus (SIN), a Semliki Forest virus (SFV), a Ross River virus (RRV), a Venezuelan equine encephalitis virus (VEEV), or an Eastern equine encephalitis virus (EEEV). In some embodiments, the alphavirus RNA replicon is of a Venezuelan equine encephalitis virus (VEEV).

In certain embodiments, a self-replicating RNA molecule comprises a polynucleotide encoding one or more nonstructural proteins nspl-4, a subgenomic promoter, such as 26S subgenomic promoter, and a gene of interest encoding a pre-fusion SARS CoV-2 S protein or the fragment thereof described herein.

A self-replicating RNA molecule can have a 5' cap (e.g., a 7-methylguanosine). This cap can enhance in vivo translation of the RNA.

The 5' nucleotide of a self-replicating RNA molecule useful with the invention can have a 5' triphosphate group. In a capped RNA this can be linked to a 7-methylguanosine via a 5'-to-5' bridge. A 5' triphosphate can enhance RIG-I binding.

A self-replicating RNA molecule can have a 3' poly-A tail. It can also include a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end.

In any of the embodiments of the present disclosure, the RNA replicon can lack (or not contain) the coding sequence (s) of at least one (or all) of the structural viral proteins (e.g., nucleocapsid protein C, and envelope proteins P62, 6K, and El). In these embodiments, the sequences encoding one or more structural genes can be substituted with one or more heterologous sequences such as, for example, a coding sequence for a pre-fusion SARS CoV-2 S protein or the fragment thereof described herein.

In certain embodiments, a self-replicating RNA vector of the application comprises one or more features to confer a resistance to the translation inhibition by the innate immune system or to otherwise increase the expression of the GOI (e.g., a pre-fusion SARS CoV-2 S protein or the fragment thereof described herein).

In certain embodiments, the RNA sequence can be codon optimized to improve translation efficiency. The RNA molecule can be modified by any method known in the art in view of the present disclosure to enhance stability and/or translation, such by adding a polyA tail, e.g., of at least 30 adenosine residues; and/or capping the 5-end with a modified ribonucleotide, e.g., 7- methylguanosine cap, which can be incorporated during RNA synthesis or enzymatically engineered after RNA transcription. In certain embodiments, an RNA replicon of the application comprises, ordered from the 5’- to 3 ’-end, (1) an alphavirus 5’ untranslated region (5’-UTR), (2) a 5’ replication sequence of an alphavirus non-structural gene nspl, (3) a downstream loop (DLP) motif of a virus species,

(4) a polynucleotide sequence encoding an autoprotease peptide, (5) a polynucleotide sequence encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4, (6) an alphavirus subgenomic promoter, (7) the polynucleotide sequence encoding the recombinant pre-fusion SARS CoV-2 S protein or the fragment or variant thereof, (8) an alphavirus 3' untranslated region (3' UTR), and (9) optionally, a poly adenosine sequence. A schematic illustration of a self-amplifying RNA replicon is shown in FIG. 7.

In certain embodiments, a self-replicating RNA vector of the application comprises a downstream loop (DLP) motif of a virus species. As used herein, a “downstream loop” or “DLP motif’ refers to a polynucleotide sequence comprising at least one RNA stem-loop, which when placed downstream of a start codon of an open reading frame (ORF) provides increased translation of the ORF compared to an otherwise identical construct without the DLP motif. As an example, members of the Alphavirus genus can resist the activation of antiviral RNA- activated protein kinase (PKR) by means of a prominent RNA structure present within in viral 26S transcripts, which allows an eIF2-independent translation initiation of these mRNAs. This structure, called the downstream loop (DLP), is located downstream from the AUG in SINV 26S mRNA. The DLP is also detected in Semliki Forest virus (SFV). Similar DLP structures have been reported to be present in at least 14 other members of the Alphavirus genus including New World (for example, MAYV, UNAV, EEEV (NA), EEEV (SA), AURAV) and Old World (SV, SFV, BEBV, RRV, SAG, GETV, MIDV, CHIKV, and ONNV) members. The predicted structures of these Alphavirus 26S mRNAs were constructed based on SHAPE (selective 2'- hydroxyl acylation and primer extension) data (Toribio et ak, Nucleic Acids Res. May 19; 44(9):4368-80, 2016), the content of which is hereby incorporated by reference). Stable stem- loop structures were detected in all cases except for CHIKV and ONNV, whereas MAYV and EEEV showed DLPs of lower stability (Toribio et ak, 2016 supra). In the case of Sindbis virus, the DLP motif is found in the first 150 nt of the Sindbis subgenomic RNA. The hairpin is located downstream of the Sindbis capsid AUG initiation codon (AUG is collated at nt 50 of the Sindbis subgenomic RNA). Previous studies of sequence comparisons and structural RNA analysis revealed the evolutionary conservation of DLP in SINV and predicted the existence of equivalent DLP structures in many members of the Alphavirus genus (see e.g., Ventoso, J. Virol. 9484- 9494, Vok 86, September 2012). Examples of a self-replicating RNA vector comprising a DLP motif are described in US Patent Application Publication US2018/0171340 and the International Patent Application Publication W02018106615, the content of which is incorporated herein by reference in its entirety. In some embodiments, a replicon RNA of the application comprises a DLP motif exhibiting at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequences set forth in SEQ ID NO: 200.

In one embodiment, the self-replicating RNA molecule also contains a coding sequence for an autoprotease peptide operably linked downstream of the DLP motif and upstream of the coding sequences of the nonstructural proteins (e.g., one or more of nspl-4) or gene of interest (e.g., a pre-fusion SARS CoV-2 S protein or the fragment thereof described herein). Examples of the autoprotease peptide include, but are not limited to, a peptide sequence selected from the group consisting of porcine teschovirus- 1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), and a combination thereof. In some embodiments, a replicon RNA of the application comprises a coding sequence for P2A having the amino acid sequence of SEQ ID NO: 202. Preferably, the coding sequence exhibits at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequences set forth in SEQ ID NO: 201.

Any of the replicons of the invention can also comprise a 5 ’ and a 3 ’ untranslated region (UTR). The UTRs can be wild type New World or Old World alphavirus UTR sequences, or a sequence derived from any of them. In various embodiments the 5’ UTR can be of any suitable length, such as about 60 nt or 50-70 nt or 40-80 nt. In some embodiments the 5’ UTR can also have conserved primary or secondary structures (e.g., one or more stem-loop(s)) and can participate in the replication of alphavirus or of replicon RNA. In some embodiments the 3’

UTR can be up to several hundred nucleotides, for example it can be 50-900 or 100-900 or 50- 800 or 100-700 or 200-700 nt. The ‘3 UTR also can have secondary structures, e.g., a step loop, and can be followed by a polyadenylate tract or poly-A tail. In any of the embodiments of the invention the 5 ’ and 3 ’ untranslated regions can be operably linked to any of the other sequences encoded by the replicon. The UTRs can be operably linked to a promoter and/or sequence encoding a heterologous protein or peptide by providing sequences and spacing necessary for recognition and transcription of the other encoded sequences. Any polyadenylation signal known to those skilled in the art in view of the present disclosure can be used. For example, the polyadenylation signal can be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human b-globin polyadenylation signal. In another embodiment, a self-replicating RNA replicon of the application comprises a modified 5’ untranslated region (5'-UTR), preferably the RNA replicon is devoid of at least a portion of a nucleic acid sequence encoding viral structural proteins. For example, the modified 5'-UTR can comprise one or more nucleotide substitutions at position 1, 2, 4, or a combination thereof. Preferably, the modified 5'-UTR comprises a nucleotide substitution at position 2, more preferably, the modified 5'-UTR has a U->G or U->A substitution at position 2. Examples of such self-replicating RNA molecules are described in US Patent Application Publication US2018/0104359 and the International Patent Application Publication WO2018075235, the content of which is incorporated herein by reference in its entirety. In some embodiments, a replicon RNA of the application comprises a 5'-UTR exhibiting at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequences set forth in SEQ ID NO: 198.

In some embodiments, an RNA replicon of the application comprises a polynucleotide sequence encoding a signal peptide sequence. Preferably, the polynucleotide sequence encoding the signal peptide sequence is located upstream of or at the 5 ’-end of the polynucleotide sequence encoding the pre-fusion SARS CoV-2 S protein or the fragment thereof. Signal peptides typically direct localization of a protein, facilitate secretion of the protein from the cell in which it is produced, and/or improve antigen expression and cross-presentation to antigen- presenting cells. A signal peptide can be present at the N-terminus of a pre-fusion SARS CoV-2 S protein or fragment thereof when expressed from the replicon, but is cleaved off by signal peptidase, e.g., upon secretion from the cell. An expressed protein in which a signal peptide has been cleaved is often referred to as the “mature protein.” Any signal peptide known in the art in view of the present disclosure can be used. For example, a signal peptide can be a cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as the Ig heavy chain gamma signal peptide SPIgG, the Ig heavy chain epsilon signal peptide SPIgE, or the short leader peptide sequence of the coronavirus. Exemplary nucleic acid sequence encoding a signal peptide is shown in SEQ ID NO: 195.

In various embodiments the RNA replicons disclosed herein can be engineered, synthetic, or recombinant RNA replicons. As non-limiting examples, an RNA replicon can be one or more of the following: 1) synthesized or modified in vitro, for example, using chemical or enzymatic techniques, for example, by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, e.g., methylation), or recombination (including homologous and site-specific recombination) of nucleic acid molecules; 2) conjoined nucleotide sequences that are not conjoined in nature; 3) engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleotide sequence; and 4) manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleotide sequence.

Any of the components or sequences of the RNA replicon can be operably linked to any other of the components or sequences. The components or sequences of the RNA replicon can be operably linked for the expression of the gene of interest in a host cell or treated organism and/or for the ability of the replicon to self-replicate. As used herein, the term “operably linked” is to be taken in its broadest reasonable context and refers to a linkage of polynucleotide elements in a functional relationship. A polynucleotide is “operably linked” when it is placed into a functional relationship with another polynucleotide. For instance, a promoter or UTR operably linked to a coding sequence is capable of effecting the transcription and expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, an operable linkage between an RNA sequence encoding a heterologous protein or peptide and a regulatory sequence (for example, a promoter or UTR) is a functional link that allows for expression of the polynucleotide of interest. Operably linked can also refer to sequences such as the sequences encoding the RdRp (e.g., nsP4), nsPl-4, the UTRs, promoters, and other sequences encoding in the RNA replicon, are linked so that they enable transcription and translation of the pre-fusion SARS CoV-2 S protein and/or replication of the replicon. The UTRs can be operably linked by providing sequences and spacing necessary for recognition and translation by a ribosome of other encoded sequences.

The immunogenicity of a pre-fusion SARS CoV-2 S protein or a fragment or variant thereof expressed by an RNA replicon can be determined by a number of assays known to persons of ordinary skill in view of the present disclosure.

Another general aspect of the application relates to a nucleic acid comprising a DNA sequence encoding an RNA replicon of the application. The nucleic acid can be, for example, a DNA plasmid or a fragment of a linearized DNA plasmid. Preferably, the nucleic acid further comprises a promoter, such as a T7 promoter, operably linked to the 5 ’-end of the DNA sequence. More preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO: 207. The nucleic acid can be used for the production of an RNA replicon of the application using a method known in the art in view of the present disclosure. For example, an RNA replicon can be obtained by in vivo or in vitro transcription of the nucleic acid. Host cells comprising a RNA replicon or a nucleic acid encoding the RNA replicon of the application also form part of the invention. The pre-fusion SARS CoV-2 S proteins or fragments or variants thereof may be produced through recombinant DNA technology involving expression of the molecules in host cells, e.g., Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6 cells, or yeast, fungi, insect cells, and the like, or transgenic animals or plants. In certain embodiments, the cells are from a multicellular organism, in certain embodiments they are of vertebrate or invertebrate origin. In certain embodiments, the cells are mammalian cells, such as human cells, or insect cells. In general, the production of a recombinant proteins, such the pre-fusion SARS CoV-2 S proteins or fragments or variants thereof of the invention, in a host cell comprises the introduction of a heterologous nucleic acid molecule encoding the protein in expressible format into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid molecule and allowing expression of the protein or fragment or variant thereof in said cell. The nucleic acid molecule encoding a protein in expressible format may be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like. The person skilled in the art is aware that various promoters can be used to obtain expression of a gene in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed.

Cell culture media are available from various vendors, and a suitable medium can be routinely chosen for a host cell to express the protein of interest, here the pre-fusion SARS CoV- 2 S proteins. The suitable medium may or may not contain serum.

A “heterologous nucleic acid molecule” (also referred to herein as ‘transgene’) is a nucleic acid molecule that is not naturally present in the host cell. It is introduced into, for instance, a vector by standard molecular biology techniques. A transgene is generally operably linked to expression control sequences. This can, for instance, be done by placing the nucleic acid encoding the transgene(s) under the control of a promoter. Further regulatory sequences may be added. Many promoters can be used for expression of atransgene(s), and are known to the skilled person, e.g., these may comprise viral, mammalian, synthetic promoters, and the like. A non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is a CMV-promoter (US 5,385,839), e.g., the CMV immediate early promoter, for instance comprising nt. -735 to +95 from the CMV immediate early gene enhancer/promoter. A polyadenylation signal, for example the bovine growth hormone polyA signal (US 5,122,458), may be present behind the transgene(s). Alternatively, several widely used expression vectors are available in the art and from commercial sources, e.g., the pcDNA and pEF vector series of Invitrogen, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc., which can be used to recombinantly express the protein of interest, or to obtain suitable promoters and/or transcription terminator sequences, polyA sequences, and the like.

The cell culture can be any type of cell culture, including adherent cell culture, e.g., cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. Nowadays, continuous processes based on perfusion principles are becoming more common and are also suitable. Suitable culture media are also well known to the skilled person and can generally be obtained from commercial sources in large quantities, or custom-made according to standard protocols. Culturing can be done for instance in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems and the like. Suitable conditions for culturing cells are known (see, e.g., Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Fiss Inc., 2000, ISBN 0-471-34889-9)).

The invention further provides compositions comprising a pre-fusion SARS CoV-2 S protein or fragment or variant thereof and/or a nucleic acid molecule, and/or a vector, as described above. The invention also provides compositions comprising a nucleic acid molecule and/or a vector, encoding such pre-fusion SARS CoV-2 S protein or fragment or variant thereof. The invention further provides immunogenic compositions comprising a pre-fusion SARS CoV- 2 S protein or fragment or variant thereof, and/or a nucleic acid molecule, and/or a vector, as described above. The invention also provides the use of a stabilized pre-fusion SARS CoV-2 S protein or fragment or variant thereof, a nucleic acid molecule, and/or a vector, according to the invention, for inducing an immune response against a SARS CoV-2 S protein or fragment or variant thereof in a subject. Further provided are methods for inducing an immune response against SARS CoV-2 S protein or fragment or variant thereof in a subject, comprising administering to the subject a pre-fusion SARS CoV-2 S protein or fragment or variant thereof, and/or a nucleic acid molecule, and/or a vector according to the invention. Also provided are pre fusion SARS CoV-2 S proteins or fragments or variants thereof, nucleic acid molecules, and/or vectors, according to the invention for use in inducing an immune response against SARS CoV-2 S protein or fragment or variant thereof in a subject. Further provided is the use of the pre-fusion SARS CoV-2 S proteins or fragments or variants thereof, and/or nucleic acid molecules, and/or vectors according to the invention for the manufacture of a medicament for use in inducing an immune response against SARS CoV-2 S protein or fragment or variant thereof in a subject. In certain embodiments, the nucleic acid molecule is DNA and/or an RNA molecule.

The pre-fusion SARS CoV-2 S proteins or fragments or variants thereof, nucleic acid molecules, or vectors of the invention may be used for prevention (prophylaxis, including post exposure prophylaxis) of SARS CoV-2 infections. In certain embodiments, the prevention may be targeted at patient groups that are susceptible for and/or at risk of SARS CoV-2 infection or have been diagnosed with a SARS CoV-2 infection. Such target groups include, but are not limited to, e.g., the elderly (e.g., > 50 years old, > 60 years old, and preferably > 65 years old), hospitalized patients, and patients who have been treated with an antiviral compound but have shown an inadequate antiviral response. In certain embodiments, the target population comprises human subjects from 2 months of age.

The pre-fusion SARS CoV-2 S proteins or fragments or variants thereof, nucleic acid molecules, and/or vectors according to the invention can be used, e.g., in stand-alone treatment and/or prophylaxis of a disease or condition caused by SARS CoV-2, or in combination with other prophylactic and/or therapeutic treatments, such as (existing or future) vaccines, antiviral agents, and/or monoclonal antibodies.

The invention further provides methods for preventing and/or treating SARS CoV-2 infection in a subject utilizing the pre-fusion SARS CoV-2 S proteins or fragments or variants thereof, nucleic acid molecules, and/or vectors according to the invention. In a specific embodiment, a method for preventing and/or treating SARS CoV-2 infection in a subject comprises administering to a subject in need thereof an effective amount of a pre-fusion SARS CoV-2 S protein or fragment or variant thereof, nucleic acid molecule, and/or a vector, as described above. A therapeutically effective amount refers to an amount of a protein, nucleic acid molecule, or vector, which is effective for preventing, ameliorating and/or treating a disease or condition resulting from infection by SARS CoV-2. Prevention encompasses inhibiting or reducing the spread of SARS CoV-2 or inhibiting or reducing the onset, development, or progression of one or more of the symptoms associated with infection by SARS CoV-2. Amelioration, as used in herein, can refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of SARS CoV-2 infection.

For administering to subjects, such as humans, the invention can employ pharmaceutical compositions comprising a pre-fusion SARS CoV-2 S protein or fragment or variant thereof, a nucleic acid molecule and/or a vector as described herein, and a pharmaceutically acceptable carrier or excipient. In the present context, the term “pharmaceutically acceptable” means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and F. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The CoV S proteins, or nucleic acid molecules, preferably are formulated and administered as a sterile solution although it can also be possible to utilize lyophilized preparations. Sterile solutions are prepared by sterile fdtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, e.g., pH 5.0 to 7.5. The CoV S proteins typically are in a solution having a suitable pharmaceutically acceptable buffer, and the composition can also contain a salt. Optionally, a stabilizing agent can be present, such as albumin. In certain embodiments, detergent is added. In certain embodiments, the CoV S proteins can be formulated into an injectable preparation.

An RNA replicon can be formulated using any suitable pharmaceutically acceptable carriers in view of the present disclosure. For example, an RNA replicon of the application can be formulated in an immunogenic composition that comprises one or more lipid molecules, preferably positively charged lipid molecules

In some embodiments, an RNA replicon of the disclosure can be formulated using one or more liposomes, lipoplexes, and/or lipid nanoparticles. In some embodiments, liposome or lipid nanoparticle formulations described herein can comprise a polycationic composition. In some embodiments, the formulations comprising a polycationic composition can be used for the delivery of the RNA replicon described herein in vivo and/or ex vitro.

Compositions and therapeutic combinations of the application can be administered to a subject by any method known in the art in view of the present disclosure, including, but not limited to, parenteral administration (e.g., intramuscular, subcutaneous, intravenous, or intradermal injection), oral administration, transdermal administration, and nasal administration. Preferably, compositions and therapeutic combinations are administered parenterally (e.g., by intramuscular injection or intradermal injection). Methods of delivery are not limited to the above described embodiments, and any means for intracellular delivery can be used.

In certain embodiments, a composition according to the invention further comprises one or more adjuvants. Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant. The terms “adjuvant” and “immune stimulant” are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to the SARS CoV-2 S proteins of the invention. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and/or aluminum phosphate; oil -emulsion compositions (or oil -in-water compositions), including squalene-water emulsions, such as MF59 (see, e.g., WO 90/14837); saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see, e.g., US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-0- deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like; eukaryotic proteins (e.g. antibodies or fragments thereof (e.g., directed against the antigen itself or CDla, CD3, CD7, CD80) and ligands to receptors (e.g., CD40L, GMCSF, GCSF, etc), which stimulate immune response upon interaction with recipient cells. In certain embodiments the compositions of the invention comprise aluminum as an adjuvant, e.g., in the form of aluminum hydroxide, aluminum phosphate, aluminum potassium phosphate, or combinations thereof, in concentrations of 0.05-5 mg, e.g., from 0.075-1.0 mg, of aluminum content per dose.

The pre-fusion SARS CoV-2 S proteins or fragments or variants thereof can also be administered in combination with or conjugated to nanoparticles, such as, e.g., polymers, liposomes, virosomes, virus-like particles. The SARS CoV-2 S proteins or fragments or variants thereof can be combined with or encapsulated in or conjugated to the nanoparticles with or without adjuvant. Encapsulation within liposomes is described, e.g., in US 4,235,877. Conjugation to macromolecules is disclosed, for example, in US 4,372,945 or US 4,474,757.

In other embodiments, the compositions do not comprise adjuvants.

In certain embodiments, the invention provides methods for making a vaccine against a SARS CoV-2 virus, comprising providing a composition according to the invention and formulating it into a pharmaceutically acceptable composition. The term “vaccine” refers to an agent or composition containing an active component effective to induce a certain degree of immunity in a subject against a certain pathogen or disease, which will result in at least a decrease (up to complete absence) of the severity, duration or other manifestation of symptoms associated with infection by the pathogen or the disease. In the present invention, the vaccine comprises an effective amount of a pre-fusion SARS CoV-2 S protein or fragment or variant thereof and/or a nucleic acid molecule encoding a pre-fusion SARS CoV-2 S protein or fragment or variant thereof, and/or a vector comprising said nucleic acid molecule, which results in an immune response against the S protein of SARS CoV-2. This provides a method of preventing serious lower respiratory tract disease leading to hospitalization and the decrease in frequency of complications such as pneumonia and bronchiolitis due to SARS CoV-2 infection and replication in a subject. The term “vaccine” according to the invention implies that it is a pharmaceutical composition, and thus typically includes a pharmaceutically acceptable diluent, carrier or excipient. It can or cannot comprise further active ingredients. In certain embodiments it can be a combination vaccine that further comprises additional components that induce an immune response against SARS CoV-2, e.g., against other antigenic proteins of SARS CoV-2, or can comprise different forms of the same antigenic component. A combination product can also comprise immunogenic components against other infectious agents, e.g., other respiratory viruses including but not limited to influenza virus or RSV. The administration of the additional active components can, for instance, be done by separate, e.g., concurrent administration, or in a prime-boost setting, or by administering combination products of the vaccines of the invention and the additional active components.

Compositions can be administered to a subject, e.g., a human subject. The total dose of the SARS CoV-2 S proteins in a composition for a single administration can, for instance, be about 0.01 pg to about 10 mg, e.g., 1 pg-l mg, e.g., 10 pg-100 pg. Determining the recommended dose will be carried out by experimentation and is routine for those skilled in the art.

Administration of the compositions according to the invention can be performed using standard routes of administration. Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transcutaneous, or mucosal administration, e.g., intranasal, oral, and the like. In one embodiment a composition is administered by intramuscular injection. The skilled person knows the various possibilities to administer a composition, e.g., a vaccine in order to induce an immune response to the antigen(s) in the vaccine.

A subject as used herein preferably is a mammal, for instance a rodent, e.g., a mouse, a cotton rat, or a non-human-primate, or a human. Preferably, the subject is a human subject.

A SARS CoV-2 S protein, a nucleic acid molecule, a vector (such as an RNA replicon) or a composition according to an embodiment of the application can be used to induce an immune response in a mammal against SARS CoV-2 virus. The immune response can include a humoral (antibody) response and/or a cell mediated response, such as a T cell response, against SARS CoV-2 virus in a human subject.

The proteins, nucleic acid molecules, vectors, and/or compositions can also be administered, either as prime, or as boost, in a homologous or heterologous prime-boost regimen. If a boosting vaccination is performed, typically, such a boosting vaccination will be administered to the same subject at a time between one week and one year, preferably between two weeks and four months, after administering the composition to the subject for the first time (which is in such cases referred to as ‘priming vaccination’). In certain embodiments, the boosting composition or vaccine is administered at least 2 weeks after the priming composition or vaccine. In certain embodiments, the boosting composition or vaccine is administered about 2 weeks to about 12 weeks after the priming composition or vaccine. In certain embodiments, the boosting composition or vaccine is administered about 4 weeks after the priming composition or vaccine. In certain embodiments, the administration comprises at least one prime and at least one booster administration.

The prime-boost administration can, for example, be a homologous prime-boost, wherein the first and second dose comprise the same antigen (e.g., the SARS-CoV-2 spike protein) expressed from the same vector (e.g., an RNA replicon). The prime-boost administration can, for example, be a heterologous prime-boost, wherein the first and second dose comprise the same antigen or a variant thereof (e.g., the SARS-CoV-2 spike protein) expressed from the same or different vector (e.g., an RNA replicon, an adenovirus, an mR A, or a plasmid). In some embodiments of a heterologous prime-boost administration, the first dose comprises an adenovirus vector comprising the SARS-CoV-2 spike protein or a variant thereof and a second dose comprising an RNA replicon vector comprising the SARS-CoV-2 spike protein or a variant thereof. In some embodiments of a heterologous prime-boost administration, the first dose comprises an RNA replicon vector comprising the SARS-CoV-2 spike protein or a variant thereof and a second dose comprising an adenovirus vector comprising the SARS-CoV-2 spike protein or a variant thereof.

In certain aspects, the RNA replicon vaccine used in a homologous prime-boost or a heterologous prime-boost administration comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 5-194 or a fragment or variant thereof. In certain embodiments, the first dose comprises an adenovirus vector comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 5-194 or a fragment or variant thereof and a second dose comprising an RNA replicon vector comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 5-194 or a fragment or variant thereof. In certain embodiments, the first dose comprises an RNA replicon vector comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 5-194 or a fragment or variant thereof and a second dose comprising an adenovirus vector comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 5-194 or a fragment or variant thereof. The SARS CoV-2 S proteins can also be used to isolate monoclonal antibodies from a biological sample, e.g., a biological sample (such as blood, plasma, or cells) obtained from an immunized animal or infected human. The invention, thus, also relates to the use of the SARS CoV-2 protein as bait for isolating monoclonal antibodies.

Also provided is the use of the pre-fusion SARS CoV-2 S proteins of the invention in methods of screening for candidate SARS CoV-2 antiviral agents, including, but not limited to, antibodies against SARS CoV-2

In addition, the proteins of the invention can be used as diagnostic tool, for example to test the immune status of an individual by establishing whether there are antibodies in the serum of such individual capable of binding to the protein of the invention. The invention, thus, also relates to an in vitro diagnostic method for detecting the presence of an ongoing or past CoV infection in a subject said method comprising the steps of a) contacting a biological sample obtained from said subject with a protein according to the invention; and b) detecting the presence of antibody-protein complexes.

The invention is further explained in the following examples. The examples do not limit the invention in any way. They merely serve to clarify the invention.

Examples

EXAMPLE 1: Instability of semi-stabilized SARS-CoV2 S protein

A plasmid corresponding to the semi-stabilized SARS-CoV2 S protein described by (Wrapp et. al., Science 2020, FurinKO+PP according to SEQ ID NO: 3) was synthesized and codon-optimized at Gene Art (Life Technologies, Carlsbad, CA). A variant with a HIS tag (based on SEQ ID NO: 3) and a variant with a C-tag were purified. The constructs were cloned into pCDNA2004 or generated by standard methods widely known within the field involving site-directed mutagenesis and PCR and sequenced. Expi293F cells were used as the expression platform. The cells were transiently transfected using ExpiFectamine (Life Technologies; Carlsbad, CA) according to the manufacturer’s instructions and cultured for 6 days at 37°C and 10% CO2. The culture supernatant was harvested and spun for 5 minutes at 300 g to remove cells and cellular debris. The spun supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use.

SARS-CoV2 S trimers were purified using a two-step purification protocol including either CaptureSelect™ C-tag affinity column for C-tagged protein, or, for HIS-tagged protein, by Complete His-tag 5 mL (Roche; Basel, Switzerland). Both proteins were further purified by size- exclusion chromatography using a HiLoad Superdex 200 16/600column (GE Healthcare). The C-tagged and HIS tagged S trimer was unstable after repeated freeze/thaw cycles (FIGs. 2A and 2B). The purified HIS-tagged S trimer and the C-tagged trimer showed decay after 1 and especially after 5 flash freezing cycles using liquid Nitrogen (FIGs 2A and 2B).

EXAMPLE 2: Stabilizing mutations analyzed with AlphaLISA and analytical SEC

In order to stabilize the labile pre-fusion conformation of SARS-CoV2 S protein, amino acid residues at position 614, 892, and 942 (numbering according to the SEQ ID NO: 1) were mutated. Plasmids coding for the recombinant SARS-CoV-2 S protein ectodomains, which were C-terminally fused to a foldon (SEQ ID NO: 4) were expressed in Expi293Fcells, and 3 days after transfection, the supernatants were tested for binding to ACE2-Fc using AlphaLISA (FIG.

3)·

For the AlphaLISA assay, SARS-CoV2 S variants in the pcDNA2004 vector containing a linker followed by a sortase A tag, followed by a Flag-tag, followed by a flexible (G4S)7 linker, and ending with a His-tag, were prepared (the sequence of the tag, which was placed at the C- terminus of the S protein, is provided in SEQ ID NO: 2). Three days after transfection, crude supernatants were diluted 300 times in AlphaLISA buffer (PBS + 0.05% Tween-20 + 0.5 mg/mL BSA). Then, 10 pL of each dilution were transferred to a 96-well plate and mixed with 40 pL acceptor beads, donor beads, and ACE2-Fc. The donor beads were conjugated to ProtA (Cat#: AS102M, Perkin Elmer; Waltham, MA), which binds to ACE2Fc. The acceptor beads were conjugated to an anti-His antibody (Cat#: AL128M, Perkin Elmer), which binds to the His-tag of the construct.

The mixture of the supernatant containing the expressed S protein, the ACE-2 -Fc, donor beads, and acceptor beads was incubated at room temperature for 2 hours without shaking. Subsequently, the chemiluminescent signal was measured with an Ensight plate reader instrument (Perkin Elmer). The average background signal attributed to mock transfected cells was subtracted from the AlphaLISA counts measured for each of the SARS-CoV-2 S variants. Subsequently, the whole data set was divided by signal measured for the SARS CoV-2 S protein having the S backbone sequence signal to normalize the signal for each of the S variants tested to the backbone.

Compared with the soluble uncleaved S variant with a C-terminal foldon domain (SEQ ID NO: 2) or the variant with the additional PP (SEQ ID NO:3), the S variants with stabilizing substitutions D614N, A892P, and A942P showed higher ACE2-Fc binding (FIG. 3).

The cell culture supernatants of transfections with a semi-stable uncleaved SARS-CoV-2 S + PP design and with a labile uncleaved SARS-CoV-2 S protein, and of variants with a single point mutation as described above (D614N, A892P, and A942P) were analyzed using analytical SEC (FIGs. 4A-4G). An ultra high-performance liquid chromatography system (Vanquish, Thermo Scientific; Waltham, MA) and pDAWN TREOS instrument (Wyatt; Santa Barbara, CA) coupled to an Optilab pT-rEX Refractive Index Detector (Wyatt), in combination with an in-line Nanostar DLS reader (Wyatt), was used for performing the analytical SEC experiment. The cleared crude cell culture supernatants were applied to a SRT-10C SEC-500 15 cm column, (Sepax Cat# 235500-4615) with the corresponding guard column (Sepax; Newark, DE) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL/min. When analyzing supernatant samples, pMALS detectors were offline and analytical SEC data was analyzed using Chrome leon 7.2.8.0 software package. The signal of supernatants of non-transfected cells was subtracted from the signal of supernatants of S transfected cells. When purified proteins were analyzed using SEC-MALS, mMALS detectors were inline and data was analyzed using Astra 7.3 software package. For the protein component, a dn/dc (mL/g) value of 0.1850 was used and for the glycan component a value of 0.1410. Compared with the semi-stable soluble uncleaved S variant with a C-terminal foldon domain +PP, the variants with additional stabilizing substitutions D614N, A892P, and especially A942P showed higher trimer content according to analytical SEC of culture supernatant (FIGs. 4 A-C). Similarly, compared with the soluble uncleaved S variant with a C-terminal foldon domain, variants with stabilizing substitutions D614N, A892P, and especially A942P showed higher trimer content according to analytical SEC of culture supernatant. The A942P mutation has a stronger stabilizing effect than the published double proline mutation in the hinge loop (compare dashed line of FIG 4B with solid line of FIG 4 E). SEC-MALS analysis was performed on the purified stabilized protein according to SEQ ID NO: 5 and showed that the peak at 5 minutes corresponds to the mass of a trimeic S protein (FIG 4G).

EXAMPLE 3: Stabilizing point mutations and disulfide bridges analyzed with AlphaLISA and analytical SEC

In order to stabilize the labile pre-fusion conformation of SARS-CoV-2 S protein, disulfide bridges were introduced between residues 880 and 888 or between residues 884 and 893, and point mutations were introduced at position 532 and 572. Similar to EXAMPLE 2, plasmids coding for the uncleaved SARS-CoV-2 S protein with or without the double proline in the hinge loop were expressed in Expi293Fcells, and 3 days after transfection the supernatants were tested for binding to ACE2-Fc using AlphaLISA as described in EXAMPLE 2 (FIG. 5). Compared with the soluble labile uncleaved S variant with a C-terminal foldon, the variants with stabilizing substitutions T572I, N532P, and with the introduction of a disulfide between residues 880 and 888 showed higher ACE2-Fc binding (FIG. 5, left panel).

In addition, compared with the soluble semi stable uncleaved S variant with a C-terminal foldon domain and the double proline, the variants with stabilizing substitutions T572I, N532P, with the introduction of a disulfide between residues 880 and 888 and with a disulfide between residues 884 and 893 showed higher ACE2-Fc binding (FIG. 5, right panel).

The cell culture supernatants of transfections with a semi stable uncleaved SARS-CoV-2 S + PP design and with a labile uncleaved SARS-CoV-2 S protein, and of variants with an introduced disulfide bridge or a single point mutation as described above (T572I, N532P, CYS880-CYS888 and CYS884-CYS893) were analyzed using analytical SEC (FIG. 6) as described in EXAMPLE 2. Compared with the semi-stable soluble uncleaved S variant with a C-terminal foldon domain + PP, the variants with stabilizing substitutions T572I, N532P, and the disulfide bridges 880C-888C and 884C-893C (FIGs. 6A-6D) showed higher trimer content according to analytical SEC of culture supernatant. Similarly, compared with the soluble uncleaved S variant, the variants with stabilizing substitutions T572I, N532P, and the variant with disulfide bridge 880C-888C showed higher trimer content according to analytical SEC of culture supernatant (FIGs. 6E-6H).

Table 1. Standard amino acids, abbreviations and properties

EXAMPLE 4: Construction and characterization ofRNA replicon expressing SARS-CoV-2 S variants

Plasmid construction

The TC-83 strain of Venezuelan Equine Encephalitis Virus (VEEV) genome sequence serves as the base sequence used to construct the SMARRT replicon. This sequence is modified by placing the Downstream LooP (DLP) from Sindbis virus upstream of the non-structural protein 1 (nsPl) with the two joined by a 2A ribosome skipping element from porcine teschovirus-1. The first 213 nucleotides of nsPl are duplicated downstream of the 5’ UTR and upstream of the DLP except for the start codon, which is mutated to TAG. This insures that all regulatory and secondary structures necessary for replication are maintained but prevents translation of this partial nspl sequence. The alphavirus structural genes are removed and EcoR V and Asc I restriction sites are placed downstream of the subgenomic promoter as a multiple cloning site (MCS) to facilitate insertion of heterologous genes of interest. 40bp of homology to the MCS is added to the 5’ and 3’ ends of each CoV2 spike antigen sequence and is cloned into the SMARRT replicon digested with EcoRV and Ascl using NEB HiFi DNA assembly master mix (cat # E2621S). All constructs are sequenced verified.

RNA transcription

Plasmids are purified using the Nucleobond xtra EF maxiprep kits (Machery-Nagel cat # 740426.10) followed by phenol/chloroform extraction and Sodium Acetate/ethanol precipitation. RNA is generated using the HiScribe T7 ARCA mRNA kit from NEB (cat # E2065S) and 1 pig of plasmid template linearized with Ndel. RNA is subsequently purified using RNeasy purification columns (Qiagen cat # 75144; Qiagen; Hilden, Germany) and is eluted in water. RNA concentration is determined using a Nanodrop spectrophotometer.

Detection of dsRNA and Spike antigen

Vero cells (ATCC, Manassas, VA, CCL-81) are cultured in DMEM supplemented with 10% fetal bovine serum (Gemini #100-106) and penicillin/streptomycin/glutamine (Gibco #10378016). The cells are electroporated in strip cuvettes with 1.5 pig ofRNA per 10⁶ cells using SF buffer (Lonza; Basel, Switzerland) and a 4D-Nucleofector. 21 h post electroporation cells are harvested for analysis by either flow cytometry or Western blot as follows. Flow cytometry: 21 hours post electroporation cells are incubated in Versene solution for 10 minutes to detach them from the plate and are washed twice in PBS containing 5% BSA. The cells are stained for surface expressed CoV2 spike protein using the antibody CR3022 directly conjugated to APC. After staining CoV2 spike on the cell surface, the cells are washed, then fixed, permeabilized, and stained for intracellular dsRNA using the J2 anti-dsRNA Ab (Scicons, #10010500) conjugated to R-PE using a Lightning-Link R-PE conjugation kit (Innova Biosciences; Cambridge, England). After staining, cells are evaluated on a LSRFortessa flow cytometer (BD) and the data are analyzed using FlowJo 10 (Tree Star, Ashland, OR).

Western blot: To analyze cells by Western blot, cells are washed with PBS following which 150 pL of lx LDS loading buffer plus reducing agent is added to each well of a 6 well plate. Whole cell lysates are transferred to a microfuge tube and are incubated at 70°C for 10 minutes. 25 pL of lysate from each sample is loaded and separated on a 4-12% Bis-Tris Gel. Proteins are transferred to a nitrocellulose membrane using an iBlot system and the membranes are probed for CoV2 spike protein with an anti-CoV2 spike antibody from Genetex (Cat# GTX632604; Genetex; Irvine, CA). The blot is then probed for actin to ensure equal loading across the different samples.

EXAMPLE 5: Dose response study for homologous prime-boost administration ofSMARRT- nCov constructs

The investigate whether the SMARRT-nCov constructs were able to elicit a humoral immune response at days 27 and 56 post administration, a dose response study for a homologous prime-boost administration of SMARRT-1158 and SMARRT-1159 constructs was conducted. SMARRT-1158, comprising a SARS-CoV-2 spike full length wild type protein (YP_009724390.1), and SMARRT-1159, comprising a SARS-CoV-2 spike protein with a wild- type signal peptide, the fiirin cleavage site removed, and stabilizing proline mutations in the hinge loop, were administered to Balb/C mice at day 0 as a priming administration at increasing dose levels of 0.1 pg, 1.0 pg, and 10 pg. The same constructs were administered at the same doses in a boosting administration at day 28 post prime administration. A DNA encoding the same spike protein as the SMARRT-1159 construct was administered as a control at a dose of 100 pg for the priming administration and 10 pg for the boosting administration. The dose schedule and experimental design is provided below in Table 2.

Table 2: Dose response study design for homologous prime-boost administration

*DNA encoding COVID-19 spike antigen (1159 construct)

% n=5/group sacrificed at day 14 and the remaining half at day 54

An ELISA assay was used to measure the spike protein specific IgG titers produced after administration of the prime and boost compositions. After administration of the prime composition, the spike protein specific IgG titers were measured at days 14 and 27, and after administration of the boost composition, the spike protein specific IgG titers were measured at days 42 and 54. As a control, the spike specific IgG titers were measured 1 day prior to the administration of the priming composition. The results are shown in FIGs. 8B-8E.

The SMARRT-1159 construct elicited higher antibody titers at days 14 and 27 compared to the SMARRT-1158 construct (FIGs. 8B and 8C). 0.1 pig of SMARRT-1159 elicited titers at similar levels to 10 pig of SMARRT-1158 (FIGs. 8B and 8C). Antibody titers elicited by SMARRT-1159 increased from day 14 to day 27 (FIGs. 8B and 8C). The DNA-1159 construct did not elicit high antibody titers (data not shown).

A second dose of the SMARRT constructs boosted the spike protein specific antibody titers when measured at 42 and 54 days (FIGs. 8C and 8D) as compared to the day 27 titers.

FIG. 9 demonstrated that the SMARRT-1159 construct was capable of producing neutralizing antibodies to the spike protein at day 27 after the administration of the priming composition.

FIGs. 10A and 10B demonstrated that similar levels of IFNy secreting cells were detected in the spleens of immunized animals 2 weeks after the first dose at day 14 (FIG. 10A) and 2 weeks after the second dose at day 54 (FIG. 10B).

Materials and methods

ELISpot assay for mouse splenocytes :

Plates were washed four times with 200 mΐ of sterile PBS in a biosafety hood. The wells of the plate were conditioned with 200 mΐ of AIM V® media (Gibco) with albumax for 2 hours.

While the plates are conditioned with the blocking buffer, a PMA/Ionomycin solution was prepared by adding 4 mΐ of PMA stock (lmg/ml) to 1.996 ml of media to create a 1:500 dilution. 200 mΐ of the 1:500 dilution was added to 9.780 ml of media to create a 1:50 dilution.

20 mΐ of Ionomycin was added to the media to create a 1:500 dilution. After preparing the PMA/Ionomycin solution, the blocking buffer was removed from the plates and the plates were patted dry on a paper towel. 100 mΐ of the PMA/Ionomycin solution, stimulations, and DMSO, were added to the wells of the plate. 100 mΐ of cells, diluted in AIM V®, were added to each well at a total concentration of 2.5 x 10⁵ cells/well. The plates were incubated at 37°C, 5% CO2 for 22 hours.

The plates were washed five times with PBS. The 1 mg/ml detection antibody, i.e., R4- 6A2 biotin) was diluted to 1 pg/ml in PBS containing 0.5% FBS. 100 mΐ of diluted detection antibody was added to each well and the plate was incubated for 2 hours at room temperature. The plates were washed five times with PBS. The secondary antibody, i.e., Streptavidin-HRP, was diluted 1 : 1000 in PBS-0.5% FBS. 100 mΐ of the secondary antibody was added to each well, and the plate was incubated for 1 hour at room temperature in the dark. The plates were washed five times. The ready to use TMB substrate was filtered, and 100 mΐ of the TMB substrate was added to each well and developed until distinct spots emerged (~10 minutes). The plates were sent for scanning and counting services.

Intracellular staining of murine splenocytes :

AIM V® plus media with co-stimulatory molecules was prepared by taking 100 ml of AIM V® tissue culture media, and adding 100 mΐ of anti-CD49d and anti-CD28 purified antibodies for a final concentration of 0.5 pg/ml. AIM V® plus media was kept on ice.

A cell activation cocktail of PMA/Ionomycin positive control media (without brefeldin A) at a 1:250 ratio was made by preparing a 500x cell activation cocktail of PMA at a concentration of 40.5 mM and Ionomycin at a concentration of 669.3 pM in DMSA. If doing pools of n = 15 groups with 0.1 ml/group; 3 mis of diluted cell activation cocktail is prepared by adding 2.988 ml of AIM V tissue culture media with 12 mΐ of the 500x cell activation cocktail to produce a 1 :250 dilution. 100 mΐ of the diluted cell activation cocktail was added to the appropriate wells of the 96 well plate.

DMSO “mock” condition media at a 1:250 dilution was prepared as follows: for 50 mice x 100 mΐ/well; a total amount of 5 mis of mock conditioned media was needed. Add 5 mis of AIM V® plus media (with co-stimulatory molecules) to 20 mΐ of DMSO and mix well. Add 100 mΐ of mock media to the appropriate wells of the 96 well plate.

SARS-CoV-2 spike-specific overlapping peptide pools were prepared and labeled. For 150 samples x 100 mΐ/well, prepare enough SAR-CoV-2 spike -specific overlapping peptide pools for 200 samples. Single cell suspensions from the mouse were prepared at a concentration of 10 x 10⁶ cells/ml. 200 mΐ of resuspended cells per mouse per condition were seeded into the round bottom of a 96-well plate to provide a final concentration of cells of 2 x 10⁶ cells/well. The plates were centrifuged at 500g for 5 minutes at 4°C and the media was decanted from the cell pellet. The cell pellet was resuspended in 100 mΐ of AIM V® Tissue culture media and stored at 4°C until stimulation condition media is added.

Once the resuspended cells were treated with the appropriate component, the 96 well plate was covered in foil and incubated at 37°C for 1 hour for the stimulation incubation.

During the incubation, the golgi plug dilution was prepared as follows noting that for each 96 well plate, enough golgi plug dilution was made for 100 wells at 0.25 mΐ/well. 19.82 ml of AIM V plus media (with co-stimulatory molecules) was added to a separate tube, and 180 mΐ of Golgi Plug was added to the tube and mixed well while on ice.

After 1 hour of the stimulation incubation, 25 mΐ/well of diluted golgi plug was added to each well, and the plate was incubated for an additional 5 hours at 37°C for a total of 6 hours of incubation time. After the 6 hours of incubation, the plate was centrifuged at 500 g for 5 minutes at 4°C. The supernatant was removed, 200 mΐ of AIM V® plus tissue culture media was added to each well, and the cells were resuspended. The plate of cells was placed at 4°C overnight, and the cells were analyzed for intracellular signaling the next day.

Extracellular and Intracellular signaling :

The plate of cells was centrifuged at 500 g for 5 minutes at 4°C. The supernatant was removed, and cells were washed by resuspending with 150 mΐ of IX PBS. Cells were then centrifuged at 500 g for 5 minutes. Following removal of PBS, cells were resuspended in 50 mΐ of FVD506 cocktail and incubated for 15 minutes at room temperature in the dark (i.e., the plate was wrapped in foil). After 15 minutes, the cells were washed twice by centrifuging at 500 x g for 5 minutes and washing in 150 mΐ cell staining buffer. After the final centrifugation, supernatants were removed, and cells were resuspended in 25 mΐ of Fc block and incubated for 15 minutes at room temperature in the dark. Next, 25 mΐ of an extracellular surface stain (CD8 FITC, CD3-APC-ef780, CD4-BV421) was added to each well. Cells were mixed and incubated for 30 minutes at 4°C in the dark.

While the cells were incubated for 30 minutes, compensation control beads were prepared by adding one drop of UltraComp beads into a polystyrene tube. 0.5 mΐ of antibody stain ( 1 compensation tube per antibody) was added to the tube, the bottom of the tube was flicked to mix the contents, and the tube was incubated at 4°C for 15 minutes in the dark. 2 ml of cell staining buffer was added to the tube, and the tube was centrifuged at 500 g for 5 minutes at 4°C. The supernatant was removed, and 300 mΐ of cell staining buffer was added to the beads. The beads were flicked to resuspend, and the compensation control beads were stored at 4°C until FACS acquisition. The beads were vortexed well prior to acquisition.

After extracellular staining, cells were centrifuged at 500 g for 5 minutes. Following removal of supernatants, cells were washed with 150 pL cell staining buffer and centrifuged at 500 g for 5 minutes. The supernatant was removed, then 200 pL of fixation and permeabilization solution was added to the cells, and the cells were resuspended and incubated for 20 minutes at 4°C in the dark. The cells were centrifuged at 500 g for 5 minutes. The supernatant was removed, then the cells were washed twice with 150 pL IX perm/wash buffer, and the cells were resuspended and centrifuged at 500 g for 5 minutes. (To make 300 mL of lx BD perm/wash buffer: 30 mL of lOx BD perm/wash buffer was added to 270 mL of distilled water. The solution was mixed well and kept on ice. (600 pL of lx perm/wash buffer per sample/per well was required)).

Supernatants were removed and 50 pL of the following intracellular cytokine stain antibody cocktail (IL-2-PE, IFNg-APC, TNFa-PE-Cy7) was added to the cells and incubated for 30 minutes at 4°C in the dark. The cells were washed with 150 pL IX perm/wash buffer. Following centrifugation at 500 x g for 5 minutes, supernatants were removed, then the cells were washed with 200 pL cell staining buffer. Following the final wash, supernatants were removed, and cells resuspended with 200 pL cell staining buffer. The samples were filtered through AcroPrep™ Advance Plates, then centrifuged at 1500rpm for 2 minutes. The cells were resuspended in staining buffer and kept on ice or in 4°C until FACS acquisition via using high- throughput sampling (EFTS) plate reader.

EXAMPLE 6: Antibody response study for heterologous prime-boost administration of adenovirus and SMARRT-nCov constructs

The primary aim of the study was to compare a 2-dose heterologous regimen of the SMARRT and Ad26 platforms expressing the prefusion stabilized spike antigen to a 2- dose homologous or single dose regimen in Balb/C mice. SMARRT-1159 or Ad26NCOV030 were administered to Balb/C mice at day 0 as a priming administration at indicated doses. The same constructs were administered at the same doses in either a homologous or heterologous boosting administration at day 28 post prime administration (FIG. 11A). A high dose of Ad26NCOV030 (10¹⁰ vp) or an empty Ad26 were included as positive and negative controls. The dose schedule and experimental design is provided below in Table 3 and FIG. 11A.

Table 3 : Study Design

An ELISA assay was used to measure the spike protein specific IgG titers produced after administration of the prime and boost compositions. After administration of the prime composition, the spike protein specific IgG titers were measured at days 14 and 27. All animals that received SMARRT-1159 elicited spike specific antibodies as early as 2 weeks that were maintained until week 4 (FIGs. 1 IB-11C). After administration of the boost, the spike protein specific IgG titers were measured at days 42 (FIG. 1 ID) and 54 (FIG. 1 IE). A second dose of the SMARRT or Ad26 constructs boosted the spike protein specific antibody titers when measured at 42 and 54 days as compared to the day 27 titers. The SMARRT-1159 - Ad26NCOV2 regimen (R-A) had significantly higher antibody response relative to the Ad26NCOV2- SMARRT-1159 (A-R) regimen, which were maintained out to day 56.

At day 56 ELISAs measuring both IgGl and IgG2 isotype levels in the serum were performed. Animals that received SMARRT-1159 for the prime had higher levels of spike- specific IgG2a isotype antibodies. As a result they also had higher IgG2a:IgGl ratios suggesting a Thl skewed response (FIGs. 12A-12B). Viral neutralization titers were measured at day 56. A trend for increased neutralization titers was observed when animals primed with SMARRT-1159 were boosted with either SMARRT-1159 or Ad26NCOV030 (FIG. 13).

Figures 14A-14B demonstrated a 2-dose heterologous or homologous regimen elicited similar levels of IFNy secreting cells in the spleens of immunized animals 4 weeks after the second dose at day 56.

References

Belouzard et al. (2009), Proc Natl Acad Sci U S A 106:5871-6. Bosch et al. (2008), J Virol 82:8887-90.

Follis et al. (2006) Virology 350:358-69.

Madu et al. (2009), J Virol 83:7411-21.

Walls et al. (2016), Nature 531:114-7.

Wrapp et. al. (2020) Science 367(6482): 1260-1263.

Hoffmann et al. (2020) BioRxiv: doi: https://doi.org/10.1101/2020.01.31.929042 Bestle et al (2020) BioRxiv doi: https://doi.Org/10.l 101/2020.04.15.042085 Hastie et al. (2017), Science 356, 923-928.

Krarup et al (2015), Nat Commun 6, 8143.

Pallesen et al. (2017), Proc Natl Acad Sci USA 114, E7348-E7357.

Rutten et al. (2020), Cell Rep. 30(13):4540-4550.

Letarov et al. (1993), Biochemistry Moscow 64: 817-823.

S-Guthe et al. (2004), J. Mol. Biol. 337: 905-915

Sequences

SEQ ID NO 1: full length S protein (underline signal peptide, double underline TM and cytoplasmic domain that is deleted in the soluble version):

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFD

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLL

ALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQP

TESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIR

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISV

TTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFS

QILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSG

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

VKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFC

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

VSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESUDLQE

LG KYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO 2: soluble S protein with furin KO, underline signal peptide, double underline linker, foldon, tags etc.):

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSRAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISV

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LG KYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 3: soluble S protein with Furin KO and double proline in the hinge loop, (underline signal peptide) double underline linker, foldon, tags etc.)

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

VKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFC

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

VSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE

LG KYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 4: foldon

GYIPEAPRDGQAYVRKDGEWVLLSTFL

SEQ ID NO 5: SEQ ID NO 2 + A942P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

NVFQTRAGCUGAEHVNNSYECDIPIGAGICASYQTQTNSPSRAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISV

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

VSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG GSGGGGSGGGGSHHHHHH

SEQ ID NO 6: SEQ ID NO 2 + A892P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 7: SEQ ID NO 2 + D614N

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 8: SEQ ID NO 2 + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

NVFQTRAGCUGAEHVNNSYECDIPIGAGICASYQTQTNSPSRAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISV TTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 9: SEQ ID NO 2 + N532P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 10: SEQ ID NO 2 + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

QILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLACTITSG

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 11: SEQ ID NO 2 + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLL

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

QILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITCG

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 12: SEQ ID NO 3 + A942P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 13: SEQ ID NO 3 + A892P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

VSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESUDLQE LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG GSGGGGSGGGGSHHHHHH

SEQ ID NO 14: SEQ ID NO 3 + D614N

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 15: SEQ ID NO 3 + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 16: SEQ ID NO 3 + N532P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSRAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISV TTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 17: SEQ ID NO 3 + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 18: SEQ ID NO 3 + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 19: SEQ ID NO 2 + A942P + A892P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLL ALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQP

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 20: SEQ ID NO 2 + A942P + D614N

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 21: SEQ ID NO 2 + A942P + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 22: SEQ ID NO 2 + A942P + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

QILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDUCAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSG

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 23: SEQ ID NO 2 + A942P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 24: SEQ ID NO 2 + A942P + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 25: SEQ ID NO 2 + A892P + D614N

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 26: SEQ ID NO 2 + A892P + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 27: SEQ ID NO 2 + A892P + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 28: SEQ ID NO 2 + A892P + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 29: SEQ ID NO 2 + A892P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 30: SEQ ID NO 2 + D614N + T572I

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 31: SEQ ID NO 2 + D614N + N532P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 32: SEQ ID NO 2 + D614N + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 33: SEQ ID NO 2 + D614N + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 34: SEQ ID NO 2 + T572I + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 35: SEQ ID NO 2 + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 36: SEQ ID NO 2 + T572I + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 37: SEQ ID NO 2 + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 38: SEQ ID NO 2 + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 39: SEQ ID NO 2 + A942P + A892P + D614N

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 40: SEQ ID NO 2 + A942P + A892P + T572I

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 41: SEQ ID NO 2 + A942P + A892P + N532P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 42: SEQ ID NO 2 + A942P + A892P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 43: SEQ ID NO 2 + A942P + A892P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 44: SEQ ID NO 2 + A942P + D614N + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 45: SEQ ID NO 2 + A942P + D614N + N532P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

VKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRUTGRLQSLQTYVTQQURAAEIRASANLAATKMSECVLGQSKRVDFC

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 46: SEQ ID NO 2 + A942P + D614N + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 47: SEQ ID NO 2 + A942P + D614N + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

QILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDUCAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITCG

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 48: SEQ ID NO 2 + A942P + T572I + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 49: SEQ ID NO 2 + A942P + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

QILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDUCAQKFNGLTVLPPLLTDEMIAQYTSALLACTITSG

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 50: SEQ ID NO 2 + A942P + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 51: SEQ ID NO 2 + A942P + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 52: SEQ ID NO 2 + A942P + N532P + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 53: SEQ ID NO 2 + A892P + D614N + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 54: SEQ ID NO 2 + A892P + D614N + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 55: SEQ ID NO 2 + A892P + D614N + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 56: SEQ ID NO 2 + A892P + D614N + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 57: SEQ ID NO 2 + A892P + T572I + N532P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 58: SEQ ID NO 2 + A892P + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 59: SEQ ID NO 2 + A892P + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 60: SEQ ID NO 2 + A892P + N532P + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 61: SEQ ID NO 2 + A892P + N532P + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 62: SEQ ID NO 2 + D614N + T572I + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 63: SEQ ID NO 2 + D614N + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 64: SEQ ID NO 2 + D614N + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 65: SEQ ID NO 2 + D614N + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 66: SEQ ID NO 2 + D614N + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 67: SEQ ID NO 2 + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 68: SEQ ID NO 2 + T572I + N532P + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 69: SEQ ID NO 2 + A942P + A892P + D614N + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 70: SEQ ID NO 2 + A942P + A892P + D614N + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 71: SEQ ID NO 2 + A942P + A892P + D614N + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 72: SEQ ID NO 2 + A942P + A892P + D614N + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 73: SEQ ID NO 2 + A942P + A892P + T572I + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 74: SEQ ID NO 2 + A942P + A892P + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 75: SEQ ID NO 2 + A942P + A892P + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 76: SEQ ID NO 2 + A942P + A892P + N532P + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

VKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRUTGRLQSLQTYVTQQURAAEIRASANLAATKMSECVLGQSKRVDFC

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 77: SEQ ID NO 2 + A942P + A892P + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 78: SEQ ID NO 2 + A942P + D614N + T572I + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 79: SEQ ID NO 2 + A942P + D614N + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 80: SEQ ID NO 2 + A942P + D614N + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 81: SEQ ID NO 2 + A942P + D614N + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 82: SEQ ID NO 2 + A942P + D614N + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 83: SEQ ID NO 2 + A942P + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 84: SEQ ID NO 2 + A942P + T572I + N532P + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

VKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRUTGRLQSLQTYVTQQURAAEIRASANLAATKMSECVLGQSKRVDFC

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 85: SEQ ID NO 2 + A892P + D614N + T572I + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 86: SEQ ID NO 2 + A892P + D614N + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 87: SEQ ID NO 2 + A892P + D614N + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 88: SEQ ID NO 2 + A892P + D614N + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 89: SEQ ID NO 2 + A892P + D614N + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 90: SEQ ID NO 2 + A892P + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 91: SEQ ID NO 2 + A892P + T572I + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 92: SEQ ID NO 2 + D614N + T572I + N532P + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

VKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRUTGRLQSLQTYVTQQURAAEIRASANLAATKMSECVLGQSKRVDFC

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 93: SEQ ID NO 2 + D614N + T572I + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 94: SEQ ID NO 2 + A942P + A892P + D614N + T572I + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 95: SEQ ID NO 2 + A942P + A892P + D614N + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 96: SEQ ID NO 2 + A942P + A892P + D614N + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 97: SEQ ID NO 2 + A942P + A892P + D614N + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 98: SEQ ID NO 2 + A942P + A892P + D614N + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 99: SEQ ID NO 2 + A942P + A892P + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 100: SEQ ID NO 2 + A942P + A892P + T572I + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 101: SEQ ID NO 2 + A942P + D614N + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 102: SEQ ID NO 2 + A942P + D614N + T572I + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 103: SEQ ID NO 2 + A892P + D614N + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

VKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRUTGRLQSLQTYVTQQURAAEIRASANLAATKMSECVLGQSKRVDFC

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 104: SEQ ID NO 2 + A892P + D614N + T572I + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 105: SEQ ID NO 2 + A942P + A892P + D614N + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 106: SEQ ID NO 2 + A942P + A892P + D614N + T572I + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 107: SEQ ID NO 3 + A942P + A892P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 108: SEQ ID NO 3 + A942P + D614N

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 109: SEQ ID NO 3 + A942P + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 110: SEQ ID NO 3 + A942P + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 111: SEQ ID NO 3 + A942P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 112: SEQ ID NO 3 + A942P + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 113: SEQ ID NO 3 + A892P + D614N

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 114: SEQ ID NO 3 + A892P + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 115: SEQ ID NO 3 + A892P + N532P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 116: SEQ ID NO 3 + A892P + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 117: SEQ ID NO 3 + A892P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 118: SEQ ID NO 3 + D614N + T572I

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 119: SEQ ID NO 3 + D614N + N532P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 120: SEQ ID NO 3 + D614N + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 121: SEQ ID NO 3 + D614N + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 122: SEQ ID NO 3 + T572I + N532P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 123: SEQ ID NO 3 + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 124: SEQ ID NO 3 + T572I + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 125: SEQ ID NO 3 + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 126: SEQ ID NO 3 + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 127: SEQ ID NO 3 + A942P + A892P + D614N

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 128: SEQ ID NO 3 + A942P + A892P + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 129: SEQ ID NO 3 + A942P + A892P + N532P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 130: SEQ ID NO 3 + A942P + A892P + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

VKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRUTGRLQSLQTYVTQQURAAEIRASANLAATKMSECVLGQSKRVDFC

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 131: SEQ ID NO 3 + A942P + A892P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 132: SEQ ID NO 3 + A942P + D614N + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 133: SEQ ID NO 3 + A942P + D614N + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 134: SEQ ID NO 3 + A942P + D614N + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 135: SEQ ID NO 3 + A942P + D614N + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 136: SEQ ID NO 3 + A942P + T572I + N532P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 137: SEQ ID NO 3 + A942P + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 138: SEQ ID NO 3 + A942P + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 139: SEQ ID NO 3 + A942P + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 140: SEQ ID NO 3 + A942P + N532P + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 141: SEQ ID NO 3 + A892P + D614N + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 142: SEQ ID NO 3 + A892P + D614N + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 143: SEQ ID NO 3 + A892P + D614N + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 144: SEQ ID NO 3 + A892P + D614N + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 145: SEQ ID NO 3 + A892P + T572I + N532P

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 146: SEQ ID NO 3 + A892P + T572I + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 147: SEQ ID NO 3 + A892P + T572I + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 148: SEQ ID NO 3 + A892P + N532P + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 149: SEQ ID NO 3 + A892P + N532P + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 150: SEQ ID NO 3 + D614N + T572I + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 151: SEQ ID NO 3 + D614N + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 152: SEQ ID NO 3 + D614N + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 153: SEQ ID NO 3 + D614N + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 154: SEQ ID NO 3 + D614N + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 155: SEQ ID NO 3 + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 156: SEQ ID NO 3 + T572I + N532P + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 157: SEQ ID NO 3 + A942P + A892P + D614N + T572I

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 158: SEQ ID NO 3 + A942P + A892P + D614N + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 159: SEQ ID NO 3 + A942P + A892P + D614N + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 160: SEQ ID NO 3 + A942P + A892P + D614N + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 161: SEQ ID NO 3 + A942P + A892P + T572I + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 162: SEQ ID NO 3 + A942P + A892P + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 163: SEQ ID NO 3 + A942P + A892P + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 164: SEQ ID NO 3 + A942P + A892P + N532P + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 165: SEQ ID NO 3 + A942P + A892P + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 166: SEQ ID NO 3 + A942P + D614N + T572I + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 167: SEQ ID NO 3 + A942P + D614N + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 168: SEQ ID NO 3 + A942P + D614N + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 169: SEQ ID NO 3 + A942P + D614N + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 170: SEQ ID NO 3 + A942P + D614N + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 171: SEQ ID NO 3 + A942P + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 172: SEQ ID NO 3 + A942P + T572I + N532P + S884C + A893C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

VKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRUTGRLQSLQTYVTQQURAAEIRASANLAATKMSECVLGQSKRVDFC

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 173: SEQ ID NO 3 + A892P + D614N + T572I + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 174: SEQ ID NO 3 + A892P + D614N + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 175: SEQ ID NO 3 + A892P + D614N + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 176: SEQ ID NO 3 + A892P + D614N + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 177: SEQ ID NO 3 + A892P + D614N + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 178: SEQ ID NO 3 + A892P + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 179: SEQ ID NO 3 + A892P + T572I + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 180: SEQ ID NO 3 + D614N + T572I + N532P + G880C + F888C

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 181: SEQ ID NO 3 + D614N + T572I + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 182: SEQ ID NO 3 + A942P + A892P + D614N + T572I + N532P

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 183: SEQ ID NO 3 + A942P + A892P + D614N + T572I + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 184: SEQ ID NO 3 + A942P + A892P + D614N + T572I + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 185: SEQ ID NO 3 + A942P + A892P + D614N + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 186: SEQ ID NO 3 + A942P + A892P + D614N + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

VKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRUTGRLQSLQTYVTQQURAAEIRASANLAATKMSECVLGQSKRVDFC

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 187: SEQ ID NO 3 + A942P + A892P + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 188: SEQ ID NO 3 + A942P + A892P + T572I + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 189: SEQ ID NO 3 + A942P + D614N + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

SEQ ID NO 190: SEQ ID NO 3 + A942P + D614N + T572I + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGACLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 191: SEQ ID NO 3 + A892P + D614N + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

VKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRUTGRLQSLQTYVTQQURAAEIRASANLAATKMSECVLGQSKRVDFC

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 192: SEQ ID NO 3 + A892P + D614N + T572I + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKUANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 193: SEQ ID NO 3 + A942P + A892P + D614N + T572I + N532P + G880C + F888C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTCGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO 194: SEQ ID NO 3 + A942P + A892P + D614N + T572I + N532P + S884C + A893C

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLUVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY

GDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLTGTGVLTESNKKFLP

FQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQNVNCTEVPVAIHADQLTPTWRVYSTGS

WTFGAGPCLQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTL

GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

LGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG RSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGG

GSGGGGSGGGGSHHHHHH

SEQ ID NO: 195 coding sequence for a short signal peptide from a Corona virus

ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGTCCAGC

SEQ ID NO: 196, 26S minimal promoter CTCTCTACGGCTAACCTG AAT G G A

SEQ ID NO: 197, T7 promoter

T AAT ACGACTCACTATAG

SEQ ID NO: 198, 5-UTR

ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA

SEQ ID NO: 199, Alpha 5' replication seq from nsPl

TAGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTT GAG GTAG AAGCCAAG CAG GTCACTG ATAATG ACCATGCTAATGCCAG AG CGTTTTCG CAT CTG G CTT C AAAACT G ATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGA

SEQ ID NO: 200, gDLP

AT AGTCAG C ATAGT ACATTT CAT CT G ACT AAT ACT ACAACACCACCACC AT G AAT AG AG G ATT CTTT AAC AT G CTCG GCCGCCGCCCCTTCCCGGCCCCCACTGCCATGTGGAGGCCGCGGAGAAGGAGGCAGGCGGCCCCG

SEQ ID NO: 201, P2A

GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT

SEQ ID NO: 202, P2A

GSGATNFSLLKQAGDVESNPGP

SEQ ID NO: 203, DLP nsp ORF encoding a 3' portion of gDLP, P2A and nspl-3

ATGAATAGAGGATTCTTTAACATGCTCGGCCGCCGCCCCTTCCCGGCCCCCACTGCCATGTGGAGGCCGCGGAGA

AGGAGGCAGGCGGCCCCGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGA

ACCCTGGACCTGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCC

CGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTC

AAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTA

TTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACT

AAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCAT

GAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGC

TGTTTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTCGC

CTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCTACCAACT

GGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGCGGTCACGTAGAG

GGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCTGTTGGCTCGACCATCTACCAC

GAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACTTACGTGGCAAGCAAAATTACACATGT

CGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAG

CCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTCTTGTGCTGCAAAGTGACAGACACATTGAACGGGGAG

AGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAGCTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATG

TCAGTGCGGACGACGCGCAAAAACTGCTGGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAA

ACACCAATACCATGAAAAATTACCTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGG

AAGATCAAGAAGATGAAAGGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAA

GGCACAAGATAACATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATT

CGTGCTGCCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGC

ACAAGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGAG

GTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAAGCC

GATGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCAGC

TACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAAGAGTGAAAAATTATCTT

GCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAAGGGCGTTATGCCGTGGAACCATA

CCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGGACTTTCAAGCTCTGAGTGAAAGTGCCAC CATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCACCATATTGCCACACATGGAGGAGCGCTGAACAC

TGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAGCACGACGGCGAATACCTGTACGACATCGACAGGAAACA

GTGCGTCAAGAAAGAACTAGTCACTGGGCTAGGGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGC

CTACGAGAGTCTGAGAACACGACCAGCCGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATC

AGGCAAGTCTGGCATCATTAAAAGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTG

CAGAAATTATAAGGGACGTCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGA

ATGGATGCAAACACCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCT

CAT AG CCATT AT AAG ACCT AAAAAG G CAGTG CTCTGCG GG G ATCCCAAACAGTG CG GTTTTTTT AACAT G ATGTG C

CTGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAAATCTGT

GACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACTAAGATTGTGATT

GACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAGGGTGGGTGAAGCAGTTG

CAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTGTATGCC

GTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCTGAACATGTGAACGTCCTACTGACCCGCACGG

AGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGATAAAAACACTGACTGCCAAGTACCCTGGGAATT

TCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGATGCCATCATGAGGCACATCTTGGAGAGACCGGACCCT

ACCGACGTCTTCCAGAATAAGGCAAACGTGTGTTGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATA

GACATGACCACTGAACAATGGAACACTGTGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTG

AACCAACTATGCGTGAGGTTCTTTGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATT

AGGAATAATCACTGGGATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTC

GCAGGTACCCACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATT

ATGATCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACACCCA

CAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGTTGTCCGTCC

CAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGATTTAGGCATCCCAG

GTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATACCATCACTATCAGCAGTGTGA

AGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATCTGAATCCCGGCGGAACCTGTGTCAGC

ATAG GTTATG GTTACG CTG AC AG G G CC AG CG AAAG CAT CATT GGTGCTATAGCGCGGCAGTT C AAGTTTT CCCG G

GTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTA

CGCACAATCCTTACAAGCTTTCATCAACCTTGACCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCA

CCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAA

GGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGA

AGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGT

TTCGGAGGTTGAAGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTA

CAAGTCAGTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAAC

CATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACT

CTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTGACAGAACC

TGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCACAAGCGATGGCA

AAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGAAATTAATGCCATGTGGCC

CGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGCATGAGCAGTATTAGGTCGAAAT

GCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCCAGA

AAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTATAGA

ATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAG

GAAGTATCTCGTGGAAACACCACCGGTAGACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGA

CACCTGAACAACCACCACTTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGG

AAGAAGAGGATAGCATAAGTTTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACG

GGCCGCCCTCTGTATCTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTG

ACACCCTGGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATG

GAGTTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAA

CACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGA

TCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTGGTCT

CCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAACAATGA SEQ ID NO: 204, nspl coding sequence

GAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAG

GTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCG

AAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACA

AGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGA

AAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACCCT

GACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAG

GATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTCGCCTACTGGATA

GG CTTT G ACACCACCCCTTTT ATGTTTAAG AACTT G GCTG G AG CAT AT CCAT CAT ACT CTACCAACTGGGCCGACGA

AACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGCGGTCACGTAGAGGGATGTCCAT

T CTT AG AA AG AAGT ATTT GAAACCATCCAACAATGTTCT ATT CTCTGTTGGCTCGACCATCTACCACGAGAAGAGG

GACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACTTACGTGGCAAGCAAAATTACACATGTCGGTGTGAGA

CTATAGTTAGTTGCGACGGGTACGTCGTTAAAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCT

ATGCTGCTACGATGCACCGCGAGGGATTCTTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTT

TTCCCGTGTGCACGTATGTGCCAGCTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGA

CGACGCGCAAAAACTGCTGGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATAC

CATGAAAAATTACCTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGA

AGATGAAAGGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGA

TAACATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTGCCC

AGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACAAGGAGC

CGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGAGGTGCGTGAA

GCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAAGCCGATGTCGAC

TTGATGTTACAAGAGGCTGGGGCC

SEQ ID NO: 205, nsp2 coding sequence

GGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCT

GTG CTTT CTCCG CAG G CTGT ACT CAAG AGT G AAAAATT AT CTT G CAT CCACCCTCTCG CTG AACAAGT C ATAGTG AT

AACACACTCTGGCCGAAAAGGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATG

CAATACCCGTCCAGGACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAG

GTACCTGCACCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAG

CGAGCACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG

GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGCCGCTC

CTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAAAGCGCAGTCA

CCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACGTCAAGAAAATGAAA

GGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACACCCCGTAGAGACCCTGTAT

ATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCCATTATAAGACCTAAAAAGGCAGTGC

TCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCCTGAAAGTGCATTTTAACCACGAGATTTGCAC

ACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAAATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACA

AAAAAATGAGAACGACGAATCCGAAAGAGACTAAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAG

GACGATCTCATTCTCACTTGTTTCAGAGGGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATG

ACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTG

TACGCACCCACCTCTGAACATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCC

GGCGACCCATGGATAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCA

GAGCATGATGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTG

TGTTGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTGTG

GATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTTTGGACTCG

ATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGGGATAACTCCCCGTCG

CCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACCCACAACTGCCTCGGGCAGTT

GCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGATCCGCGCATAAACCTAGTACCTGTAA

ACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACACCCACAGAGTGACTTTTCTTCATTCGTCAGCAAA

TTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGTTGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGAC CGGCCTGAGGCTACCTTCAGAGCTCGGCTGGATTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTG

TTAATGTGAGGACCCCATATAAATACCATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGAC

CAAGAAAGCTTGTCTGCATCTGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAG

CGAAAGCATCATTGGTGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAG

ACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTT

GACCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGT

SEQ ID NO: 206, nsp3 coding sequence

GCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGC

AAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGAT

CGAAGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAA

AGTTTCGGAGGTTGAAGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAA

TTACAAGTCAGTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGA

ACCATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGA

CTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTGACAGAA

CCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCACAAGCGATGG

CAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGAAATTAATGCCATGTG

GCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGCATGAGCAGTATTAGGTCGA

AATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCC

AGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTAT

AGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCC

AAGGAAGTATCTCGTGGAAACACCACCGGTAGACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGG

GGACACCTGAACAACCACCACTTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAG

AGGAAGAAGAGGATAGCATAAGTTTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATT

CACGGGCCGCCCTCTGTATCTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCAT

ACTTGACACCCTGGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAG

TATGGAGTTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACA

AGAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGG

GTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTG

GTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAACAATGA

CGGTTTGATGCGGGTGCA

SEQ ID NO: 207, nsp4 coding sequence

T AC AT CTTTTCCTCCG ACACCG GT CAAG G G CATTT ACAACAAAAAT CAGT AAG G CAAACG GTG CTATCCG AAGT G G

TGTTGGAGAGGACCGAATTGGAGATTTCGTATGCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGA

AATTACAGTTAAATCCCACACCTGCTAACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAA

CAGCTAGACGTATTCTGCAAGGCCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGC

ATCCTGTTCCTTTGTATTCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCC

ATGTTGAAAGAGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGA

CGGAGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCCTATT

TGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAGCTGCCACAA

AAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCA

AGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGGCTTACTGAAGAAAACGTGG

TAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTTGCAG

GACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAACAAAACATACTGAA

GAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCGCTAGCAACAGCGTATCTGTGCGGAATCCACCGAGA

GCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGAACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGAC

GCTATTATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGG

ACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTG

AGGCGGCTTTCGGCGAAATTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCT

GGAATGTTCCTCACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAA CCGGATCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTATTTCT GTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAAAGGCTGTTTA AGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGCATGAAGAGTCAACA CGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTATGAAACCGTAGGAACTTCC ATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGCTACCTGAGAGGGGCCCCTATAACTCT CTACGGC

SEQ ID NO: 208, 3'-UTR AT AC AG C AG C AATT G G CAAG CT G CTT AC AT AG AACTCG CG G CG ATT GGCATGCCG CTTT AAAATTTTT ATTTT ATTT

TT CTTTT CTTTT CCG AATCG G ATTTT GTTTTT AAT ATTT C

SEQ ID NO: 209, polyA site

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

Claims

Claims I/We claim:

1. An RNA replicon encoding a recombinant pre-fusion SARS CoV-2 S protein or a fragment or variant thereof, wherein the recombinant pre-fusion SARS CoV-2 S protein or fragment or variant thereof comprises an S 1 and an S2 domain, and comprises at least one mutation selected from the group consisting of a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 into proline (P), a mutation of the amino acid at position 892, a mutation of the amino acid at position 614, a mutation of the amino acid at position 572, a mutation of the amino acid at position 532, a disulfide bridge between residues 880 and 888, and a disulfide bridge between residues 884 and 893, wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO: 1.

2. The RNA replicon according to claim 1, wherein the amino acid at position 892 is not alanine (A), the amino acid at position 614 is not aspartic acid (D) or glycine (G), the amino acid at position 532 is not asparagine (N), and/or amino acid at position 572 is not threonine (T).

3. The RNA replicon according to claim 1 or 2, wherein the recombinant pre-fusion SARS CoV-2 S protein or fragment or variant thereof comprises at least a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 into P, and a mutation selected from the group consisting of a mutation of the amino acid at position 892, a mutation of the amino acid at position 614, a mutation of the amino acid at position 572, a mutation of the amino acid at position 532, a disulfide bridge between residues 880 and 888 and a disulfide bridge between residues 884 and 893.

4. The RNA replicon according to claim 3, wherein the recombinant pre-fusion SARS CoV- 2 S protein or fragment or variant thereof comprises at least a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 into P, and a mutation selected from the group consisting of a mutation of the amino acid at position 892, a mutation of the amino acid at position 614, a mutation of the amino acid at position 572, a mutation of the amino acid at position 532, a disulfide bridge between residues 880 and 888 and a disulfide bridge between residues 884 and 893, provided that the recombinant pre-fusion SARS CoV-2 S protein does not comprise both the disulfide bridge between residues 880 and 888 and the disulfide bridge between residues 884 and 893.

5. The RNA replicon according to any one of claims 1-4, wherein the recombinant pre fusion SARS CoV-2 S protein or fragment or variant thereof comprises a disulfide bridge between residues 880 and 888.

6. The RNA replicon according to any one of claims 1-5, wherein the recombinant pre fusion SARS CoV-2 S protein or fragment or variant thereof comprises a mutation of the amino acid at position 942 into P.

7. The RNA replicon according to any one of claims 1-6, wherein the recombinant pre fusion SARS CoV-2 S protein or fragment or variant thereof comprises a mutation of the amino acid at position 892 into P.

8. The RNA replicon according to any one of claims 1-7, wherein the recombinant pre fusion SARS CoV-2 S protein or fragment or variant thereof comprises a mutation of the amino acid at position 614 into N.

9. The RNA replicon according to any one of claims 1-8, wherein the recombinant pre fusion SARS CoV-2 S protein or fragment or variant thereof comprises a mutation of the amino acid at position 532 into P.

10. The RNA replicon according to any one of claims 1-9, wherein the recombinant pre fusion SARS CoV-2 S protein or fragment or variant thereof comprises a mutation of the amino acid at position 572 into isoleucine (I).

11. The RNA replicon according to any one of claims 1-10, wherein the recombinant pre fusion SARS CoV-2 S protein or fragment or variant thereof comprises a mutation of the amino acid at position 942 into P, a disulfide bridge between the amino acid residues at positions 880 and 888 and a mutation of the amino acid at position 641 into N.

12. The RNA replicon according to any one of claims 1-11, wherein the recombinant pre fusion SARS CoV-2 S protein or fragment or variant thereof further comprises a deletion of the ftirin cleavage site.

13. The RNA replicon according to claim 12, wherein the deletion of the furin cleavage site comprises a mutation of the amino acid at position 682 into serine (S) and/or a mutation of the amino acid at position 685 into glycine (G).

14. The RNA replicon according to any one of claims 1-13, wherein the recombinant pre fusion SARS CoV-2 S protein or fragment or variant thereof further comprises a mutation of the amino acids at position 986 and 987 into P.

15. The RNA replicon according to any one of claims 1-14, wherein the recombinant pre fusion SARS CoV-2 S protein or fragment or variant thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5-194 or a fragment or variant thereof.

16. The RNA replicon according to any one of claims 1-15, wherein the recombinant pre fusion SARS CoV-2 S protein or fragment or variant thereof does not comprise a signal peptide or a tag sequence.

17. The RNA replicon according to any one of claims 1-15, wherein the recombinant pre fusion SARS CoV-2 S protein or fragment or variant thereof comprises a truncated S2 domain.

18. The RNA replicon according to claim 17, wherein the transmembrane and cytoplasmic domain of the recombinant pre-fusion SARS CoV-2 S protein or fragment or variant thereof have been removed.

19. The RNA replicon according to claim 17 or 18, wherein a heterologous trimerization domain of the recombinant pre-fusion SARS CoV-2 S protein or fragment or variant thereof has been linked to the truncated S2 domain.

20. The RNA replicon according to claim 19, wherein the heterologous trimerization domain is a foldon domain comprising the amino acid sequence of SEQ ID NO:4.

21. The RNA replicon according to any one of claims 1-20, comprising, ordered from the 5’- to 3 ’-end:

(1) a 5’ untranslated region (5’-UTR) required for nonstructural protein-mediated amplification of an RNA virus;

(2) a polynucleotide sequence encoding at least one, preferably all, of non-structural proteins of the RNA virus;

(3) a subgenomic promoter of the RNA virus;

22. The RNA replicon according to claim 21, comprising, ordered from the 5’- to 3’-end,

(1) an alphavirus 5’ untranslated region (5’-UTR),

(2) a 5’ replication sequence of an alphavirus non-structural gene nspl,

(3) a downstream loop (DLP) motif of a virus species,

(4) a polynucleotide sequence encoding an autoprotease peptide,

(6) an alphavirus subgenomic promoter,

(8) an alphavirus 3' untranslated region (3' UTR), and

(9) optionally, a poly adenosine sequence.

23. The RNA replicon of claim 22, wherein the DLP motif is from a virus species selected from the group consisting of Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MTDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), and Buggy Creek virus.

24. The RNA replicon of claim 22 or 23, wherein the autoprotease peptide is selected from the group consisting of porcine tesehovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof, preferably, the autoprotease peptide comprising the peptide sequence of P2A.

25. An RNA replicon, comprising, ordered from the 5’ - to 3 ’-end,

(1) a 5’-UTR having the polynucleotide sequence of SEQ ID NO: 198,

(2) a 5’ replication sequence having the polynucleotide sequence of SEQ ID NO: 199,

(3) a DLP motif comprising the polynucleotide sequence of SEQ ID NO:200,

(4) a polynucleotide sequence encoding a P2A sequence of SEQ ID NO: 202,

(5) a polynucleotide sequence encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4 having the amino acid sequences encoded by the polynucleotide sequences of SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 207, respectively,

(6) a subgenomic promoter having polynucleotide sequence of SEQ ID NO: 196,

(7) a polynucleotide sequence encoding a pre-fusion SARS CoV-2 S protein having the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 5-194, or a fragment or variant thereof, and

(8) a 3' UTR having the polynucleotide sequence of SEQ ID NO:208.

26. The RNA replicon of claim 25, wherein:

(a) the polynucleotide sequence encoding the P2A sequence comprises SEQ ID NO: 201,

(b) the polynucleotide sequence encoding the alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4 comprises SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 207, respectively, and (c) the RNA replicon further comprises a poly adenosine sequence, preferably the poly adenosine sequence has the SEQ ID NO:209, at the 3 ’-end of the replicon.

27. A nucleic acid comprising a DNA sequence encoding the RNA replicon of any one of claims 1-26, preferably, the nucleic acid further comprises a T7 promoter operably linked to the 5 ’-end of the DNA sequence, more preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO: 197.

28. A composition comprising the RNA replicon of any one of claims 1-26.

29. A vaccine against COVID-19 comprising the RNA replicon of any one of claims 1-26.

30. A method for vaccinating a subject against COVID-19, the method comprising administering to the subject the vaccine according to claim 29.

31. A method for reducing infection and/or replication of SARS-CoV-2 in a subject, comprising administering to the subject a composition according to claim 28 or a vaccine according to claim 29.

32. The method of claim 30 or 31, wherein the composition or vaccine is administered as part of a prime-boost administration regimen.

33. The method of claim 32, wherein the prime-boost administration regimen is a homologous prime-boost administration regimen.

34. The method of claim 32, wherein the prime-boost administration regimen is a heterologous prime-boost administration regimen.

35. The method of claim 34, wherein the heterologous prime-boost administration regimen comprises a prime-administration of the vaccine of claim 29 to prime the immune response and a boost-administration of a vaccine comprising an adenoviral vector encoding a recombinant pre fusion SARS CoV-2S protein or fragment thereof to boost the immune response.

36. The method of claim 34, wherein the heterologous prime-boost administration regimen comprises a prime-administration of a vaccine comprising an adenoviral vector encoding a recombinant pre-fusion SARS CoV-2S protein or fragment thereof to prime the immune response and a boost-administration of the vaccine of claim 29 to boost the immune response.

37. The method of any one of claims 34-36, wherein the RNA replicon and adenoviral vector encode the same recombinant pre-fusion SARS CoV-2S protein or fragment thereof or a variant thereof.

38. The method of any one of claims 32-37, wherein the boost-administration is administered at least about 2 weeks after the prime-administration.

39. The method of any one of claims 32-37, wherein the boost-administration is administered about 2 weeks to about 12 weeks after the prime-administration.

40. The method of claim 38 or 39, wherein the boost-administration is administered about 4 weeks after the prime-administration.

41. An isolated host cell comprising the nucleic acid according to claim 27.

42. An isolated host cell comprising the RNA replicon of any one of claims 1-26.

43. A method of making an RNA replicon, comprising transcribing the nucleic acid according to claim 27 in vivo or in vitro.