SARS-COV-2 SURROGATE VIRUS NEUTRALIZATION ASSAY TEST KIT

Abstract

A kit, composition and method for detection of antibodies to severe acute respiratory syndrome related coronavirus (SARSr-CoV), and for diagnosis of SARSr-CoV infection.

Description

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims priority to Singapore Patent Application No. 10202002784P filed Mar. 25, 2020 and Singapore Patent Application No. 10202004468Q filed on May 14, 2020.

[0002] The foregoing applications, and all documents cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced herein (including without limitation all literature documents, patents, published patent applications cited herein) ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Any Genbank sequences mentioned in this disclosure are incorporated by reference with the Genbank sequence to be that of the earliest effective filing date of this disclosure.

[0003] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present disclosure.

FIELD OF THE INVENTION

[0004] The present disclosure relates to the detection of antibodies to severe acute respiratory syndrome-related coronavirus (SARSr-CoV), and the diagnosis of SARSr-CoV infection.

BACKGROUND OF THE INVENTION

[0005] The coronavirus disease 2019 (COVID-19) outbreak, started in Wuhan, China, has spread rapidly to other cities in China, and affected more than 160 countries with more than 332,930 infections and 14,509 deaths as of 23 Mar. 2020. The etiological agent is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is genetically 80% identical to severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1) which caused the 2002-2003 outbreak of severe acute respiratory syndrome [3].

[0006] Current diagnosis is based on clinical history and chest radiographic findings, and confirmation currently relies on nucleic acid-based assays.

[0007] There is an urgent need for reliable and easy-to-use assays for the detection of SARS-CoV-2, to expand the tools available to combat this worst pandemic in mankind's modern history.

[0008] Unlike molecular tests, serological tests are more difficult to develop and often suffer from specificity issues. The current "gold standard" is virus neutralization test (VNT) which requires the use of live virus in a specialized biocontainment facility, biosafety level 3 (BSL3) lab. The test process is not only expensive and at a biosafety risk, but also slow, typically taking 3-5 days to get results.

[0009] Several groups are developing ELISA-based tests to meet the need for BSL2-based tests which can be completed within hours rather than days. The present inventor was the first to use ELISA and VNT assays to facilitate contact tracing in Singapore [30]. Since then, several other groups have developed ELISA tests for SARS-CoV-2 infection [31, 32]. Most groups are using indirect ELISA format (coating ELISA plate with antigen, followed by binding with test sera, and HRP-conjugated secondary antibodies) (FIG. 1). This is the simplest/cheapest serological assay format, hence widely adopted. But it usually suffers from specificity issues. ELISA tests are currently used for "front line" screenings and there is usually a need for confirmation by the more specific and reliable VNT assays [30].

[0010] All current ELISA tests are species- (mostly human) and subtype- (IgG or IgM) specific. This can be a major drawback for surveillance studies beyond human sera. There is an urgent need for serological surveillance of different wildlife species to understand the missing link which might be responsible for the transmission of the virus from animal(s) to humans. It is a huge undertaking to develop specific ELISAs for all of the many wildlife species to be surveyed.

SUMMARY OF THE INVENTION

[0011] In a first aspect, the present disclosure provides a kit which may comprise: (i) a polypeptide encoded by a SARSr-CoV or a fragment thereof, (ii) a polypeptide which binds specifically to the polypeptide or fragment of (i), and (iii) means for detecting interaction between the polypeptide or fragment of (i), and the polypeptide of (ii).

[0012] Also provided is a kit which may comprise: (i) a spike protein or a fragment thereof encoded by a SARSr-CoV, and (ii) an ACE2 protein or a fragment thereof which binds specifically to the spike protein or fragment of (i).

[0013] In some embodiments, the kit may further comprise means for detecting interaction between the spike protein or fragment of (i) and the ACE2 protein or fragment of (ii).

[0014] In some embodiments, the spike protein or fragment of (i) or the ACE2 protein or fragment of (ii) may be conjugated to a detection entity. In some embodiments, the spike protein or fragment of (i) or the ACE2 protein or fragment of (ii) may be immobilised on a solid support.

[0015] In some embodiments,

[0016] (a) the spike protein or fragment of (i) is conjugated to a detectable entity, and wherein the ACE2 or fragment of (ii) is immobilised on a solid support; or (b) the ACE2 or fragment of (ii) is conjugated to a detectable entity, and wherein the spike protein or fragment of (i) is immobilised on a solid support.

[0017] In some embodiments, the SARSr-CoV may be SARS-CoV-2 or a variant thereof.

[0018] In some embodiments, the spike protein or fragment may comprise or consist of the S1 subunit. In some embodiments, the spike protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 12 or 27. In some embodiments, the spike protein or fragment may comprise or consist of the receptor binding domain (RBD) of the spike protein. In some embodiments the spike protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 13 or 26. In some embodiments, the the spike protein or fragment may comprise or consist of the receptor binding motif (RBM) of the spike protein. In some embodiments the spike protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14.

[0019] In some embodiments, the ACE2 protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 15, 16 or 29. In some embodiments, the ACE2 protein or fragment may comprise or consist of the extracellular domain. In some embodiments, the ACE2 protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 17 or 30.

[0020] In some embodiments, the detection entity may be a horseradish peroxidase.

[0021] The present disclosure also provides a composition which may comprise:

[0022] (a) a solid support, and

[0023] (b) (i) a polypeptide encoded by a SARSr-CoV or a fragment thereof, and/or (ii) a polypeptide which binds specifically to the polypeptide or fragment of (i).

[0024] The present disclosure also provides a composition which may comprise:

[0025] (a) a solid support, and

[0026] (b) (i) a spike protein or a fragment thereof encoded by a SARSr-CoV, and/or (ii) an ACE2 protein or a fragment thereof which binds specifically to the spike protein or fragment of (i).

[0027] In some embodiments, the composition may further comprise means for detecting interaction between the spike protein or fragment of (i) and the ACE2 protein or fragment of (ii).

[0028] In some embodiments, the spike protein or fragment of (i) or the ACE2 protein or fragment of (ii) may be immobilised on the solid support.

[0029] In some embodiments, the spike protein or fragment of (i) or the ACE2 protein or fragment of (ii) may be conjugated to a detection entity.

[0030] In some embodiments,

[0031] (a) the spike protein or fragment of (i) is conjugated to a detectable entity, and wherein the ACE2 or fragment of (ii) is immobilised on the solid support; or

[0032] (b) the ACE2 or fragment of (ii) is conjugated to a detectable entity, and wherein the spike protein or fragment of (i) is immobilised on the solid support.

[0033] In some embodiments, the SARSr-CoV may be SARS-CoV-2 or a variant thereof.

[0034] In some embodiments, the spike protein or fragment may comprise or consist of the S1 subunit. In some embodiments, the spike protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 12 or 27. In some embodiments, the spike protein or fragment may comprise or consist of the receptor binding domain (RBD) of the spike protein. In some embodiments, the spike protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 13 or 26. In some embodiments, the the spike protein or fragment may comprise or consist of the receptor binding motif (RBM) of the spike protein. In some embodiments the spike protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14.

[0035] In some embodiments, the ACE2 protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 15, 16 or 29. In some embodiments, the ACE2 protein or fragment may comprise or consist of the extracellular domain. In some embodiments, the ACE2 protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 17 or 30.

[0036] In some embodiments the detection entity may be a horseradish peroxidase.

[0037] The present disclosure also provides a polypeptide which may comprise the amino acid sequence of a polypeptide encoded by a SARSr-CoV or a fragment thereof, wherein the polypeptide may be conjugated to a detection entity.

[0038] The present disclosure also provides a polypeptide which may comprise the amino acid sequence of a spike protein or a fragment thereof encoded by a SARSr-CoV, wherein the polypeptide may be conjugated to a detection entity.

[0039] In some embodiments, the SARSr-CoV may be SARS-CoV-2 or a variant thereof.

[0040] In some embodiments, the spike protein or fragment may comprise or consist of the S1 subunit. In some embodiments, the spike protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 12 or 27. In some embodiments, the spike protein or fragment may comprise or consist of the receptor binding domain (RBD) of the spike protein. In some embodiments, the spike protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 13 or 26. In some embodiments, the the spike protein or fragment may comprise or consist of the receptor binding motif (RBM) of the spike protein. In some embodiments the spike protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14.

[0041] The present disclosure also provides a polypeptide which may bind specifically to a polypeptide encoded by a SARSr-CoV or a fragment thereof, wherein the polypeptide may be conjugated to a detection entity.

[0042] The present disclosure also provides a polypeptide which may comprise the amino acid sequence of an ACE2 protein or a fragment thereof, wherein the polypeptide may be conjugated to a detection entity.

[0043] In some embodiments, the ACE2 protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 15, 16 or 29. In some embodiments, the ACE2 protein or fragment may comprise or consist of the extracellular domain. In some embodiments, the ACE2 protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 17 or 30.

[0044] In some embodiments, the detection entity may be a horseradish peroxidase.

[0045] The present disclosure also provides a nucleic acid encoding a polypeptide according to the present disclosure.

[0046] The present disclosure also provides a vector which may comprise the nucleic acid according to the present disclosure.

[0047] The present disclosure also provides a cell which may comprise the nucleic acid or vector according to the present disclosure.

[0048] The present disclosure also provides the use of the kit, composition or polypeptide according to the present disclosure, in a method for detecting the presence of antibodies to a SARSr-CoV in a sample.

[0049] In some embodiments, the SARSr-CoV may be SARS-CoV-2 or a variant thereof.

[0050] The present disclosure also provides the use of the kit, composition or polypeptide according to the present disclosure, in a method for determining whether a subject is or has been infected with a SARSr-CoV.

[0051] In some embodiments, the SARSr-CoV may be SARS-CoV-2 or a variant thereof.

[0052] In some embodiments, the subject may be a mammal. In some embodiments, the subject may be a human.

[0053] The present disclosure also provides a method of analysing a sample for the presence of antibodies to a SARSr-CoV, which may comprise:

[0054] contacting the sample with: (i) a polypeptide encoded by a SARSr-CoV or a fragment thereof, and (ii) a polypeptide which binds specifically to the polypeptide or fragment of (i), and

[0055] determining the level of interaction between the polypeptide or fragment of (i) and the polypeptide of (ii).

[0056] In some embodiments, the SARSr-CoV may be SARS-CoV-2 or a variant thereof.

[0057] In some embodiments, the sample may be a blood sample, a lymph sample, a saliva sample, a synovial fluid sample. In some embodiments, the sample may be serum.

[0058] The present disclosure also provides a method of determining whether a subject is or has been infected with a SARSr-CoV, which may comprise:

[0059] contacting a sample from the subject with: (i) a polypeptide encoded by a SARSr-CoV or a fragment thereof, and (ii) a polypeptide which binds specifically to the polypeptide or fragment of (i), and

[0060] determining the level of interaction between the polypeptide or fragment of (i) and the polypeptide of (ii).

[0061] In some embodiments, the SARSr-CoV may be SARS-CoV-2 or a variant thereof.

[0062] In some embodiments, the subject may be a mammal. In some embodiments, the subject may be a human.

[0063] In some embodiments, the sample may be a blood sample, a lymph sample, a saliva sample, a synovial fluid sample. In some embodiments, the sample may be serum.

[0064] The present disclosure also provides a method of analysing a sample for the presence of antibodies to a SARSr-CoV, which may comprise:

[0065] contacting the sample with: (i) a spike protein or a fragment thereof encoded by a SARSr-CoV, and (ii) an ACE2 protein or a fragment thereof which binds specifically to the spike protein or fragment of (i), and

[0066] determining the level of interaction between the spike protein or fragment of (i) and the ACE2 or fragment of (ii).

[0067] The present disclosure also provides a method of determining whether a subject is or has been infected with a SARSr-CoV, which may comprise:

[0068] contacting a sample obtained from the subject with: (i) a spike protein or a fragment thereof encoded by a SARSr-CoV, and (ii) an ACE2 protein or a fragment thereof which binds specifically to the spike protein or fragment of (i), and

[0069] determining the level of interaction between the spike protein or fragment of (i) and the ACE2 or fragment of (ii).

[0070] In some embodiments, the spike protein or fragment of (i) or the ACE2 protein or fragment of (ii) may be conjugated to a detection entity.

[0071] In some embodiments, the spike protein or fragment of (i) or the ACE2 protein or fragment of (ii) may be immobilised on a solid support.

[0072] In some embodiments, the spike protein or fragment of (i) may be conjugated to a detectable entity, and wherein the ACE2 or fragment of (ii) may be immobilised on a solid support.

[0073] In some embodiments, the SARSr-CoV may be SARS-CoV-2 or a variant thereof.

[0074] In some embodiments, the subject may be a mammal. In some embodiments, the subject may be a human.

[0075] In some embodiments, the sample may be a blood sample, a lymph sample, a saliva sample, a synovial fluid sample. In some embodiments, the sample may be serum.

[0076] In some embodiments, the spike protein or fragment may comprise or consist of the S1 subunit. In some embodiments, the spike protein or fragment comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 12 or 27. In some embodiments, the spike protein or fragment may comprise or consist of the receptor binding domain (RBD) of the spike protein. In some embodiments the spike protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOs: 13 or 26. In some embodiments, the the spike protein or fragment may comprise or consist of the receptor binding motif (RBM) of the spike protein. In some embodiments the spike protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14.

[0077] In some embodiments, the ACE2 protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 15, 16 or 29. In some embodiments, the ACE2 protein or fragment may comprise or consist of the extracellular domain. In some embodiments, the ACE2 protein or fragment may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 17 or 30.

[0078] In some embodiments, the detection entity may be a horseradish peroxidase.

[0079] Other features and advantages of the instant disclosure will be apparent from the following detailed description and examples which should not be construed as limiting. The contents of all references, GenBank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0080] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U. S.C. .sctn. 112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0081] It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0083] FIG. 1. Schematic representation of the principle of indirect ELISA. 1) Antigen coating; 2) Binding of antigen-specific antibody; 3) Binding of HRP-conjugated secondary antibody; 4) Colour development with HRP substrate (TMB).

[0084] FIGS. 2A and 2B. Schematic representation of the principle of virus neutralization. (2A) Most virus-neutralizing antibodies act by blocking virus attachment although some can also act by blocking uncoating. (2B) Surrogate virus neutralization mimics the action of most neutralizing antibodies by blocking receptor-viral protein interaction in an ELISA plate well.

[0085] FIGS. 3A and 3B. Graphs showing the results of analysis of direct binding to hACE2 coated onto an ELISA plate for (3A) recombinant HRP-conjugated SARS-CoV-2 spike protein RBD, (3B) recombinant HRP-conjugated SARS-CoV-2 spike protein S1 subunit, and recombinant HRP-conjugated SARS-CoV-2 nucleoprotein.

[0086] FIGS. 4A and 4B. Graphs showing the results of analysis of serum samples by surrogate virus neutralisation test (sVNT) using immobilised human ACE2 protein and (4A) HRP-conjugated SARS-CoV-2 spike protein RBD or (4B) HRP-conjugated SARS-CoV-2 spike protein S1 subunit.

[0087] FIG. 5. Graph showing the results of analysis of 74 serum samples from PCR-confirmed COVID-19 patients and 11 negative (healthy) human serum samples using the sVNT using immobilised human ACE2 protein and HRP-conjugated SARS-CoV-2 spike protein RBD.

[0088] FIGS. 6A to 6F. Schematic representations and graphs showing the principle and initial validation of the SARS-CoV-2 surrogate virus neutralization test (sVNT). (6A) Mechanism of conventional virus neutralization test (VNT). Anti-SARS-CoV-2 neutralizing antibodies block SARS-CoV-2 Spike protein from binding to hACE2 receptor proteins on the host cell surface. (6B) In the sVNT assay, anti-SARS-CoV-2 neutralizing antibodies block HRP-conjugated RBD protein from binding to the hACE2 protein pre-coated on an ELISA plate. (6C) Binding of HRP-conjugated SARS-CoV-2 N, S1 and RBD proteins to hACE2. (6D) Inhibition of SARS-CoV-2 RBD-hACE2 interaction by COVID-19 patient sera. (6E) Binding of HRP-conjugated SARS-CoV RBD to hACE2. (6F) Inhibition of SARS-CoV RBD-hACE2 interaction by SARS patient sera.

[0089] FIGS. 7A to 7D. Bar charts showing isotype-independent neutralization by human sera with different levels of IgM and IgG antibodies. (7A) High IgM/Low IgG (n=5); (7B) Low IgM/High IgG (n=3); (7C) Low IgM/Low IgG (n=9); (7D) High IgM/High IgG (n=5). The IgM and IgG levels were determined by isotype-specific capture ELISA.

[0090] FIGS. 8A to 8G. Graphs showing species-independent and virus-specific neutralization. (8A) Rabbit anti-SARS-CoV-2 RBD sera from immunization (n=3). (8B) Ferret anti-SARS-CoV sera from infection (n=2). (8C) Rabbit anti-SARS-CoV sera from immunization (n=2). (8D) SARS-CoV-2 sVNT using different coronavirus sera: human COVID-19 sera (n=10), human SARS sera sampled in 2003 (n=7, <1 year), human SARS-CoV sera sampled in 2020 (n=10, >17 years), human OC43 sera (n=8), human 229E/NL63 sera (n=10), MERS-CoV sera from experimentally infected alpaca (n=4). (8E) Comparative analysis of homologous and heterologous NAb levels for the 2003 SARS serum panel. (8F) Comparative analysis of homologous and heterologous NAb levels for the 2020 SARS serum panel. (8G) Comparative analysis of homologous N-specific antibodies in the three serum cohorts. SARS-CoV-2 N protein indirect ELISA for COVID-19 sera and SARS-CoV N protein indirect ELISA for the two SARS serum panels.

[0091] FIGS. 9A to 9C. Correlation between sVNT and VNT and sVNT testing with two COVID-19 patient cohorts from two different nations. (9A) Correlation analysis for 13 COVID-19 sera with different levels of SARS-CoV-2 antibodies by VNT and sVNT at 70% inhibition. Testing of healthy control and COVID-19 serum cohorts in Singapore (9B) (COVID-19 n=77, control n=75) and Nanjing, China (9C) (COVID-19 n=50, control n=50).

[0092] FIG. 10. Table showing correlation between VNT and sVNT for different COVID-19 sera. Neutralization titers were obtained from two biological replicates each with two technical replicates. sVNT titers at 90%, 70%, 50% and 30% inhibition, respectively, are shown.

[0093] FIG. 11. Graph showing titration of COVID-19 sera with 417 different levels of SARS-CoV-2 antibodies. Serum samples were two-fold diluted starting at 1:10. Percentage inhibition was plotted at each dilution point for two negative controls and 13 COVID-19 sera from PCR positive patients.

DETAILED DESCRIPTION OF THE INVENTION

[0094] The present invention is based on the principle of using surrogate molecules of live SARSr-CoV in assays for the detection of the presence of neutralising antibody to SARSr-CoV. The invention strips the traditional virus neutralisation test back to its molecular elements, based on antibodies blocking the molecular interaction of the cell entry receptor (ACE2) and the key viral spike protein S1 domain (RBD) (FIG. 2B).

[0095] Advantageously, analysis of samples using the articles and in accordance with methods of the present disclosure is more reliable than ELISA-based analysis, and is more versatile, not being limited to the detection of certain antibody isotypes or the detection of antibodies in samples derived from a limited number of species. Rather, the articles and methods of the present disclosure can be employed to detect neutralising antibodies to SARSr-CoV across all antibody isotypes and in samples derived from any species. As the disclosed articles and methods provide for more sensitive and specific analysis, there is no need to confirm results by virus neutralisation tests, as is typically required for ELISA-based analysis.

[0096] Moreover, the analysis of samples using the articles and in accordance with methods of the present disclosure is associated with the advantage over virus neutralisation tests that it does not employ live virus or infectious material, and is therefore safe and does not require the use of BSL3 facilities. Furthermore, the use of the articles and methods of the present disclosure is less expensive and time-consuming than virus neutralisation tests, with results obtainable in a matter of hours.

[0097] The present disclosure concerns severe acute respiratory syndrome-related coronavirus (SARSr-CoV). The virology of SARSr-CoV and epidemiology of disease associated with SARSr-CoV infection are reviewed, for example, in Cheng et al., Clin Microbiol Rev (2007) 20(4): 660-694 and de Wit et al., Nat Rev Microbiol (2016) 14: 523-534, both of which are hereby incorporated by reference in their entirety.

[0098] SARSr-CoV is a species of coronavirus of the genus Betacoronavirus and subgenus Sarbecoronavirus that infects humans, bats and certain other mammals. It is an enveloped positive-sense single-stranded RNA virus.

[0099] Two strains of SARSr-CoV have caused serious outbreaks of severe respiratory diseases in humans: SARS-CoV, which caused an outbreak of severe acute respiratory syndrome (SARS) between 2002 and 2003, and SARS-CoV-2, which has caused the coronavirus disease 2019 (COVID-19) pandemic. There are hundreds of strains of SARSr-CoV known only to infect non-human species; bats are a major reservoir of many strains of SARS-related coronaviruses.

[0100] In some embodiments in accordance with the various aspects of the present disclosure, the SARSr-CoV is a strain of SARSr-CoV that causes coronavirus disease in humans.

[0101] In some embodiments, the SARSr-CoV is SARS-CoV or a variant thereof, or SARS-CoV-2 or a variant thereof. In some embodiments, the SARSr-CoV is SARS-CoV-2 or a variant thereof.

[0102] In some embodiments herein, "SARS-CoV" refers to the SARSr-CoV strain having the nucleotide sequence with a GenBank Accession No.: AY278488.2 ("SARS coronavirus BJ01, complete genome") see, e.g., Wu et al., Genomics Proteomics Bioinformatics 1 (2), 131-144 (2003). A variant of SARS-CoV may comprise a nucleotide sequence having at least 80% sequence identity, e.g. one of at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the nucleotide sequence with a GenBank Accession No.: AY278488.2.

[0103] As used herein, "sequence identity" refers to the percent of nucleotides/amino acid residues in a subject sequence that are identical to nucleotides/amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using the publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780 software. When using such softwares, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0104] In some embodiments herein, "SARS-CoV-2" refers to the SARSr-CoV strain having the nucleotide sequence with a GenBank Accession No.: MN996527.1 ("Severe acute respiratory syndrome coronavirus 2 isolate WIV02, complete genome"), reported in Zhou et al., Nature (2020) 579: 270-273. A variant of SARS-CoV-2 may comprise a nucleotide sequence having at least 80% sequence identity, e.g. one of at least 81%, 82%, 83%, 84%, 85%, 86%, 8'7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the nucleotide sequence with a GenBank Accession No.: MN996527.1.

[0105] In preferred embodiments, the SARSr-CoV is SARS-CoV-2 or a variant thereof.

[0106] The present disclosure also concerns polypeptides encoded by a SARSr-CoV.

[0107] Herein, a polypeptide refers to any molecule which may comprise three or more amino acid residues joined by peptide bonds. A polypeptide according to the present disclosure encompasses peptides (e.g. tripeptides, oligopeptides, etc.), and also polypeptides which may comprise chemical modification, such e.g. glycosylation (glycopolypeptides), phosphorylation, hydroxylation, sulfonation, palmitoylation, and disulfide formation. Polypeptides may also be referred to as proteins.

[0108] Constituent proteins and glycoproteins encoded by the SARSr-CoV genome are described e.g. in Masters, Adv Virus Res. (2006) 66:193-292 and Song et al., Viruses (2019) 11(1): 59, both of which are hereby incorporated by reference in their entirety. The SARSr-CoV genome encodes four major structural proteins: the spike (S) protein, the envelope (E) protein, the membrane (M) protein, and the nucleocapsid (N) protein.

[0109] The spike protein of SARS-CoV may have the amino acid sequence shown in SEQ ID NO: 1. The envelope protein of SARS-CoV may have the amino acid sequence shown in SEQ ID NO: 2. The membrane protein of SARS-CoV may have the amino acid sequence shown in SEQ ID NO: 3. The nucleocapsid protein of SARS-CoV may have the amino acid sequence shown in SEQ ID NO: 4.

[0110] The spike protein of SARS-CoV-2 may have the amino acid sequence shown in SEQ ID NO: 5. The envelope protein of SARS-CoV-2 may have the amino acid sequence shown in SEQ ID NO: 6. The membrane protein of SARS-CoV-2 may have the amino acid sequence shown in SEQ ID NO: 7. The nucleocapsid protein of SARS-CoV-2 may have the amino acid sequence shown in SEQ ID NO: 8.

[0111] In some embodiments, a polypeptide encoded by a SARS-CoV may comprise or consist of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a polypeptide encoded by a SARS-CoV may comprise or consist of the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, a polypeptide encoded by a SARS-CoV may comprise or consist of the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, a polypeptide encoded by a SARS-CoV may comprise or consist of the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0112] In some embodiments, a polypeptide encoded by a SARS-CoV-2 may comprise or consist of the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, a polypeptide encoded by a SARS-CoV-2 may comprise or consist of the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, a polypeptide encoded by a SARS-CoV-2 may comprise or consist of the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, a polypeptide encoded by a SARS-CoV-2 may comprise or consist of the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0113] In some embodiments, a polypeptide encoded by a SARSr-CoV is selected from a spike protein, an envelope protein, a membrane protein and a nucleocapsid protein, or a fragment of a spike protein, envelope protein, membrane protein or nucleocapsid protein. In some embodiments, a polypeptide encoded by a SARSr-CoV is selected from a spike protein, an envelope protein or a membrane protein, or a fragment of a spike protein, envelope protein or membrane protein. In some embodiments, a polypeptide encoded by a SARSr-CoV is a spike protein or a fragment of a spike protein.

[0114] As used herein, a "fragment" of a reference protein/polypeptide may be of any length (by number of amino acids), although may optionally be at least 25% of the length of the reference protein/polypeptide and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the reference protein/polypeptide.

[0115] The SARSr-CoV spike protein comprises S1 and S2 subunits. The S1 subunit comprises a minimal receptor-binding domain (RBD) through which the SARSr-CoV binds to ACE2 expressed by host cells. Although virus neutralization can be achieved by other type of antibodies, receptor/entry blocking antibodies represent a substantial majority of neutralization antibodies for any given virus [33].

[0116] The RBD has been shown to be sufficient for binding to ACE2 in in vitro experiments [34]. From SARS studies, it has been shown that most neutralizing antibodies to SARS-CoV are directed against the RBD region [35].

[0117] The RBD in turn comprises the receptor binding motif (RBM), which is the region of the RBD that contacts ACE2.

[0118] In some embodiments, a polypeptide encoded by a SARSr-CoV may comprise or consist of the S1 subunit of a spike protein. In some embodiments, a polypeptide encoded by a SARSr-CoV may comprise or consist of the RBD of a spike protein. In some embodiments, a polypeptide encoded by a SARSr-CoV may comprise or consist of the RBM of a spike protein.

[0119] The S1 subunit of the spike protein of SARS-CoV may have the amino acid sequence shown in SEQ ID NO: 9. The RBD of the spike protein of SARS-CoV may have the amino acid sequence shown in SEQ ID NOs: 10 or 28. The RBM of the spike protein of SARS-CoV may have the amino acid sequence shown in SEQ ID NO: 11.

[0120] The S1 subunit of the spike protein of SARS-CoV-2 may have the amino acid sequence shown in SEQ ID NOs: 12 or 27. The RBD of the spike protein of SARS-CoV-2 may have the amino acid sequence shown in SEQ ID NOs: 13 or 26. The RBM of the spike protein of SARS-CoV-2 may have the amino acid sequence shown in SEQ ID NO: 14.

[0121] In some embodiments, a polypeptide encoded by a SARSr-CoV (e.g. a spike protein or a fragment thereof encoded by a SARSr-CoV) may comprise or consist of the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, a polypeptide encoded by a SARSr-CoV (e.g. a spike protein or a fragment thereof encoded by a SARSr-CoV) may comprise or consist of the amino acid sequence of SEQ ID NOs: 10 or 28, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 10 or 28. In some embodiments, a polypeptide encoded by a SARSr-CoV (e.g. a spike protein or a fragment thereof encoded by a SARSr-CoV) may comprise or consist of the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 11.

[0122] In some embodiments, a polypeptide encoded by a SARSr-CoV (e.g. a spike protein or a fragment thereof encoded by a SARSr-CoV) may comprise or consist of the amino acid sequence of SEQ ID NOs: 12 or 27, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 12 or 27. In some embodiments, a polypeptide encoded by a SARSr-CoV (e.g. a spike protein or a fragment thereof encoded by a SARSr-CoV) may comprise or consist of the amino acid sequence of SEQ ID NOs: 13 or 26, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 13 or 26. In some embodiments, a polypeptide encoded by a SARSr-CoV (e.g. a spike protein or a fragment thereof encoded by a SARSr-CoV) may comprise or consist of the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 14.

[0123] The present disclosure also concerns polypeptides which bind specifically to polypeptides/fragments thereof encoded by a SARSr-CoV.

[0124] As used herein, "specific binding" refers to interaction which is selective for the relevant molecule, and which can be discriminated from non-specific binding. A polypeptide that binds specifically to a given molecule preferably binds the molecule with greater affinity, and/or with greater duration than it binds to other molecules to which the polypeptide does not bind specifically.

[0125] The ability of a given polypeptide to bind specifically to a given molecule can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442), Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay. Through such analysis binding to a given molecule can be measured and quantified. In some embodiments, the binding may be the response detected in a given assay.

[0126] In some embodiments, the extent of binding of the polypeptide to a non-target molecule is less than about 10% of the level of binding to the molecule to which the polypeptide binds specifically, as measured e.g. by ELISA, SPR, Bio-Layer Interferometry or by RIA. Alternatively, specific binding may be reflected in terms of binding affinity, wherein the polypeptide binds to the molecule to which the polypeptide binds specifically with a dissociation constant (KD) that is at least 0.1 order of magnitude (i.e. 0.1.times.10.sup.n, where n is an integer representing the order of magnitude) greater than the K.sub.D of the polypeptide towards a non-target molecule. This may optionally be one of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

[0127] A polypeptide which binds specifically to a polypeptide/fragment encoded by a SARSr-CoV may be e.g. an interaction partner for the polypeptide/fragment encoded by a SARSr-CoV, or an antigen-binding molecule which binds specifically to the polypeptide/fragment encoded by a SARSr-CoV.

[0128] An interaction partner for a polypeptide/fragment encoded by a SARSr-CoV may be any polypeptide which associates with the polypeptide/fragment. The association may involve covalent interaction (e.g. disulfide bonding) and/or non-covalent interaction (e.g. electrostatic interaction (e.g. ionic bonding, hydrogen bonding), Van der Waals forces) between the polypeptide/fragment encoded by a SARSr-CoV and the interaction partner.

[0129] As used herein, an "antigen-binding molecule" refers to a molecule which is capable of specific binding to a target polypeptide, and encompasses e.g. monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g. Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.) and aptamers.

[0130] In some embodiments, an interaction partner for a polypeptide/fragment encoded by a SARSr-CoV is a polypeptide expressed by a cell to which the SARSr-CoV binds, or a fragment thereof. In some embodiments, an interaction partner is a receptor molecule expressed at the cell surface of a cell to which the SARSr-CoV binds, or a fragment thereof.

[0131] In some embodiments, a polypeptide which binds specifically to a polypeptide/fragment encoded by a SARSr-CoV may comprise or consist of the amino acid sequence of an ACE2 or a fragment thereof, or comprise or consist of an amino acid sequence having at least 60% sequence identity, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of an ACE2 or a fragment thereof.

[0132] Angiotensin-converting enzyme 2 (ACE2) is a single-pass type I transmembrane carboxypeptidase which attaches to the cell membrane of cells of the outer surface tissues of lungs, arteries, heart, kidney, and intestines. The structure and function of ACE2 is described e.g. in Hamming et al., J Pathol (2004) 203(2): 631-637, which is hereby incorporated by reference in its entirety. ACE2 has been identified to be the entry point into cells for SARS-CoV and SARS-CoV-2, via interaction with the spike protein. The SARSr-CoV spike protein binds to the extracellular domain of ACE2. ACE2 has been identified as the key cell entry receptor for both SARS-CoV and SARS-CoV-2 [3, 7].

[0133] In this specification "ACE2" refers to ACE2 from any species and includes ACE2 isoforms, fragments, variants (including mutants) or homologues from any species. In some embodiments, the ACE2 is ACE2 from a mammal (e.g. a therian, placental, epitherian, preptotheria, archontan, primate (rhesus, cynomolgous, non-human primate or human)). In some embodiments, the ACE2 is ACE2 from a human, bat, pangolin, civet or pig. Isoforms, fragments, variants or homologues of ACE2 may optionally be characterised as having at least 70% sequence identity, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature ACE2 isoform from a given species, e.g. human.

[0134] Human ACE2 isoform 1's amino acid sequence is set forth in SEQ ID NOs: 15 or 29. Human ACE2 isoform 2's amino acid sequence is shown in SEQ ID NO: 16. The extracellular domain of human ACE2 isoform 1 is shown in SEQ ID NOs: 17 or 30.

[0135] The bat ACE2 may have the amino acid sequence shown in SEQ ID NOs: 18 or 31. The extracellular domain of bat ACE2 may have the amino acid sequence shown in SEQ ID NOs: 19 or 32.

[0136] Pangolin ACE2 may have the amino acid sequence shown in SEQ ID NO: 20. The extracellular domain of pangolin ACE2 may have the amino acid sequence shown in SEQ ID NO: 21.

[0137] Civet ACE2 may have the amino acid sequence shown in SEQ ID NO: 22. The extracellular domain of civet ACE2 may have the amino acid sequence shown in SEQ ID NO: 23.

[0138] Pig ACE2 may have the amino acid sequence shown in SEQ ID NOs: 24 or 33. The extracellular domain of pig ACE2 may have the amino acid sequence shown in SEQ ID NOs: 25 or 34.

[0139] In some embodiments, an ACE2 or fragment thereof may comprise or consist of the amino acid sequence of SEQ ID NOs: 15 or 29, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 15 or 29.

[0140] In some embodiments, an ACE2 or fragment thereof may comprise or consist of the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0141] In some embodiments, an ACE2 or fragment thereof may comprise or consist of the amino acid sequence of SEQ ID NOs: 17 or 30, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 17 or 30.

[0142] In some embodiments, an ACE2 or fragment thereof may comprise or consist of the amino acid sequence of SEQ ID NOs: 18 or 31, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 18 or 31.

[0143] In some embodiments, an ACE2 or fragment thereof may comprise or consist of the amino acid sequence of SEQ ID NOs: 19 or 32, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 19 or 32.

[0144] In some embodiments, an ACE2 or fragment thereof may comprise or consist of the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0145] In some embodiments, an ACE2 or fragment thereof may comprise or consist of the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 21.

[0146] In some embodiments, an ACE2 or fragment thereof may comprise or consist of the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 22.

[0147] In some embodiments, an ACE2 or fragment thereof may comprise or consist of the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0148] In some embodiments, an ACE2 or fragment thereof may comprise or consist of the amino acid sequence of SEQ ID NOs: 24 or 33, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 24 or 33.

[0149] In some embodiments, an ACE2 or fragment thereof may comprise or consist of the amino acid sequence of SEQ ID NOs: 25 or 34, or an amino acid sequence having at least 70% sequence identity, e.g. one of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 25 or 34.

[0150] In some embodiments, the ACE2 or fragment according to the present disclosure is selected in accordance with the kits, compositions, uses and methods of the present disclosure to display (i) specific binding to the polypeptide/fragment thereof encoded by a SARSr-CoV (e.g. a spike protein or a fragment thereof encoded by a SARSr-CoV), and (ii) an affinity of binding to the polypeptide/fragment thereof encoded by a SARSr-CoV providing for sufficient sensitivity for the reliable detection of the presence in a sample of antibodies to the polypeptide/fragment thereof encoded by a SARSr-CoV (e.g. a spike protein or a fragment thereof encoded by a SARSr-CoV).

[0151] The present disclosure provides kits and compositions which may comprise a polypeptide encoded by a SARSr-CoV or a fragment thereof (e.g. a spike protein or a fragment thereof encoded by a SARSr-CoV), and/or a polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment (e.g. an ACE2 protein or a fragment thereof which binds specifically to a spike protein or a fragment thereof encoded by a SARSr-CoV).

[0152] In some embodiments in accordance with such aspects the kits and compositions may further comprise a solid support.

[0153] The solid support may be any solid support to which a polypeptide can readily be immobilised (e.g. by adsorption or conjugation), and which is suitable for the analysis of antibody containing samples, e.g. blood derived samples such as serum samples in accordance with the present disclosure. Suitable solid supports for use in such kits and compositions are well known to the skilled person.

[0154] In some embodiments, a solid support may comprise or consist of polystyrene, polypropylene, polycarbonate, cyclo-olefin, glass or quartz. In some embodiments, a solid support may be a microtiter (or "multiwell") plate or microarray plate. In some embodiments, a solid support may be a bead, e.g. a magnetic bead.

[0155] A polypeptide according to the present disclosure may be immobilised on (or `coated` on) a solid support according to the present disclosure in accordance with methods well known to the skilled person. A polypeptide may be covalently or non-covalently immobilised on a solid support.

[0156] For example, a polypeptide may be immobilised on a solid support by applying a solution of a polypeptide in buffer solution under conditions suitable for, and for sufficient time to allow, the polypeptide to adsorb to the surface of the solid support. Alternatively, a polypeptide may be conjugated to a solid support, e.g. through a covalent bond.

[0157] In some embodiments, articles of the present disclosure may further comprise means for detecting interaction between a polypeptide encoded by a SARSr-CoV or a fragment thereof (e.g. a spike protein or a fragment thereof encoded by a SARSr-CoV) and a polypeptide which binds specifically to the polypeptide encoded by the SARSr-CoV or fragment (e.g. an ACE2 protein or a fragment thereof which binds specifically to a spike protein or a fragment thereof encoded by the SARSr-CoV).

[0158] The means for detecting interaction can be any suitable means. For example, the means could employ an antibody capable of specifically binding to the polypeptide complex formed by interaction between the polypeptide encoded by a SARSr-CoV or a fragment thereof and the polypeptide which binds specifically to the polypeptide encoded by the SARSr-CoV or fragment, or could employ a reporter of interaction between the polypeptide encoded by a SARSr-CoV or a fragment thereof and the polypeptide which binds specifically to the polypeptide encoded by the SARSr-CoV or fragment.

[0159] In some embodiments, the means for detecting interaction may employ a detection entity. By way of illustration, in the experimental examples hereinbelow SARS-CoV-2 S1 and RBD polypeptides are conjugated to a horseradish peroxidase (HRP) moiety. After washing to remove unbound SARS-CoV-2 S1 and RBD polypeptide, the level of horseradish peroxidase activity is indicative of the amount of S 1/RBD bound to the immobilised ACE2.

[0160] Accordingly, in some embodiments in accordance with the various aspects of the present disclosure the polypeptide encoded by a SARSr-CoV or a fragment thereof (e.g. a spike protein or a fragment thereof encoded by a SARSr-CoV), and/or the polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment (e.g. an ACE2 protein or a fragment thereof which binds specifically to a spike protein or a fragment thereof encoded by a SARSr-CoV) are/is conjugated to a detection entity for use to detect interaction between the polypeptide encoded by a SARSr-CoV or a fragment thereof and the polypeptide which binds specifically to the polypeptide encoded by the SARSr-CoV or fragment.

[0161] A detection entity may, for example, be a detectable moiety, e.g. a fluorescent, luminescent, immuno-detectable, radio, chemical, nucleic acid or polypeptide label. In some embodiments, a detection entity may be a moiety having detectable activity, e.g. an enzymatic activity on a given substrate. Examples of detection entities having detectable activity include e.g. horseradish peroxidase (HRP) and luciferase moieties.

[0162] In some embodiments wherein a detection entity is a moiety having detectable activity, the kit or composition according to the present disclosure may further comprise reagents required for analysis of the detectable activity.

[0163] In some embodiments, the kit or composition according to the disclosure may employ detection by chemiluminescence. That is, in some embodiments, the detectable activity is chemiluminescence. Assays based on detection of chemiluminescence are described e.g. in Kricka et al., Analytica chimica acta, (2003), 500(1): 279-286 and Chen et al., Chinese Journal of Analytical Chemistry (2012) 40(1): 3-10, both of which are hereby incorporated by reference in their entirety.

[0164] In some embodiments a detection entity may e.g. be a chemical entity which produces electromagnetic radiation upon appropriate excitation. In some embodiments, a chemical entity may be an acridinium compound (e.g. an acridinium ester or acridinium sulfonamide ester), which may be excited using alkaline hydrogen peroxide.

[0165] In some embodiments a detection entity may e.g. be an enzyme entity which catalyses the production of electromagnetic radiation from a luminescent chemical. In some embodiments, an enzyme entity may be HRP, which may be used to catalyse decomposition of luminol in the presence of hydrogen peroxide. In some embodiments, an enzyme entity may be alkaline phosphatase, which may be used to catalyse decomposition of AMPPD.

[0166] In some embodiments, the polypeptide encoded by a SARSr-CoV or a fragment thereof (e.g. a spike protein or a fragment thereof (e.g. RBD) encoded by a SARSr-CoV) is provided in the kit or composition of the present disclosure such that in use to analyse a sample, the quantity or concentration of the polypeptide encoded by a SARSr-CoV or a fragment thereof (e.g. a spike protein or a fragment thereof (e.g. RBD) encoded by a SARSr-CoV) is (a) less than or equal to (in molar ratio) the quantity/concentration of neutralising antibodies to the SARSr-CoV in the sample being analysed using the kit or composition, and/or (b) sufficient to produce a detectable signal of interaction between the polypeptide encoded by a SARSr-CoV or a fragment thereof (e.g. a spike protein or a fragment thereof (e.g. RBD) encoded by a SARSr-CoV) and a polypeptide which binds specifically to the polypeptide encoded by the SARSr-CoV or fragment (e.g. an ACE2 protein or a fragment thereof which binds specifically to a spike protein or a fragment thereof encoded by the SARSr-CoV) in the absence of neutralising antibodies to the SARSr-CoV in the sample being analysed using the kit or composition.

[0167] The skilled person is able to determine suitable quantities/concentrations of polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof where the compositions and kits are put to such use e.g. by determination of/reference to the quantity/concentration of neutralising antibodies to the SARSr-CoV in samples determined or otherwise known to contain neutralising antibodies to the SARSr-CoV. For example, in some embodiments a suitable quantity/concentration of the polypeptide may be less than or equal to (in molar ratio) the average (e.g. the mean) quantity/concentration of neutralising antibodies to the SARSr-CoV in reference sample(s) containing neutralising antibodies to the SARSr-CoV. In some embodiments, serial dilution of the sample to be tested may be used to determine a suitable quantity of a polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof.

[0168] The skilled person is also able to determine suitable quantities/concentrations of polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof where the compositions and kits are put to such use e.g. by determination of/reference to the minimal quantity/concentration required to produce a detectable signal of interaction between the polypeptide encoded by a SARSr-CoV or a fragment thereof (e.g. a spike protein or a fragment thereof (e.g. RBD) encoded by a SARSr-CoV) and the polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment (e.g. ACE2) in the absence of neutralising antibodies to the SARSr-CoV in the sample.

[0169] The present disclosure also provides the constituent polypeptides of the kits and compositions described herein. The polypeptides may be provided in isolated or substantially purified form.

[0170] In some embodiments the polypeptides of the present disclosure may comprise one or more linker sequences between amino acid sequences, e.g. between the amino acid sequence of a spike protein or a fragment thereof encoded by a SARSr-CoV and a detection entity.

[0171] Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence. Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and/or serine residues.

[0172] In some embodiments, the linker sequence may comprise at least one glycine residue and/or at least one serine residue. In some embodiments the linker sequence may consist of glycine and serine residues. In some embodiments, the linker sequence may have a length of 1-2, 1-3, 1-4, 1-5 or 1-10 amino acids.

[0173] The polypeptides of the present disclosure may additionally comprise further amino acids or sequences of amino acids. For example, the polypeptide may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing or purification of the polypeptide. For example, the polypeptide may comprise a sequence encoding a His, (e.g. 6.times. His), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N- or C-terminus of the polypeptide.

[0174] The polypeptides of the present disclosure may additionally comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides.

[0175] The signal peptide may be present at the N-terminus of the polypeptide, and may be present in the newly synthesised polypeptide. The signal peptide provides for efficient trafficking and secretion of the polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature polypeptide secreted from the cell expressing the polypeptide.

[0176] Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

[0177] Polypeptides according to the disclosure may be prepared according to methods for the production of polypeptides known to the skilled person.

[0178] Polypeptides may be prepared by chemical synthesis, e.g. liquid or solid phase synthesis. For example, peptides/polypeptides can by synthesised using the methods described in, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby incorporated by reference in its entirety.

[0179] Alternatively, polypeptides may be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146, both of which are hereby incorporated by reference in their entirety.

[0180] For recombinant production according to the disclosure, any cell suitable for the expression of polypeptides may be used. The cell may be a prokaryote or eukaryote. In some embodiments the cell is a prokaryotic cell, such as a cell of archaea or bacteria. In some embodiments the bacteria may be Gram-negative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia coli. In some embodiments, the cell is a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell, e.g. CHO, HEK (e.g. HEK293), HeLa or COS cells. In some embodiments, the cell is a CHO cell that transiently or stably expresses the polypeptides.

[0181] In some cases the cell is not a prokaryotic cell because some prokaryotic cells do not allow for the same folding or post-translational modifications as eukaryotic cells. In addition, very high expression levels are possible in eukaryotes and proteins can be easier to purify from eukaryotes using appropriate tags. Specific plasmids may also be utilised which enhance secretion of the protein into the media.

[0182] In some embodiments polypeptides may be prepared by cell-free-protein synthesis (CFPS), e.g. using a system described in Zemella et al. Chembiochem (2015) 16(17): 2420-2431, which is hereby incorporated by reference in its entirety.

[0183] Production may involve culture or fermentation of a eukaryotic cell modified to express the polypeptide of interest. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air/oxygen and/or growth factors. Secreted proteins can be collected by partitioning culture media/fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted polypeptide. Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein above).

[0184] Bioreactors include one or more vessels in which cells may be cultured. Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.

[0185] Following culturing the cells that express the polypeptide, the polypeptide of interest may be isolated. Any suitable method for separating proteins from cells known in the art may be used. In order to isolate the polypeptide it may be necessary to separate the cells from nutrient medium. If the polypeptide is secreted from the cells, the cells may be separated by centrifugation from the culture media that contain the secreted polypeptide of interest. If the polypeptide of interest is collected within the cell, protein isolation may comprise centrifugation to separate cells from cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption e.g. by sonification, rapid freeze-thaw or osmotic lysis.

[0186] It may then be desirable to isolate the polypeptide of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins.

[0187] Other methods for distinguishing different proteins are known in the art, for example ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation, or may be performed subsequently to precipitation.

[0188] Once the polypeptide of interest has been isolated from culture it may be desired or necessary to concentrate the polypeptide. A number of methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilisation.

[0189] The present disclosure provides a nucleic acid encoding a polypeptide according to the present disclosure. It will be appreciated that "a nucleic acid" encompasses a plurality of such nucleic acids.

[0190] In some embodiments, the nucleic acid is purified or isolated, e.g. from other nucleic acid, or naturally-occurring biological material. In some embodiments the nucleic acid(s) comprise or consist of DNA and/or RNA.

[0191] The present disclosure also provides a vector which may comprise the nucleic acid according to the present disclosure.

[0192] The nucleotide sequence may be contained in a vector, e.g. an expression vector. A "vector" as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the disclosure.

[0193] The term "operably linked" may include the situation where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g. promoter and/or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) may then be translated into a desired peptide(s)/polypeptide(s).

[0194] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes).

[0195] In some embodiments, the vector may be a eukaryotic vector, e.g. a vector which may comprises the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. that may comprises a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0196] The present disclosure also provides a cell which may comprises or expresses a polypeptide according to the present disclosure. Also provided is a cell which may comprises or expresses a nucleic acid or vector according to the present disclosure. The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the genus Bos), horse (including any animal in the family Equidae), donkey, and non-human primate).

[0197] The present disclosure also provides a method for producing a cell which may comprises a nucleic acid or vector according to the present disclosure, which may comprise introducing a nucleic acid or vector according to the present disclosure into a cell. In some embodiments, introducing an isolated nucleic acid or vector according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction).

[0198] The present disclosure also provides a method for producing a cell which may express or comprise a polypeptide according to the present disclosure, which may comprise introducing a nucleic acid or vector according to the present disclosure in a cell. In some embodiments, the methods additionally comprise culturing the cell under conditions suitable for expression of the nucleic acid or vector by the cell. In some embodiments, the methods are performed in vitro.

[0199] The present disclosure also provides cells obtained or obtainable by the methods according to the present disclosure.

[0200] The present disclosure also concerns methods using and uses of the kits, compositions and polypeptides of the present disclosure.

[0201] The articles of the present disclosure are useful for detecting antibodies to a polypeptide encoded by a SARSr-CoV or a fragment thereof (e.g. a spike protein or a fragment thereof encoded by a SARSr-CoV).

[0202] In particular, the articles of the present disclosure are useful for detecting the presence of antibodies which reduce or inhibit binding of a polypeptide encoded by a SARSr-CoV or fragment thereof to a polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment. Such antibodies may be referred to as neutralising antibodies.

[0203] By way of further explanation, with reference to the experimental examples hereinbelow, the presence of antibodies which inhibit binding of SARS-CoV-2 spike protein S1 subunit and SARS-CoV-2 spike protein RBD are detected in samples based on the determination of a reduced level of interaction between the SARS-CoV-2 spike protein S1 subunit or SARS-CoV-2 spike protein RBD with ACE2 immobilised on the solid support. The presence of neutralising antibodies in the sample is inferred from the determination of a reduced level of interaction relative to a control condition lacking neutralising antibodies to SARS-CoV-2 spike protein S1 subunit or SARS-CoV-2 spike protein RBD.

[0204] Accordingly, the articles of the present disclosure are useful in methods for detecting the presence of antibodies to a SARSr-CoV in a sample. It will be appreciated that the methods are useful for the detection of the presence of antibodies to the spike protein or a fragment thereof encoded by a SARSr-CoV employed in the kit/composition.

[0205] By way of illustration, the assays exemplified herein employ SARS-CoV-2 spike protein S1 subunit or SARS-CoV-2 spike protein RBD, and are therefore useful for the detection of antibodies to SARS-CoV-2 spike protein S1 subunit or SARS-CoV-2 spike protein RBD.

[0206] Detection of the presence of antibodies to a SARSr-CoV in a sample may be indicative of an ongoing or previous infection with the SARSr-CoV. Detection of the presence of antibodies to a SARSr-CoV in a sample may be indicative of an ongoing or previous immune response, particularly a humoral immune response, to the SARSr-CoV.

[0207] Accordingly, the present disclosure provides methods for determining whether a subject is or has been infected with a SARSr-CoV, and also provides methods for determining whether a subject is mounting/has mounted an immune response (e.g. a humoral immune response) to a SARSr-CoV.

[0208] The methods may generally comprise analysing a sample in order to determine whether its contents reduce or inhibit interaction between a polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof and a polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment (e.g. ACE2), relative to the level of interaction observed for an appropriate negative control condition.

[0209] An appropriate negative control condition may e.g. employ an equivalent sample known to lack antibodies capable of reducing or inhibiting interaction between a polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof and a polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment (e.g. ACE2). For example, a control sample may be from a known naive subject, e.g. a subject known not to have been infected with the SARSr-CoV.

[0210] Determination of reduced or inhibited interaction indicates the presence of neutralising antibodies to the polypeptide encoded by a SARSr-CoV or fragment thereof (e.g. the spike protein or fragment thereof) in the sample.

[0211] As used herein, "reduced" or "inhibited" interaction may be a level of interaction which is less than 1 times, e.g. .ltoreq.0.99 times, .ltoreq.0.95 times, .ltoreq.0.9 times, .ltoreq.0.85 times, .ltoreq.0.8 times, .ltoreq.0.75 times, .ltoreq.0.7 times, .ltoreq.0.65 times, .ltoreq.0.6 times, .ltoreq.0.55 times, .ltoreq.0.5 times, .ltoreq.0.45 times, .ltoreq.0.4 times, .ltoreq.0.35 times, .ltoreq.0.3 times, .ltoreq.0.25 times, .ltoreq.0.2 times, .ltoreq.0.15 times, .ltoreq.0.1 times, .ltoreq.0.05 times, or .ltoreq.0.01 times the level of interaction between the polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof and the polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment (e.g. ACE2) observed in the negative control condition.

[0212] Alternatively, "reduced" or "inhibited" interaction may be expressed in terms of percentage inhibition of the level of interaction between the polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof and the polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment (e.g. ACE2) observed in the negative control condition. In such instances, "reduced" or "inhibited" interaction may refer to percentage inhibition of greater than 0%, e.g. greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or 100% of the level of interaction between the polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof and the polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment (e.g. ACE2) observed in the negative control condition.

[0213] In some embodiments, aspects of the present disclosure may comprise:

[0214] contacting a sample obtained from the subject with: (i) a polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof, and (ii) a polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment (e.g. ACE2), and

[0215] determining the level of interaction between the polypeptide or fragment of (i) and the polypeptide of (ii).

[0216] In some embodiments, aspects of the present disclosure may comprise:

[0217] (a) contacting a sample obtained from the subject with (i) a polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof,

[0218] (b) contacting the mixture formed by step (a) with (ii) a polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment (e.g. ACE2), and

[0219] (c) determining the level of interaction between the polypeptide or fragment of (i) and the polypeptide of (ii).

[0220] In some embodiments, aspects of the present disclosure may comprise:

[0221] (a) contacting a sample obtained from the subject with (i) a polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment (e.g. ACE2),

[0222] (b) contacting the mixture formed by step (a) with (ii) a polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof, and

[0223] (c) determining the level of interaction between the polypeptide or fragment of (i) and the polypeptide of (ii).

[0224] In some embodiments, "contacting" samples/mixtures with polypeptides according to the present disclosure may comprise mixing such samples/mixture and compositions (e.g. solutions) which may comprise the polypeptides, e.g. in an appropriate container or on an appropriate solid support.

[0225] In some embodiments, "contacting" samples/mixtures with polypeptides according to the present disclosure may comprise applying a sample/mixture to a solid support to which the polypeptides are immobilised.

[0226] In some embodiments, the aspects may comprise contacting the sample with a composition which comprises a polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof (RBD)), wherein the polypeptide is provided at a quantity or concentration which is (a) less than or equal to (in terms of molar ratio) the quantity/concentration of neutralising antibodies to the SARSr-CoV in the sample, and/or (b) sufficient to produce a detectable signal of interaction between the polypeptide or fragment of (i) and the polypeptide or fragment of (ii) in the absence of neutralising antibodies to the SARSr-CoV in the sample.

[0227] In some embodiments, the aspects may comprise determining the presence or absence of neutralising antibodies to the SARSr-CoV in the sample. In some embodiments, the aspects may comprise determining the quantity and/or concentration of neutralising antibodies to the SARSr-CoV in the sample.

[0228] The skilled person is able to determine suitable quantities/concentrations of polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof to be employed in such methods e.g. by determination of/reference to the quantity/concentration of neutralising antibodies to the SARSr-CoV in samples determined or otherwise known to contain neutralising antibodies to the SARSr-CoV. For example, in some embodiments a suitable quantity/concentration of the polypeptide may be less than or equal to (in molar ratio) the average (e.g. the mean) quantity/concentration of neutralising antibodies to the SARSr-CoV in reference sample(s) containing neutralising antibodies to the SARSr-CoV.

[0229] The skilled person is also able to determine suitable quantities/concentrations of polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof to be employed in such methods e.g. by determination of/reference to the minimal quantity/concentration required to produce a detectable signal of interaction between the polypeptide encoded by a SARSr-CoV or a fragment thereof (e.g. a spike protein or a fragment thereof (e.g. RBD) encoded by a SARSr-CoV) and the polypeptide which binds specifically to the polypeptide encoded by a SARSr-CoV or fragment (e.g. ACE2) in the absence of neutralising antibodies to the SARSr-CoV in the sample.

[0230] In some embodiments, the aspects may employ a range of different quantities/concentrations of polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof for the analysis of a given sample. That is, in some embodiments, aspects of the present disclosure may comprise performing the analysis with a range of different quantities/concentrations of the polypeptide encoded by a SARSr-CoV (e.g. a spike protein) or fragment thereof for the analysis of aliquots of a given same sample. The range of different quantities/concentrations of the polypeptide may be a dilution series, and may e.g. by prepared by serial dilution of a solution of the polypeptide.

[0231] In some embodiments, the aspects may further comprise one or more of the following:

[0232] obtaining a sample (e.g. a blood sample) from a subject;

[0233] preparing a blood-derived sample (e.g. a serum sample) from a blood sample obtained from a subject;

[0234] providing a composition or kit according to the present disclosure;

[0235] applying a blood-derived sample (e.g. a serum sample) to a composition according to the present disclosure;

[0236] incubating the sample with the polypeptides/composition for sufficient time and under appropriate conditions for the formation of antibody:antigen complexes;

[0237] aspiration of the sample;

[0238] washing to remove unbound proteins;

[0239] detection of interaction between the polypeptide or fragment of (i) and the polypeptide or fragment of (ii).

[0240] In some embodiments, the aspects may further comprise determining the level (e.g. the percentage) of inhibition of interaction between the polypeptide or fragment of (i) and the polypeptide or fragment of (ii).

[0241] In some embodiments, the aspects may further comprise comparing the level (e.g. the percentage) of inhibition of interaction between the polypeptide or fragment of (i) and the polypeptide or fragment of (ii) as observed to a reference threshold value for determining that the sample contains neutralising antibodies to the SARSr-CoV. In some embodiments, the aspects may further comprise determining on the basis of such comparison whether a sample contains or does not contain neutralising antibodies to the SARSr-CoV (i.e. determining the presence or absence of neutralising antibodies to the SARSr-CoV in the sample).

[0242] Appropriate reagents, buffers and washing steps to be employed in such methods are well known to the person skilled in the art of molecular biology, and can moreover be inferred by reference to the experimental examples herein and Bossart et al., J Viol Meth (2007) 142: 29-40, which is hereby incorporated by reference in its entirety.

[0243] In some aspects, the articles and methods of the present disclosure are employed for the diagnosis of infection with a SARSr-CoV, e.g. SARS-CoV-2. In some aspects, the articles and methods are employed for the diagnosis of a disease caused by infection with a SARSr-CoV, e.g. COVID-19.

[0244] Aspects of the present disclosure concern the analysis of samples from subjects. The sample may be of, or may be derived from, any product produced by a subject. A sample may be taken from any tissue or bodily fluid, e.g. a blood sample (including blood-derived samples), serum sample, lymph sample, saliva sample, synovial fluid sample. A blood-derived sample may be a selected fraction of a patient's blood, e.g. a selected cell-containing fraction, or a plasma or serum fraction.

[0245] The sample may be any sample containing the antibody products of a humoral immune response. In some embodiments the sample obtained from a subject is an antibody-containing sample or antibody-containing fraction thereof. The sample is preferably a blood sample or a blood-derived sample, more preferably a serum sample.

[0246] Samples may be collected (e.g. by venesection), processed and/or stored (e.g. frozen and stored at -80.degree. C.) according to known techniques, and in accordance with the analysis to be subsequently performed on the sample.

[0247] A sample may be or may have been collected from a subject of interest. The subject according to the various aspects of the present disclosure may be any animal. In some embodiments, the subject is a mammal (e.g. a therian, placental, epitherian, preptotheria, archontan, primate (rhesus, cynomolgous, non-human primate or human)). In some embodiments, the subject is a human, bat, pangolin, civet or pig. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient.

[0248] The subject may be suspected of being infected with a SARSr-CoV, or may be suspected of having a disease caused by infection with a SARSr-CoV. The subject may be suspected of having previously been infected with a SARSr-CoV, or may be suspected of having previously had a disease caused by infection with a SARSr-CoV.

[0249] The subject may have been diagnosed with infection with a SARSr-CoV or may have been diagnosed with a disease caused by infection with a SARSr-CoV. The subject may have experienced infection with a SARSr-CoV or may have had a disease caused by infection with a SARSr-CoV.

[0250] In particular embodiments, the articles and methods of the present disclosure provide for the detection of the presence in a sample (e.g. a patient-derived sample) of neutralising antibodies to a SARSr-CoV (e.g. SARS-CoV-2), based on detection of blocking of the interaction/binding between an ACE2 protein or fragment thereof and a spike protein or fragment thereof encoded by the SARSr-CoV (e.g. the RBD of the SARSr-CoV) by such neutralising antibodies.

[0251] More particularly, preferred embodiments provide for the detection of the presence in a sample (e.g. a sample derived from a COVID-19 patient) of neutralising antibodies to a SARS-CoV-2, based on detection of blocking of the interaction/binding between an ACE2 protein or fragment thereof and the RBD of the SARS-CoV-2 by such neutralising antibodies.

[0252] Even where such neutralising antibodies are present in a sample, they will often be present at low levels (i.e. there will be a limited quantity of the antibody). In such instances, if there is an over-supply (i.e. an excess) of spike protein/fragment thereof (e.g. RBD) in the reaction mix, the quantity of antibodies in a test sample may be insufficient to bind to all of the spike protein/fragment thereof (e.g. RBD) available, and the surplus spike protein/fragment thereof (e.g. RBD) will bind to the ACE2 protein/fragment thereof yielding signal, and thus resulting in a false-negative result.

[0253] Therefore, in preferred embodiments in accordance with the preset disclosure, the quantity of spike protein/fragment thereof (e.g. RBD) is present in the reaction mix at a level which is (i) sufficient to provide a detectable level of signal (i.e. on interaction with the ACE2 protein/fragment thereof) in the absence of neutralising antibodies to the SARSr-CoV to enable the correct determination of true negative samples lacking neutralising antibodies to the SARSr-CoV, but (ii) not more, in terms of molar ratio (i.e. no molar excess of), than the amount of neutralising antibodies to the SARSr-CoV in the sample, such that all of the spike protein/fragment thereof (e.g. RBD) present in the reaction mix will be neutralized by the antibodies available in the sample, and false-negative results will be avoided.

[0254] It will be appreciated that one way to achieve the optimal quantity of the spike protein/fragment thereof (e.g. RBD) in the reaction mix is to prepare a solution (e.g. using a suitable buffer) containing the soluble spike protein/fragment thereof (e.g. RBD) at an appropriate concentration (e.g. by serial dilution of a stock solution), and to use an appropriate volume of the spike protein/fragment thereof (e.g. RBD)-containing solution in the reaction mix.

[0255] Another way to achieve the optimal quantity of the spike protein/fragment thereof (e.g. RBD) in the reaction mix is to provide an appropriate quantity of the spike protein/fragment thereof (e.g. RBD) to the reaction mix e.g. in a form immobilized on a solid support, e.g. in the form of the spike protein/fragment thereof (e.g. RBD) immobilized on beads.

[0256] The person of ordinary skill in the art would readily recognize permutations of the embodiments of particular interest described above may be devised, and that the articles and methods of the present disclosure can be calibrated and therefore be made quantitative or at least semi-quantitative.

[0257] In some embodiments in accordance with the various aspects of the present disclosure, the kits, compositions and methods may be characterised by reference to certain functional properties.

[0258] In some embodiments, a kit, composition and/or method according to the present disclosure may possess one or more of the following properties:

[0259] (i) Ability to detect the presence of neutralising antibodies to the SARSr-CoV in a sample with greater than 90% (e.g. one of greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or 100%) specificity;

[0260] (ii) Ability to detect the presence of neutralising antibodies to the SARSr-CoV in a sample with greater than 90% (e.g. one of greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or 100%) sensitivity;

[0261] (iii) Ability to detect the presence of neutralising antibodies to the SARSr-CoV in a sample without employing the relevant live SARSr-CoV;

[0262] (iv) Ability to detect the presence of neutralising antibodies to the SARSr-CoV irrespective of antibody isotype;

[0263] (v) Ability to detect the presence of neutralising antibodies to the SARSr-CoV in a sample irrespective of the species of the subject from which the sample is derived; and

[0264] (vi) Ability to arrive at a determination of the presence or absence of neutralising antibodies to the SARSr-CoV in a sample within less than 12 hours (e.g. one of less than 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours or 1 hour).

[0265] The disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0266] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the disclosure in diverse forms thereof.

[0267] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventor will not be bound by any of these theoretical explanations.

[0268] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated. Also, where a polypeptide-encoding nucleic acid sequence is disclosed herein equivalent polypeptide-encoding sequences as a result of degeneracy of the genetic code are also expressly contemplated.

[0269] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" in relation to a numerical value is optional and means for example +/-10%.

[0270] Methods disclosed herein may be performed, or products may be present, in vitro, ex vivo, or in vivo. The term "in vitro" is intended to encompass experiments with materials, biological substances, cells and/or tissues in laboratory conditions or in culture whereas the term "in vivo" is intended to encompass experiments and procedures with intact multi-cellular organisms. In some embodiments, methods performed in vivo may be performed on non-human animals. "Ex vivo" refers to something present or taking place outside an organism, e.g. outside the human or animal body, which may be on tissue (e.g. whole organs) or cells taken from the organism.

[0271] For standard molecular biology techniques, see Sambrook, J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.

[0272] Aspects and embodiments of the present disclosure will now be discussed with reference to the accompanying figures and the following examples. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference in their entirety. While the disclosure has been described in conjunction with the exemplary embodiments described below, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the disclosure are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the disclosure.

EXAMPLES

Example 1

Surrogate Virus Neutralization Test

[0273] In this Example, the inventor presents a novel surrogate virus neutralization test (COVID-sVNT).

1.1 Materials and Methods

[0274] Recombinant His-tagged human ACE2 (hACE2, Cat #: 10108-H08H) was purchased from SinoBiologics. Recombinant SARS-CoV-2 nucleoprotein (NP) (Cat #: Z03488), spike protein S1 subunit (Cat #: Z03501) and spike protein RBD (Cat #: Z03479) were purchased from GenScript and conjugated with horse radish peroxidase (HRP) through customer service by GenScript.

[0275] 1) Indirect ELISA: RBD protein (50 ng) was coated in a 96-well ELISA plate. Test serum was incubated at 1:100 dilution, after washing to remove unbound protein, followed by incubation with HRP-conjugated anti-human antibody (at a dilution of 1:10,000). After washing, HRP substrate was added for colour development.

[0276] 2) Surrogate virus-host binding assay: hACE2 was coated onto wells of a 96-well plate at a concentration of 100 ng/well. After washing to remove unbound protein, HRP-conjugated recombinant nucleoprotein, S1 or RBD protein (20-100 ng) was then added to assess specific binding. After washing, HRP substrate was added for colour development.

[0277] 3) Surrogate virus neutralization test (sVNT): hACE2 was coated onto wells of a 96-well plate at a concentration of 100 ng/well. In a separate plate, HRP-conjugated recombinant nucleoprotein, S1 or RBD protein (20-100 ng) was preincubated with different dilutions of test sera. The serum-HRP-protein mixes were then added to the hACE2 coated plate to assess specific inhibition/neutralization. After washing, HRP substrate was added for colour development.

1.2 Results

[0278] Six serum samples were used in this proof of concept study: two SARS-CoV-2 positive serum samples (COVID-55, COVID-63), two SARS-CoV positive serum samples (SARS-2, SARS-7), one negative serum sample (NEGATIVE), and one unknown serum sample (TEST-007).

[0279] The results of the above two assays are shown in the table 1 below.

TABLE-US-00001 TABLE 1 Results of indirect ELISA and sVNT Sample ELISA.sup.1 sVNT-RBD.sup.2 sVNT-S1.sup.2 VNT.sup.3 COVID-55 1.49 +ve +ve +ve COVID-63 1.62 +ve +ve +ve TEST-007 1.04 -ve -ve -ve SARS-2 0.20 -ve -ve -ve SARS-7 0.25 -ve -ve -ve NEGATIVE 0.10 -ve -ve -ve .sup.1Average from two independent experiments .sup.2Inhibition/neutralization at 1:40 or greater dilution .sup.3Neutralisiton at 1:20 or greater dilution

[0280] In the surrogate virus-host binding assay, when different HRP-protein conjugates were added to the hACE2 coated plate, the HRP-NP displayed no binding at all (FIG. 3B) whereas both HRP-S1 and HRP-RBD displayed significant binding (FIGS. 3A and 3B), demonstrating that the recombinant S1 and RBD proteins were functional, and suitable to be used in surrogate virus neutralization assays.

[0281] As a proof of concept, the inventor conducted the sVNT using the same panel of sera used for the indirect ELISA.

[0282] As shown in FIGS. 4A and 4B, only the two SARS-CoV-2 positive sera showed significant inhibition/neutralization in either RBD- (FIG. 4A) or S1- (FIG. 4B) based sVNT. The assay demonstrated excellent specificity, with the inhibition curves for COVID-19 samples in FIGS. 4A and 4B being completely dose and dilution dependent.

[0283] The inventor next performed analysis of 74 serum samples obtained from COVID-19 patients having been tested positive by PCR for SARS-CoV-2 infection, and 11 negative (healthy) human serum samples, using the version of the sVNT employing recombinant HRP-conjugated SARS-CoV-2 spike protein RBD.

[0284] The results are shown in FIG. 5. The sVNT was able to reliably distinguish samples containing neutralising antibodies to SARS-CoV-2 from samples not containing such antibodies.

[0285] It is known that not all COVID-19 patients induce strong neutralising antibody responses to SARS-CoV-2, accounting for the results in samples determined by sVNT to be weakly-positive.

1.3 Conclusion

[0286] In conclusion, the inventor has developed an assay capable of detecting neutralising antibodies to SARS-CoV-2 with high sensitivity and specificity.

[0287] Importantly, the surrogate virus neutralization test is more specific than the widely-used indirect ELISA assay, as evidenced by the TEST-007 sample giving a positive reading in the indirect ELISA assay, but being shown by the surrogate virus neutralization test to be negative (as confirmed by the live virus neutralisation test).

Example 2

SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) Based on Antibody-Mediated Blockage of ACE2-Spike (RBD) Protein Interaction

[0288] There is an urgent need for a robust serological test to detect neutralizing antibodies to SARS-CoV-2. Such a test is not only important for contact tracing, but for determining infection rate, herd immunity and predicted humoral protection. The current gold standard is a virus neutralization test (VNT) requiring live virus and a biosafety level 3 (BSL3) laboratory. On the other hand, the ELISA- or lateral flow-based assays are for the detection of binding antibodies, which does not directly correlate with their neutralizing ability. Here the inventor describes a SARS-CoV-2 surrogate virus neutralization test (sVNT) that is designed to detect total neutralizing antibodies in an isotype- and species-independent manner. This simple and rapid test is based on antibody-mediated blockage of virus-host interaction between the ACE2 receptor protein and the receptor binding domain (RBD) of the viral spike protein. The test has been validated with two COVID-19 patient cohorts in two different countries, achieving 100% specificity and 95-100% sensitivity, and is capable of differentiating antibody responses from other known human coronaviruses. Importantly, the sVNT does not require BSL3 containment, thereby making the test immediately accessible to the global community.

2.1 Introduction

[0289] The COVID-19 outbreak was first recognized in December 2019 in Wuhan, China.sup.1, which has since spread to all parts of the world resulting in a total 2,160,207 infections with 146,088 deaths as of 18 Apr. 2020.sup.2. The causative agent was identified as 2019-nCoV, subsequently designated SARS-CoV-2.sup.3,4, which belongs to the species SARS-related coronavirus (SARSr-CoV), same as for SARS-CoV, the causative agent of the SARS outbreak 17 years ago.sup.5.

[0290] While molecular detection, such as polymerase chain reaction (PCR) and next generation sequencing (NGS), played and continue to play an important role in acute diagnosis and monitoring of genetic changes of the virus, there is now an urgent need for a reliable and versatile serological or antibody test. Such a test is needed for retrospective contact tracing, investigation of asymptomatic infection rate, accurate determination of case fatality rate, assessment of herd immunity and humoral protective immunity in recovered patients and recipients of vaccine candidates, and in the search for the natural reservoir host and intermediate host(s) [6]. Research laboratories and pharmaceutical companies are racing to produce antibody tests that can detect SARS-CoV-2 infection with sufficient specificity and sensitivity [6]. There are two types of antibody tests one can aim for. The first type is the virus neutralization test (VNT) which detects neutralizing antibodies (NAbs) in a patient's blood. VNT requires handling live SARS-CoV-2 in a specialized biosafety level 3 (BSL3) containment facility which is tedious and time consuming, taking 2-4 days to complete. Pseudovirus-based virus neutralization test (pVNT) is similar, but still requires the use of live viruses and cells although handled in a BSL2 laboratory [7, 8]. All other assays, such as ELISA and lateral flow rapid tests, represent the second assay type which detect only binding antibodies, and not NAbs [6, 9-11].

[0291] In this Example the inventor describes a surrogate virus neutralization test (sVNT) which detects NAbs, but without the need to use any live virus or cells and can be completed in 1-2 hours in a BSL2 lab. Using purified receptor binding domain (RBD) protein from the viral spike (S) protein and the host cell receptor ACE2, the test is designed to mimic the virus-host interaction by direct protein-protein interaction in a test tube or an ELISA plate well. This highly specific interaction can then be neutralized, i.e., blocked by highly specific NAbs in patient or animal sera in the same manner as in a conventional VNT.

2.2 Materials and Methods

[0292] Human embryonic kidney (HEK293T) cells (ATCC# CRL-3216) and African green monkey kidney, clone E6 (Vero-E6) cells (ATCC# CRL-1586) were maintained in Dulbecco's modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. SARS-CoV-2, isolate BetaCoV/Singapore/2/2020 (Accession ID EPI_ISL_406973), was used for virus neutralization test on Vero-E6 cells as described previously [26].

[0293] In Singapore, COVID-19 patient sera used in this study was from the Singapore PROTECT study as described [13]. Sera from recovered SARS patients from 2003 were as previously described [15]. For SARS recall sampling in 2020, the inventor obtained blood from consenting individuals previously admitted for SARS (ethics approval number: NHG DSRB E 2020/00091). The hCoV serum panel included post-infection samples from subjects confirmed CoV 229/NL63 and CoV OC43 positive using the SeeGene RV12 respiratory multiplex kit in a previous study (ethics approval number: NUS-IRB 11-3640) [27]. Negative control sera were obtained from residual serum samples from previous unrelated studies.

[0294] In Nanjing, China, COVID-19 convalescent sera were collected with written informed consent and approved by the ethics committee of the Second Hospital of Nanjing (ethics approval number: 2020-LS-ky003). Rabbit anti-SARS-CoV-2 RBD sera were purchased from GenScript. Rabbit and ferret anti-SARS-CoV sera, and alpaca anti-MERS-CoV sera were as described in previous studies [28, 29].

Direct Binding and sVNT Assay

[0295] For direct binding, hACE2 protein (GenScript, Cat #: Z03484) was coated at 100 ng/well in 100 mM carbonate-bicarbonate coating buffer (pH 9.6). HRP-conjugated SARS-CoV-2 nucleocapsid (briefly referred to as N hereinafter, GenScript, Cat #: Z03488), S1 (GenScript, Cat #: Z03501), RBD (GenScript, Cat #: Z03479) or HRP-conjugated SARS-CoV-RBD (customer-made by GenScript) was added to the hACE2 coated plate at different concentration in OptEIA assay diluent (BD) for 1 h at room temperature. Unbound HRP-conjugated antigens were removed by five phosphate buffered saline, 0.05% tween-20 (PBST) washes. Colorimetric signal was developed on the enzymatic reaction of HRP with chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB) (Invitrogen). Equal volume of TMB stop solution (KPL) was added to stop the reaction, and the absorbance reading at 450 nm and 570 nm were acquired using Cytation 5 microplate reader (BioTek). For the surrogate neutralization test (sVNT), 6 ng of HRP-RBD (from SARS-CoV or SARS-CoV-2) was pre-incubated with test serum at the final dilution of 1:20 for 1 h at 37.degree. C., followed by hACE2 incubation for 1 h at room temperature. Inhibition (%)=(1-Sample OD value/Negative Control OD value).times.100.

Indirect ELISA

[0296] SARS-CoV-2 N protein and SARS-CoV N protein were expressed from the pcDNA3.1 SARS-CoV-2 N and pDualGC SARS-CoV N transfected HEK293T cells and purified using Ni Sepharose (GE healthcare). For indirect ELISA, 100 ng of each protein was coated onto MaxiSORP ELISA plate (Nunc) using 100 mM carbonate buffer and blocked with BD OptEIA (BD). COVID-19 and SARS patient sera were tested at a dilution of 1:50 and detected by Goat-anti-human IgG-HRP (Santa Cruz) at 1:10,000 dilution. The chromogenic signal was developed using TMB substrate (Invitrogen) and the reaction was stop with TMB stop solution (KPL). Absorbance readings at 450 and 570 nm were obtained using Cytation 5 microplate reader (Bio-Tek).

Capture ELISA

[0297] 96-well Maxisorp plates (Nunc) were coated with 10 .mu.g/ml of anti-human IgM (SeraCare) or anti-human IgG (Jackson labs) in bicarbonate buffer overnight at 4.degree. C. Wells were blocked using BD OptEIA assay diluent (BD) for 1 h at 37.degree. C., and heat-inactivated sera diluted 1:50 were next added and incubated for 1 h at 37.degree. C. Following extensive washing, SARS-CoV-2-RBD-HRP (GenScript) diluted 4 .mu.g/ml was added and incubated for 30 min at 37.degree. C. Chromogenic reaction was quantified following the addition of TMB substrate (Invitrogen) and stop solution (KPL SeraCare). The absorbance of the samples was measured at 450 nm and the background at 570 nm. Negative controls consisted of 37 naive human sera. Results are presented as fold-change over average reading of negative controls.

Statistical Analysis

[0298] Statistical analysis was perform using GraphPad Prism software with the Kruskal-Wallis test to compare multiple groups, followed by Dunn's multiple comparisons test. Data were considered significant if * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

2.3 Results

Biochemical Simulation of Virus-Receptor Interaction and Antibody-Mediated Neutralization

[0299] Immediately after SARS-CoV-2 was identified as the causative agent of the COVID-19 outbreak, it was shown that the human angiotensin converting enzyme-2 (hACE2) is the main functional receptor for viral entry [3]. The inventor hypothesized that the virus-receptor binding can be mimicked in vitro via a protein-protein interaction using purified recombinant hACE2 and the RBD of the SARS-CoV-2 S protein. This interaction can be blocked by virus NAbs present in the test serum, using the same principle as a conventional VNT conducted using live virus inside a BSL3 facility (FIGS. 6A and 6B).

[0300] Direct binding was demonstrated using different SARS-CoV-2 proteins conjugated with horseradish peroxidase (HRP). There is a dose-dependent specific binding between hACE2 and RBD or S1, but not with the nucleocapsid (N) protein, with the RBD producing the best binding characteristics (FIG. 6C). The HRP-RBD protein was chosen for subsequent studies. It was then demonstrated that the specific RBD-hACE2 binding can be blocked or neutralized by COVID-19 sera in a dose-dependent manner, but not by sera from healthy controls (FIG. 6D). To prove that the same principle works with the closely related SARS-CoV, which also uses hACE2 as the entry receptor [12], similar experiments were repeated and proved that the SARS-CoV RBD performed in an almost identical manner in this new test format (FIGS. 6E and 6F), termed surrogate virus neutralization test (sVNT).

Isotype- and Species-Independent Neutralization

[0301] One of the advantages of the sVNT is its ability to detect total antibodies in patient sera, in contrast to most SARS-CoV-2 antibody tests published or marketed, which are almost all isotype-specific, mostly for IgM or IgG, with some for IgA [9-11]. From a panel of 77 COVID-19 positive sera from patients in Singapore, the inventor has designated four groups based on IgM or IgG ELISA levels, determined by in-house capture ELISA assays (see Methods), present in the patient convalescent sera: a) high IgM/low IgG; b) low IgM/high IgG; c) low IgM/low IgG; and d) high IgM/high IgG. All groups showed strong neutralization activity in the sVNT (FIGS. 7A-7D), demonstrating the isotype-independent performance of the assay. It is worth to note that for panel c with low IgM/IgG, the % inhibition in sVNT is still significant at 70-75%, demonstrating its superior sensitivity as this group of sera were deemed negative or weakly positive with isotype-specific capture ELISA based on IgM or IgG alone.

[0302] The inventor then tested different animal sera in the sVNT assays to demonstrate species-independent performance. Results from three independent rabbits immunized with the SARS-CoV-2 RBD protein, demonstrate very potent neutralizing activity in the SARS-CoV-2 sVNT (FIG. 8A). Similarly, sera from ferrets infected with SARS-CoV (FIG. 8B) and rabbits immunized with inactivated SARS-CoV (FIG. 8C) also showed an efficient dose-dependent inhibition of the hACE2-SARS-CoV RBD interaction in the SARS-CoV sVNT.

Specificity Against Other hCoVs and Comparison of SARS Sera Collected in 2003 vs 2020

[0303] To demonstrate specificity, the inventor tested different panels of sera against other known human coronaviruses (hCoVs) and confirmed that the SARS-CoV-2 sVNT can differentiate antibody responses between COVID-19 and other coronavirus infections (FIG. 8D). For SARS sera, there is some level of cross reactivity as expected from their close genetical relatedness and previous published studies [3, 7]. But the difference in neutralization is statistically significant, and hence the sVNT can be used to differentiate SARS-CoV-2 infection from past SARS infection. For human sera from patients with 229/NL63 or OC43 infection and alpaca sera from experimental MERS-CoV infection, there is no detectable cross neutralization.

[0304] During the investigation of potential cross reactivity between SARS sera and SARS-CoV-2 virus, several important observations were made. Firstly, despite the lack of cross neutralization by SARS sera against the live SARS-CoV-2 virus in VNT observed by us and other groups [13], some level of cross neutralization in sVNT was detected (FIG. 8D), indicating sVNT is more sensitive than VNT. Secondly, SARS NAbs are detectable for at least 17 years in recovered patients (FIG. 8F). Thirdly, the cross neutralization level is higher in the 2020 SARS sera than the 2003 samples (FIG. 8D) although the homologous neutralizing level of the 2020 SARS sera (FIG. 8F) is lower than the 2003 SARS sera (FIG. 8E). Lastly, the N-specific antibody level was found to be much lower in the 2020 SARS sera than the 2003 samples (FIG. 8G).

Correlation Between Live Virus VNT and Biochemical sVNT

[0305] A panel of COVID-19 sera with different levels of SARS-CoV-2 NAbs as shown by sVNT (FIG. 11) were chosen for a comparative and correlation study between the live virus based VNT and the RBD-hACE2 based sVNT. The results demonstrate good overall correlation between the two assays (FIG. 9A and FIG. 10). The SARS-CoV-2 sVNT is more sensitive than VNT. At the 50% inhibition cutoff, which is considered a stringent cutoff as evident from the titration curves in FIG. 11, all COVID-19 patient sera showed neutralization at 1:20 or greater with the COVID-19 Patient 13 serum reaching a neutralization titer equal to or greater than 640 (FIG. 11).

Validation with Two Cohorts of Positive and Negative Sera from Two Countries

[0306] To validate the performance of the SARS-CoV-2 sVNT, two different cohorts of positive and negative sera were analysed. The assay was performed in two different countries by two independent groups to further assure reliability and reproducibility. For the first cohort, 77 sera from PCR-confirmed COVID-19 patients in Singapore collected on days 14-33 after symptom onset and 75 healthy control sera were tested. All control sera were negative, resulting in a 100% specificity. Using a cutoff at 20% inhibition, the assay sensitivity is at 100%. The sensitivity decreases to 95.6% when a 40% cutoff is used (FIG. 9B). For the second cohort, 50 sera each of healthy controls and PCR-confirmed COVID-19 patients in Nanjing, China, sampled on days 27-61 after symptom onset were tested. The specificity is 100%. The sensitivity is 98% and 96% using a 20% and 40% cutoff, respectively (FIG. 9C).

2.3 Discussion

[0307] We are more than 100 days into the COVID-19 outbreak and attention worldwide, both in the scientific community and for policy makers, has shifted focus from acute diagnostic strategy and capacity to the use of serology for the "exit strategy", relying on accurate assessment of infection prevalence at the individual and population (herd) level. Discussion and debate on the role of serology has intensified greatly in this context [6].

[0308] While there are many COVID-19 lab-based or point-of-care (POC) antibody test kits commercially available, none are capable of measuring NAbs. VNT or pVNT remain the only platform for detection of NAbs. Both require live virus and cells, highly skilled operators, are less sensitive in general, and take days to obtain results. VNT and pVNT are thus not suitable for mass production and testing, even in the most developed nations.

[0309] The World Health Organization (WHO) has recently cautioned that positive results from antibody tests do not equal to protective immunity [14] due to two aspects or obstacles. Firstly, most, if not all, current testing done at large scale is for detection of binding antibodies only and does not measure NAbs; secondly, the presence of NAbs may or may not correlate with protection. While the second aspect will take much more in-depth scientific and clinical research to resolve in the specific context of COVID-19 infection, past experiences with viral infection in general argue that in most recovered patients NAb level is a good indicator of protective immunity, despite the fact that some patients may not obey this "rule of thumb" [15, 16]. In this Example, the inventor describes a novel sVNT platform to tackle the first obstacle.

[0310] The data presented here demonstrated that sVNT is as specific as, and more sensitive than VNT. The results obtained from sVNT correlates well with VNT. The major advantage of sVNT is that it can be rapidly conducted in most research or clinical labs without the need to use live biological materials and biosafety containment. The sVNT is also amenable to high throughput testing and/or fully automated testing after minimal adaptation.

[0311] Another advantage of sVNT is its ability to detect SARS-CoV-2 antibodies in a species-independent manner. As the origin of SARS-CoV-2 and early transmission event remain elusive, the sVNT assay will be ideally suited for "virus hunting" as past studies have amply demonstrated that serological surveys are more superior than molecular detection as the virus-specific antibodies last much longer in animals than the viral genetic material [17-19]. Sampling serum for antibody detection is also more reliable than other sampling approaches used for molecular detection as the target tissues can vary from virus to virus [20-22].

[0312] In addition, sVNT offers a key advantage over most ELISA or POC tests in its ability to detect total NAbs in an isotype-independent manner. This will not only simplify the testing strategy, but also further increase test sensitivity. As shown in FIG. 7C for the serum panel of COVID-19 patients showing low IgM and IgG in the isotype-specific ELISAs, the sVNT assay still detected significant level of NAbs. Although the mechanism needs further investigation, there are at least two possibilities: the presence of other Ig isotypes or neutralization synergy or cooperativity from the combination of different isotype antibodies targeting different neutralization critical epitopes, as previously observed for HIV and other viruses [23-25].

[0313] Results obtained for the two SARS serum panels are very interesting. The long lasting NAbs 17 years after initial infection is encouraging news for recovered COVID-19 patients considering the close relationship of the two viruses. The mechanism and biological significance of the increased cross neutralization towards SARS-COV-2 coupled with the decrease/disappearance of N-specific antibodies 17 years after infection warrants further investigation in the context of better understanding SARSr-CoV immune response dynamics.

[0314] In summary, the inventor has addressed the challenge of COVID-19 serology with a new approach that enables the detection of NAbs in an easy, safe, rapid and inexpensive manner with enhanced specificity and sensitivity. The data indicate that their performance is generally well correlated. Its application can cover many aspects of COVID-19 investigation from contact tracing, sero-prevalence survey, reservoir/intermediate animal tracking to assessment of herd immunity, longevity of protective immunity and efficacy of different vaccine candidates.

[0315] Exemplary sequences of the disclosure are set forth below.

TABLE-US-00002 SEQ ID NO: DESCRIPTION SEQUENCE 1 SARS-CoV MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFY spike SNVTGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIR protein ACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKN KDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDTWGTSAAAY FVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSIAEIDKGIYQTSNFRVVPSGDVV RFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDL CFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKY RYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFEL LNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPK TSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVF QTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNT NIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAE QDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQ YGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFA MQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTL VKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLA ATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAY FPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEE LDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPW YVWLGFIAGLIAIVMVTILLCCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGVKLHYT 2 SARS-CoV MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPTVYVYSRVK envelope NLNSSEGVPDLLV protein 3 SARS-CoV MADNGTITVEELKQLLEQWNLVIGFLFLAWIMLLQFAYSNRNRFLYIIKLVFLWLLWPVTLAC membrane FVLAAVYRINWVTGGIAIAMACIVGLMWLSYFVASFRLFARTRSMWSFNPETNILLNVPLRGT protein IVTRPLMESELVIGAVIIRGHLRMAGHSLGRCDIKDLPKEITVATSRTLSYYKLGASQRVGTD SGFAAYNRYRIGNYKLNTDHAGSNDNIALLVQ 4 SARS-CoV MSDNGPQSNQRSAPRITFGGPTDSTDNNQNGGRNGARPKQRRPQGLPNNTASWFTALTQHGKE nucleocapsid ELRFPRGQGVPINTNSGPDDQIGYYRRATRRVRGGDGKMKELSPRWYFYYLGTGPEASLPYGA protein NKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQGTTLPKGFYAEGSRGGSQASSRSSS RSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRLNQLESKVSGKGQQQQGQTVTKKSA AEASKKPRQKRTATKQYNVTQAFGRRGPEQTQGNFGDQDLIRQGTDYKHWPQIAQFAPSASAF FGMSRIGMEVTPSGTWLTYHGAIKLDDKDPQFKDNVILLNKHIDAYKTFPPTEPKKDKKKKTD EAQPLPQRQKKQPTVTLLPAADMDDFSRQLQNSMSGASADSTQA 5 SARS-CoV-2 MFLLTTKRTMFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDL spike FLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSL protein LIVNNATNVVEKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE GKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLA LHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSF TVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLY NSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGC VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSY GFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKK FLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVP VAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRA RSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDST ECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPS KPSKRSFIEDLLFNKVTLADAGFEKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQY TSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQD SLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITG RLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVF LHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNE VAKNLNESLIDLQELGKYEQYEKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCG SCCKFDEDDSEPVLKGVKLHYT 6 SARS-CoV-2 MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVK envelope NLNSSRVPDLLV protein 7 SARS-CoV-2 MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVTLA membrane CFVLAAVYRINVVITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILLNVPLH protein GTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVA GDSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ 8 SARS-CoV-2 MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKED nucleocapsid LKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGAN protein KDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSR SRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAA EASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFF GMSRIGMEVTPSGTWLTYTGAEKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADE TQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA 9 SARS-CoV SDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTF spike DNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFA protein S1 VSKPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQP subunit IDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDTVVGTSAAAYFVGYLKPTTFML KYDENGTITDAVDCSQNPLAELKCSVKSFEEDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGE VFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVK GDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNEDATSTGNYNYKYRYLRHGKLRPFER DISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLEKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFG GVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHV DTSYECDIPIGAGICASYHTVSLLR 10 SARS-CoV RVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYG spike VSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDAT protein STGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPY RBD RVVVLSFELLNAPATVCGPKLSTDLIKNQCVNF 11 SARS-CoV NTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTT spike TGIGYQPY protein RBM 12 SARS-CoV-2 SQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTVVFHAIHVSGTN spike GTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN protein S1 DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGY subunit FKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAA AYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESI VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLND LCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYL YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSF ELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRD PQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSN VFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR 13 SARS-CoV-2 RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG spike VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSK protein VGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQP RBD YRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF 14 SARS-CoV-2 NSNNLDSKVGGNYNYLYRLFRKSNLKREERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQP spike TNGVGYQPY protein RBM 15 Human ACE2 MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMN isoform 1 NAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTI YSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNE MARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEEKPLYEHLHAYVRAKLMNAY PSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNEDVTDAMVDQAWDAQRIFKEAEKFF VSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGH IQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLK QALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLF HVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWT LALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSEKVRISLKSALGD KAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSD IIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVG IVILIFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDDVQTSF 16 Human ACE2 MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMN isoform 2 NAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTI YSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNE MARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEEKPLYEHLHAYVRAKLMNAY PSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNEDVTDAMVDQAWDAQRIFKEAEKFF VSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGH IQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLK QALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLF HVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLL 17 Human ACE2 QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLA isoform 1 QMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLL extracellular LEPGLNEEMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGD domain YEVNGVDGYDYSRGQLIEDVEHTFEEEKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGD MWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSM LTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNG ANEGFHEAVGEEMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEK WRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLY QFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPL LNYFEPLFTVVLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSS VAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRS RINDAFRLNDNSLEFLGIQPTLGPPNQPPVS 18 Bat MSGSSWFLLSLVAVTAAQSTTEDLAKKFLDDFNSEAENLSHQSSLASWEYNTNISDENVQKMD (Rhinolophus EAGAKWSDFYEKQSKLAKNFSLEEHENDTVKLQLQILQQSGSPVLSEDKSKRLNSILNAMSTI ferminequillum) YSTGKVCKPNNPQECLLLEPGLDNIMGTSKDYNERLWAWEGWRAEVGKQLRPLYEEYVVLKNE ACE2 MARGYHYEDYGDYWRRDYETEGSPDLEYSRDQLEKDVERIFAEIKPLYEQLHAYVRTKLMDTY PFHISPTGCLPAHLLGDMWGRFWTNLYPLTVPFGQKPNIDVTDAMLNQNWDAKRIFKEAEKFL VSIGLPNMTEGFWNNSMLTDPGDGRKVVCHPTAWDLGKGDFRIKMCTKVTMEDFLTAHHEMGH IQYDMAYASQPYLLRNGANEGFHEAVGEVMSLSVATPEHLKTMGLLSSDFLEDNETEINFLFK QALNIVGTLPLTYMLEKWRWMVFKGEIPKEEWMKKWWEMKRKIVGVVEPVPHDETYCDPASLF HVANDYSFIRYYTRTIFEFQFHEALCRIAKHDGPLHKCGISNSTDAGEKLHQMLSVGKSQPWT SVLKDFVGSKNMDVGPLLRYFEPLYTWLTEQNRKSFVGWNTDWSPYADQSIKVWISLKSALGE KAYEWNNNEMYLFRSSVAYAMREYFLKTKNQTILFGEEDVWVSNLKPRISFNFYVTSPRNLSD IIPRPEVEGAIRMSRSRINDAFRLDDNSLEFLGIQPTLGPPYQPPVTIWLIVFGVVMAVVVVG IVVLIITGIRDRRKKDQARSEENPYSSVDLSKGENNPGFQNGNDVQTSF 19 Bat QSTTEDLAKKFLDDFNSEAENLSHQSSLASWEYNTNISDENVQKMDEAGAKWSDFYEKQSKLA (Rhinolophus KNFSLEEHENDTVKLQLQILQQSGSPVLSEDKSKRLNSILNAMSTIYSTGKVCKPNNPQECLL ferrumequinum) LEPGLDNEVIGTSKDYNERLWAWEGWRAEVGKQLRPLYEEYVVLKNEMARGYHYEDYGDYWRR ACE2 DYETEGSPDLEYSRDQLIKDVERIFAEIKPLYEQLHAYVRTKLMDTYPFHISPTGCLPAHLLG extracellular DMWGREWTNLYPLTVPFGQKPNEDVTDAMLNQNVVDAKRIFKEAEKFLVSIGLPNMTEGFWNN domain SMLTDPGDGRKVVCHPTAWDLGKGDFREKMCTKVTMEDFLTAHHEMGHIQYDMAYASQPYLLR NGANEGFHEAVGEVMSLSVATPEHLKTMGLLSSDFLEDNETEINFLFKQALNIVGTLPLTYML EKWRWMVFKGEIPKEEWMKKWWEMKRKIVGVVEPVPHDETYCDPASLFHVANDYSFIRYYTRT IFEFQFHEALCRIAKHDGPLHKCGISNSTDAGEKLHQMLSVGKSQPWTSVLKDEVGSKNMDVG PLLRYEEPLYTWLTEQNRKSFVGWNTDWSPYADQSIKVWISLKSALGEKAYEWNNNEMYLFRS SVAYAMREYFLKTKNQTILFGEEDVWVSNLKPRISFNFYVTSPRNLSDIIPRPEVEGAIRMSR SRINDAFRLDDNSLEFLGIQPTLGPPYQPPVT 20 Pangolin MSGSSWLLLSLVAVTAAQSTSDEEAKTFLEKFNSEAEELSYQSSLASWNYNTNITDENVQKMN (Manis VAGAKWSTFYEEQSKIAKNYQLQNIQNDTIKRQLQALQLSGSSALSADKNQRLNTILNTMSTI javanica) YSTGKVCNPGNPQECSLLEPGLDNIMESSKDYNERLWAWEGWRSEVGKQLRPLYEEYVVLKNE ACE2 MARANHYEDYGDYWRGDYEAEGANGYNYSRDHLIEDVEHIFTQIKPLYEHLHAYVRAKLMDNY PSHISPTGCLPAHLLGDMWGREWTNLYPLTVPFRQKPNIDVTDAMVNQTWDANRIFKEAEKFF VSVGLPKMTQTFWENSMLTEPGDGRKVVCHPTAWDLGKHDFREKMCTKVTMDDFLTAHHEMGH IQYDMAYAMQPYLLRNGANEGFHEAVGEIMSLSAATPKHLKNIGLLPPDFYEDNETEINFLLK QALTIVGTLPFTYMLEKWRWMVESGQIPKEQWMKKWWEMKREIVGVVEPVPHDETYCDPASLF HVANDYSFIRYYTRTIYQFQFQEALCQTAKHEGPLHKCDISNSAEAGQKLLQMLSLGKSKPWT LALERVVGTKNMDVRPLLNYFEPLLTWLKEQNKNSFVGWNTDWSPYAAQSEKVRISLKSALGE KAYEWNDSEMYLFRSSVAYAMREYFSKVKKQTIPFEDECVRVSDLKPRVSFIFFVTLPKNVSA VIPRAEVEEAIRISRSRINDAFRLDDNSLEFLGIQPTLQPPYQPPVTIWLIVEGVVMGVVVVG IVVLIFTGIRDRKKKDQARSEQNPYASVDLSKGENNPGFQNVDDVQTSF 21 Pangolin QSTSDEEAKTFLEKFNSEAEELSYQSSLASWNYNTNITDENVQKMNVAGAKWSTFYEEQSKIA (Manis KNYQLQNIQNDTEKRQLQALQLSGSSALSADKNQRLNTILNTMSTIYSTGKVCNPGNPQECSL javanica) LEPGLDNEVIESSKDYNERLWAWEGWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRG ACE2 DYEAEGANGYNYSRDHLIEDVEHIFTQIKPLYEHLHAYVRAKLMDNYPSHISPTGCLPAHLLG extracellular DMWGRFWTNLYPLTVPFRQKPNIDVTDAMVNQTWDANRIFKEAEKFFVSVGLPKMTQTFWENS domain MLTEPGDGRKVVCHPTAWDLGKHDFRIKMCTKVTMDDFLTAHHEMGHIQYDMAYAMQPYLLRN GANEGFHEAVGEIMSLSAATPKHLKNIGLLPPDFYEDNETEINFLLKQALTIVGTLPFTYMLE KWRWMVESGQIPKEQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVANDYSFIRYYTRTI YQFQFQEALCQTAKHEGPLHKCDISNSAEAGQKLLQMLSLGKSKPWTLALERVVGTKNMDVRP LLNYFEPLLTWLKEQNKNSFVGWNTDWSPYAAQSIKVRISLKSALGEKAYEWNDSEMYLFRSS VAYAMREYFSKVKKQTIPILDECVRVSDLKPRVSFIFFVTLPKNVSAVIPRAEVEEAIRISRS

RINDAFRLDDNSLEFLGIQPTLQPPYQPPVT 22 Civet MSGSFWLLLSFAALTAAQSTTEELAKTFLETFNYEAQELSYQSSVASWNYNTNITDENAKNMN (Paguma EAGAKWSAYYEEQSKLAQTYPLAEIQDAKEKRQLQALQQSGSSVLSADKSQRLNTILNAMSTI larvata) YSTGKACNPNNPQECLLLEPGLDNIMENSKDYNERLWAWEGWRAEVGKQLRPLYEEYVALKNE ACE2 MARANNYEDYGDYWRGDYEEEWTGGYNYSRNQLIQDVEDTFEQEKPLYQHLHAYVRAKLMDTY PSRISRTGCLPAHLLGDMWGRFWTNLYPLTVPFGQKPNIDVTDAMVNQNWDARRIFKEAEKFF VSVGLPNMTQGFWENSMLTEPGDGRKVVCHPTAWDLGKGDFRIKMCTKVTMDDFLTAHHEMGH IQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPNHLKTIGLLSPAFSEDNETEINFLLK QALTIVGTLPFTYMLEKWRWMVFKGAIPKEQWMQKWWEMKRNIVGVVEPVPHDETYCDPASLF HVANDYSFIRYYTRTIYQFQFQEALCQIAKHEGPLHKCDISNSTEAGKKLLEMLSLGRSEPWT LALERVVGAKNMNVTPLLNYFEPLFTWLKEQNRNSFVGWDTDWRPYSDQSIKVRISLKSALGE KAYEWNDNEMYLERSSIAYAMREYESKVKNQTIPFVEDNVWVSDLKPRISENFFVTFSNNVSD VIPRSEVEDAIRMSRSRINDAFRLDDNSLEFLGIEPTLSPPYRPPVTIWLIVFGVVMGAIVVG IVLLIVSGIRNRRKNDQAGSEENPYASVDLNKGENNPGFQHADDVQTSF 23 Civet QSTTEELAKTFLETFNYEAQELSYQSSVASWNYNTNITDENAKNMNEAGAKWSAYYEEQSKLA (Paguma QTYPLAEIQDAKIKRQLQALQQSGSSVLSADKSQRLNTILNAMSTIYSTGKACNPNNPQECLL larvata) LEPGLDNEVIENSKDYNERLWAWEGWRAEVGKQLRPLYEEYVALKNEMARANNYEDYGDYWRG ACE2 DYEEEWTGGYNYSRNQLIQDVEDTFEQEKPLYQHLHAYVRAKLMDTYPSRISRTGCLPAHLLG extracellular DMWGREWTNLYPLTVPFGQKPNIDVTDAMVNQNWDARRIFKEAEKFFVSVGLPNMTQGFWENS domain MLTEPGDGRKVVCHPTAWDLGKGDFREKMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRN GANEGFHEAVGEIMSLSAATPNHLKTIGLLSPAFSEDNETEINFLLKQALTIVGTLPFTYMLE KWRWMVFKGAIPKEQWMQKWWEMKRNIVGVVEPVPHDETYCDPASLFHVANDYSFIRYYTRTI YQFQFQEALCQIAKHEGPLHKCDISNSTEAGKKLLEMLSLGRSEPWTLALERVVGAKNMNVTP LLNYFEPLFTWLKEQNRNSFVGWDTDWRPYSDQSIKVRISLKSALGEKAYEWNDNEMYLFRSS IAYAMREYFSKVKNQTIPFVEDNVWVSDLKPRISFNFFVTFSNNVSDVIPRSEVEDAIRMSRS RINDAFRLDDNSLEFLGIEPTLSPPYRPPVT 24 Pig MSGSFWLLLSLIPVTAAQSTTEELAKTFLEKFNLEAEDLAYQSSLASWNYNTNITDENIQKMN (Sus scrofa) DARAKWSAFYEEQSRIAKTYPLDEIQTLILKRQLQALQQSGTSGLSADKSKRLNTILNTMSTI ACE2 YSSGKVLDPNNPQECLVLEPGLDEIMENSKDYSRRLWAWESWRAEVGKQLRPLYEEYVVLENE MARANNYEDYGDYWRGDYEVTGTGDYDYSRNQLMEDVERTFAEEKPLYEHLHAYVRAKLMDAY PSRISPTGCLPAHLLGDMWGREWTNLYPLTVPFGEKPSIDVTEAMVNQSWDAIRIFEEAEKFF VSIGLPNMTQGFWNNSMLTEPGDGRKVVCHPTAWDLGKGDFREKMCTKVTMDDFLTAHHEMGH IQYDMAYAIQPYLLRNGANEGFHEAVGEIMSLSAATPHYLKALGLLPPDFYEDSETEINFLLK QALTIVGTLPFTYMLEKWRWMVFKGEIPKEQWMQKWWEMKREIVGVVEPLPHDETYCDPACLF HVAEDYSFIRYYTRTIYQFQFHEALCRTAKHEGPLYKCDISNSTEAGQKLLQMLSLGKSEPWT LALENIVGVKTMDVKPLLSYFEPLLTWLKAQNGNSSVGWNTDWTPYADQSEKVRISLKSALGK EAYEWNDNEMYLFRSSIAYAMRNYFSSAKNETIPFGAEDVWVSDLKPRISENFFVTSPANMSD IIPRSDVEKAISMSRSRINDAFRLDDNTLEFLGIQPTLGPPDEPPVTVWLIEFGVVMGLVVVG IVVLIFTGIRDRRKKKQASSEENPYGSMDLSKGESNSGFQNGDDIQTSF 25 Pig QSTTEELAKTFLEKFNLEAEDLAYQSSLASWNYNTNITDENIQKMNDARAKWSAFYEEQSRIA (Sus scrofa) KTYPLDEIQTLILKRQLQALQQSGTSGLSADKSKRLNTILNTMSTIYSSGKVLDPNNPQECLV ACE2 LEPGLDEEVIENSKDYSRRLWAWESWRAEVGKQLRPLYEEYVVLENEMARANNYEDYGDYWRG extracellular DYEVTGTGDYDYSRNQLMEDVERTFAEIKPLYEHLHAYVRAKLMDAYPSRISPTGCLPAHLLG domain DMWGRFWTNLYPLTVPFGEKPSIDVTEAMVNQSWDAIRIFEEAEKFFVSIGLPNMTQGFWNNS MLTEPGDGRKVVCHPTAWDLGKGDFREKMCTKVTMDDFLTAHHEMGHIQYDMAYAIQPYLLRN GANEGFHEAVGEMSLSAATPHYLKALGLLPPDFYEDSETEINFLLKQALTIVGTLPFTYMLEK WRWMVFKGEIPKEQWMQKWWEMKREIVGVVEPLPHDETYCDPACLFHVAEDYSFIRYYTRTIY QFQFHEALCRTAKHEGPLYKCDISNSTEAGQKLLQMLSLGKSEPWTLALENIVGVKTMDVKPL LSYFEPLLTWLKAQNGNSSVGWNTDWTPYADQSEKVRISLKSALGKEAYEWNDNEMYLFRSSI AYAMRNYFSSAKNETIPFGAEDVWVSDLKPRISENFEVTSPANMSDIIPRSDVEKAISMSRSR INDAFRLDDNTLEFLGIQPTLGPPDEPPVT 26 SARS-CoV-2 PNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF spike TNVYDSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF protein RKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSPELLH RBD APATVCGPKKS 27 SARS-CoV-2 MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAQCVNLTTRTQLPPAYTNSFTRGVYY spike PDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKREDNPVLPFNDGVYFASTEKSNII protein S1 RGWIEGTTLDSKTQSLLIVNNATNVVEKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSAN subunit NCTFEYVSQPFLMDLEGKQGNEKNLREFVFKNEDGYFKIYSKHTPINLVRDLPQGFSALEPLV DLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVD CALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAW NRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNEN ENGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTS NQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGA GICASYQTQTNSPRRAR 28 SARS-CoV MNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCF spike SNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRY protein RBD LRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLN APATYLSLNTAAAL 29 Human ACE2 MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKENHEAEDLEYQSSLASWNYNTNITEENVQNMN NAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTI YSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNE MARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEEKPLYEHLHAYVRAKLMNAY PSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNEDVTDAMVDQAWDAQRIFKEAEKFF VSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGH IQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLK QALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLF HVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWT LALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSEKVRISLKSALGD RAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISENFFVTAPKNVSD IIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVG IVILIFTGIRDRKKKNKARSGENPYASEDISKGENNPGFQNTDDVQTSF 30 ACE2 QSTIEEQAKTFLDKENHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLA Human QMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLL extracellular LEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGD domain YEVNGVDGYDYSRGQLIEDVEHTFEEEKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGD MWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSM LTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNG ANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEK WRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLY QFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPL LNYFEPLFTVVLKDQNKNSFVGWSTDWSPYADQSEKVRISLKSALGDRAYEWNDNEMYLFRSS VAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRS RINDAFRLNDNSLEFLGIQPTLGPPNQPPVS 31 Bat MSSSSWLLLSLVAVTTAQFTTEDLAKIFLDEFNSEAENLSYQSSLASWDYNTNINDENVQKMD (Rhinolophus EAGAKWSAFYEEQSKLAKNYPLEQIQNVTVKLQLQILQQSGSPVLSEDKSKRLNSILNAMSTI sinicus) YSTGKVCKPNKPHECLLLEPGLDNIMGTSKDYSERLWAWEGWRAEVGKQLRPLYEEYVVLKNE ACE2 MARGYHYEDYGDYWRRDYETEESPGPGYSRDQLMKDVERIFTEIKPLYEHLHAYVRAKLMDTY PFHISPTGCLPAHLLGDMWGRFWTNLYPLTVPFGQKPNIDVTDEMLKQGWDADRIFKEAEKFF VSVGLPNMTEGFWNNSMLTEPGDGRKVVCHPTAWDLGKGDFREKMCTKVTMEDFLTAHHEMGH IQYDMAYASQPYLLRNGANEGFHEAVGEVMSLSVATPKHLKTMGLLSPDFREDNETEINFLLK QALNIVGTLPFTYMLEKWRWMVFKGEIPKEEWMKKWWEMKRKIVGVVEPVPHDETYCDPASLF HVANDYSFIRYYTRTIFEFQFHEALCRIAQHDGPLHKCDISNSTDAGKKLHQMLSVGKSQAWT KTLEDIVDSRNMDVGPLLRYFEPLYTWLQEQNRKSYVGWNTDWSPYSDQSEKVRISLKSALGE NAYEWNDNEMYLFRSSVAYAMREYFLKEKHQTILFGAENVWVSNLKPRISENFHVTSPGNLSD IIPRPEVEGAIRMSRSRINDAFRLDDNSLEFLGIQPTLGPPYQPPVTIWLIVFGVVMAVVVVG IVVLIITGIRDRRKTDQARSEENPYSSVDLSKGENNPGFQNGDDVQTSF 32 Bat QFTTEDLAKIFLDEFNSEAENLSYQSSLASWDYNTNINDENVQKMDEAGAKWSAFYEEQSKLA (Rhinolophus KNYPLEQIQNVTVKLQLQILQQSGSPVLSEDKSKRLNSILNAMSTIYSTGKVCKPNKPHECLL sinicus) LEPGLDNIMGTSKDYSERLWAWEGWRAEVGKQLRPLYEEYVVLKNEMARGYHYEDYGDYWRRD ACE2 YETEESPGPGYSRDQLMKDVERIFTEEKPLYEHLHAYVRAKLMDTYPFHISPTGCLPAHLLGD extracellular MWGREWTNLYPLTVPFGQKPNIDVTDEMLKQGWDADRIFKEAEKFFVSVGLPNMTEGFWNNSM domain LTEPGDGRKVVCHPTAWDLGKGDFRIKMCTKVTMEDFLTAHHEMGHIQYDMAYASQPYLLRNG ANEGFHEAVGEVMSLSVATPKHLKTMGLLSPDFREDNETEINFLLKQALNIVGTLPFTYMLEK WRWMVFKGEIPKEEWMKKWWEMKRKIVGVVEPVPHDETYCDPASLFHVANDYSFIRYYTRTIF EFQFHEALCRIAQHDGPLHKCDISNSTDAGKKLHQMLSVGKSQAWTKTLEDIVDSRNMDVGPL LRYFEPLYTWLQEQNRKSYVGWNTDWSPYSDQSEKVRISLKSALGENAYEWNDNEMYLFRSSV AYAMREYFLKEKHQTILFGAENVWVSNLKPRISFNFHVTSPGNLSDIIPRPEVEGAIRMSRSR INDAFRLDDNSLEFLGIQPTLGPPYQPPVT 33 Pig MSGSFWLLLSLIPVTAAQSTTEELAKTFLEKFNLEAEDLAYQSSLASWTINTNITDENIQKMN (Sus scrofa) DARAKWSAFYEEQSRIAKTYPLDEIQTLILKRQLQALQQSGTSGLSADKSKRLNTILNTMSTI ACE2 YSSGKVLDPNNPQECLVLEPGLDEIMENSKDYSRRLWAWESWRAEVGKQLRPLYEEYVVLENE MARANNYEDYGDYWRGDYEVTGTGDYDYSRNQLMEDVERTFAEEKPLYEHLHAYVRAKLMDAY PSRISPTGCLPAHLLGDMWGRFWTNLYPLTVPFGEKPSEDVTEAMVNQSWDAIRIFEEAEKFF VSIGLPNMTQGFWNNSMLTEPGDGRKVVCHPTAWDLGKGDFREKMCTKVTMDDFLTAHHEMGH IQYDMAYAIQPYLLRNGANEGFHEAVGEIMSLSAATPHYLKALGLLPPDEYEDSETEINFLLK QALTIVGTLPFTYMLEKWRWMVFKGEIPKEQWMQKWWEMKREIVGVVEPLPHDETYCDPACLF HVAEDYSFIRYYTRTIYQFQFHEALCRTAKHEGPLYKCDISNSTEAGQKLLQMLSLGKSEPWT LALENIVGVKTMDVKPLLSYFEPLLTWLKAQNGNSSVGWNTDWTPYADQSIKVRISLKSALGE DAYEWNDNEMYLFRSSIAYAMRNYFSSAKNETIPFGAVDVWVSDLKPRISENFFVTSPANMSD IIPRSDVEKAISMSRSRINDAFRLDDNTLEFLGIQPTLGPPDEPPVTVWLIIFGVVMGLVVVG IVVLIFTGIRDRRKKKQASSEENPYGSMDLSKGESNSGFQNGDDIQTSF 34 Pig QSTTEELAKTFLEKFNLEAEDLAYQSSLASWTINTNITDENIQKMNDARAKWSAFYEEQSRIA (Sus scrofa) KTYPLDEIQTLILKRQLQALQQSGTSGLSADKSKRLNTILNTMSTIYSSGKVLDPNNPQECLV ACE2 LEPGLDEEVIENSKDYSRRLWAWESWRAEVGKQLRPLYEEYVVLENEMARANNYEDYGDYWRG extracellular DYEVTGTGDYDYSRNQLMEDVERTFAEIKPLYEHLHAYVRAKLMDAYPSRISPTGCLPAHLLG domain DMWGREWTNLYPLTVPFGEKPSIDVTEAMVNQSWDAIRIFLEAEKFFVSIGLPNMTQGFWNNS MLTEPGDGRKVVCHPTAWDLGKGDFREKMCTKVTMDDFLTAHHEMGHIQYDMAYAIQPYLLRN GANEGFHEAVGEIMSLSAATPHYLKALGLLPPDFYEDSETEINFLLKQALTIVGTLPFTYMLE KWRWMVFKGEIPKEQWMQKWWElVIKREIVGVVEPLPHDETYCDPACLFHVAEDYSFIRYYTR TIYQFQFHEALCRTAKHEGPLYKCDISNSTEAGQKLLQMLSLGKSEPWTLALENIVGVKTMDV KPLLSYFEPLLTWLKAQNGNSSVGWNTDWTPYADQSIKVRISLKSALGEDAYEWNDNEMYLFR SSIAYAMRNYFSSAKNETIPFGAVDVWVSDLKPRISFNFFVTSPANMSDIIPRSDVEKAISMS RSRINDAFRLDDNTLEFLGIQPTLGPPDEPPVT

[0316] Having thus described in detail preferred embodiments of the present disclosure, it is to be understood that the disclosure defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present disclosure.

REFERENCES

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Sequence CWU 1

1

3411255PRTSARS-CoV 1Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu1 5 10 15Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln 20 25 30His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg 35 40 45Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser 50 55 60Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Asp Asn Pro Val65 70 75 80Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn 85 90 95Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln 100 105 110Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys 115 120 125Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met 130 135 140Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr145 150 155 160Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser 165 170 175Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly 180 185 190Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp 195 200 205Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu 210 215 220Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro225 230 235 240Ala Gln Asp Thr Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr 245 250 255Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile 260 265 270Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys 275 280 285Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr305 310 315 320Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser 325 330 335Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala 370 375 380Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly385 390 395 400Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser 420 425 430Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu 435 440 445Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly 450 455 460Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp465 470 475 480Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val 485 490 495Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly 500 505 510Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn 515 520 525Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg 530 535 540Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp545 550 555 560Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys 565 570 575Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser 580 585 590Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr 595 600 605Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr 610 615 620Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu625 630 635 640His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile 645 650 655Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys 660 665 670Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala 675 680 685Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile 690 695 700Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys705 710 715 720Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu 725 730 735Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile 740 745 750Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys 755 760 765Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe 770 775 780Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile785 790 795 800Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met 805 810 815Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile 820 825 830Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr 835 840 845Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala 850 855 860Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe865 870 875 880Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn 885 890 895Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala 900 905 910Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly 915 920 925Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu 930 935 940Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn945 950 955 960Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp 965 970 975Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln 980 985 990Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala 995 1000 1005Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp 1010 1015 1020Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala 1025 1030 1035Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln 1040 1045 1050Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys 1055 1060 1065Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser 1070 1075 1080Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr 1085 1090 1095Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly 1100 1105 1110Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp 1115 1120 1125Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser 1130 1135 1140Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val 1145 1150 1155Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys 1160 1165 1170Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr 1175 1180 1185Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile 1190 1195 1200Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu Cys Cys 1205 1210 1215Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys Gly 1220 1225 1230Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys 1235 1240 1245Gly Val Lys Leu His Tyr Thr 1250 1255276PRTSARS-CoV 2Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser1 5 10 15Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala 20 25 30Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn 35 40 45Val Ser Leu Val Lys Pro Thr Val Tyr Val Tyr Ser Arg Val Lys Asn 50 55 60Leu Asn Ser Ser Glu Gly Val Pro Asp Leu Leu Val65 70 753221PRTSARS-CoV 3Met Ala Asp Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Gln Leu Leu1 5 10 15Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Ala Trp Ile Met 20 25 30Leu Leu Gln Phe Ala Tyr Ser Asn Arg Asn Arg Phe Leu Tyr Ile Ile 35 40 45Lys Leu Val Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys Phe 50 55 60Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Val Thr Gly Gly Ile Ala65 70 75 80Ile Ala Met Ala Cys Ile Val Gly Leu Met Trp Leu Ser Tyr Phe Val 85 90 95Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe Asn 100 105 110Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu Arg Gly Thr Ile Val 115 120 125Thr Arg Pro Leu Met Glu Ser Glu Leu Val Ile Gly Ala Val Ile Ile 130 135 140Arg Gly His Leu Arg Met Ala Gly His Ser Leu Gly Arg Cys Asp Ile145 150 155 160Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu Ser 165 170 175Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Gly Thr Asp Ser Gly Phe 180 185 190Ala Ala Tyr Asn Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr Asp 195 200 205His Ala Gly Ser Asn Asp Asn Ile Ala Leu Leu Val Gln 210 215 2204422PRTSARS-CoV 4Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile1 5 10 15Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly 20 25 30Arg Asn Gly Ala Arg Pro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn 35 40 45Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Glu 50 55 60Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Gly65 70 75 80Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Val Arg 85 90 95Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr 100 105 110Tyr Leu Gly Thr Gly Pro Glu Ala Ser Leu Pro Tyr Gly Ala Asn Lys 115 120 125Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys 130 135 140Asp His Ile Gly Thr Arg Asn Pro Asn Asn Asn Ala Ala Thr Val Leu145 150 155 160Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly 165 170 175Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg 180 185 190Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro 195 200 205Ala Arg Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu 210 215 220Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Val Ser Gly Lys Gly Gln225 230 235 240Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser 245 250 255Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Gln Tyr Asn Val Thr 260 265 270Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly 275 280 285Asp Gln Asp Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln 290 295 300Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg305 310 315 320Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr His Gly 325 330 335Ala Ile Lys Leu Asp Asp Lys Asp Pro Gln Phe Lys Asp Asn Val Ile 340 345 350Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu 355 360 365Pro Lys Lys Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro 370 375 380Gln Arg Gln Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp385 390 395 400Met Asp Asp Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser 405 410 415Ala Asp Ser Thr Gln Ala 42051282PRTSARS-CoV-2 5Met Phe Leu Leu Thr Thr Lys Arg Thr Met Phe Val Phe Leu Val Leu1 5 10 15Leu Pro Leu Val Ser Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln 20 25 30Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro 35 40 45Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe 50 55 60Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser65 70 75 80Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn 85 90 95Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly 100 105 110Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile 115 120 125Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe 130 135 140Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser145 150 155 160Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr 165 170 175Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln 180 185 190Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly 195 200 205Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp 210 215 220Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile225 230 235 240Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser 245 250 255Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala 260 265 270Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr 275 280 285Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro 290 295 300Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly305 310 315 320Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val 325 330 335Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 340 345 350Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 355 360 365Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 370 375 380Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys385 390 395 400Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 405 410 415Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 420 425 430Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 435 440 445Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 450 455 460Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu465 470 475 480Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn

485 490 495Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 500 505 510Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 515 520 525Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys 530 535 540Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val545 550 555 560Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg 565 570 575Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu 580 585 590Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr 595 600 605Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val 610 615 620Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro625 630 635 640Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala 645 650 655Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp 660 665 670Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn 675 680 685Ser Pro Arg Arg Ala Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr 690 695 700Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser705 710 715 720Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu 725 730 735Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys 740 745 750Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe 755 760 765Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp 770 775 780Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr785 790 795 800Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro 805 810 815Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe 820 825 830Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp 835 840 845Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe 850 855 860Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala865 870 875 880Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr 885 890 895Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala 900 905 910Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn 915 920 925Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln 930 935 940Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val945 950 955 960Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser 965 970 975Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg 980 985 990Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly 995 1000 1005Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg 1010 1015 1020Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met 1025 1030 1035Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly 1040 1045 1050Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly 1055 1060 1065Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn 1070 1075 1080Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe 1085 1090 1095Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val 1100 1105 1110Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn 1115 1120 1125Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn 1130 1135 1140Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys 1145 1150 1155Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val 1160 1165 1170Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile 1175 1180 1185Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn 1190 1195 1200Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr 1205 1210 1215Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu 1220 1225 1230Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met Thr Ser 1235 1240 1245Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys 1250 1255 1260Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys 1265 1270 1275Leu His Tyr Thr 1280675PRTSARS-CoV-2 6Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser1 5 10 15Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala 20 25 30Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn 35 40 45Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn 50 55 60Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val65 70 757222PRTSARS-CoV-2 7Met Ala Asp Ser Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Lys Leu1 5 10 15Leu Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Thr Trp Ile 20 25 30Cys Leu Leu Gln Phe Ala Tyr Ala Asn Arg Asn Arg Phe Leu Tyr Ile 35 40 45Ile Lys Leu Ile Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys 50 55 60Phe Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Ile Thr Gly Gly Ile65 70 75 80Ala Ile Ala Met Ala Cys Leu Val Gly Leu Met Trp Leu Ser Tyr Phe 85 90 95Ile Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe 100 105 110Asn Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu His Gly Thr Ile 115 120 125Leu Thr Arg Pro Leu Leu Glu Ser Glu Leu Val Ile Gly Ala Val Ile 130 135 140Leu Arg Gly His Leu Arg Ile Ala Gly His His Leu Gly Arg Cys Asp145 150 155 160Ile Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu 165 170 175Ser Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Ala Gly Asp Ser Gly 180 185 190Phe Ala Ala Tyr Ser Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr 195 200 205Asp His Ser Ser Ser Ser Asp Asn Ile Ala Leu Leu Val Gln 210 215 2208419PRTSARS-CoV-2 8Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr1 5 10 15Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro65 70 75 80Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln145 150 155 160Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala 195 200 205Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln225 230 235 240Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile305 310 315 320Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu385 390 395 400Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415Thr Gln Ala9654PRTSARS-CoV 9Ser Asp Leu Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn1 5 10 15Tyr Thr Gln His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu 20 25 30Ile Phe Arg Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro 35 40 45Phe Tyr Ser Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Asp 50 55 60Asn Pro Val Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu65 70 75 80Lys Ser Asn Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn 85 90 95Lys Ser Gln Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile 100 105 110Arg Ala Cys Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser 115 120 125Lys Pro Met Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe 130 135 140Asn Cys Thr Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser145 150 155 160Glu Lys Ser Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn 165 170 175Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val 180 185 190Val Arg Asp Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys 195 200 205Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala 210 215 220Phe Ser Pro Ala Gln Asp Thr Trp Gly Thr Ser Ala Ala Ala Tyr Phe225 230 235 240Val Gly Tyr Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn 245 250 255Gly Thr Ile Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu 260 265 270Leu Lys Cys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln 275 280 285Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro 290 295 300Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys305 310 315 320Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val 325 330 335Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys 340 345 350Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn 355 360 365Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile 370 375 380Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro385 390 395 400Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp 405 410 415Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His 420 425 430Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser 435 440 445Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro 450 455 460Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro465 470 475 480Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr 485 490 495Val Cys Gly Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val 500 505 510Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser 515 520 525Ser Lys Arg Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp 530 535 540Phe Thr Asp Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile545 550 555 560Ser Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn 565 570 575Ala Ser Ser Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp 580 585 590Val Ser Thr Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile 595 600 605Tyr Ser Thr Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile 610 615 620Gly Ala Glu His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly625 630 635 640Ala Gly Ile Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg 645 65010222PRTSARS-CoV 10Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr Asn1 5 10 15Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser Val 20 25 30Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp65 70 75 80Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly Gln 85 90 95Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Met 100 105 110Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr 115 120 125Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg 130 135 140Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys145 150 155 160Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr 165 170 175Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val 180 185 190Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly Pro 195 200 205Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe 210 215 2201171PRTSARS-CoV 11Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys1 5 10 15Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile 20 25 30Ser Asn Val Pro Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala 35 40 45Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr 50 55 60Gly Ile Gly Tyr Gln Pro Tyr65 7012673PRTSARS-CoV-2 12Ser Gln Cys Val Asn Leu Thr

Thr Arg Thr Gln Leu Pro Pro Ala Tyr1 5 10 15Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg 20 25 30Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser 35 40 45Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr 50 55 60Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe65 70 75 80Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr 85 90 95Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr 100 105 110Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe 115 120 125Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu 130 135 140Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser145 150 155 160Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn 165 170 175Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr 180 185 190Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe 195 200 205Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr 210 215 220Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly225 230 235 240Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly 245 250 255Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr 260 265 270Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys 275 280 285Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser 290 295 300Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile305 310 315 320Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala 325 330 335Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp 340 345 350Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr 355 360 365Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr 370 375 380Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro385 390 395 400Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp 405 410 415Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys 420 425 430Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn 435 440 445Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly 450 455 460Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu465 470 475 480Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr 485 490 495Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val 500 505 510Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn 515 520 525Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn 530 535 540Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr545 550 555 560Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr 565 570 575Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr 580 585 590Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val 595 600 605Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr 610 615 620Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly625 630 635 640Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala 645 650 655Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala 660 665 670Arg13223PRTSARS-CoV-2 13Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn1 5 10 15Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp65 70 75 80Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr145 150 155 160Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 2201472PRTSARS-CoV-2 14Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu1 5 10 15Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile 20 25 30Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu 35 40 45Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr 50 55 60Asn Gly Val Gly Tyr Gln Pro Tyr65 7015805PRTHomo sapiens 15Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala1 5 10 15Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala65 70 75 80Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu145 150 155 160Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu225 230 235 240His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu305 310 315 320Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe385 390 395 400His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met465 470 475 480Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu545 550 555 560Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met625 630 635 640Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn705 710 715 720Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val 740 745 750Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg 755 760 765Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile 770 775 780Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp785 790 795 800Val Gln Thr Ser Phe 80516555PRTHomo sapiens 16Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala1 5 10 15Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala65 70 75 80Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu145 150 155 160Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu225 230 235 240His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu305 310 315 320Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe385 390 395 400His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met465 470 475 480Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Leu545 550 55517723PRTHomo sapiens 17Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn1 5 10 15His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala 35 40 45Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln 50 55 60Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu65 70 75 80Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser 85 90 95Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr 100 105 110Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135

140Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg145 150 155 160Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala 165 170 175Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp 195 200 205Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser225 230 235 240Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280 285Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly305 310 315 320Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His385 390 395 400Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu 405 410 415Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr465 470 475 480Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490 495Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly 530 535 540Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys545 550 555 560Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp 580 585 590Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys625 630 635 640Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala 645 650 655Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys 660 665 670Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg 675 680 685Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser 690 695 700Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro705 710 715 720Pro Val Ser18805PRTRhinolophus ferrumequinum 18Met Ser Gly Ser Ser Trp Phe Leu Leu Ser Leu Val Ala Val Thr Ala1 5 10 15Ala Gln Ser Thr Thr Glu Asp Leu Ala Lys Lys Phe Leu Asp Asp Phe 20 25 30Asn Ser Glu Ala Glu Asn Leu Ser His Gln Ser Ser Leu Ala Ser Trp 35 40 45Glu Tyr Asn Thr Asn Ile Ser Asp Glu Asn Val Gln Lys Met Asp Glu 50 55 60Ala Gly Ala Lys Trp Ser Asp Phe Tyr Glu Lys Gln Ser Lys Leu Ala65 70 75 80Lys Asn Phe Ser Leu Glu Glu Ile His Asn Asp Thr Val Lys Leu Gln 85 90 95Leu Gln Ile Leu Gln Gln Ser Gly Ser Pro Val Leu Ser Glu Asp Lys 100 105 110Ser Lys Arg Leu Asn Ser Ile Leu Asn Ala Met Ser Thr Ile Tyr Ser 115 120 125Thr Gly Lys Val Cys Lys Pro Asn Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140Glu Pro Gly Leu Asp Asn Ile Met Gly Thr Ser Lys Asp Tyr Asn Glu145 150 155 160Arg Leu Trp Ala Trp Glu Gly Trp Arg Ala Glu Val Gly Lys Gln Leu 165 170 175Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190Gly Tyr His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Arg Asp Tyr Glu 195 200 205Thr Glu Gly Ser Pro Asp Leu Glu Tyr Ser Arg Asp Gln Leu Ile Lys 210 215 220Asp Val Glu Arg Ile Phe Ala Glu Ile Lys Pro Leu Tyr Glu Gln Leu225 230 235 240His Ala Tyr Val Arg Thr Lys Leu Met Asp Thr Tyr Pro Phe His Ile 245 250 255Ser Pro Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270Arg Phe Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Gly Gln Lys 275 280 285Pro Asn Ile Asp Val Thr Asp Ala Met Leu Asn Gln Asn Trp Asp Ala 290 295 300Lys Arg Ile Phe Lys Glu Ala Glu Lys Phe Leu Val Ser Ile Gly Leu305 310 315 320Pro Asn Met Thr Glu Gly Phe Trp Asn Asn Ser Met Leu Thr Asp Pro 325 330 335Gly Asp Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350Lys Gly Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Glu Asp 355 360 365Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380Tyr Ala Ser Gln Pro Tyr Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe385 390 395 400His Glu Ala Val Gly Glu Val Met Ser Leu Ser Val Ala Thr Pro Glu 405 410 415His Leu Lys Thr Met Gly Leu Leu Ser Ser Asp Phe Leu Glu Asp Asn 420 425 430Glu Thr Glu Ile Asn Phe Leu Phe Lys Gln Ala Leu Asn Ile Val Gly 435 440 445Thr Leu Pro Leu Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460Lys Gly Glu Ile Pro Lys Glu Glu Trp Met Lys Lys Trp Trp Glu Met465 470 475 480Lys Arg Lys Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ala Asn Asp Tyr Ser Phe 500 505 510Ile Arg Tyr Tyr Thr Arg Thr Ile Phe Glu Phe Gln Phe His Glu Ala 515 520 525Leu Cys Arg Ile Ala Lys His Asp Gly Pro Leu His Lys Cys Gly Ile 530 535 540Ser Asn Ser Thr Asp Ala Gly Glu Lys Leu His Gln Met Leu Ser Val545 550 555 560Gly Lys Ser Gln Pro Trp Thr Ser Val Leu Lys Asp Phe Val Gly Ser 565 570 575Lys Asn Met Asp Val Gly Pro Leu Leu Arg Tyr Phe Glu Pro Leu Tyr 580 585 590Thr Trp Leu Thr Glu Gln Asn Arg Lys Ser Phe Val Gly Trp Asn Thr 595 600 605Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Trp Ile Ser Leu 610 615 620Lys Ser Ala Leu Gly Glu Lys Ala Tyr Glu Trp Asn Asn Asn Glu Met625 630 635 640Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Glu Tyr Phe Leu 645 650 655Lys Thr Lys Asn Gln Thr Ile Leu Phe Gly Glu Glu Asp Val Trp Val 660 665 670Ser Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Tyr Val Thr Ser Pro 675 680 685Arg Asn Leu Ser Asp Ile Ile Pro Arg Pro Glu Val Glu Gly Ala Ile 690 695 700Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asp Asp Asn705 710 715 720Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Tyr Gln 725 730 735Pro Pro Val Thr Ile Trp Leu Ile Val Phe Gly Val Val Met Ala Val 740 745 750Val Val Val Gly Ile Val Val Leu Ile Ile Thr Gly Ile Arg Asp Arg 755 760 765Arg Lys Lys Asp Gln Ala Arg Ser Glu Glu Asn Pro Tyr Ser Ser Val 770 775 780Asp Leu Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Gly Asn Asp785 790 795 800Val Gln Thr Ser Phe 80519723PRTRhinolophus ferrumequinum 19Gln Ser Thr Thr Glu Asp Leu Ala Lys Lys Phe Leu Asp Asp Phe Asn1 5 10 15Ser Glu Ala Glu Asn Leu Ser His Gln Ser Ser Leu Ala Ser Trp Glu 20 25 30Tyr Asn Thr Asn Ile Ser Asp Glu Asn Val Gln Lys Met Asp Glu Ala 35 40 45Gly Ala Lys Trp Ser Asp Phe Tyr Glu Lys Gln Ser Lys Leu Ala Lys 50 55 60Asn Phe Ser Leu Glu Glu Ile His Asn Asp Thr Val Lys Leu Gln Leu65 70 75 80Gln Ile Leu Gln Gln Ser Gly Ser Pro Val Leu Ser Glu Asp Lys Ser 85 90 95Lys Arg Leu Asn Ser Ile Leu Asn Ala Met Ser Thr Ile Tyr Ser Thr 100 105 110Gly Lys Val Cys Lys Pro Asn Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125Pro Gly Leu Asp Asn Ile Met Gly Thr Ser Lys Asp Tyr Asn Glu Arg 130 135 140Leu Trp Ala Trp Glu Gly Trp Arg Ala Glu Val Gly Lys Gln Leu Arg145 150 155 160Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Gly 165 170 175Tyr His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Arg Asp Tyr Glu Thr 180 185 190Glu Gly Ser Pro Asp Leu Glu Tyr Ser Arg Asp Gln Leu Ile Lys Asp 195 200 205Val Glu Arg Ile Phe Ala Glu Ile Lys Pro Leu Tyr Glu Gln Leu His 210 215 220Ala Tyr Val Arg Thr Lys Leu Met Asp Thr Tyr Pro Phe His Ile Ser225 230 235 240Pro Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255Phe Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270Asn Ile Asp Val Thr Asp Ala Met Leu Asn Gln Asn Trp Asp Ala Lys 275 280 285Arg Ile Phe Lys Glu Ala Glu Lys Phe Leu Val Ser Ile Gly Leu Pro 290 295 300Asn Met Thr Glu Gly Phe Trp Asn Asn Ser Met Leu Thr Asp Pro Gly305 310 315 320Asp Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335Gly Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Glu Asp Phe 340 345 350Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365Ala Ser Gln Pro Tyr Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380Glu Ala Val Gly Glu Val Met Ser Leu Ser Val Ala Thr Pro Glu His385 390 395 400Leu Lys Thr Met Gly Leu Leu Ser Ser Asp Phe Leu Glu Asp Asn Glu 405 410 415Thr Glu Ile Asn Phe Leu Phe Lys Gln Ala Leu Asn Ile Val Gly Thr 420 425 430Leu Pro Leu Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445Gly Glu Ile Pro Lys Glu Glu Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460Arg Lys Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr465 470 475 480Cys Asp Pro Ala Ser Leu Phe His Val Ala Asn Asp Tyr Ser Phe Ile 485 490 495Arg Tyr Tyr Thr Arg Thr Ile Phe Glu Phe Gln Phe His Glu Ala Leu 500 505 510Cys Arg Ile Ala Lys His Asp Gly Pro Leu His Lys Cys Gly Ile Ser 515 520 525Asn Ser Thr Asp Ala Gly Glu Lys Leu His Gln Met Leu Ser Val Gly 530 535 540Lys Ser Gln Pro Trp Thr Ser Val Leu Lys Asp Phe Val Gly Ser Lys545 550 555 560Asn Met Asp Val Gly Pro Leu Leu Arg Tyr Phe Glu Pro Leu Tyr Thr 565 570 575Trp Leu Thr Glu Gln Asn Arg Lys Ser Phe Val Gly Trp Asn Thr Asp 580 585 590Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Trp Ile Ser Leu Lys 595 600 605Ser Ala Leu Gly Glu Lys Ala Tyr Glu Trp Asn Asn Asn Glu Met Tyr 610 615 620Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Glu Tyr Phe Leu Lys625 630 635 640Thr Lys Asn Gln Thr Ile Leu Phe Gly Glu Glu Asp Val Trp Val Ser 645 650 655Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Tyr Val Thr Ser Pro Arg 660 665 670Asn Leu Ser Asp Ile Ile Pro Arg Pro Glu Val Glu Gly Ala Ile Arg 675 680 685Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asp Asp Asn Ser 690 695 700Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Tyr Gln Pro705 710 715 720Pro Val Thr20805PRTManis javanica 20Met Ser Gly Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala1 5 10 15Ala Gln Ser Thr Ser Asp Glu Glu Ala Lys Thr Phe Leu Glu Lys Phe 20 25 30Asn Ser Glu Ala Glu Glu Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45Asn Tyr Asn Thr Asn Ile Thr Asp Glu Asn Val Gln Lys Met Asn Val 50 55 60Ala Gly Ala Lys Trp Ser Thr Phe Tyr Glu Glu Gln Ser Lys Ile Ala65 70 75 80Lys Asn Tyr Gln Leu Gln Asn Ile Gln Asn Asp Thr Ile Lys Arg Gln 85 90 95Leu Gln Ala Leu Gln Leu Ser Gly Ser Ser Ala Leu Ser Ala Asp Lys 100 105 110Asn Gln Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125Thr Gly Lys Val Cys Asn Pro Gly Asn Pro Gln Glu Cys Ser Leu Leu 130 135 140Glu Pro Gly Leu Asp Asn Ile Met Glu Ser Ser Lys Asp Tyr Asn Glu145 150 155 160Arg Leu Trp Ala Trp Glu Gly Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205Ala Glu Gly Ala Asn Gly Tyr Asn Tyr Ser Arg Asp His Leu Ile Glu 210 215 220Asp Val Glu His Ile Phe Thr Gln Ile Lys Pro Leu Tyr Glu His Leu225 230 235 240His Ala Tyr Val Arg Ala Lys Leu Met Asp Asn Tyr Pro Ser His Ile 245 250 255Ser Pro Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270Arg Phe Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Arg Gln Lys 275 280 285Pro Asn Ile Asp Val Thr Asp Ala Met Val Asn Gln Thr Trp Asp Ala 290 295 300Asn Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu305 310 315 320Pro Lys Met Thr Gln Thr Phe Trp Glu Asn Ser Met Leu Thr Glu Pro 325 330 335Gly Asp Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350Lys

His Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380Tyr Ala Met Gln Pro Tyr Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe385 390 395 400His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415His Leu Lys Asn Ile Gly Leu Leu Pro Pro Asp Phe Tyr Glu Asp Asn 420 425 430Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460Ser Gly Gln Ile Pro Lys Glu Gln Trp Met Lys Lys Trp Trp Glu Met465 470 475 480Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ala Asn Asp Tyr Ser Phe 500 505 510Ile Arg Tyr Tyr Thr Arg Thr Ile Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525Leu Cys Gln Thr Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540Ser Asn Ser Ala Glu Ala Gly Gln Lys Leu Leu Gln Met Leu Ser Leu545 550 555 560Gly Lys Ser Lys Pro Trp Thr Leu Ala Leu Glu Arg Val Val Gly Thr 565 570 575Lys Asn Met Asp Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Leu 580 585 590Thr Trp Leu Lys Glu Gln Asn Lys Asn Ser Phe Val Gly Trp Asn Thr 595 600 605Asp Trp Ser Pro Tyr Ala Ala Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620Lys Ser Ala Leu Gly Glu Lys Ala Tyr Glu Trp Asn Asp Ser Glu Met625 630 635 640Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Glu Tyr Phe Ser 645 650 655Lys Val Lys Lys Gln Thr Ile Pro Phe Glu Asp Glu Cys Val Arg Val 660 665 670Ser Asp Leu Lys Pro Arg Val Ser Phe Ile Phe Phe Val Thr Leu Pro 675 680 685Lys Asn Val Ser Ala Val Ile Pro Arg Ala Glu Val Glu Glu Ala Ile 690 695 700Arg Ile Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asp Asp Asn705 710 715 720Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gln Pro Pro Tyr Gln 725 730 735Pro Pro Val Thr Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val 740 745 750Val Val Val Gly Ile Val Val Leu Ile Phe Thr Gly Ile Arg Asp Arg 755 760 765Lys Lys Lys Asp Gln Ala Arg Ser Glu Gln Asn Pro Tyr Ala Ser Val 770 775 780Asp Leu Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Val Asp Asp785 790 795 800Val Gln Thr Ser Phe 80521723PRTManis javanica 21Gln Ser Thr Ser Asp Glu Glu Ala Lys Thr Phe Leu Glu Lys Phe Asn1 5 10 15Ser Glu Ala Glu Glu Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30Tyr Asn Thr Asn Ile Thr Asp Glu Asn Val Gln Lys Met Asn Val Ala 35 40 45Gly Ala Lys Trp Ser Thr Phe Tyr Glu Glu Gln Ser Lys Ile Ala Lys 50 55 60Asn Tyr Gln Leu Gln Asn Ile Gln Asn Asp Thr Ile Lys Arg Gln Leu65 70 75 80Gln Ala Leu Gln Leu Ser Gly Ser Ser Ala Leu Ser Ala Asp Lys Asn 85 90 95Gln Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr 100 105 110Gly Lys Val Cys Asn Pro Gly Asn Pro Gln Glu Cys Ser Leu Leu Glu 115 120 125Pro Gly Leu Asp Asn Ile Met Glu Ser Ser Lys Asp Tyr Asn Glu Arg 130 135 140Leu Trp Ala Trp Glu Gly Trp Arg Ser Glu Val Gly Lys Gln Leu Arg145 150 155 160Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala 165 170 175Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Ala 180 185 190Glu Gly Ala Asn Gly Tyr Asn Tyr Ser Arg Asp His Leu Ile Glu Asp 195 200 205Val Glu His Ile Phe Thr Gln Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220Ala Tyr Val Arg Ala Lys Leu Met Asp Asn Tyr Pro Ser His Ile Ser225 230 235 240Pro Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255Phe Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Arg Gln Lys Pro 260 265 270Asn Ile Asp Val Thr Asp Ala Met Val Asn Gln Thr Trp Asp Ala Asn 275 280 285Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300Lys Met Thr Gln Thr Phe Trp Glu Asn Ser Met Leu Thr Glu Pro Gly305 310 315 320Asp Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335His Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365Ala Met Gln Pro Tyr Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His385 390 395 400Leu Lys Asn Ile Gly Leu Leu Pro Pro Asp Phe Tyr Glu Asp Asn Glu 405 410 415Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Ser 435 440 445Gly Gln Ile Pro Lys Glu Gln Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr465 470 475 480Cys Asp Pro Ala Ser Leu Phe His Val Ala Asn Asp Tyr Ser Phe Ile 485 490 495Arg Tyr Tyr Thr Arg Thr Ile Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510Cys Gln Thr Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525Asn Ser Ala Glu Ala Gly Gln Lys Leu Leu Gln Met Leu Ser Leu Gly 530 535 540Lys Ser Lys Pro Trp Thr Leu Ala Leu Glu Arg Val Val Gly Thr Lys545 550 555 560Asn Met Asp Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Leu Thr 565 570 575Trp Leu Lys Glu Gln Asn Lys Asn Ser Phe Val Gly Trp Asn Thr Asp 580 585 590Trp Ser Pro Tyr Ala Ala Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605Ser Ala Leu Gly Glu Lys Ala Tyr Glu Trp Asn Asp Ser Glu Met Tyr 610 615 620Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Glu Tyr Phe Ser Lys625 630 635 640Val Lys Lys Gln Thr Ile Pro Phe Glu Asp Glu Cys Val Arg Val Ser 645 650 655Asp Leu Lys Pro Arg Val Ser Phe Ile Phe Phe Val Thr Leu Pro Lys 660 665 670Asn Val Ser Ala Val Ile Pro Arg Ala Glu Val Glu Glu Ala Ile Arg 675 680 685Ile Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asp Asp Asn Ser 690 695 700Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gln Pro Pro Tyr Gln Pro705 710 715 720Pro Val Thr22805PRTPaguma larvata 22Met Ser Gly Ser Phe Trp Leu Leu Leu Ser Phe Ala Ala Leu Thr Ala1 5 10 15Ala Gln Ser Thr Thr Glu Glu Leu Ala Lys Thr Phe Leu Glu Thr Phe 20 25 30Asn Tyr Glu Ala Gln Glu Leu Ser Tyr Gln Ser Ser Val Ala Ser Trp 35 40 45Asn Tyr Asn Thr Asn Ile Thr Asp Glu Asn Ala Lys Asn Met Asn Glu 50 55 60Ala Gly Ala Lys Trp Ser Ala Tyr Tyr Glu Glu Gln Ser Lys Leu Ala65 70 75 80Gln Thr Tyr Pro Leu Ala Glu Ile Gln Asp Ala Lys Ile Lys Arg Gln 85 90 95Leu Gln Ala Leu Gln Gln Ser Gly Ser Ser Val Leu Ser Ala Asp Lys 100 105 110Ser Gln Arg Leu Asn Thr Ile Leu Asn Ala Met Ser Thr Ile Tyr Ser 115 120 125Thr Gly Lys Ala Cys Asn Pro Asn Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140Glu Pro Gly Leu Asp Asn Ile Met Glu Asn Ser Lys Asp Tyr Asn Glu145 150 155 160Arg Leu Trp Ala Trp Glu Gly Trp Arg Ala Glu Val Gly Lys Gln Leu 165 170 175Arg Pro Leu Tyr Glu Glu Tyr Val Ala Leu Lys Asn Glu Met Ala Arg 180 185 190Ala Asn Asn Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205Glu Glu Trp Thr Gly Gly Tyr Asn Tyr Ser Arg Asn Gln Leu Ile Gln 210 215 220Asp Val Glu Asp Thr Phe Glu Gln Ile Lys Pro Leu Tyr Gln His Leu225 230 235 240His Ala Tyr Val Arg Ala Lys Leu Met Asp Thr Tyr Pro Ser Arg Ile 245 250 255Ser Arg Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270Arg Phe Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Gly Gln Lys 275 280 285Pro Asn Ile Asp Val Thr Asp Ala Met Val Asn Gln Asn Trp Asp Ala 290 295 300Arg Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu305 310 315 320Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Glu Pro 325 330 335Gly Asp Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350Lys Gly Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe385 390 395 400His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Asn 405 410 415His Leu Lys Thr Ile Gly Leu Leu Ser Pro Ala Phe Ser Glu Asp Asn 420 425 430Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460Lys Gly Ala Ile Pro Lys Glu Gln Trp Met Gln Lys Trp Trp Glu Met465 470 475 480Lys Arg Asn Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ala Asn Asp Tyr Ser Phe 500 505 510Ile Arg Tyr Tyr Thr Arg Thr Ile Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525Leu Cys Gln Ile Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540Ser Asn Ser Thr Glu Ala Gly Lys Lys Leu Leu Glu Met Leu Ser Leu545 550 555 560Gly Arg Ser Glu Pro Trp Thr Leu Ala Leu Glu Arg Val Val Gly Ala 565 570 575Lys Asn Met Asn Val Thr Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590Thr Trp Leu Lys Glu Gln Asn Arg Asn Ser Phe Val Gly Trp Asp Thr 595 600 605Asp Trp Arg Pro Tyr Ser Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620Lys Ser Ala Leu Gly Glu Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met625 630 635 640Tyr Leu Phe Arg Ser Ser Ile Ala Tyr Ala Met Arg Glu Tyr Phe Ser 645 650 655Lys Val Lys Asn Gln Thr Ile Pro Phe Val Glu Asp Asn Val Trp Val 660 665 670Ser Asp Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Phe Ser 675 680 685Asn Asn Val Ser Asp Val Ile Pro Arg Ser Glu Val Glu Asp Ala Ile 690 695 700Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asp Asp Asn705 710 715 720Ser Leu Glu Phe Leu Gly Ile Glu Pro Thr Leu Ser Pro Pro Tyr Arg 725 730 735Pro Pro Val Thr Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Ala 740 745 750Ile Val Val Gly Ile Val Leu Leu Ile Val Ser Gly Ile Arg Asn Arg 755 760 765Arg Lys Asn Asp Gln Ala Gly Ser Glu Glu Asn Pro Tyr Ala Ser Val 770 775 780Asp Leu Asn Lys Gly Glu Asn Asn Pro Gly Phe Gln His Ala Asp Asp785 790 795 800Val Gln Thr Ser Phe 80523723PRTPaguma larvata 23Gln Ser Thr Thr Glu Glu Leu Ala Lys Thr Phe Leu Glu Thr Phe Asn1 5 10 15Tyr Glu Ala Gln Glu Leu Ser Tyr Gln Ser Ser Val Ala Ser Trp Asn 20 25 30Tyr Asn Thr Asn Ile Thr Asp Glu Asn Ala Lys Asn Met Asn Glu Ala 35 40 45Gly Ala Lys Trp Ser Ala Tyr Tyr Glu Glu Gln Ser Lys Leu Ala Gln 50 55 60Thr Tyr Pro Leu Ala Glu Ile Gln Asp Ala Lys Ile Lys Arg Gln Leu65 70 75 80Gln Ala Leu Gln Gln Ser Gly Ser Ser Val Leu Ser Ala Asp Lys Ser 85 90 95Gln Arg Leu Asn Thr Ile Leu Asn Ala Met Ser Thr Ile Tyr Ser Thr 100 105 110Gly Lys Ala Cys Asn Pro Asn Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125Pro Gly Leu Asp Asn Ile Met Glu Asn Ser Lys Asp Tyr Asn Glu Arg 130 135 140Leu Trp Ala Trp Glu Gly Trp Arg Ala Glu Val Gly Lys Gln Leu Arg145 150 155 160Pro Leu Tyr Glu Glu Tyr Val Ala Leu Lys Asn Glu Met Ala Arg Ala 165 170 175Asn Asn Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Glu 180 185 190Glu Trp Thr Gly Gly Tyr Asn Tyr Ser Arg Asn Gln Leu Ile Gln Asp 195 200 205Val Glu Asp Thr Phe Glu Gln Ile Lys Pro Leu Tyr Gln His Leu His 210 215 220Ala Tyr Val Arg Ala Lys Leu Met Asp Thr Tyr Pro Ser Arg Ile Ser225 230 235 240Arg Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255Phe Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270Asn Ile Asp Val Thr Asp Ala Met Val Asn Gln Asn Trp Asp Ala Arg 275 280 285Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Glu Pro Gly305 310 315 320Asp Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335Gly Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Asn His385 390 395 400Leu Lys Thr Ile Gly Leu Leu Ser Pro Ala Phe Ser Glu Asp Asn Glu 405 410 415Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445Gly Ala Ile Pro Lys Glu Gln Trp Met Gln Lys Trp Trp Glu Met Lys 450 455 460Arg Asn Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr465 470 475 480Cys Asp Pro Ala Ser Leu Phe His Val Ala Asn Asp

Tyr Ser Phe Ile 485 490 495Arg Tyr Tyr Thr Arg Thr Ile Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510Cys Gln Ile Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525Asn Ser Thr Glu Ala Gly Lys Lys Leu Leu Glu Met Leu Ser Leu Gly 530 535 540Arg Ser Glu Pro Trp Thr Leu Ala Leu Glu Arg Val Val Gly Ala Lys545 550 555 560Asn Met Asn Val Thr Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575Trp Leu Lys Glu Gln Asn Arg Asn Ser Phe Val Gly Trp Asp Thr Asp 580 585 590Trp Arg Pro Tyr Ser Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605Ser Ala Leu Gly Glu Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620Leu Phe Arg Ser Ser Ile Ala Tyr Ala Met Arg Glu Tyr Phe Ser Lys625 630 635 640Val Lys Asn Gln Thr Ile Pro Phe Val Glu Asp Asn Val Trp Val Ser 645 650 655Asp Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Phe Ser Asn 660 665 670Asn Val Ser Asp Val Ile Pro Arg Ser Glu Val Glu Asp Ala Ile Arg 675 680 685Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asp Asp Asn Ser 690 695 700Leu Glu Phe Leu Gly Ile Glu Pro Thr Leu Ser Pro Pro Tyr Arg Pro705 710 715 720Pro Val Thr24805PRTSus scrofa 24Met Ser Gly Ser Phe Trp Leu Leu Leu Ser Leu Ile Pro Val Thr Ala1 5 10 15Ala Gln Ser Thr Thr Glu Glu Leu Ala Lys Thr Phe Leu Glu Lys Phe 20 25 30Asn Leu Glu Ala Glu Asp Leu Ala Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45Asn Tyr Asn Thr Asn Ile Thr Asp Glu Asn Ile Gln Lys Met Asn Asp 50 55 60Ala Arg Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Arg Ile Ala65 70 75 80Lys Thr Tyr Pro Leu Asp Glu Ile Gln Thr Leu Ile Leu Lys Arg Gln 85 90 95Leu Gln Ala Leu Gln Gln Ser Gly Thr Ser Gly Leu Ser Ala Asp Lys 100 105 110Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125Ser Gly Lys Val Leu Asp Pro Asn Asn Pro Gln Glu Cys Leu Val Leu 130 135 140Glu Pro Gly Leu Asp Glu Ile Met Glu Asn Ser Lys Asp Tyr Ser Arg145 150 155 160Arg Leu Trp Ala Trp Glu Ser Trp Arg Ala Glu Val Gly Lys Gln Leu 165 170 175Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Glu Asn Glu Met Ala Arg 180 185 190Ala Asn Asn Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205Val Thr Gly Thr Gly Asp Tyr Asp Tyr Ser Arg Asn Gln Leu Met Glu 210 215 220Asp Val Glu Arg Thr Phe Ala Glu Ile Lys Pro Leu Tyr Glu His Leu225 230 235 240His Ala Tyr Val Arg Ala Lys Leu Met Asp Ala Tyr Pro Ser Arg Ile 245 250 255Ser Pro Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270Arg Phe Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Gly Glu Lys 275 280 285Pro Ser Ile Asp Val Thr Glu Ala Met Val Asn Gln Ser Trp Asp Ala 290 295 300Ile Arg Ile Phe Glu Glu Ala Glu Lys Phe Phe Val Ser Ile Gly Leu305 310 315 320Pro Asn Met Thr Gln Gly Phe Trp Asn Asn Ser Met Leu Thr Glu Pro 325 330 335Gly Asp Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350Lys Gly Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380Tyr Ala Ile Gln Pro Tyr Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe385 390 395 400His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro His 405 410 415Tyr Leu Lys Ala Leu Gly Leu Leu Pro Pro Asp Phe Tyr Glu Asp Ser 420 425 430Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460Lys Gly Glu Ile Pro Lys Glu Gln Trp Met Gln Lys Trp Trp Glu Met465 470 475 480Lys Arg Glu Ile Val Gly Val Val Glu Pro Leu Pro His Asp Glu Thr 485 490 495Tyr Cys Asp Pro Ala Cys Leu Phe His Val Ala Glu Asp Tyr Ser Phe 500 505 510Ile Arg Tyr Tyr Thr Arg Thr Ile Tyr Gln Phe Gln Phe His Glu Ala 515 520 525Leu Cys Arg Thr Ala Lys His Glu Gly Pro Leu Tyr Lys Cys Asp Ile 530 535 540Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Leu Gln Met Leu Ser Leu545 550 555 560Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Ile Val Gly Val 565 570 575Lys Thr Met Asp Val Lys Pro Leu Leu Ser Tyr Phe Glu Pro Leu Leu 580 585 590Thr Trp Leu Lys Ala Gln Asn Gly Asn Ser Ser Val Gly Trp Asn Thr 595 600 605Asp Trp Thr Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620Lys Ser Ala Leu Gly Lys Glu Ala Tyr Glu Trp Asn Asp Asn Glu Met625 630 635 640Tyr Leu Phe Arg Ser Ser Ile Ala Tyr Ala Met Arg Asn Tyr Phe Ser 645 650 655Ser Ala Lys Asn Glu Thr Ile Pro Phe Gly Ala Glu Asp Val Trp Val 660 665 670Ser Asp Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ser Pro 675 680 685Ala Asn Met Ser Asp Ile Ile Pro Arg Ser Asp Val Glu Lys Ala Ile 690 695 700Ser Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asp Asp Asn705 710 715 720Thr Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asp Glu 725 730 735Pro Pro Val Thr Val Trp Leu Ile Ile Phe Gly Val Val Met Gly Leu 740 745 750Val Val Val Gly Ile Val Val Leu Ile Phe Thr Gly Ile Arg Asp Arg 755 760 765Arg Lys Lys Lys Gln Ala Ser Ser Glu Glu Asn Pro Tyr Gly Ser Met 770 775 780Asp Leu Ser Lys Gly Glu Ser Asn Ser Gly Phe Gln Asn Gly Asp Asp785 790 795 800Ile Gln Thr Ser Phe 80525723PRTSus scrofa 25Gln Ser Thr Thr Glu Glu Leu Ala Lys Thr Phe Leu Glu Lys Phe Asn1 5 10 15Leu Glu Ala Glu Asp Leu Ala Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30Tyr Asn Thr Asn Ile Thr Asp Glu Asn Ile Gln Lys Met Asn Asp Ala 35 40 45Arg Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Arg Ile Ala Lys 50 55 60Thr Tyr Pro Leu Asp Glu Ile Gln Thr Leu Ile Leu Lys Arg Gln Leu65 70 75 80Gln Ala Leu Gln Gln Ser Gly Thr Ser Gly Leu Ser Ala Asp Lys Ser 85 90 95Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Ser 100 105 110Gly Lys Val Leu Asp Pro Asn Asn Pro Gln Glu Cys Leu Val Leu Glu 115 120 125Pro Gly Leu Asp Glu Ile Met Glu Asn Ser Lys Asp Tyr Ser Arg Arg 130 135 140Leu Trp Ala Trp Glu Ser Trp Arg Ala Glu Val Gly Lys Gln Leu Arg145 150 155 160Pro Leu Tyr Glu Glu Tyr Val Val Leu Glu Asn Glu Met Ala Arg Ala 165 170 175Asn Asn Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190Thr Gly Thr Gly Asp Tyr Asp Tyr Ser Arg Asn Gln Leu Met Glu Asp 195 200 205Val Glu Arg Thr Phe Ala Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220Ala Tyr Val Arg Ala Lys Leu Met Asp Ala Tyr Pro Ser Arg Ile Ser225 230 235 240Pro Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255Phe Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Gly Glu Lys Pro 260 265 270Ser Ile Asp Val Thr Glu Ala Met Val Asn Gln Ser Trp Asp Ala Ile 275 280 285Arg Ile Phe Glu Glu Ala Glu Lys Phe Phe Val Ser Ile Gly Leu Pro 290 295 300Asn Met Thr Gln Gly Phe Trp Asn Asn Ser Met Leu Thr Glu Pro Gly305 310 315 320Asp Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335Gly Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365Ala Ile Gln Pro Tyr Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro His Tyr385 390 395 400Leu Lys Ala Leu Gly Leu Leu Pro Pro Asp Phe Tyr Glu Asp Ser Glu 405 410 415Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445Gly Glu Ile Pro Lys Glu Gln Trp Met Gln Lys Trp Trp Glu Met Lys 450 455 460Arg Glu Ile Val Gly Val Val Glu Pro Leu Pro His Asp Glu Thr Tyr465 470 475 480Cys Asp Pro Ala Cys Leu Phe His Val Ala Glu Asp Tyr Ser Phe Ile 485 490 495Arg Tyr Tyr Thr Arg Thr Ile Tyr Gln Phe Gln Phe His Glu Ala Leu 500 505 510Cys Arg Thr Ala Lys His Glu Gly Pro Leu Tyr Lys Cys Asp Ile Ser 515 520 525Asn Ser Thr Glu Ala Gly Gln Lys Leu Leu Gln Met Leu Ser Leu Gly 530 535 540Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Ile Val Gly Val Lys545 550 555 560Thr Met Asp Val Lys Pro Leu Leu Ser Tyr Phe Glu Pro Leu Leu Thr 565 570 575Trp Leu Lys Ala Gln Asn Gly Asn Ser Ser Val Gly Trp Asn Thr Asp 580 585 590Trp Thr Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605Ser Ala Leu Gly Lys Glu Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620Leu Phe Arg Ser Ser Ile Ala Tyr Ala Met Arg Asn Tyr Phe Ser Ser625 630 635 640Ala Lys Asn Glu Thr Ile Pro Phe Gly Ala Glu Asp Val Trp Val Ser 645 650 655Asp Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ser Pro Ala 660 665 670Asn Met Ser Asp Ile Ile Pro Arg Ser Asp Val Glu Lys Ala Ile Ser 675 680 685Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asp Asp Asn Thr 690 695 700Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asp Glu Pro705 710 715 720Pro Val Thr26200PRTSARS-CoV-2 26Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr1 5 10 15Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 20 25 30Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 35 40 45Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 50 55 60Asn Val Tyr Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile65 70 75 80Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro 85 90 95Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp 100 105 110Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys 115 120 125Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 130 135 140Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe145 150 155 160Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln 165 170 175Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala 180 185 190Thr Val Cys Gly Pro Lys Lys Ser 195 20027710PRTSARS-CoV-2 27Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr1 5 10 15Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala 20 25 30Ala His Ser Ala Phe Ala Gln Cys Val Asn Leu Thr Thr Arg Thr Gln 35 40 45Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro 50 55 60Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe65 70 75 80Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser 85 90 95Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn 100 105 110Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly 115 120 125Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile 130 135 140Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe145 150 155 160Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser 165 170 175Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr 180 185 190Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln 195 200 205Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly 210 215 220Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp225 230 235 240Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile 245 250 255Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser 260 265 270Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala 275 280 285Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr 290 295 300Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro305 310 315 320Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly 325 330 335Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val 340 345 350Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 355 360 365Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 370 375 380Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser385 390 395 400Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 405 410 415Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 420 425 430Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 435 440 445Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 450 455 460Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu465 470 475 480Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 485 490 495Ile Tyr Gln Ala Gly

Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 500 505 510Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 515 520 525Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 530 535 540Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys545 550 555 560Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val 565 570 575Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg 580 585 590Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu 595 600 605Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr 610 615 620Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val625 630 635 640Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro 645 650 655Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala 660 665 670Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp 675 680 685Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn 690 695 700Ser Pro Arg Arg Ala Arg705 71028203PRTSARS-CoV 28Met Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr1 5 10 15Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys 20 25 30Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe 35 40 45Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser 50 55 60Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln65 70 75 80Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu 85 90 95Pro Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile 100 105 110Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg 115 120 125His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe 130 135 140Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp145 150 155 160Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln 165 170 175Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala 180 185 190Thr Tyr Leu Ser Leu Asn Thr Ala Ala Ala Leu 195 20029805PRTHomo sapiens 29Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala1 5 10 15Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala65 70 75 80Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu145 150 155 160Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu225 230 235 240His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu305 310 315 320Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe385 390 395 400His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met465 470 475 480Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu545 550 555 560Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620Lys Ser Ala Leu Gly Asp Arg Ala Tyr Glu Trp Asn Asp Asn Glu Met625 630 635 640Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn705 710 715 720Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val 740 745 750Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg 755 760 765Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile 770 775 780Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp785 790 795 800Val Gln Thr Ser Phe 80530723PRTHomo sapiens 30Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn1 5 10 15His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn 20 25 30Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala 35 40 45Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln 50 55 60Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu65 70 75 80Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser 85 90 95Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr 100 105 110Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135 140Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg145 150 155 160Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala 165 170 175Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp 195 200 205Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser225 230 235 240Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280 285Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly305 310 315 320Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His385 390 395 400Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu 405 410 415Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr465 470 475 480Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490 495Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly 530 535 540Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys545 550 555 560Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp 580 585 590Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605Ser Ala Leu Gly Asp Arg Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys625 630 635 640Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala 645 650 655Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys 660 665 670Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg 675 680 685Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser 690 695 700Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro705 710 715 720Pro Val Ser31805PRTRhinolophus sinicus 31Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Thr1 5 10 15Ala Gln Phe Thr Thr Glu Asp Leu Ala Lys Ile Phe Leu Asp Glu Phe 20 25 30Asn Ser Glu Ala Glu Asn Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45Asp Tyr Asn Thr Asn Ile Asn Asp Glu Asn Val Gln Lys Met Asp Glu 50 55 60Ala Gly Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Lys Leu Ala65 70 75 80Lys Asn Tyr Pro Leu Glu Gln Ile Gln Asn Val Thr Val Lys Leu Gln 85 90 95Leu Gln Ile Leu Gln Gln Ser Gly Ser Pro Val Leu Ser Glu Asp Lys 100 105 110Ser Lys Arg Leu Asn Ser Ile Leu Asn Ala Met Ser Thr Ile Tyr Ser 115 120 125Thr Gly Lys Val Cys Lys Pro Asn Lys Pro His Glu Cys Leu Leu Leu 130 135 140Glu Pro Gly Leu Asp Asn Ile Met Gly Thr Ser Lys Asp Tyr Ser Glu145 150 155 160Arg Leu Trp Ala Trp Glu Gly Trp Arg Ala Glu Val Gly Lys Gln Leu 165 170 175Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190Gly Tyr His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Arg Asp Tyr Glu 195 200 205Thr Glu Glu Ser Pro Gly Pro Gly Tyr Ser Arg Asp Gln Leu Met Lys 210 215 220Asp Val Glu Arg Ile Phe Thr Glu Ile Lys Pro Leu Tyr Glu His Leu225 230 235 240His Ala Tyr Val Arg Ala Lys Leu Met Asp Thr Tyr Pro Phe His Ile 245 250 255Ser Pro Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270Arg Phe Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Gly Gln Lys 275 280 285Pro Asn Ile Asp Val Thr Asp Glu Met Leu Lys Gln Gly Trp Asp Ala 290 295 300Asp Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu305 310 315 320Pro Asn Met Thr Glu Gly Phe Trp Asn Asn Ser Met Leu Thr Glu Pro 325 330 335Gly Asp Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350Lys Gly Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Glu Asp 355 360 365Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380Tyr Ala Ser Gln Pro Tyr Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe385 390 395 400His Glu Ala Val Gly Glu Val Met Ser Leu Ser Val Ala Thr Pro Lys 405 410 415His Leu Lys Thr Met Gly Leu Leu Ser Pro Asp Phe Arg Glu Asp Asn 420 425 430Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Asn Ile Val Gly 435 440 445Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460Lys Gly Glu Ile Pro Lys Glu Glu Trp Met Lys Lys Trp Trp Glu Met465 470 475 480Lys Arg Lys Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ala Asn Asp Tyr Ser Phe 500 505 510Ile Arg Tyr Tyr Thr Arg Thr Ile Phe Glu Phe Gln Phe His Glu Ala 515 520

525Leu Cys Arg Ile Ala Gln His Asp Gly Pro Leu His Lys Cys Asp Ile 530 535 540Ser Asn Ser Thr Asp Ala Gly Lys Lys Leu His Gln Met Leu Ser Val545 550 555 560Gly Lys Ser Gln Ala Trp Thr Lys Thr Leu Glu Asp Ile Val Asp Ser 565 570 575Arg Asn Met Asp Val Gly Pro Leu Leu Arg Tyr Phe Glu Pro Leu Tyr 580 585 590Thr Trp Leu Gln Glu Gln Asn Arg Lys Ser Tyr Val Gly Trp Asn Thr 595 600 605Asp Trp Ser Pro Tyr Ser Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620Lys Ser Ala Leu Gly Glu Asn Ala Tyr Glu Trp Asn Asp Asn Glu Met625 630 635 640Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Glu Tyr Phe Leu 645 650 655Lys Glu Lys His Gln Thr Ile Leu Phe Gly Ala Glu Asn Val Trp Val 660 665 670Ser Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe His Val Thr Ser Pro 675 680 685Gly Asn Leu Ser Asp Ile Ile Pro Arg Pro Glu Val Glu Gly Ala Ile 690 695 700Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asp Asp Asn705 710 715 720Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Tyr Gln 725 730 735Pro Pro Val Thr Ile Trp Leu Ile Val Phe Gly Val Val Met Ala Val 740 745 750Val Val Val Gly Ile Val Val Leu Ile Ile Thr Gly Ile Arg Asp Arg 755 760 765Arg Lys Thr Asp Gln Ala Arg Ser Glu Glu Asn Pro Tyr Ser Ser Val 770 775 780Asp Leu Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Gly Asp Asp785 790 795 800Val Gln Thr Ser Phe 80532723PRTRhinolophus sinicus 32Gln Phe Thr Thr Glu Asp Leu Ala Lys Ile Phe Leu Asp Glu Phe Asn1 5 10 15Ser Glu Ala Glu Asn Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp Asp 20 25 30Tyr Asn Thr Asn Ile Asn Asp Glu Asn Val Gln Lys Met Asp Glu Ala 35 40 45Gly Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Lys Leu Ala Lys 50 55 60Asn Tyr Pro Leu Glu Gln Ile Gln Asn Val Thr Val Lys Leu Gln Leu65 70 75 80Gln Ile Leu Gln Gln Ser Gly Ser Pro Val Leu Ser Glu Asp Lys Ser 85 90 95Lys Arg Leu Asn Ser Ile Leu Asn Ala Met Ser Thr Ile Tyr Ser Thr 100 105 110Gly Lys Val Cys Lys Pro Asn Lys Pro His Glu Cys Leu Leu Leu Glu 115 120 125Pro Gly Leu Asp Asn Ile Met Gly Thr Ser Lys Asp Tyr Ser Glu Arg 130 135 140Leu Trp Ala Trp Glu Gly Trp Arg Ala Glu Val Gly Lys Gln Leu Arg145 150 155 160Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Gly 165 170 175Tyr His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Arg Asp Tyr Glu Thr 180 185 190Glu Glu Ser Pro Gly Pro Gly Tyr Ser Arg Asp Gln Leu Met Lys Asp 195 200 205Val Glu Arg Ile Phe Thr Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220Ala Tyr Val Arg Ala Lys Leu Met Asp Thr Tyr Pro Phe His Ile Ser225 230 235 240Pro Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255Phe Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270Asn Ile Asp Val Thr Asp Glu Met Leu Lys Gln Gly Trp Asp Ala Asp 275 280 285Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro 290 295 300Asn Met Thr Glu Gly Phe Trp Asn Asn Ser Met Leu Thr Glu Pro Gly305 310 315 320Asp Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335Gly Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Glu Asp Phe 340 345 350Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365Ala Ser Gln Pro Tyr Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380Glu Ala Val Gly Glu Val Met Ser Leu Ser Val Ala Thr Pro Lys His385 390 395 400Leu Lys Thr Met Gly Leu Leu Ser Pro Asp Phe Arg Glu Asp Asn Glu 405 410 415Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Asn Ile Val Gly Thr 420 425 430Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445Gly Glu Ile Pro Lys Glu Glu Trp Met Lys Lys Trp Trp Glu Met Lys 450 455 460Arg Lys Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr465 470 475 480Cys Asp Pro Ala Ser Leu Phe His Val Ala Asn Asp Tyr Ser Phe Ile 485 490 495Arg Tyr Tyr Thr Arg Thr Ile Phe Glu Phe Gln Phe His Glu Ala Leu 500 505 510Cys Arg Ile Ala Gln His Asp Gly Pro Leu His Lys Cys Asp Ile Ser 515 520 525Asn Ser Thr Asp Ala Gly Lys Lys Leu His Gln Met Leu Ser Val Gly 530 535 540Lys Ser Gln Ala Trp Thr Lys Thr Leu Glu Asp Ile Val Asp Ser Arg545 550 555 560Asn Met Asp Val Gly Pro Leu Leu Arg Tyr Phe Glu Pro Leu Tyr Thr 565 570 575Trp Leu Gln Glu Gln Asn Arg Lys Ser Tyr Val Gly Trp Asn Thr Asp 580 585 590Trp Ser Pro Tyr Ser Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605Ser Ala Leu Gly Glu Asn Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Glu Tyr Phe Leu Lys625 630 635 640Glu Lys His Gln Thr Ile Leu Phe Gly Ala Glu Asn Val Trp Val Ser 645 650 655Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe His Val Thr Ser Pro Gly 660 665 670Asn Leu Ser Asp Ile Ile Pro Arg Pro Glu Val Glu Gly Ala Ile Arg 675 680 685Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asp Asp Asn Ser 690 695 700Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Tyr Gln Pro705 710 715 720Pro Val Thr33805PRTSus scrofa 33Met Ser Gly Ser Phe Trp Leu Leu Leu Ser Leu Ile Pro Val Thr Ala1 5 10 15Ala Gln Ser Thr Thr Glu Glu Leu Ala Lys Thr Phe Leu Glu Lys Phe 20 25 30Asn Leu Glu Ala Glu Asp Leu Ala Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45Thr Ile Asn Thr Asn Ile Thr Asp Glu Asn Ile Gln Lys Met Asn Asp 50 55 60Ala Arg Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Arg Ile Ala65 70 75 80Lys Thr Tyr Pro Leu Asp Glu Ile Gln Thr Leu Ile Leu Lys Arg Gln 85 90 95Leu Gln Ala Leu Gln Gln Ser Gly Thr Ser Gly Leu Ser Ala Asp Lys 100 105 110Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125Ser Gly Lys Val Leu Asp Pro Asn Asn Pro Gln Glu Cys Leu Val Leu 130 135 140Glu Pro Gly Leu Asp Glu Ile Met Glu Asn Ser Lys Asp Tyr Ser Arg145 150 155 160Arg Leu Trp Ala Trp Glu Ser Trp Arg Ala Glu Val Gly Lys Gln Leu 165 170 175Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Glu Asn Glu Met Ala Arg 180 185 190Ala Asn Asn Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205Val Thr Gly Thr Gly Asp Tyr Asp Tyr Ser Arg Asn Gln Leu Met Glu 210 215 220Asp Val Glu Arg Thr Phe Ala Glu Ile Lys Pro Leu Tyr Glu His Leu225 230 235 240His Ala Tyr Val Arg Ala Lys Leu Met Asp Ala Tyr Pro Ser Arg Ile 245 250 255Ser Pro Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270Arg Phe Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Gly Glu Lys 275 280 285Pro Ser Ile Asp Val Thr Glu Ala Met Val Asn Gln Ser Trp Asp Ala 290 295 300Ile Arg Ile Phe Glu Glu Ala Glu Lys Phe Phe Val Ser Ile Gly Leu305 310 315 320Pro Asn Met Thr Gln Gly Phe Trp Asn Asn Ser Met Leu Thr Glu Pro 325 330 335Gly Asp Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350Lys Gly Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380Tyr Ala Ile Gln Pro Tyr Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe385 390 395 400His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro His 405 410 415Tyr Leu Lys Ala Leu Gly Leu Leu Pro Pro Asp Phe Tyr Glu Asp Ser 420 425 430Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460Lys Gly Glu Ile Pro Lys Glu Gln Trp Met Gln Lys Trp Trp Glu Met465 470 475 480Lys Arg Glu Ile Val Gly Val Val Glu Pro Leu Pro His Asp Glu Thr 485 490 495Tyr Cys Asp Pro Ala Cys Leu Phe His Val Ala Glu Asp Tyr Ser Phe 500 505 510Ile Arg Tyr Tyr Thr Arg Thr Ile Tyr Gln Phe Gln Phe His Glu Ala 515 520 525Leu Cys Arg Thr Ala Lys His Glu Gly Pro Leu Tyr Lys Cys Asp Ile 530 535 540Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Leu Gln Met Leu Ser Leu545 550 555 560Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Ile Val Gly Val 565 570 575Lys Thr Met Asp Val Lys Pro Leu Leu Ser Tyr Phe Glu Pro Leu Leu 580 585 590Thr Trp Leu Lys Ala Gln Asn Gly Asn Ser Ser Val Gly Trp Asn Thr 595 600 605Asp Trp Thr Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620Lys Ser Ala Leu Gly Glu Asp Ala Tyr Glu Trp Asn Asp Asn Glu Met625 630 635 640Tyr Leu Phe Arg Ser Ser Ile Ala Tyr Ala Met Arg Asn Tyr Phe Ser 645 650 655Ser Ala Lys Asn Glu Thr Ile Pro Phe Gly Ala Val Asp Val Trp Val 660 665 670Ser Asp Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ser Pro 675 680 685Ala Asn Met Ser Asp Ile Ile Pro Arg Ser Asp Val Glu Lys Ala Ile 690 695 700Ser Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asp Asp Asn705 710 715 720Thr Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asp Glu 725 730 735Pro Pro Val Thr Val Trp Leu Ile Ile Phe Gly Val Val Met Gly Leu 740 745 750Val Val Val Gly Ile Val Val Leu Ile Phe Thr Gly Ile Arg Asp Arg 755 760 765Arg Lys Lys Lys Gln Ala Ser Ser Glu Glu Asn Pro Tyr Gly Ser Met 770 775 780Asp Leu Ser Lys Gly Glu Ser Asn Ser Gly Phe Gln Asn Gly Asp Asp785 790 795 800Ile Gln Thr Ser Phe 80534723PRTSus scrofa 34Gln Ser Thr Thr Glu Glu Leu Ala Lys Thr Phe Leu Glu Lys Phe Asn1 5 10 15Leu Glu Ala Glu Asp Leu Ala Tyr Gln Ser Ser Leu Ala Ser Trp Thr 20 25 30Ile Asn Thr Asn Ile Thr Asp Glu Asn Ile Gln Lys Met Asn Asp Ala 35 40 45Arg Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Arg Ile Ala Lys 50 55 60Thr Tyr Pro Leu Asp Glu Ile Gln Thr Leu Ile Leu Lys Arg Gln Leu65 70 75 80Gln Ala Leu Gln Gln Ser Gly Thr Ser Gly Leu Ser Ala Asp Lys Ser 85 90 95Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Ser 100 105 110Gly Lys Val Leu Asp Pro Asn Asn Pro Gln Glu Cys Leu Val Leu Glu 115 120 125Pro Gly Leu Asp Glu Ile Met Glu Asn Ser Lys Asp Tyr Ser Arg Arg 130 135 140Leu Trp Ala Trp Glu Ser Trp Arg Ala Glu Val Gly Lys Gln Leu Arg145 150 155 160Pro Leu Tyr Glu Glu Tyr Val Val Leu Glu Asn Glu Met Ala Arg Ala 165 170 175Asn Asn Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val 180 185 190Thr Gly Thr Gly Asp Tyr Asp Tyr Ser Arg Asn Gln Leu Met Glu Asp 195 200 205Val Glu Arg Thr Phe Ala Glu Ile Lys Pro Leu Tyr Glu His Leu His 210 215 220Ala Tyr Val Arg Ala Lys Leu Met Asp Ala Tyr Pro Ser Arg Ile Ser225 230 235 240Pro Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255Phe Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Gly Glu Lys Pro 260 265 270Ser Ile Asp Val Thr Glu Ala Met Val Asn Gln Ser Trp Asp Ala Ile 275 280 285Arg Ile Phe Glu Glu Ala Glu Lys Phe Phe Val Ser Ile Gly Leu Pro 290 295 300Asn Met Thr Gln Gly Phe Trp Asn Asn Ser Met Leu Thr Glu Pro Gly305 310 315 320Asp Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys 325 330 335Gly Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe 340 345 350Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365Ala Ile Gln Pro Tyr Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro His Tyr385 390 395 400Leu Lys Ala Leu Gly Leu Leu Pro Pro Asp Phe Tyr Glu Asp Ser Glu 405 410 415Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr 420 425 430Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys 435 440 445Gly Glu Ile Pro Lys Glu Gln Trp Met Gln Lys Trp Trp Glu Met Lys 450 455 460Arg Glu Ile Val Gly Val Val Glu Pro Leu Pro His Asp Glu Thr Tyr465 470 475 480Cys Asp Pro Ala Cys Leu Phe His Val Ala Glu Asp Tyr Ser Phe Ile 485 490 495Arg Tyr Tyr Thr Arg Thr Ile Tyr Gln Phe Gln Phe His Glu Ala Leu 500 505 510Cys Arg Thr Ala Lys His Glu Gly Pro Leu Tyr Lys Cys Asp Ile Ser 515 520 525Asn Ser Thr Glu Ala Gly Gln Lys Leu Leu Gln Met Leu Ser Leu Gly 530 535 540Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Ile Val Gly Val Lys545 550 555 560Thr Met Asp Val Lys Pro Leu Leu Ser Tyr Phe Glu Pro Leu Leu Thr 565 570 575Trp Leu Lys Ala Gln Asn Gly Asn Ser Ser Val Gly Trp Asn Thr Asp 580 585 590Trp Thr Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605Ser Ala Leu Gly Glu Asp Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620Leu Phe Arg Ser Ser Ile Ala Tyr Ala Met Arg Asn Tyr Phe Ser Ser625 630 635 640Ala Lys Asn Glu Thr Ile Pro Phe Gly Ala Val Asp Val Trp Val Ser 645 650 655Asp Leu Lys

Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ser Pro Ala 660 665 670Asn Met Ser Asp Ile Ile Pro Arg Ser Asp Val Glu Lys Ala Ile Ser 675 680 685Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asp Asp Asn Thr 690 695 700Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asp Glu Pro705 710 715 720Pro Val Thr

Claims

1. A kit for detecting neutralizing antibodies to a severe acute respiratory syndrome-related coronavirus (SARSr-COV), comprising: (i) a SARS-COV-1 or -2 spike protein or an ACE2-binding fragment of a SARS-COV-1 or -2 spike protein, (ii) a human ACE2 protein or a SARS-CoV-1 or -2 receptor binding domain (RBD) binding fragment of a human ACE2 protein thereof which binds specifically to the spike protein or the fragment thereof of (i), and a detection entity comprising a horseradish peroxidase, for detecting interaction between the spike protein or the ACE2-binding fragment of (i) and the human ACE2 protein or RBD binding fragment of (ii), wherein the spike protein or the ACE2-binding fragment of (i) is conjugated to the detection entity; and the RBD binding fragment of (ii) is immobilised on a solid support.

2. (canceled)

3. (canceled)

4. The kit according to claim 1, wherein the kit is for detecting neutralizing antibodies to SARSr-COV in a sample and the sample is a blood sample, a lymph sample, a saliva sample, or a synovial fluid sample and the SARSr-CoV is SARS-CoV-2.

5. The kit according to claim 1, wherein the SARSr-CoV is SARS-CoV-2.

6. The kit according to claim 1, wherein the spike protein or the ACE2-binding fragment of (i) comprises an S1 subunit that comprises an amino acid sequence comprising SEQ ID NO: 12 or 27.

7. (canceled)

8. The kit according to claim 1, wherein the kit is for performing a method of analysing a sample for the presence of neutralizing antibodies to a SARSr-CoV, comprising: contacting the sample with (i) the spike protein or the ACE2-binding fragment, and (ii) the human ACE2 protein or the RBD binding fragment, and determining the level of interaction between the spike protein or ACE2-binding fragment of (i) and the human ACE2 protein or the RBD binding fragment of (ii), and wherein the spike protein or the ACE2-binding fragment of (i) is at a quantity that is: (a) sufficient to produce a detectable signal of interaction between the spike protein or the ACE2-binding fragment of (i) and the human ACE2 protein or the RBD binding fragment of (ii) in the absence of the sample, and/or (b) less than or equal to, in molar ratio, the quantity of the antibodies to the SARSr-CoV in the sample, and/or (c) a minimal quantity required to produce a detectable signal of interaction between the spike protein or the ACE2-binding fragment of (i) and the ACE2 protein or the RBD binding fragment of (ii) in the absence of the sample.

9. The kit according to claim 1, wherein the RBD comprises an amino acid sequence comprising SEQ ID NO: 13 or 26.

10. The kit according to claim 1, wherein the ACE2 protein or the RBD-binding fragment of (ii) comprises an ACE2 protein extracellular domain having an amino acid sequence comprising SEQ ID NO: 17 or 30.

11. (canceled)

12. The kit according to claim 8, wherein the spike protein or the ACE2-binding fragment of (i) comprises an S1 subunit that comprises an amino acid sequence comprising SEQ ID NO: 12 or 27.

13. The kit according to claim 1, wherein the kit is for performing a method of analysing a sample for the presence of neutralizing antibodies to a SARSr-CoV, comprising: contacting the sample with (i) the spike protein or the ACE2-binding fragment, and (ii) the human ACE2 protein or the RBD binding fragment, and determining the level of interaction between the spike protein or ACE2-binding fragment of (i) and the human ACE2 protein or the RBD binding fragment of (ii).

14. The kit according to claim 13, wherein the sample is a blood sample, a lymph sample, a saliva sample, or a synovial fluid sample.

15. The kit according to claim 13, wherein the SARSr-CoV is SARS-CoV-2.

16. (canceled)

17. The kit according to claim 8, wherein the RBD comprises an amino acid sequence comprising SEQ ID NO: 13 or 26.

18. The kit according to claim 8, wherein the ACE2 protein or the RBD-binding fragment of (ii) comprises an ACE2 protein extracellular domain having an amino acid sequence comprising SEQ ID NO: 17 or 30.

19. The kit according to claim 13, wherein the spike protein or the ACE2-binding fragment of (i) comprises an S1 subunit that comprises an amino acid sequence comprising SEQ ID NO: 12 or 27.

20. (canceled)

21. The kit according to claim 8, wherein the sample is a blood sample, a lymph sample, a saliva sample, or a synovial fluid sample.

22. The kit according to claim 13, wherein the RBD comprises an amino acid sequence comprising SEQ ID NO: 13 or 26.

23. The kit according to claim 13, wherein the ACE2 protein or the RBD binding fragment of (ii) comprises an ACE2 protein extracellular domain having an amino acid sequence comprising SEQ ID NO: 17 or 30.

24. (canceled)

25. The kit according to claim 13, wherein the spike protein or the ACE2-binding fragment of (i) is at a quantity that is (a) sufficient to produce a detectable signal of interaction between the spike protein or the ACE2-binding fragment (i) and the ACE2 protein or the RBD binding fragment of (ii) in the absence of the sample, and (b) less than or equal to, in molar ratio, the quantity of the antibodies to the SARSr-CoV in the sample.

26. The kit according to claim 13, wherein the spike protein or the ACE2-binding fragment of (i) is at a minimal quantity required to produce a detectable signal of interaction between the spike protein or the ACE2-binding fragment of (i) and the ACE2 protein or the RBD binding fragment of (ii) in the absence of the sample.

27. The kit according to claim 8, wherein the spike protein or the ACE2-binding fragment of (i) is at a quantity that is: (a) sufficient to produce a detectable signal of interaction between the spike protein or the ACE2-binding fragment of (i) and the ACE2 protein or the RBD binding fragment of (ii) in the absence of the sample.

28. The kit according to claim 8, wherein the spike protein or the ACE2-binding fragment of (i) is at a quantity that is: (c) a minimal quantity required to produce a detectable signal of interaction between the spike protein or the ACE2-binding fragment of (i) and the ACE2 protein or the RBD binding fragment of (ii) in the absence of the sample.

29. The kit according to claim 27, wherein the sample is a blood sample, a lymph sample, a saliva sample, or a synovial fluid sample and the SARSr-CoV is SARS-CoV-2.

30. The kit according to claim 28, wherein the sample is a blood sample, a lymph sample, a saliva sample, or a synovial fluid sample and the SARSr-CoV is SARS-CoV-2.

31. The kit according to claim 29, wherein the spike protein or the ACE2-binding fragment of (i) comprises an S1 subunit that comprises the amino acid sequence set forth in SEQ ID NO: 12 or 27, or the RBD comprises an amino acid sequence comprising SEQ ID NO: 13 or 26, or the ACE2 protein or the RBD binding fragment of (ii) comprises an ACE2 protein extracellular domain having an amino acid sequence comprising SEQ ID NO: 17 or 30.

32. The kit according to claim 30, wherein the spike protein or the ACE2-binding fragment of (i) comprises an S1 subunit that comprises the amino acid sequence set forth in SEQ ID NO: 12 or 27, or the RBD comprises an amino acid sequence comprising SEQ ID NO: 13 or 26, or the ACE2 protein or the RBD binding fragment of (ii) comprises an ACE2 protein extracellular domain having an amino acid sequence comprising SEQ ID NO: 17 or 30.

33. The kit according to claim 13, wherein the sample is a blood sample, a lymph sample, a saliva sample, or a synovial fluid sample and the SARSr-CoV is SARS-CoV-2.

34. The kit according to claim 8, wherein the sample is a blood sample, a lymph sample, a saliva sample, or a synovial fluid sample and the SARSr-CoV is SARS-CoV-2.

35. The kit according to claim 1, wherein the kit is for detecting neutralizing antibodies to SARSr-COV in a sample and the sample is a blood sample, a lymph sample, a saliva sample, or a synovial fluid sample.

36. The kit according to claim 8, wherein the SARSr-CoV is SARS-CoV 2.

37. The kit according to claim 4, wherein the spike protein or the ACE2-binding fragment of (i) comprises an S1 subunit that comprises the amino acid sequence set forth in SEQ ID NO: 12 or 27, or the RBD comprises an amino acid sequence comprising SEQ ID NO: 13 or 26, or the ACE2 protein or the RBD binding fragment of (ii) comprises an ACE2 protein extracellular domain having an amino acid sequence comprising SEQ ID NO: 17 or 30.