METHODS AND PRODUCTS FOR SEROLOGICAL ANALYSIS OF SARS-COV-2 INFECTION

FIELD OF THE INVENTION [0001] This invention is generally in the field of cells, immune complexes, kits, and methods relating to SARS-CoV-2.

BACKGROUND OF THE INVENTION [0002] About four months after the initial description of atypical pneumonia cases in Wuhan in

December 2019, COVID-19 has become a major pandemic threat. In April 2020, about half of the human population was under confinement, 2 million infections had been officially diagnosed, with 121 ,000 fatalities and 0.5 million recovered cases. COVID-19 is caused by SARS-CoV-2 ^{1 2} , a betacoronavirus displaying 80% nucleotide homology with Severe Acute Respiratory Syndrome virus (now termed SARS-CoV-1 ), that was responsible for an outbreak of 8,000 estimated cases in 2003.

[0003] PCR-based tests are widely used for COVID-19 diagnosis and for detection and quantification of SARS-CoV2 RNA ³ _’ ⁴⁵ . These virological assays are instrumental to monitor individuals with active infections. The average virus RNA load is 10 ⁵ copies per nasal or oropharyngeal swab at day 5 post symptom onsets and may reach 10 ⁸ copies ⁶ . A decline occurs after days 10-11 , but viral RNA can be detected up to day 28 post-onset in recovered patients at a time when antibodies (Abs) are most often readily detectable ⁶⁷ . Disease severity correlates with viral loads, and elderly patients, who are particularly sensitive to infection, display higher viral loads ⁶⁷ . [0004] Serological assays are also being implemented. Anti-Spike (S) and Nucleoprotein (N) humoral responses in COVID-19 patients are assessed, because the two proteins are highly immunogenic. The viral spike (S) protein allows viral binding and entry into target cells. S binding to a cellular receptor, angiotensin-converting enzyme 2 (ACE2) for SARS-CoV-1 and -CoV-2, is followed by S cleavage and priming by the cellular protease TMPRSS2 or other endosomal proteases ⁸ . S genes from SARS-CoV-1 and -CoV2 share 76% amino-acid similarity ² . One noticeable difference between the two viruses is the presence of a furin cleavage site in SARS- CoV-2, which is suspected to enhance viral infectivity ² . The structures of S from SARS-CoV-1 and Co-V-2 in complex with ACE2 have been elucidated ^9-11 . S consists of three S1-S2 dimers, displaying different conformational changes upon virus entry leading to fusion ⁹ _’ ^{10 12} . Some anti-S antibodies, including those targeting the receptor binding domain (RBD), display a neutralizing activity, but their relative frequency among the generated anti-SARS-CoV-2 antibodies during infection remains poorly characterized. The nucleoprotein N is highly conserved between SARS- CoV1 and -CoV2 (96% amino-acid homology). N plays a crucial role in subgenomic viral RNA transcription and viral replication and assembly.

[0005] Serological assays are currently being performed using in-house, pre-commercial versions or commercially available ELISA-based diagnostics tests ⁶ _’ ⁷ _’ ^13-15 . Other techniques, including point-of-care and auto-tests are also becoming available. In hospitalized patients, seroconversion is typically detected between 5-14 days post symptom onset, with a median time of 5-12 days for anti-S IgM and 14 days for IgG and IgA ⁶ _’ ⁷ _’ ^13-16 . The kinetics of anti-N response was described to be similar to that of anti-S, although N responses might appear earlier ^15-17 . Anti-SARS-CoV-2 antibody titers correlate with disease severity, likely reflecting higher viral replication rates and/or immune activation in patients with severe outcome. Besides N and S, antibody responses to other viral proteins (mainly ORF9b and NSP5) were also identified by antibody microarray ¹⁷ .

[0006] Neutralization titers observed in individuals infected with other coronaviruses, such as MERS- CoV, are considered to be relatively low ^{6 18} . With SARS-CoV-2, neutralizing antibodies (Nabs) have been detected in symptomatic individuals ⁶ _’ ⁸ _’ ¹⁹²⁰ , and their potency seems to be associated with high levels of antibodies. Neutralization is assessed using plaque neutralization assays, microneutralization assays, or inhibition of infection with viral pseudotypes carrying the S protein ⁶ _’ ^{8 19-21} . Of note, potent monoclonal NAbs that target RBD have been cloned from infected individuals ²² . Whether asymptomatic infections, which are currently often undocumented ²³ , and most likely represent the majority of SARS-CoV-2 cases, lead to protective immunity, and whether this immunity is mediated by NAbs, remain outstanding questions.

[0007] A need exists for more sensitive antibody-based assays for SARS-CoV-2 infection. This disclosure meets this and other needs.

SUMMARY OF THE INVENTION

[0008] It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their antibody response profile. To this end the inventors have developed an assay, termed S-Flow, which allows identification and quantification of anti-Spike SARS-CoV-2 antibodies by flow-cytometry. A comparison of S-Flow to alternative assays using samples taken from 491 pre-epidemic individuals, 51 patients from Hopital Bichat (Paris), 209 pauci-symptomatic individuals in the French Oise region, 200 contemporary Oise blood donors, and 162 hospital staff is presented in the examples. Overall, the results obtained with the S-Flow assays were similar to the other assays, with surprising and unexpected improvements in sensitivity. The S-Flow assay allowed an earlier detection of seroconversion in five hospitalized patients with critical COVID-19 and better identification of SARS-CoV-2 infected individuals in a cohort of blood donors. Overall, the data demonstrate that the S-Flow assay outperforms other assays to reveal SARS-cov-2 sero-conversion. The specificity and sensitivity of the disclosed S-Flow assay is >99%.

[0009] Accordingly, a first aspect of the present invention provides 293T cells expressing a SARS- CoV-2 S protein on their surface. In another aspect, the invention provides Jurkat cells expressing a SARS-CoV-2 S protein on their surface. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on a plasmid in the cell. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence integrated into the genome of the cell. In a particular embodiment, the SARS-CoV-2 S protein has an amino acid sequence at least 95% identical to SEQ ID NO: 1. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence having a nucleotide sequence at least 80% identical to SEQ ID NO: 2. In a particular embodiment, the 293T cells are cell line 293T-S, deposited at the CNCM on May 5, 2020, under registration number CNCM I-5509.

[0010] In another aspect, the present invention provides immune complexes comprising a SARS- CoV-2 S protein present on the surface of a 293T cell or Jurkat cells and an antibody bound to the SARS-CoV-2 S protein. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on a plasmid in the cell. In a particular embodiment, the SARS- CoV-2 S protein is expressed from a coding sequence integrated into the genome of the cell. In a particular embodiment, the SARS-CoV-2 S protein has an amino acid sequence at least 95% identical to SEQ ID NO: 1. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence having a nucleotide sequence at least 80% identical to SEQ ID NO: 2. In a particular embodiment, the 293T cells are cell line 293T-S, deposited at the CNCM on May 5, 2020, under registration number CNCM i-5509. In a particular embodiment, the anti- SARS-CoV-2 S protein antibody is a patient antibody. In a particular embodiment, the immune complex further comprises a secondary antibody bound to the anti-SARS-CoV-2 S protein antibody. In some embodiments the secondary antibody is labeled.

[0011] In another aspect, the present invention provides kits for detecting anti-SARS-CoV-2 S protein antibodies in a biological sample, comprising 293T cells expressing a SARS-CoV-2 S protein on their surface. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on a plasmid in the cell. In a particular embodiment, the SARS-CoV- 2 S protein is expressed from a coding sequence integrated into the genome of the cell. In a particular embodiment, the SARS-CoV-2 S protein has an amino acid sequence at least 95% identical to SEQ ID NO: 1. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence having a nucleotide sequence at least 80% identical to SEQ ID NO: 2. In a particular embodiment, the 293T cells are cell line 293T-S, deposited at the CNCM on May 5, 2020, under registration number CNCM I-5509. In a particular embodiment, the kit further comprises a control anti-SARS-CoV-2 S protein antibody. In a particular embodiment, the control anti-SARS-CoV-2 S protein antibody is a patient antibody. In a particular embodiment, the kit further comprises secondary antibodies that bind to the anti-SARS-CoV-2 S protein antibodies.

[0012] In another aspect, this present invention provides in vitro methods for detecting antibodies against a SARS-CoV-2 S protein in a biological sample. In a particular embodiment, the methods comprise providing eukaryotic cells expressing a SARS-CoV-2 S protein on their surface; providing a biological sample from a patient; contacting the eukaryotic cells with the biological sample; and assaying for binding between the SARS-CoV-2 S protein present on the surface of the eukaryotic cells and antibodies present in the sample. In a particular embodiment, the methods comprise providing 293T cells expressing a SARS-CoV-2 S protein on their surface; providing a biological sample from a patient; contacting the 293T cells with the biological sample; and assaying for binding between the SARS-CoV-2 S protein present on the surface of the 293T cells and antibodies present in the sample. In another embodiment, the method comprises providing Jurkat cells expressing a SARS-CoV-2 S protein on their surface; providing a biological sample from a patient; contacting the Jurkat cells with the biological sample; and assaying for binding between the SARS-CoV-2 S protein present on the surface of the Jurkat cells and antibodies present in the sample. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on a plasmid in the cell. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence integrated into the genome of the cell. In a particular embodiment, the SARS-CoV-2 S protein has an amino acid sequence at least 95% identical to SEQ ID NO: 1. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence having a nucleotide sequence at least 80% identical to SEQ ID NO: 2. In a particular embodiment, the 293T cells are cell line 293T-S, deposited at the CNCM on May 5, 2020, under registration number CNCM i-5509. In a particular embodiment, assaying for binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample is performed by a method comprising visualizing antigen-antibody complexes. In a particular embodiment, assaying for binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells comprises contacting the 293T cells or Jurkat cells with a secondary antibody against antibodies present in the sample and visualizing antigen-antibody complexes. In a particular embodiment, binding of antibodies present in the sample is detected on the surface of at least 20% of the 293T cells or Jurkat cells of the invention, contacted with the sample. In a particular embodiment, binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample is detected using a fluorescent activated cell sorting (FACS) assay.

[0013] In another aspect, the present invention provides in vitro methods for diagnosing a SARS- CoV-2 infection in a patient. The methods may comprise providing eukaryotic cells expressing a SARS-CoV-2 S protein on their surface; providing a biological sample from a patient; contacting the eukaryotic cells with the biological sample; and assaying for binding between the SARS-CoV- 2 S protein present on the surface of the eukaryotic cells and antibodies present in the sample. The methods may comprise providing 293T cells expressing a SARS-CoV-2 S protein on their surface; providing a biological sample from a patient; contacting the 293T cells with the biological sample; and assaying for binding between the SARS-CoV-2 S protein present on the surface of the 293T cells and antibodies present in the sample. The method may comprise providing Jurkat cells expressing a SARS-CoV-2 S protein on their surface; providing a biological sample from a patient; contacting the Jurkat cells with the biological sample; and assaying for binding between the SARS-CoV-2 S protein present on the surface of the Jurkat cells and antibodies present in the sample. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on a plasmid in the cell. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence integrated into the genome of the cell. In a particular embodiment, the SARS-CoV-2 S protein has an amino acid sequence at least 95% identical to SEQ ID NO: 1. In some embodiments the SARS-CoV-2 S protein is expressed from a coding sequence having a nucleotide sequence at least 80% identical to SEQ ID NO: 2. In a particular embodiment, the 293T cells are cell line 293T-S, deposited at the CNCM on May 5, 2020, under registration number CNCM I-5509. In a particular embodiment, detection of binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample indicates that the patient is infected or was infected with SARS-CoV-2. In a particular embodiment, the absence of detection of binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample indicates that the patient is not infected or was not infected with SARS-CoV-2. In a particular embodiment, detection of binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample indicates that the patient is infected or was infected with SARS-CoV-2; and the absence of detection of binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample indicates that the patient is not infected or was not infected with SARS- CoV-2. In a particular embodiment, binding of antibodies present in the sample is detected on the surface of at least 20% of the 293T cells or Jurkat cells of the invention contacted with the sample, indicating that the patient is infected or was infected with SARS-CoV-2. In a particular embodiment, binding of antibodies present in the sample is not detected on the surface of at least 20% of the 293T cells or Jurkat cells of the invention contacted with the sample, indicating that the patient is not infected or was not infected with SARS-CoV-2. In a particular embodiment, assaying for binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample is performed by a method comprising visualizing antigen-antibody complexes. In a particular embodiment, assaying for binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells comprises contacting the 293T cells or Jurkat cells with a secondary antibody against antibodies present in the sample and visualizing antigen-antibody complexes. In a particular embodiment, binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample is detected using a fluorescent activated cell sorting (FACS) assay.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Fig. 1. Summary of Assays. Certain characteristics of the ELISA N ELISA tri-S, S-Flow, and LIPS assays.

[0015] Fig. 2. S-Flow Assay. Schematic representation of the S-Flow assay.

[0016] Fig. 3. S-Flow Data. Representative examples of S-Flow data. Anti-S IgG levels in the serum of two hospitalized patients (B1 and B2) were measured. Cells transfected with a control plasmid indicate background levels (293T) and cells transfected with a S-expressing plasmid (293T-S) identify the specific signal.

[0017] Fig. 4A and 4B. Evaluation of S-Flow on COVID-19 Patients. A) Antibody titers determined by serial dilution for 2 patients (B1 and B2). Dashed lines indicated background levels and plain line specific signal. B) Levels of anti-S IgG and IgM antibodies in nine hospitalized patients (B1 - B9).

[0018] Fig. 5. Summary of the Cohorts. Certain characteristics of assayed cohorts. [0019] Fig. 6. SARS-Cov-2 Antibodies in Pre-epidemic Individuals. Results of ELISA N, ELISA tri-S, S-Flow, LIPS S1 , and LIPS N assaying of sera from pre-epidemic individuals sampled between 2017 and 2019. Pre-epidemic samples were used to determine the cut-off of each assay, which is indicated by a dashed line and a grey area. ELISAs were set to 95% specificity. The number of positive samples is indicated. Each dot represents a sample.

[0020] Fig. 7. SARS-CoV-2 Antibodies in Symptomatic Individuals. Results of ELISA N, ELISA tri-S, S-Flow, LIPS S1 , and LIPS N assaying of sera from pauci- symptomatic individuals from the Crepy-en-Vallois epidemic cluster (bottom row) and hospitalized patients with confirmed COVID-19 (top row). Pre-epidemic samples were used to determine the cut-off of each assay, which is indicated by a dashed line and a grey area. ELISAs were set to 95% specificity. The number of positive samples is indicated. Each dot represents a sample.

[0021] Fig. 8. SARS-CoV-2 Antibodies in Healthy Blood Donors. Results of ELISA N, ELISA tri- S, S-Flow, LIPS S1 , and LIPS N assaying of sera from healthy blood donors. Pre-epidemic samples were used to determine the cut-off of each assay, which is indicated by a dashed line and a grey area. ELISAs were set to 95% specificity. The number of positive samples is indicated. Each dot represents a sample.

[0022] Fig. 9. Antibody Detection in Five Hospitalized Patients. Kinetics of seroconversion in 5 hospitalized patients with at least 5 longitudinal samples. All patients were admitted in intensive care unit. Each line represents a participant. Dashed lines and grey areas indicate assays cut-off of positivity.

[0023] Fig. 10A and 10B. Correlations Between Assays. Data from pauci-symptomatic individuals and hospitalized patients (n=329) were pooled to compare assays. A) Data obtained with an assay were correlated to all other tests. Dashed lines indicate assays cut-offs for positivity. Values in light grey areas are positive in one assay and values in dark grey areas are positive in the two assays. Each dot represents a participant. B) Pearson correlation coefficient (R ² ) of each comparison. Values are color-coded, white corresponding to the lower value and dark grey the highest. All correlations are significant (p>0.0001).

[0024] Fig. 11. Capacity of secondary antibodies to specifically detect IgG or IgA. Increasing concentrations of a purified anti-spike immunoglobin cloned either as IgG or IgA were tested for binding to anti-lgG-FC-AF647 (A) or anti-lgG-FV-AF488 (B).

[0025] Fig. 12. Comparison of simplex and multiplex assays. Dots indicate positive COVID-19 samples.

[0026] Fig. 13. Schematic representation of Spike protein from Wuhan ancestral strain (B.1- G), a D614G reference strain (B.1-B), B.1.1.7 and B.1.351. DETAILED DESCRIPTION OF THE INVENTION

[0027] It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their antibody response profile. The examples describe a pilot study to assess the levels of anti-SARS-CoV-2 antibodies in samples taken from 491 pre- epidemic individuals, 51 patients from Hopital Bichat (Paris), 209 pauci-symptomatic individuals in the French Oise region and 200 contemporary Oise blood donors. Various assays are compared, and a novel S-Flow assay is described.

SARS-CoV-2 S Protein, variants and homologous thereof

[0028] As used herein, the SARS-CoV-2 S protein preferably comprises or consists of the amino acid sequence of SEQ ID NO:1 corresponding to Genbank: QHD43416.1 (spike protein of the Wuhan strain B.1-G, of. Zhou et al ² ).

[0029] In some embodiments, the SARS-CoV-2 S protein comprises or consists of an homologous amino acid sequence thereof, that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 .

[0030] In some embodiments, the SARS-CoV-2 S protein comprises or consists of an homologous amino acid sequence thereof, that has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 15, 16, 17, 18, 19 or 20 amino acid changes relative to SEQ ID NO: 1 . In some embodiments, the SARS-CoV-2 S Protein homologous comprises or consists of an amino acid sequence that has no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid changes relative to SEQ ID NO: 1 .

[0031] In a preferred embodiment, the SARS-CoV-2 S protein is encoded by a nucleotide sequence that comprises or consists of nucleotides 21563 to 25384 of Genbank: MN908947.3 (SEQ ID NO: 2).

[0032] In some embodiments, the SARS-CoV-2 S protein is encoded by an homologous nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2.

[0033] In some embodiments, the SARS-CoV-2 S protein is encoded by an homologous nucleotide sequence that is codon-optimized, such as a codon optimized sequence of SEQ ID NO: 20, encoding SEQ ID NO:1 . [0034] The SARS-CoV-2 S protein used in the invention can be any variants which appeared subsequently to the first identified viral strain disclosed in Zhou et al., 2020 ² . In particular, it can be the SARS-CoV-2 S protein of the following SARS-CoV-2 variant strains:

[0035] i. the hCoV-19 / France / ARA-104350/2020 (GISAID ID: EPI_ISL_683350) strain of lineage B.1-B (this strain has at least the D614G mutation in its spike protein; it is considered today as the circulating wild-type strain in Europe, by comparison with the variants cited below); its spike protein is of SEQ ID NO:9, and can be encoded by SEQ ID NO:7. ii. the viral strain called “English variant” hCoV-19 / France / ARA-SC2118 / 2020 (GISAID ID: EPI ISL 900512) of lineage B.1.1.7; its spike protein is of SEQ ID NO:10, and can be encoded by SEQ ID NO:4. iii. the South African strain (501 Y.V2.HV001) of lineage B.1.351 ; its spike protein is of SEQ ID NO:11 , and can be encoded by SEQ ID NO:5. and iv. the Brazilian variant strain of lineage P.1 ; its spike protein is of SEQ ID NO:12, and can be encoded by SEQ ID NO:8, see Faria, N. R. et al ³⁹ . for more details. The following table summarizes the listed sequences of the Spike proteins and variants thereof, as well as their encoding sequence, that are useful in the context of the invention.

The cells of the invention can express any of the S protein variants that have been identified so far (Jacob JJ. Et al, 2020 ³⁸ ), as well as those that will be identified later.

[0036] In another embodiment, the SARS-CoV-2 S protein used in or expressed by the cells of the invention is the mutant protein Spike of lineage B.1 -B having SEQ ID NO:9, or the mutant protein Spike of lineage B.1 .1 .7 having SEQ ID NO:10, or the mutant protein Spike of lineage B.1 .351 of SEQ ID NO:11 , or the mutant protein Spike of lineage P.1 of SEQ ID NO:12. [0037] In some embodiments, the SARS-CoV-2 S protein comprises or consists of an homologous amino acid sequence thereof, that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9-12.

[0038] In some embodiments, the SARS-CoV-2 S protein comprises or consists of an homologous amino acid sequence thereof, that has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 15, 16, 17, 18, 19 or 20 amino acid changes relative to SEQ ID NO: 9-12. In some embodiments, the SARS-CoV-2 S Protein homologous comprises or consists of an amino acid sequence that has no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid changes relative to SEQ ID NO: 9-12.

[0039] In another embodiment, the SARS-CoV-2 S protein used in or expressed by the cells of the invention is the mutated D614G protein Spike mutant protein of lineage B.1-B. encoded by SEQ ID NO:7, or the mutant protein Spike of lineage B.1 .1 .7 encoded by SEQ ID NO:4, or the mutant protein Spike of lineage B.1 .351 encoded by SEQ ID NO:5, or the mutant protein Spike of lineage

P.1 encoded by SEQ ID NO:8.

[0040] In some embodiments, the SARS-CoV-2 S protein is encoded by an homologous nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8.

Cells Expressing SARS-CoV-2 S Protein

[0041] In a first aspect, the present invention provides 293T cells expressing a SARS-CoV-2 S protein on their surface. A skilled artisan will appreciate that in certain embodiments any 293T cells known in the art may be used. In one embodiment the 293T cells are HEK293T cells. In a preferred embodiment the 293T cells are from ATCC (ATCC® CRL-3216™).

[0042] In another aspect, the invention provides Jurkat cells expressing a SARS-CoV-2 S protein on their surface. A skilled artisan will appreciate that in certain embodiments any Jurkat cells known in the art may be used.

[0043] Suitable methods of growing and maintaining 293T cells are well known in the art. In a nonlimiting example, 293T cells may be split every 2-3 days using DMEM medium supplemented with 10% fetal calf serum and 1% Penicillin streptomycin (complete medium). [0044] In a particular embodiment, the SARS-CoV-2 S protein present in the cells of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 1 . In some embodiments the SARS-CoV-2 S protein comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments the SARS-CoV-2 S protein comprises or consists of an amino acid sequence that has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes relative to SEQ ID NO: 1 . In a particular embodiment, the SARS-CoV-2 S protein comprises or consists of an amino acid sequence that has no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid changes relative to SEQ ID NO: 1 .

[0045] In a more particular embodiment, the SARS-CoV-2 S present in the cells of the invention is a variant of SEQ ID NO:1 such as the Spike protein of SEQ ID NO:9-12, as disclosed above. In some embodiments, the SARS-CoV-2 S protein comprises or consists of an homologous amino acid sequence thereof, that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9-12. In some embodiments the SARS-CoV-2 S protein comprises or consists of an amino acid sequence that has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes relative to any of SEQ ID NO: 9-12. In a particular embodiment, the SARS-CoV-2 S protein comprises or consists of an amino acid sequence that has no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid changes relative to any of SEQ ID NO:9-12.

[0046] In some embodiments, the SARS-CoV-2 S protein is encoded by a nucleotide sequence that is codon-optimized, such as the codon optimized sequences of SEQ ID NO: 20, or the codon optimized sequences coding for the variant Spike proteins, such as SEQ ID NO: 4 or 5 or 7 or 8. In a particular embodiment, the SARS-CoV-2 S protein is encoded by a nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20 or 4 or 5 or 7 or 8.

[0047] In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on a plasmid in the cell. In a preferred embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on the phCMV backbone (GenBank: AJ318514), in place of the VSV-G gene. 293T Cells may be transfected with Lipofectamine 2000 (Life technologies).

[0048] In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence integrated into the genome of the cell.

[0049] In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present in a lentiviral vector that is integrated into the genome of the cell. In some embodiments the lentiviral vector is a pLV-Puro vector. For example, the SARS-CoV-2 S protein can be expressed from SEQ ID NO:13 corresponding to the lentiviral vector pLV-EF1a-Spike_B.1- GJRES-Puromycin or SEQ ID NO:14 corresponding to the lentiviral vector pLV-EF1a-Spike_B.1- GJRES-Hygromycin. In a particular embodiment, the 293T cells are cell line 293T-S, deposited at the CNCM on May 5, 2020, under registration number CNCM I-5509.

[0050] In a more particular embodiment, the 293 T cells of the invention express the SARS-CoV-2 S protein of SEQ ID NO:1 or any variant thereof as disclosed above (for example SEQ ID NO:9- 12), or homologous sequences thereof, as disclosed above.

[0051] They can be obtained by using the expression plasmids having the SEQ ID NO:15 (expression plasmid phCMV_Spike_B.1-G), SEQ ID NO:16 (expression plasmid phCMV_Spike_B.1-B), SEQ ID NO:17 (expression plasmid phCMV_Spike_B.1 .1 .7), SEQ ID NO:18 (expression plasmid phCMV_Spike_B.1 .351), or SEQ ID NO:19 (expression plasmid phCMV_Spike_P.1).

SARS-CoV-2 S Protein Immune Complexes

[0052] In another aspect, the present invention provides immune complexes comprising a SARS- CoV-2 S protein present on the surface of a 293T cell or Jurkat cells and an antibody bound to the SARS-CoV-2 S protein. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on a plasmid in the cell. In a preferred embodiment, the SARS- CoV-2 S protein is expressed from a coding sequence present on the phCMV backbone (GenBank: AJ318514), in place of the VSV-G gene. In a particular embodiment, the SARS-CoV- 2 S protein is expressed from a coding sequence integrated into the genome of the cell. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present in a lentiviral vector that is integrated into the genome of the cell. In a particular embodiment, the lentiviral vector is a pLV-Puro vector. For example, the SARS-CoV-2 S protein can be expressed from SEQ ID NO:13 corresponding to the lentiviral vector pLV-EF1a-Spike_B.1-G_IRES- Puromycin or SEQ ID NO:14 corresponding to the lentiviral vector pLV-EF1 a-Spike_B.1 -GJRES- Hygromycin. They can also be obtained by using the expression plasmids having the SEQ ID NO:15 (expression plasmid phCMV_Spike_B.1-G), SEQ ID NO:16 (expression plasmid phCMV_Spike_B.1-B), SEQ ID NO:17 (expression plasmid phCMV_Spike_B.1 .1 .7), SEQ ID NO:18 (expression plasmid phCMV_Spike_B.1 .351), or SEQ ID NO:19 (expression plasmid phCMV_Spike_P.1).

[0053] In a preferred embodiment, the 293T cells are cell line 293T-S, deposited at the CNCM on May 5, 2020, under registration number CNCM I-5509.

[0054] In a preferred embodiment, said immune complexes contain the 293T cells or Jurkat cells of the invention, in particular the cells 293T-S, deposited at the CNCM on May 5, 2020, under registration number CNCM i-5509.

[0055] The antibody contained in said immune complexes may be an antibody generated by a patient’s immune system following infection with SARS-CoV-2. Alternatively, the antibody may be an antibody from any other source known in the art. In a particular embodiment, the antibody is generated by introducing (e.g., by injection) a SARS-CoV-2 S protein or antigenic fragment thereof into a mammal.

[0056] In a particular embodiment, the antibody is a polyclonal antibody. In a particular embodiment, the antibody is a monoclonal antibody. In a particular embodiment, the antibody is an IgG antibody. In a particular embodiment, the antibody is an IgM antibody. In a particular embodiment, the antibody is an IgA antibody.

[0057] In a particular embodiment, the antibody contained in said immune complexes is a chimeric antibody and/or fragment of an antibody (e.g., Fab, Fv, scFv) directed against the SARS-CoV-2 S protein.

[0058] In a particular embodiment, the anti-SARS-CoV-2 S protein antibody present in the immune complex is a patient antibody. In a particular embodiment, the anti-SARS-CoV-2 S protein antibody is present in isolated patient serum.

[0059] In a particular embodiment, the anti-SARS-CoV-2 S protein antibody present in the immune complex is labeled.

[0060] In a particular embodiment, the SARS-CoV-2 S protein present in the immune complex is expressed from a coding sequence present on a plasmid in the cell. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence integrated into the genome of the cell.

[0061] In a particular embodiment, the SARS-CoV-2 S protein present in the immune complex comprises or consists of the amino acid sequence of SEQ ID NO: 1 . In some embodiments the SARS-CoV-2 S protein comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments the SARS-CoV-2 S protein comprises or consists of an amino acid sequence that has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes relative to SEQ ID NO: 1 . In a particular embodiment, the SARS-CoV-2 S protein comprises or consists of an amino acid sequence that has no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid changes relative to SEQ ID NO: 1 .

[0062] In a more particular embodiment, the SARS-CoV-2 S present in the immune complex is SEQ ID NO:1 or any variant thereof, or homologous sequences thereof, as disclosed above.

[0063] In a more particular embodiment, the SARS-CoV-2 S present in the immune complex of the invention is a variant of SEQ ID NO:1 such as the Spike protein of SEQ ID NO:9-12, as disclosed above. In some embodiments, the SARS-CoV-2 S protein comprises or consists of an homologous amino acid sequence thereof, that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9-12. In some embodiments the SARS-CoV-2 S protein comprises or consists of an amino acid sequence that has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes relative to any of SEQ ID NO: 9-12. In a particular embodiment, the SARS-CoV-2 S protein comprises or consists of an amino acid sequence that has no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid changes relative to any of SEQ ID NO:9-12.

[0064] In a particular embodiment, the SARS-CoV-2 S protein present in the immune complex is encoded by a nucleotide sequence that comprises or consists of SEQ ID NO: 2 or 20 or 4 or 5 or 7 or 8. In some embodiments the SARS-CoV-2 S protein is encoded by a nucleotide sequence that is codon-optimized, such as a codon optimized variant of SEQ ID NO:20 or 4 or 5 or 7 or 8. In a particular embodiment, the SARS-CoV-2 S protein is encoded by a nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or 20 or 4 or 5 or 7 or 8.

[0065] In a particular embodiment, the immune complex further comprises a secondary antibody bound to the anti-SARS-CoV-2 S protein antibody. In a particular embodiment, the secondary antibody is labeled.

[0066] Preferably, the secondary antibody binds to human immunoglobulins. Kits

[0067] In another aspect, the present invention provides kits for detecting anti-SARS-CoV-2 S protein antibodies in a biological sample, said kits comprising at least 293T cells or Jurkat cells expressing a SARS-CoV-2 S protein on their surface, such as those disclosed above. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on a plasmid in the cell. In a preferred embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on the phCMV backbone (GenBank: AJ318514), in place of the VSV- G gene. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence integrated into the genome of the cell. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present in a lentiviral vector that is integrated into the genome of the cell. In a particular embodiment, the lentiviral vector is a pLV-Puro vector. For example, the SARS-CoV-2 S protein can be expressed from SEQ ID NO:13 corresponding to the lentiviral vector pLV-EF1a-Spike_B.1-G_IRES-Puromycin or SEQ ID NO:14 corresponding to the lentiviral vector pLV-EF1a-Spike_B.1-G_IRES-Hygromycin. They can also be obtained by using the expression plasmids having the SEQ ID NO:15 (expression plasmid phCMV_Spike_B.1-G), SEQ ID NO:16 (expression plasmid phCMV_Spike_B.1-B), SEQ ID NO:17 (expression plasmid phCMV_Spike_B.1 .1 .7), SEQ ID NO:18 (expression plasmid phCMV_Spike_B.1 .351), or SEQ ID NO:19 (expression plasmid phCMV_Spike_P.1).

[0068] In a preferred embodiment, the 293T cells contained in the kit of the invention are cell line 293T-S, deposited at the CNCM on May 5, 2020, under registration number CNCM I-5509.

[0069] In a particular embodiment, the SARS-CoV-2 S protein expressed by the cells in the kit has an amino acid sequence at least 95% identical to SEQ ID NO: 1 . In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence having a nucleotide sequence at least 80% identical to SEQ ID NO: 2. In a particular embodiment, the kit further comprises a control anti-SARS-CoV-2 S protein antibody. In a particular embodiment, the control anti-SARS- CoV-2 S protein antibody is a patient antibody. In a particular embodiment, the kit further comprises secondary antibodies that bind to the anti-SARS-CoV-2 S protein antibodies.

[0070] In a more particular embodiment, the SARS-CoV-2 S protein expressed by the cells in the kit of the invention is SEQ ID NO:1 or any variant thereof, or homologous sequences thereof, as disclosed above.

[0071] In a preferred embodiment, the SARS-CoV-2 S protein expressed by the cells in the kit comprises or consists of the amino acid sequence of SEQ ID NO: 1 . In a particular embodiment, the SARS-CoV-2 S protein expressed by the cells in the kit comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 . In a particular embodiment, the SARS-CoV-2 S protein expressed by the cells in the kit comprises or consists of an amino acid sequence that has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes relative to SEQ ID NO: 1. In a particular embodiment, the SARS-CoV-2 S protein expressed by the cells in the kit comprises or consists of an amino acid sequence that has no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid changes relative to SEQ ID NO: 1 .

[0072] In a more particular embodiment, the SARS-CoV-2 S expressed by the cells in the kit of the invention is a variant of SEQ ID NO:1 such as the Spike protein of SEQ ID NO:9-12, as disclosed above. In some embodiments, the SARS-CoV-2 S protein comprises or consists of an homologous amino acid sequence thereof, that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9-12. In some embodiments, the SARS-CoV-2 S protein expressed by the cells in the kit of the invention comprises or consists of an amino acid sequence that has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes relative to any of SEQ ID NO: 9-12. In a particular embodiment, the SARS-CoV-2 S protein expressed by the cells in the kit of the invention comprises or consists of an amino acid sequence that has no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid changes relative to any of SEQ ID NO:9-12.

[0073] In a particular embodiment, the SARS-CoV-2 S protein expressed by the cells in the kit of the invention is encoded by a nucleotide sequence that comprises or consists of SEQ ID NO: 2 or 20 or 4 or 5 or 7 or 8. In some embodiments the SARS-CoV-2 S protein is encoded by a nucleotide sequence that is codon-optimized, such as a codon optimized variant of SEQ ID NO:20 or 4 or 5 or 7 or 8. In a particular embodiment, the SARS-CoV-2 S protein expressed by the cells in the kit of the invention is encoded by a nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or 20 or 4 or 5 or 7 or 8.

[0074] In a preferred embodiment, the kit of the invention may also contain an antibody generated by a patient’s immune system following infection with SARS-CoV-2. Alternatively, the antibody may be an antibody from any other source known in the art. In some embodiments the antibody is generated by introducing (e.g., by injection) a SARS-CoV-2 S protein or antigenic fragment thereof into a mammal.

[0075] In a particular embodiment, the antibody is a polyclonal antibody. In a particular embodiment, the antibody is a monoclonal antibody. In a particular embodiment, the antibody is an IgG antibody. In a particular embodiment, the antibody is an IgM antibody. In a particular embodiment, the antibody is an IgA antibody.

[0076] In a particular embodiment, the antibody is a chimeric antibody and/or fragment of an antibody (e.g., Fab, Fv, scFv) directed against the SARS-CoV-2 S protein.

[0077] In a particular embodiment, the anti-SARS-CoV-2 S protein antibody is a patient antibody. In a particular embodiment, the anti-SARS-CoV-2 S protein antibody is present in isolated patient serum.

[0078] In a particular embodiment, the anti-SARS-CoV-2 S protein antibody is labeled.

[0079] In a particular embodiment, the kit of the invention further comprises a secondary antibody that can bind to an anti-SARS-CoV-2 S protein antibody. In a particular embodiment, the secondary antibody is labeled.

[0080] Preferably, the secondary antibody binds to human immunoglobulins.

[0081] In one embodiment, the secondary antibody or an antibody fragment that binds to human immunoglobulins binds specifically to IgG, IgA, and IgM. In one embodiment, the antibody or an antibody fragment that binds to human immunoglobulins binds specifically to IgG, IgA, or IgM. In a more particular embodiment, the kit of the invention contains several sets of secondary antibodies, similarly or differentially labelled, in order to detect simultaneously human IgG and IgA, or human IgG and IgM, or human IgA and IgM, or human IgA, IgG and IgM. This kit can then be used for multiplex analysis of the antibodies present in the patient sample, as shown in example 2.

[0082] In a particular embodiment, the kit comprises a control serum sample from a patient having a known anti-SARS-CoV-2 S protein antibody titer.

[0083] In a preferred embodiment, the kit of the invention also contains the control cell line 293T- CTRL, deposited at the CNCM on May 5, 2020, under registration number CNCM I-5508.

Methods For Detecting Antibodies Against a SARS-CoV-2 S Protein

[0084] In another aspect, the present invention provides in vitro methods for detecting antibodies against a SARS-CoV-2 S protein in a biological sample. In a particular embodiment, the methods comprise providing eukaryotic cells expressing a SARS-CoV-2 S protein on their surface; providing a biological sample from a patient; contacting the cells with the biological sample; and assaying for binding between the SARS-CoV-2 S protein present on the surface of the cells and antibodies present in the sample. The skilled artisan will appreciate that any suitable type of eukaryotic cells may be used. In a preferred embodiment, the eukaryotic cells are mammalian cells. In a preferred embodiment, the eukaryotic cells are primate cells. In a more preferred embodiment, the eukaryotic cells are human cells.

[0085] In a particular embodiment, the methods of the invention use the cells of the invention, the immune complexes of the invention and/or the kits of the invention, as disclosed above. In specific embodiment, the methods comprise providing Jurkat cells expressing a SARS-CoV-2 S protein on their surface; providing a biological sample from a patient; contacting the cells with the biological sample; and assaying for binding between the SARS-CoV-2 S protein present on the surface of the cells and antibodies present in the sample. In specific embodiment, the methods comprise providing 293T cells expressing a SARS-CoV-2 S protein on their surface; providing a biological sample from a patient; contacting the 293T cells with the biological sample; and assaying the presence of the immune complexes of the invention. In particular, the methods comprise assaying the binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on a plasmid in the cell. In a preferred embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on the phCMV backbone (GenBank: AJ318514), in place of the VSV-G gene. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence integrated into the genome of the cell. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present in a lentiviral vector that is integrated into the genome of the cell. In some embodiments the lentiviral vector is a pLV-Puro vector. For example, the SARS-CoV-2 S protein can be expressed from SEQ ID NO:13 corresponding to the lentiviral vector pLV-EF1 a-Spike_B.1-G_IRES-Puromycin or SEQ ID NO:14 corresponding to the lentiviral vector pLV-EF1 a-Spike_B.1-G_IRES-Hygromycin. They can also be obtained by using the expression plasmids having the SEQ ID NO:15 (expression plasmid phCMV_Spike_B.1-G), SEQ ID NO:16 (expression plasmid phCMV_Spike_B.1-B), SEQ ID NO:17 (expression plasmid phCMV_Spike_B.1 .1 .7), SEQ ID NO:18 (expression plasmid phCMV_Spike_B.1 .351), or SEQ ID NO:19 (expression plasmid phCMV_Spike_P.1).

[0086] In a preferred embodiment, the 293T cells are cell line 293T-S, deposited at the CNCM on May 5, 2020, under registration number CNCM I-5509. In a particular embodiment, the SARS- CoV-2 S protein has an amino acid sequence at least 90% identical to SEQ ID NO: 1. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence having a nucleotide sequence at least 60% identical to SEQ ID NO: 2. [0087] In a preferred embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of the amino acid sequence of SEQ ID NO: 1 .

[0088] In a more particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods is SEQ ID NO:1 or any variant thereof, or any homologous sequences thereof, as disclosed above.

[0089] In a particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In a particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of an amino acid sequence that has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes relative to SEQ ID NO: 1. In a particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of an amino acid sequence that has no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid changes relative to SEQ ID NO: 1 .

[0090] In a more particular embodiment, the SARS-CoV-2 S carried or expressed by the cells used in these methods is a variant of SEQ ID NO:1 such as the Spike protein of SEQ ID NO:9-12, as disclosed above. In some embodiments, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of an homologous amino acid sequence thereof, that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9-12. In some embodiments the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of an amino acid sequence that has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes relative to any of SEQ ID NO: 9-12. In a particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of an amino acid sequence that has no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid changes relative to any of SEQ ID NO:9-12.

[0091 ] In a particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods is encoded by a nucleotide sequence that comprises or consists of SEQ ID NO: 2 or 20 or 4 or 5 or 7 or 8. In some embodiments the SARS-CoV-2 S protein is encoded by a nucleotide sequence that is codon-optimized, such as a codon optimized variant of SEQ ID NO:20 or 4 or 5 or 7 or 8. In a particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods is encoded by a nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or 20 or 4 or 5 or 7 or 8.

[0092] In a particular embodiment, assaying for binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample is performed by a method comprising visualizing antigen-antibody complexes. In a particular embodiment, assaying for binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells comprises contacting the 293T cells or Jurkat cells with a secondary antibody against antibodies present in the sample and visualizing antigen-antibody complexes. In a particular embodiment, the binding of antibodies present in the sample is detected on the surface of at least 20% of the 293T cells or Jurkat cells of the invention contacted with the sample. In a particular embodiment, the binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample is detected using a fluorescent activated cell sorting (FACS) assay.

[0093] In a particular embodiment, the binding of antibodies present in the sample is detected on the surface of at least 20%, at least 30%, at least 40%, or at least 50% of the 293T cells or Jurkat cells of the invention contacted with the sample. In a particular embodiment, the binding of antibodies present in the sample is not detected on the surface of at least 20% of the 293T cells or Jurkat cells of the invention contacted with the sample. In a particular embodiment, the binding of antibodies present in the sample is not detected on the surface of at least 20%, at least 30%, at least 40%, or at least 50% of the 293T cells or Jurkat cells of the invention contacted with the sample.

[0094] In other terms, the methods of the invention conclude that the sample contains a significant amount of anti-SARS-CoV-2 antibodies if at least 20%, at least 30%, at least 40%, or at least 50% of the 293T cells or Jurkat cells of the invention, contacted with the patient’s sample, are bound to secondary antibodies, preferably to labelled secondary antibodies, indirectly via the first patient’s antibody.

[0095] The antibody detected by means of the methods of the invention may be an antibody generated by a patient’s immune system following infection with SARS-CoV-2. Alternatively, the antibody may be an antibody from any other source known in the art. In a particular embodiment, the antibody is generated by introducing (e.g., by injection) a SARS-CoV-2 S protein or antigenic fragment thereof into a mammal. [0096] In a particular embodiment, the antibody detected by means of the methods of the invention is a polyclonal antibody. In a particular embodiment, the antibody detected by means of the methods of the invention is a monoclonal antibody. In a particular embodiment, the antibody detected by means of the methods of the invention is a IgG antibody. In a particular embodiment, the antibody is an IgM antibody. In a particular embodiment, the antibody is an IgA antibody.

[0097] In a particular embodiment, the antibody detected by means of the methods of the invention is a chimeric antibody and/or fragment of an antibody (e.g., Fab, Fv, scFv) directed against the SARS-CoV-2 S protein.

[0098] In a particular embodiment, the anti-SARS-CoV-2 S protein antibody detected by the methods of the invention is a patient antibody. In a particular embodiment, the anti-SARS-CoV-2 S protein antibody detected by the methods of the invention is present in isolated patient serum.

[0099] In a particular embodiment, the anti-SARS-CoV-2 S protein antibody is labeled.

[0100] In a particular embodiment, the methods of the invention require to use a secondary antibody that will bind to the anti-SARS-CoV-2 S protein antibody of the patient, once bound to the cell of the invention. In some embodiments, the secondary antibody is labeled.

[0101 ] Preferably, said secondary antibody binds to human immunoglobulins.

[0102] In one embodiment, said secondary antibody or an antibody fragment that binds to human immunoglobulins binds specifically to IgG, IgA, and IgM. In a more particular embodiment, the methods of the invention use several sets of secondary antibodies, similarly or differentially labelled, in order to detect simultaneously human IgG and IgA, or human IgG and IgM, or human IgA and IgM, or human IgA, IgG and IgM. These methods can then be used for detecting simultaneously, in a multiplex assay, the antibodies present in the patient sample, as shown in example 2.

[0103] In a preferred embodiment, the methods of the invention also use the control cell line 293T- CTRL, deposited at the CNCM on May 5, 2020, under registration number CNCM I-5508. The percentage of labelled control cells can for example be used to highlight and calculate specific binding, when compared to the labelled cells of the invention expressing the S protein.

Methods for Diagnosing a SARS-CoV-2 Infection

[0104] In another aspect, this present invention provides methods for diagnosing a SARS-CoV-2 infection in a patient. These methods may comprise providing eukaryotic cells expressing a SARS-CoV-2 S protein on their surface; providing a biological sample from a patient; contacting the cells with the biological sample; and assaying for binding between the SARS-CoV-2 S protein present on the surface of the cells and antibodies present in the sample. The skilled artisan will appreciate that any suitable type of eukaryotic cells may be used. In a preferred embodiment, the eukaryotic cells are mammalian cells. In a preferred embodiment the eukaryotic cells are primate cells. In a more preferred embodiment, the eukaryotic cells are human cells.

[0105] The methods may use the cells of the invention, the immune complexes of the invention and/or the kits of the invention, as disclosed above. As a matter of fact, these kits comprise 293T cells or Jurkat cells expressing a SARS-CoV-2 S protein on their surface. The methods of the invention then comprise providing a biological sample from a patient; contacting the 293T cells or Jurkat cells with the biological sample; and assaying the binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on a plasmid in the cell. In a preferred embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present on the phCMV backbone (GenBank: AJ318514), in place of the VSV-G gene. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence integrated into the genome of the cell. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence present in a lentiviral vector that is integrated into the genome of the cell. In a particular embodiment, the lentiviral vector is a pLV- Puro vector. In a preferred embodiment, the 293T cells are cell line 293T-S, deposited at the CNCM on May 5, 2020, under registration number CNCM I-5509. For example, the SARS-CoV- 2 S protein can be expressed from SEQ ID NO:13 corresponding to the lentiviral vector pLV- EF1 a-Spike_B.1 -GJRES-Puromycin or SEQ ID NO:14 corresponding to the lentiviral vector pLV- EF1a-Spike_B.1-G_IRES-Hygromycin. They can also be obtained by using the expression plasmids having the SEQ ID NO:15 (expression plasmid phCMV_Spike_B.1-G), SEQ ID NO:16 (expression plasmid phCMV_Spike_B.1-B), SEQ ID NO:17 (expression plasmid phCMV_Spike_B.1 .1 .7), SEQ ID NO:18 (expression plasmid phCMV_Spike_B.1 .351), or SEQ ID NO:19 (expression plasmid phCMV_Spike_P.1).

[0106] In a particular embodiment, the SARS-CoV-2 S protein has an amino acid sequence at least 90% identical to SEQ ID NO: 1. In a particular embodiment, the SARS-CoV-2 S protein is expressed from a coding sequence having a nucleotide sequence at least 60% identical to SEQ ID NO: 2.

[0107] In a more particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods is SEQ ID NO:1 or any variant thereof, or any homologous sequences thereof, as disclosed above. [0108] In a preferred embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of the amino acid sequence of SEQ ID NO: 1. In a particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In a particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of an amino acid sequence that has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes relative to SEQ ID NO: 1 . In a particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of an amino acid sequence that has no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid changes relative to SEQ ID NO: 1 .

[0109] In a more particular embodiment, the SARS-CoV-2 S carried or expressed by the cells used in these methods is a variant of SEQ ID NO:1 such as the Spike protein of SEQ ID NO:9-12, as disclosed above. In some embodiments, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of an homologous amino acid sequence thereof, that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9-12. In some embodiments the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of an amino acid sequence that has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes relative to any of SEQ ID NO: 9-12. In a particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods comprises or consists of an amino acid sequence that has no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid changes relative to any of SEQ ID NO:9-12.

[0110] In a particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods is encoded by a nucleotide sequence that comprises or consists of SEQ ID NO: 2 or 20 or 4 or 5 or 7 or 8. In some embodiments the SARS-CoV-2 S protein is encoded by a nucleotide sequence that is codon-optimized, such as a codon optimized variant of SEQ ID NO:20 or 4 or 5 or 7 or 8. In a particular embodiment, the SARS-CoV-2 S protein carried or expressed by the cells used in these methods is encoded by a nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or 20 or 4 or 5 or 7 or 8.

[0111] In a particular embodiment, the detection of the binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample indicates that the patient is infected or was infected with SARS-CoV-2. In a particular embodiment, the absence of detection of the binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample indicates that the patient is not infected or was not infected with SARS-CoV-2. In a particular embodiment, the detection of the binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample indicates that the patient is infected or was infected with SARS-CoV-2; and the absence of detection of binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample indicates that the patient is not infected or was not infected with SARS- CoV-2. In a particular embodiment, the binding of antibodies present in the sample is detected on the surface of at least 20% of the 293T cells or Jurkat cells of the invention contacted with the sample, indicating that the patient is infected or was infected with SARS-CoV-2. In a particular embodiment, the binding of antibodies present in the sample is detected on the surface of at least 20%, at least 30%, at least 40%, or at least 50% of the 293T cells or Jurkat cells of the invention contacted with the sample, indicating that the patient is infected or was infected with SARS-CoV- 2. In a particular embodiment, the binding of antibodies present in the sample is not detected on the surface of at least 20% of the 293T cells or Jurkat cells of the invention contacted with the sample, indicating that the patient is not infected or was not infected with SARS-CoV-2 In a particular embodiment, the binding of antibodies present in the sample is not detected on the surface of at least 20%, at least 30%, at least 40%, or at least 50% of the 293T cells or Jurkat cells of the invention contacted with the sample, indicating that the patient is not infected or was not infected with SARS-CoV-2.

[0112] In other terms, the methods of the invention conclude that the patient is infected or was infected with SARS-CoV-2 if at least 20%, at least 30%, at least 40%, or at least 50% of the 293T cells of the invention, contacted with the patient’s sample, are bound to secondary antibodies, preferably to labelled secondary antibodies, indirectly via the patient’s antibodies.

[0113] In a particular embodiment, assaying the binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample is performed by a method comprising visualizing antigen-antibody complexes. In a particular embodiment, assaying for binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells comprises contacting the 293T cells or Jurkat cells of the invention with a secondary antibody against antibodies present in the sample and visualizing antigen-antibody complexes. In a particular embodiment, the binding between the SARS-CoV-2 S protein present on the surface of the 293T cells or Jurkat cells and antibodies present in the sample is detected using a fluorescent activated cell sorting (FACS) assay.

[0114] The antibody that is detected may be an antibody generated by a patient’s immune system following infection with SARS-CoV-2. Alternatively, the antibody may be an antibody from any other source known in the art. In a particular embodiment, the antibody is generated by introducing (e.g., by injection) a SARS-CoV-2 S protein or antigenic fragment thereof into a mammal.

[0115] In some embodiments the antibody is a polyclonal antibody. In some embodiments the antibody is a monoclonal antibody. In some embodiments, the antibody is a IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody is an IgA antibody.

[0116] In some embodiments the antibody is a chimeric antibody and/or fragment of an antibody (e.g., Fab, Fv, scFv) directed against the SARS-CoV-2 S protein.

[0117] In a particular embodiment, the anti-SARS-CoV-2 S protein antibody which is detected by the methods of the invention is a patient’s antibody. In a particular embodiment, the anti-SARS- CoV-2 S protein antibody which is detected by the methods of the invention is present in an isolated patient serum.

[0118] In a particular embodiment, the methods of the invention need using a secondary antibody that can bind to the anti-SARS-CoV-2 S protein antibody. In a particular embodiment, said secondary antibody is labeled.

[0119] Preferably, said secondary antibody binds to human immunoglobulins.

[0120] In one embodiment, the secondary antibody or an antibody fragment that binds to human immunoglobulins binds specifically to IgG, IgA, and IgM. In a more particular embodiment, the methods of the invention use several sets of secondary antibodies, similarly or differentially labelled, in order to detect simultaneously human IgG and IgA, or human IgG and IgM, or human IgA and IgM, or human IgA, IgG and IgM. These methods can then be used for detecting simultaneously, in a multiplex assay, the antibodies present in the patient sample, as shown in example 2.

[0121] In a preferred embodiment, the diagnostic methods of the invention also use the control cell line 293T-CTRL, deposited at the CNCM on May 5, 2020, under registration number CNCM I- 5508. The percentage of labelled control cells can for example be used to highlight and calculate specific binding, when compared to the labelled cells of the invention expressing the S protein. DEFINITIONS

[0122] As used herein, the term “antibody” or “antibodies” is meant to include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, fragments thereof, such as F(ab')2 and Fab fragments, single-chain variable fragments (scFvs), single-domain antibody fragments (VHHs or Nanobodies), bivalent antibody fragments (diabodies), as well as any recombinantly and synthetically produced binding partners.

[0123] In some embodiments, the antibody is a camelid VHH.

[0124] In a preferred embodiment, the antibody is an alpaca VHH.

[0125] For the purposes of the present invention, the expression “chimeric antibody” is understood to mean, in relation to an antibody of a particular animal species or of a particular class of antibody, an antibody of a given animal species and/or class of antibody comprising all or part of a heavy chain and/or of a light chain of an antibody of another animal species and/or of another class of antibody.

[0126] Purified proteins can be used to produce antibodies by conventional techniques. Recombinant or synthetic proteins or peptides can also be used to produce antibodies by conventional techniques.

[0127] The antibodies used in the invention can be synthetic, semi-synthetic, monoclonal, or polyclonal and can be made by techniques well known in the art. Such antibodies specifically bind to proteins and polypeptides via the antigen-binding sites of the antibody (as opposed to non-specific binding). Purified or synthetic proteins and peptides can be employed as immunogens in producing antibodies immunoreactive therewith. The proteins and peptides contain antigenic determinants or epitopes that elicit the formation of antibodies.

[0128] These antigenic determinants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon protein folding (C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded proteins have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the protein and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes can be identified by any of the methods known in the art. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.

[0129] Antibodies are defined to be specifically binding if they bind proteins or polypeptides with a Ka of greater than or equal to about 10 ⁷ M- ¹ . Affinities of binding partners or antibodies can be readily determined using conventional techniques, for example those described by Scatchard et al., Ann. N.Y. Acad. ScL, 51 :660 (1949).

[0130] Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, alpaca, camels, rabbits, mice, or rats, using procedures that are well known in the art. In general, a purified protein or polypeptide that is appropriately conjugated is administered to the host animal typically through parenteral injection. The immunogenicity can be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immunizations, small samples of serum are collected and tested for reactivity to proteins or polypeptides. Examples of various assays useful for such determination include those described in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures, such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radio-immunoprecipitation, enzyme-linked immunosorbent assays (ELISA), dot blot assays, and sandwich assays. See U.S. Pat. Nos. 4,376,110 and 4,486,530.

[0131] Monoclonal antibodies can be readily prepared using well known procedures. See, for example, the procedures described in U.S. Pat. Nos. RE 32,011 , 4,902,614, 4,543,439, and 4,411 ,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKeam, and Bechtol (eds.), 1980.

[0132] For example, the host animals, such as mice, can be injected intraperitoneally at least once and preferably at least twice at about 3 week intervals with isolated and purified proteins or conjugated polypeptides, for example a peptide comprising or consisting of the specific amino acids set forth above. Mouse sera are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse. Approximately two to three weeks later, the mice are given an intravenous boost of the protein or polypeptide. Mice are later sacrificed, and spleen cells fused with commercially available myeloma cells, such as Ag8.653 (ATCC), following established protocols. Briefly, the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell. The fusing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG). Fusion is plated out into plates containing media that allows for the selective growth of the fused cells. The fused cells can then be allowed to grow for approximately eight days. Supernatants from resultant hybridomas are collected and added to a plate that is first coated with goat anti-mouse Ig. Following washes, a label, such as a labeled protein or polypeptide, is added to each well followed by incubation. Positive wells can be subsequently detected. Positive clones can be grown in bulk culture and supernatants are subsequently purified over a Protein A column (Pharmacia).

[0133] The monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al. , "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 3:1 -9 (1990). Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, 7:394 (1989).

[0134] Antigen-binding fragments of such antibodies, which can be produced by conventional techniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab')2 fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.

[0135] The monoclonal antibodies herein disclosed include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies can be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment can comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, May, 1993). Procedures to generate antibodies transgenically can be found in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806.

[0136] Antibodies produced by genetic engineering methods, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, can be used. Such chimeric and humanized monoclonal antibodies can be produced by genetic engineering using standard DNA techniques known in the art, for example using methods described in Robinson et al. International Publication No. WO 87/02671 ; Akira, et al. European Patent Application 0184187; Taniguchi, M., European Patent Application 0171496; Morrison et al. European Patent Application 0173494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 0125023; Better et al., Science 240:1041 1043, 1988; Liu et al., PNAS 84:3439 3443, 1987; Liu et al., J. Immunol. 139:3521 3526, 1987; Sun et al. PNAS 84:214218, 1987; Nishimura et al., Cane. Res. 47:999 1005, 1987; Wood et al., Nature 314:446 449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80:1553 1559, 1988); Morrison, S. L., Science 229:1202 1207, 1985; Oi et al., BioTechniques 4:214, 1986; Winter U.S. Pat. No. 5,225,539; Jones et al., Nature 321 :552 525, 1986; Verhoeyan et al., Science 239:1534, 1988; and Beidler et al., J. Immunol. 141 :40534060, 1988.

[0137] In connection with synthetic and semi-synthetic antibodies, such terms are intended to cover but are not limited to antibody fragments, isotype switched antibodies, humanized antibodies (e.g., mouse-human, human-mouse), hybrids, antibodies having plural specificities, and fully synthetic antibody-like molecules.

[0138] In one embodiment, the kits and methods of the invention invention can use single-domain antibodies (sdAb), also known as NANOBODIES. A sdAb is a fragment consisting of a single monomeric variable antibody domain that can bind selectively to a specific antigen. In one embodiment, the sdAbs are from heavy-chain antibodies found in camelids (VHH fragments), or cartilaginous fishes (VNAR fragments), or are obtained by splitting dimeric variable domains into monomers.

[0139] As used herein, the term “label” designates any label that is selected from a chemiluminescent label, an enzyme label, a fluorescence label, and a radioactive (e.g., iodine) label. In a preferred embodiment, the secondary antibody used in the immune complexes, the kits and methods of the invention is a labeled antibody or antibody fragment that binds to human immunoglobulins.

[0140] Preferred labels include a fluorescent label, such as FITC, a chromophore label, an affinity- ligand label, an enzyme label, such as alkaline phosphatase, horseradish peroxidase, or b galactosidase, an enzyme cofactor label, a hapten conjugate label, such as digoxigenin or dinitrophenyl, a Raman signal generating label, a magnetic label, a spin label, an epitope label, such as the FLAG or HA epitope, a luminescent label, a heavy atom label, a nanoparticle label, an electrochemical label, a light scattering label, a spherical shell label, semiconductor nanocrystal label, wherein the label can allow visualization with or without a secondary detection molecule.

[0141] Preferred labels include suitable enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; members of a binding pair that are capable of forming complexes such as streptavidin/biotin, avidin/biotin or an antigen/antibody complex including, for example, rabbit IgG and anti-rabbit IgG; fluorophores such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine, eosin, green fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue, Texas Red, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanide complexes such as those including Europium and Terbium, cyanine dye family members, such as Cy3 and Cy5, molecular beacons and fluorescent derivatives thereof, as well as others known in the art; a luminescent material such as luminol; light scattering or plasmon resonant materials such as gold or silver particles or quantum dots; or radioactive material include 14C, 1231, 1241, 1251, 32P, 33P, 35S, or 3H.

[0142] As used herein, the term “biological sample” refers to any sample containing antibodies from a subject. Antibodies may be contained in a solid tissue, in fluids and/or excretions of said subject. Said fluid is for example blood, serum, plasma, saliva, nasal swabs or urine. In a preferred embodiment, said biological sample is a blood sample of said subject, bone marrow or spleen or skin biopsies, or any other cells. Such a blood sample may be obtained by a completely harmless blood collection from the subject and thus allows for a non-invasive diagnosis.

[0143] As used herein, the term “subject” refers to any mammal, preferably a human. Said subject may be a healthy individual or an individual presenting symptoms from the COVID disease.

EXAMPLES

1 . EXAMPLE 1 : set up of the IgM/lgG S-FLOW assay

1. 1. Materials

A. Cohorts

[0144] Pre-epidemic sera originated from two pre-epidemic healthy donors’ sources: 200 sera from the Diagmicoll cohort collection of ICAReB platform29 approved by CPP Ile-de-France I, sampled before november 2019. 200 anonymized samples from blood donors recruited in March 2017 at the Val d’Oise sites of Etablissement Frangais du Sang (EFS, the French blood agency). The ICAReB platform (BRIF code n°BB-0033-00062) of Institut Pasteur collects and manages bioresources following ISO 9001 and NF S 96-900 quality standards 29.

[0145] COVID-19 cases were from included at Hopital Bichat-Claude-Bernard in the French COVID- 19 cohort. Some of the patients have been previously described ²⁴ Each participant provided written consent to participate to the study, which was approved by the regional investigational review board (IRB; Comite de Protection des Personnes Ile-de-France VII, Paris, France) and performed according to the European guidelines and the Declaration of Helsinki.

[0146] Pauci-symptomatic individuals: On Feb 24, 2020, a patient from Crepy-en-Valois (Oise region, northern France) was admitted to a hospital in Paris with confirmed SARS-CoV-2 infection. As part of an epidemiological investigation around this case, a cluster of COVID-19 cases, based around a high school with an enrolment of 1200 pupils, was identified. On March 3-4, students at the high school, their parents, teachers and staff (administrative staff, cleaners, catering staff) were invited to participate to the investigation. A 5 mL blood sample was taken from 209 individuals who reported fever or mild respiratory symptoms (cough or dyspnea) since mid-January 2020. The median age was 18 years (interquartile range: 17-45), and 65 % were female.

[0147] Samples from blood donors were collected by EFS (Lille, France) in Clermont (Oise) on March 20 and Noyon (Oise) on March 24, both cities are located at 60 kilometers from Crepy-en- Valois.

[0148] All sera were heat-inactivated 30-60 min at 56°C, aliquoted and conserved at 4°C for short term use or frozen.

B. ELISA-N

[0149] A codon-optimized nucleotide fragment encoding full length nucleoprotein was synthetized and cloned into pETM11 expression vector (EMBL). The His-tagged SARS-CoV-2 N protein was bacterially expressed in E. coli BL21 (DE3) and purified as a soluble dimeric protein by affinity purification using a Ni-NTA Protino column (Macherey Nagel) and gel filtration using a Hiload 16/60 superdex 200 pg column (HE Healthcare). 96-well ELISA plates were coated overnight with N in PBS (50 ng/well in 50 mI). After washing 4 times with PBS-0.1% Tween 20 (PBST), 100 mI of diluted sera (1 :200) in PBST-3% milk were added and incubated 1 h at 37Ό. After washing 3 times with PBST, plates were incubated with 8,000-fold diluted peroxydase-conjugated goat anti-human IgG (Southern Biotech) for 1 h. Plates were revealed by adding 100 mI of HRP chromogenic substrate (TMB, Eurobio Scientific) after 3 washing steps in PBST. After 30 min incubation, optical densities were measured at 405 nm (OD 405). OD measured at 620 nm was subtracted from values at 405 nm for each sample.

C. ELISA tri-S

[0150] A codon-optimized nucleotide fragment encoding a stabilized version of the SARS-CoV-2 S ectodomain (amino acid 1 to 1208) followed by a foldon trimerization motif and tags (8xHisTag, StrepTag, and AviTag) was synthetized and cloned into pcDNA™3.1/Zeo(+) expression vector (Thermo Fisher Scientific). Trimeric S (tri-S) glycoproteins were produced by transient co transfection of exponentially growing Freestyle™ 293-F suspension cells (Thermo Fisher Scientific, Waltham, MA) using polyethylenimine (PEI)-precipitation method as previously described ³⁰ . Recombinant tri-S proteins were purified by affinity chromatography using the Ni Sepharose® Excel Resin according to manufacturer’s instructions (ThermoFisher Scientific). Protein purity was evaluated by in-gel protein silver-staining using Pierce® Silver Stain kit (ThermoFisher Scientific) following SDS-PAGE in reducing and non-reducing conditions using NuPAGE™ 3-8% Tris-Acetate gels (Life Technologies). High-binding 96-well ELISA plates (Costar, Corning) were coated overnight with 125 ng/well of purified tri-S proteins in PBS. After washings with PBS-0.1% Tween 20 (PBST), plate wells were blocked with PBS-1% Tween 20- 5%sucrose-3% milk powder for 2 h. After PBST washings, 1 :100-diluted sera in PBST-1% BSA and 7 consecutive 1 :4 dilutions were added and incubated 2 h. After PBST washings, plates were incubated with 1 ,000-fold diluted peroxydase-conjugated goat anti-human IgG/lgM/lgA (Immunology Jackson ImmunoReseach, 0.8 pg/ml final) for 1 h. Plates were revealed by adding 100 mI of HRP chromogenic substrate (ABTS solution, Euromedex) after PBST washings. Optical densities were measured at 405nm (OD405nm) following a 30 min incubation. Experiments were performed in duplicate at room temperature and using HydroSpeed™ microplate washer and Sunrise™ microplate absorbance reader (Tecan Mannedorf, Switzerland). Area under the curve (AUC) values were determined by plotting the logio of the dilution factor values (x axis) required to obtain OD405nm values (y axis). AUC calculation and Receiving Operating Characteristics (ROC) analyses were performed using GraphPad Prism software (v8.4.1 , GraphPad Prism Inc.).

D. S-Flow Assay

[0151] HEK293T (referred as 293T) cells were from ATCC (ATCC® CRL-3216™) and tested negative for mycoplasma. Cells were split every 2-3 days using DMEM medium supplemented with 10% fetal calf serum and 1% Penicillin streptomycin (complete medium). A codon optimized version of the SARS-Cov-2 S gene (GenBank: QHD43416.1 , SEQ ID NO:1)\ was transferred into the phCMV backbone (GenBank: AJ318514), by replacing the VSV-G gene. 293T Cells were transfected with S or a control plasmid using Lipofectamine 2000 (Life technologies). One day after, transfected cells were detached using PBS-EDTA and transferred into U-bottom 96-well plates (50,000 cell/well). Cell were incubated at 4°C for 30 min with sera (1 :300 dilution, unless otherwise specified) in PBS containing 0.5% BSA and 2 mM EDTA, washed with PBS, and stained using either anti-lgG AF647 (ThermoFisher) or Anti-lgM (PE by Jackson ImmunoResearch or AF488 by ThermoFisher). Cells were washed with PBS and fixed 10 min using 4% PFA. Data were acquired on an Attune Nxt instrument (Life Technologies). In less than 0.5% of the samples tested, we detected a signal in control 293T cells, likely corresponding to antibodies binding to other human surface antigens. Specific binding was calculated with the formula: 100 x (% binding on 293T-S - binding on control cells)/(100 - binding on control cells). We generated stably- expressing 293T S cells during completion of this study, which yielded similar results.

[0152] A 293T genetically modified cell line, named 293T-S, genetically modified with the pLV - SARS-cov-2 S - Puro vector of SEQ ID NO:13, was deposited with the Collection Nationale de Cultures de Microorganismes on May 5, 2020, under registration number CNCM I-5509.

[0153] A 293T genetically modified cell line, named 293T-CTRL, genetically modified with a pLV - Empty - Puro vector, was deposited with the Collection Nationale de Cultures de Microorganismes on May 5, 2020, under registration number CNCM I-5508.

[0154] Representative procedures for the assay are as follows. [0155] Reagents:

293T-S cells (293T cells expressing the Spike protein of SARS-cov-2), 293T-CTLR cells (293T cells expressing a Empty transgene),

Complete medium: DMEM (Gibco) + 10%FCS + 1% PenStrep (Gibco), PBS-EDTA : PBS (Gibco) + 2 mM EDTA (Sigma), - U-Bottom 96 well plate,

Staining Buffer: PBS (Gibco) + 0.5% BSA (Sigma) + 2 mM EDTA (Sigma), PBS (Gibco), anti-Hu IgG Alexafluor 647 antibody (ref: A21445, Invitrogen), and PFA 2%: dilution 1 :1 of PFA 4% (ref: J61899, Alfa Aesar) and PBS (Gibco). [0156] Step-by-step protocol:

1 ) prepare the 293T-S and 293T-CTLR cells: a) remove the medium from the culture flask, b) wash with 10ml_ of PBS EDTA, c) remove PBS-EDTA and leave the falsk in the incubator for 5min to detach cells, d) Recover cells with 5mL of Complete medium, re-seed 1 mL (in 12mL final) of cells to maintain the culture and keep 4mL for the assay, and e) Count the cells;

2) seed 50,000 cells per well of a 96 well plate. Each samples must be interrogated using a well of 293T-S and separate well of 293-E cells;

3) Spin 2min@2000rpm;

4) Remove the supernatant using a multi-channel;

5) Incubate 30min at 4°C with 50uL of serum diluted 1 :300 in Staining Buffer;

6) add 150uL of PBS;

7) Spin 2min@2000rpm;

8) Remove the supernatant using a multi-channel;

9) Incubate 30min at 4°C with 35uL of anti-Hu AlexaFluor 647 antibody diluted 1 :600 in Staining Buffer;

10) add 150uL of PBS;

11) Spin 2min@2000rpm;

12) Remove the supernatant using a multi-channel;

13) add 40uL of PFA 2% and incubate 15min at room temperature;

14) add 120uL of PBS;

15) acquired 90uL using a cytometer within 24h;

16) To analyze the data, normalized the % or S positive cells observed in the 293-S using the frequency of % or S positive cells observed in the 293-E using the formula:

(((% of S+ 293T-S)-(% of S+ 293T-CTLR))/ (100-(% of S+ 293T-CTLR))) ^* 100.

E. LIPS Assay

[0157] T en recombinant antigens were designed based on the viral genome sequence of the SARS- CoV-2 strain France/IDF0372/2020 (accession no EPI_ISL_406596) obtained from GISAID

31 database . Five targeted different domains of S: Full S1 sub-unit (residues 1-698), N-terminal domain of S1 (S1 -NTD, residues 1 -305), domain connecting the S1 -NTD to the RBD (S1 -CD, residues 307-330 and 529-700 connected by a GGGSGG linker, SEQ ID NO:6), Full S2 sub-unit (residues 686-1208), and S441 -685. For constructs that did not contain an endogenous signal peptide (residues 1 -14) i.e. S1-CD and S2 constructs, an exogenous signal peptide coming from a human kappa light chain (METDTLLLWVLLLWVPGSTG, SEQ ID NO:3) was added to ensure efficient protein secretion into the media. Five additional recombinant antigens, targeting overlapping domains of N, were designed: Full N (residues 1 -419), N-terminal domain (residues 1 -209), C-terminal domain (residues 233-419), N120-419 and N 111 -419. The LIPS assay was

32 designed as described with minor modifications. Expression vectors were synthesized by GenScript Company, using as backbone the pcDNA3.1 (+) plasmid, with codon usage optimized for human cells. HEK-293F cells were grown in suspension and transfected with PolyEthylenlmine (PEI-25 kDa, Polyscience Inc., USA). Valproic acid (2.2 mM) was added at day 1 to boost expression. Recombinant proteins were harvested at day 3 in supernatants or crude cell

3 lysates. Luciferase activity was quantified with a Centro XS LB 960 luminometer (Berthold

Technologies, France). 10 ⁸ LU of antigens were engaged per reaction. S1 and C-terminal domain (residues 233-419) were selected for analysing the cohorts. To increase sensitivity, the cohorts were tested at a final dilution of 1 :10 of sera.

F. Data Processing and Analysis

[0158] Flow cytometry data were analyzed with FlowJo v10 software (TriStar). Calculations were performed using Excel 365 (Microsoft). Figures were drawn on Prism 8 (GraphPad Software). Statistical analysis was performed using Prism 8.

1.2. Description of Serological Tests

[0159] We first designed four tests to assess the levels of anti-SARS-CoV-2 antibodies in human sera. Their characteristics are summarized in Figure 1 .

[0160] ELISAs. The two ELISAs are classical tests, using as target antigens the full-length N protein (ELISA N) or the extracellular domain of S in a trimerized form (ELISA tri-S). The two recombinant antigens were produced in E. Coli (N) or in human cells (S).

[0161 ] The ELISA N assay is a classical indirect test for the detection of total immunoglobulins, using plates coated with a purified His-tagged SARS-CoV 2 N protein. Titration curves of sera from 22 COVID- 19 patients and 4 pre-pandemic sera initially led to the determination that a dilution of 1 :200 was of optimal sensitivity and specificity, and was later used for testing of large cohorts. [0162] The ELISA tri-S, for trimeric S, allows for the detection of IgG antibodies directed against the SARS-CoV-2 Spike. We developed an ELISA using as antigen a purified, recombinant and tagged form of the S glycoprotein ectodomain, which was stabilized and trimerized using a foldon motif. Serum IgG from pre-epidemic (n=100), pauci-symptomatic (n=209), and hospitalized individuals (n=159) were titrated using serum dilutions ranging from 1 :100 to 1 :1 ,638,400 (figure S1 of Grzelak et al ³⁷ ). Receiving-operating characteristic analyses using either total area under the curve or single optical density measurements indicated that the 1 :400 dilution provides the best sensitivity and specificity values and was therefore used in subsequent analyses (figure S1 of Grzelak et al ³⁷ ). Of note, the tri-S ELISA also permitted the titration of anti-S IgM and IgA antibodies in human sera (figure S1 of Grzelak et al ³⁷ ).

[0163] S-Flow. The third assay, termed S-Flow, is based on the recognition of the S protein expressed at the surface of 293T cells (293T-S cells). We reasoned that in-situ expression of S will allow detection of antibodies binding to various conformations and domains of the viral glycoprotein. We verified that S was functionally active, by mixing 293T-S cells with target cells expressing ACE2. Large and numerous syncytia were detected, indicating that S binds to its receptor and performs fusion (not shown). The principle of the S-Flow assay is depicted on Fig. 2. 293T-S cells are incubated with dilutions of sera to be tested. Antibody binding is detected by adding a fluorescent secondary antibody (anti-lgG or anti- IgM). The signal is measured by flow- cytometry using an automated 96-well plate holder. The background signal is measured in 293T cells lacking S and subtracted in order to define a specific signal and a cut-off for positivity.

[0164] T o establish the specificity of the assay, we first analyzed a series of 40 sera collected before 2019, from the Institut Pasteur biobank (ICAReB). All sera were negative (not shown), strongly suggesting that antibodies against other coronaviruses circulating in France were not detected. We then measured the sensitivity of the assay, by assessing the reactivity of sera from Covid-19 patients hospitalized at Hopital Bichat (table S1 of Grzelak et al ³⁷ ). An example of binding with two patients’ sera (B1 and B2) is depicted on Fig. 3. Serial dilutions allowed for the determination of a titer, which reached a value of 24,600 and 2,700 for B1 and B2, respectively (Fig. 3). Of note, the median fluorescence intensity (MFI) of the signal decreased with the dilution, indicating that MFI, in addition to the % of positive cells, provides a quantitative measurement of the levels of specific antibodies. We thus selected a single dilution (1 :300) to analyze large numbers of samples. We then analyzed samples from 9 patients (B1 -B9) (Fig. 4A). We observed an increase of the IgG response over time, with positivity appearing 6 days after symptoms onset. Serial dilutions indicated that antibody titers raised over time (not shown). We observed similar patterns with the IgM and IgG responses (Fig. 4B). The absence of an earlier IgM response may be due to the lower sensitivity of the secondary anti-lgM antibodies tested or because of a short delay between the two responses, which has been already observed in COVID-19 patients. Addressing this question will require the analysis of a higher number of individuals. We also tested a secondary anti-whole Ig antibody, but it did not prove more sensitive than the anti- IgG. We thus tested the different cohorts with the secondary anti-lgG.

[0165] LIPS. The fourth assay, termed LIPS (Luciferase Immunoprecipitation Assay) is based on the use of antigens made of viral proteins (or domains) fused to nanoluciferase (nanoluc) (Fig. S3 of Grzelak et al ³⁷ ). The objective was to develop an assay that is able to test large diverse cohorts and evaluate the range of antibody responses against a set of viral proteins or domains. This opens the possibility to select the best antigens for high throughput binding assays. Each antigen is used at the same molar concentration, based on a standardization by luciferase activity of the amount of Ag engaged in each reaction. This allows for easy direct comparison of the Ab responses (amplitude and kinetic) against each antigen. A panel of 10 different S and N-derived antigens were first evaluated with a set of 34 pre-epidemic human sera were along with those of with 6 COVID hospitalized patients (Fig. S3 of Grzelak et al ³⁷ ). Two patients were sampled at 3 different time points. The strongest signals in COVID patients’ sera compared to background of pre-epidemic sera were identified with S1 , S2 and N (C-term part) antigens (Fig. S3 of Grzelak et al ³⁷ ). Additional investigations on a limited panel of sera sampled in pauci-symptomatic patients showed that S2 responses were, regarding the diagnostic sensitivity and quantitative responses, similar to full S responses evaluated by S-Flow (Fig. S3 of Grzelak et al ³⁷ ). To avoid redundancy, we focused LIPS analysis to N, selecting it for its sensitivity regarding an intracellular viral protein not targeted by NAbs and S1 as it is described as a target of most NAbs. To establish the specificity of the assay, we first analyzed the same series of 40 sera we used for S-Flow and found all of the sera to be negative (Fig. S3 of Grzelak et al ³⁷ ). We also measured the kinetic of apparition of antibodies in the same longitudinal samples from 5 patients (Fig. 9 and Table 2). We observed an increase of response overtime, with positivity appearing 7-10 days after symptoms onset. Of note, the protein A/G beads used for precipitation of the immune complexes do not bind efficiently to IgM or IgA. Protein L, which has a higher affinity binding to IgA, has not yet been tested.

1.3. Description of the Groups

[0166] We screened different cohorts to evaluate the performance of the four assays and corresponding antigens (Table 2).

[0167] We first used sera from up to 491 pre-epidemic individuals, collected before 2019, to assess the specificity of the tests. We then measured antibody levels in 51 hospitalized COVI D-19 patients from Hopital Bichat (Paris), to determine the sensitivity of the tests and analyze the kinetics of seroconversion. The clinical and virological characteristics of four of these patients have been

24 recently described . We next studied the prevalence of SARS-COV-2 positive individuals in a cohort of pauci-symptomatic individuals in Crepy-en-Valois, a city of 15,000 inhabitants in Oise. On 24 February 2020, a staff member from a high school in Crepy-en-Valois was admitted to an hospital in Paris with confirmed SARS-CoV-2 infection. On March 3-4, students from the high school, parents of the students, teachers and staff were invited to participate to an epidemiological investigation around this case. 209 blood samples were collected from individuals reporting mild signs compatible with COVID-19 (fever, cough or dyspnea). Finally, we tested 200 sera from blood donors from the Etablissement Frangais du Sang (EFS) in Lille (France). The blood samples were donated in two cities, Clermont (10,000 inhabitants) on March 20 and Noyon (13,000 inhabitants) on March 24, each located at about 60 kilometers from Crepy-en-Valois.

1.4. Comparison of the Assays

[0168] Results obtained with sera from each category of individuals are presented on Fig. 6 and 7. The pre- epidemic samples served as negative controls. With the four assays, signals were consistently negative (S-Flow and LIPS S1 ) or low (ELISAs and LIPS N). This strongly suggested that a prior exposure to human seasonal coronaviruses associated to the “common cold” (such as HCoV-OC43, HCoV-229E, HCoV- HKU-1 or HCoV-NL63) does not induce an obvious cross reaction with our assays. This was expected, since these prevalent viruses are distantly related to SARS-CoV-2 atthe protein level. With each assay, we established cut-off thresholds. For ELISA N, the cut-off was set at 95% percentile of 491 pre- epidemic sera. For ELISA tri-S, the cut-off was established as the mean + 2 standard deviations (SD) of the 100 pre-epidemic samples analyzed, which corresponds to 95% specificity. For the S-Flow, we established a cut-off that corresponded to a signal >20% of cells positive by flow cytometry. For the LIPS assays, the cut- off was based on internal controls. S-Flow and LIPS S1 cut-offs eliminated all pre- epidemic samples analyzed.

[0169] Having established these cut-off levels, we analyzed samples from 51 patients from Hopital Bichat. Some of the patients were analyzed at different time points, representing a total of up to 161 samples. The percentage of positive samples varied between 65 and 72%, with a mean of

64%. The fact that not all patients were seropositive reflected the various sampling times from each individual. To study more precisely the kinetics of seroconversion, we selected 5 patients with more than five longitudinal samples and known dates of symptom onsets (Fig. 9). In these patients, seroconversion was detected between 5-10 days post symptom onsets with ELISA-N, LIPS-N, ELISA tri-S and S-Flow. The LIPS S1 assay became positive with a slower kinetic, and one of the patients remained just below the cut-off. For some patients, the LIPS N and ELISA N signals appeared before the LIPS S1 and ELISA tri-S, which suggest different kinetics of N- and S/S1 responses independently of the sensitivity of the test.

[0170] We then tested the 209 sera obtained from pauci-symptomatic individuals in Oise. Positivity rates varied from 27% to 36% between the assays, with a mean of 32% (Fig.7 and Table 3).

This range of variation was more marked than with hospitalized patients, likely because pauci- symptomatic COVID-19 individuals display lower viral loads than those requiring hospitalization and may generate lower levels and different patterns of antibodies. To our knowledge, these figures represent one of the first evaluations of SARS-CoV-2 prevalence in pauci-symptomatic individuals within a cluster of severe cases. The fact that only one third of the individuals were tested positive suggests that some of them may not have seroconverted at the time of sampling, and/or that other viruses or environmental causes were responsible for the reported symptoms.

[0171] We next examined SARS-CoV-2 seroprevalence in samples collected from blood donors on March 20-24, 2020. Eligibility criteria for blood donation included an absence of recent signs of infection or antibiotic treatment. The donors can thus be considered as asymptomatic individuals with stringent criteria. The donors were negative with ELISA-N and LIPS assays. With S-Flow, 6 donors were positive, including two with a strong signal. These 6 positive and 10 negative donors were then tested with ELISA tri-S, and only the two strong responders scored positive. Therefore, the positivity rate in this cohort was low (1 -3% with the two most sensitive assays). This suggests that the virus had not circulated to a large extent in a radius of 60 kilometers around the initial clusters. It is also likely that asymptomatic infection induces low and delayed seroconversion. Further studies are warranted to evaluate SARS-CoV-2 prevalence in denser population environments.

1.5. Correlations Between Assays

[0172] We performed a side-by-side comparison of the assays using the three cohorts. For a given assay, we first scored the number of positive samples measured with the other assays (Fig. 3 of Grzelak 2020 ³⁷ ). With hospitalized patients, roughly similar numbers of positive cases were obtained with the four assays, with the exception of LIPS S1 , confirming that this assay is less sensitive, probably because it does not catch antibodies targeting other S domains. However, combining the LIPS S1 and N results gave similar detection rates than any of the three other tests. With the cohort of pauci-symptomatic individuals, the S-Flow and ELISA tri-S yielded very close results and higher detection rates than the other tests. In blood donors, positive cases were only detected with these two tests.

[0173] We then mixed results obtained with the three cohorts and calculated correlation rates between each assay (Fig. 10). The dot plots indicate that sera with high antibody levels are generally caught by the four assays. Important differences are however observed with samples with a low antibody concentration, reflecting both the choice of the antigens and the intrinsic different sensitivities of the assays.

[0174] These results have been published in Grzelak et al, 2020 ³⁷ , whose data are incorporated herein by reference. In particular, the results of figures 2, 3 and 5 of Grzelak et al, 2020 ³⁷ are incorporated by reference.

1.6. Discussion

[0175] We have designed four serological assays to detect anti-SARS-CoV2 antibodies. The first two assays are ELISA detecting anti-N and anti-S responses. The S-Flow assay allows to identify and score the levels of antibodies binding to all domains and conformations of S expressed at the cell surface. The LIPS assays target different domains of S and N, and allow for the detailed profiling of the humoral responses. We have evaluated their performance and compared their results with two neutralisation assays, a reference MNT assay and a pseudovirus neutralisation assay. [0176] Each assay presents advantages and drawbacks. ELISAs are widely used, either as in-house or commercial tests, and can be easily performed in routine diagnostic laboratories in large quantities. They can be performed at a high scale. The S-Flow assay captures all anti-S antibodies and provides excellent sensitivity but requires access to a cell culture system and flow cytometry equipment. Thus, it would be less adapted to high-throughput screenings. The LIPS assay allows the testing of different target antigens in a liquid phase assay, also preserving as much as possible conformational epitopes and it appears to be as sensitive as ELISA and S-Flow for some of the antigens tested. It requires access to a bioluminescence detection instrument. The two neutralisation assays require cell culture systems, with MNT using infectious virus and necessitating access to a BSL3 facility, whereas pseudovirus neutralisation is adaptable to high- throughput screenings.

[0177] Serological diagnostic tests are complementary to viral detection by RT-PCR for diagnostic purposes in patients. Results from our study and others indicate that in severe and critical cases (hospitalized patients), seroconversion is detectable as soon as 5-14 days post symptom onset

^{6,7,13 15} . In such cases, antibody titers can reach high levels, and the different assays gave similar results. Detection of anti-N and anti-full S responses demonstrated similar rates of seroconversion, whereas the S1 response was delayed. The anti-N response appeared slightly more rapidly than S/S1 responses for a given type of test, which could be of interest to develop routine diagnostics tests, if confirmed.

[0178] At the population level, serological tests are used in surveys to identify persons who have been infected. Regarding the identification of pauci-symptomatic or asymptomatic individuals, we consistently observed similar levels of seroprevalence, again with different sensitivities depending on the assay. ELISA tri-S, S-Flow and the combined LIPS S1+N gave higher detection rates than ELISA-N. Combining ELISA N and S assays may also increase the sensitivity of detection. In our cohort of 209 pauci-symptomatic individuals, only a minor fraction of individuals was tested by RT- PCR (not shown).

[0179] It has been reported that in 175 convalescent patients with mild symptoms, N Abs are detected

20 from day 10-15 after disease onset in a large fraction of patients . The titers of NAb correlated

20 with the titers of anti-S antibodies (targeting S, RBD, and S2 regions) . A critical question is the detection of antibodies, their neutralisation potential in asymptomatic individuals, and, more generally, the correlates of protection. In our pilot study with 200 healthy blood donors, the ELISA N and LIP S1 +N assays were negative, whereas six individuals scored positive with S-Flow. When reanalyzed with the ELISA tri-S, two of the six individuals were positive. These results indicate that the most sensitive assays are required for identification of asymptomatic SARS-CoV- 2 infected individuals, who will likely mount a weaker response than patients experiencing a mild or severe infection.

[0180] The S-Flow test of the invention has been successfully used to analyze the antibody response (IgG, IgA and IgM) to SARS-COV-2 in sera of asymptomatic and mildly symptomatic COVD-19 individuals. The data are available in Dufloo et al., MedRxiv 2020 ³⁵ and are incorporated herein by reference (see figure 3 of Dufloo et al.,).

[0181 ] The S-Flow test of the invention has also been successfully used to analyze the level of IgGs and IgMs in male and female participants being mildly infected with SARS-COV-2, up to 6 months after symptom onset (Grzelak et al, 2021 ³⁶ ). The data are incorporated herein by reference. In particular, Figure 1 of Grzelak et al shows that the S-Flow test of the invention is the more sensitive test over three different assays (Lateral Flow Assay from Biosynex™ detecting IgG and IgM against the RBD, ELISA EDI™ detecting IgG against N, and neutralization (ID20)) and that performance of the S-Flow test is the same at 6 months and 3 months.

2. EXAMPLE 2: set up of multiplex IgG/lgA S-Flow assay

2.1 . Methods

[0182] Human embryonic kidney (HEK) 293T (referred to as 293T) from the American Type Culture Collection (ATCC) (ATCC CRL- 3216) were engineered to stably express a codon-optimized version of the SARS-CoV-2 S gene (SEQ ID NO:2) encoding the protein Spike from the Wuhan strain (GenBank: QHD43416.1 , SEQ ID NO:1 ) together with a puromycin resistance gene. Cells were split every 2 to 3 days using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (complete medium) and 0.1pg/mL of Puromycin to maintain transgene expression. The day of the assay, cells were detached using PBS-EDTA and transferred into U-bottom 96-well plates (50,000 cells per well). Cells were incubated at 4°C for 30 min with sera (1 :300 dilution) or antibodies (at the indicated dilution) in PBS containing 0.5% BSA and 2 mM EDTA. Then, cells were washed with PBS, and stained using anti-lgG Alexa Fluor 647 (1 :600; Ref: 109-605-170; Jackson ImmunoResearch) and or anti- IgA Alexa 488 (1 :200; Ref: 109-545-011 ; Jackson ImmunoResearch. After 30min of staining at 4Ό, cells were washed with PBS and fixed for 10 min using 4% paraformaldehyde (PFA). Data were acquired on an Attune NxT instrument (Life Technologies). Specific binding was calculated with the following formula: 100 * (% binding on 293T-S - binding on control cells)/(100 - binding on control cells). 2.2. Results

[0183] We first evaluated the capacity of each of the secondary antibodies to specifically detect IgG or IgA. We tested increasing concentrations of a purified anti-spike immunoglobin cloned either as IgG or IgA. The anti-lgG Alexa Fluor 647 antibody detects IgG to Spike-expressing cells at low concentration (Figure 11 A). Cross-reactivity to IgA is limited, with only 20% of cells being positive when the staining is performed with 10pg/mL of IgA. Comparison of binding curves shows that this secondary antibody recognizes IgGs >10,000 times more efficiently than IgA (Figure 11 A). Similarly, the anti-IgA Alexa Fluor 488 secondary antibody is highly specific of IgA (Figure 11 B).

[0184] Then, we compared the multiplex assay to our historical simplex IgG and IgA assays, using a panel of positive and negative sera (Figure 12). We used a monoclonal anti-spike antibody as standard to calculate a normalized binding unit (BU). The multiplex assay performs slightly better to detect IgG and provides similar data on IgA as compared to the historical assay (Figure 12). The improved capacity to detect IgG is due to the use of different anti-human IgG secondary antibodies in the simplex and multiplex assays. This suggests that the multiplex assay is as efficient as the simplex assay, with no abnormal cross-reactions.

3. EXAMPLE 3: Set-up of an S-flow assay to detect antibody binding to the spike of SARS-CoV-2 variants B.1 .1 .7, B.1 .351 and P.1 (see also in Planas et al, ³⁵ )

3.1 . Methods

[0185] To induce ectopic expression of S from SARS-CoV-2 variants, we first added in silico the B.1.1.7 spike mutations (D69-70, DU144, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H, see SEQ ID NO:4 and figure 13) or the B.1 .351 spike mutations (L18F, D80A, D215G, D242-244, K417N, E484K, N501Y, D614G and A701V, see SEQ ID NO:5 and Figure 13) or the P.1 spike mutations (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, see SEQ ID NO:8) into the codon-optimized Wuhan reference strain (SEQ ID NO:2), ordered these synthetic genes (GeneArt, Thermo Fisher Scientific) and transferred them into the phCMV backbone (GenBank: AJ318514), by replacing the VSV-G gene. The D614G S-protein was generated by introducing the corresponding mutation in the Wuhan reference strain using Q5 Site-Directed Mutagenesis Kit (NEB). Then, 293T cells from the American Type Culture Collection (CRL-3216) were transfected with the different S-protein expression plasmids or a control plasmid using Lipofectamine 2000 (Life technologies). These plasmids had the following sequences : SEQ ID NO:15 (expression plasmid phCMV_Spike_B.1-G), SEQ ID NO:16 (expression plasmid phCMV_Spike_B.1-B), SEQ ID NO:17 (expression plasmid phCMV_Spike_B.1 .1 .7), SEQ ID NO:18 (expression plasmid phCMV_Spike_B.1 .351), or SEQ ID NO:19 (expression plasmid phCMV_Spike_P.1).

[0186] One day after, transfected cells were detached using PBS-EDTA and transferred into U- bottom 96-well plates (50,000 cells per well). Cells were incubated at 4 °C for 30 min with sera

(1:300 dilution) or nasal swabs (1:50 dilution) in PBS containing 0.5% BSA and 2 mM EDTA, washed with PBS and stained using anti-lgG AF647 (1 :600 dilution; Thermo Fisher). Cells were washed with PBS and fixed for 10 min using 4% paraformaldehyde. Data were acquired on an Attune Nxt instrument (Life Technologies). Stainings were also performed on control (293T- empty) cells. Results were analyzed with FlowJo 10.7.1 (Becton Dickinson). The specific binding was calculated as follows: 100 * (percentage binding 293T S protein - percentage binding 293T- empty)/(100 - percentage binding 293T-empty).

3.2. Results

[0187] The assay was validated using samples from convalescent individuals at 3-, 6- and 9-months post infections, and samples from vaccinated individuals up to 6 weeks post-injection. The data are available in Planas et al., Nat Med 2021 ³⁴ and are incorporated herein by reference.

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