WO2021240425A1 - Procédé de détection rapide d'anticorps contre sras-cov-2 à l'aide d'une protéine de fusion nucléospicule recombinante - Google Patents

Procédé de détection rapide d'anticorps contre sras-cov-2 à l'aide d'une protéine de fusion nucléospicule recombinante Download PDF

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WO2021240425A1
WO2021240425A1 PCT/IB2021/054637 IB2021054637W WO2021240425A1 WO 2021240425 A1 WO2021240425 A1 WO 2021240425A1 IB 2021054637 W IB2021054637 W IB 2021054637W WO 2021240425 A1 WO2021240425 A1 WO 2021240425A1
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cov
seq
sars
antibodies against
ligand
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Sudarsanareddy LOKIREDDY
Madhusudhana Rao Nalam
Sridhar Rao Kunchala
Rakesh Kumar Mishra
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Oncosimis Biotech Private Limited
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention relates to a method for rapid detection of antibodies against SARS- CoV-2 in a biological sample. More specifically, the method involves novel recombinant nucleospike fusion protein for rapid detection of antibodies against SARS-CoV-2 in a biological sample by bio-layer interferometry assay.
  • Coronaviruses are a group of highly diverse, enveloped, positive sense, and single stranded RNA viruses. They are known to cause diseases of respiratory, enteric, hepatic, and neurological systems in both humans and animals. Coronaviruses are zoonotic, i.e. can be transmitted between animals and people. The novel coronavirus designated assevere acute respiratory syndrome- Coronavirus-2 (SARS-Cov-2), has caused the worst pandemic of 21 st century so far affecting 213 countries and territories around the world.
  • SARS-Cov-2 coronavirus-2
  • SARS-CoV-2 The basic structure of SARS-CoV-2 is similar to any other coronavirus comprising of spike glycoprotein (S), membrane protein (M), envelope protein and nucleocapsid protein (N).
  • SARS-CoV-2 spikes are composed of trimmers of spike glycoprotein.
  • the spike glycoprotein has two functional domains: S1 and S2.
  • S1 domain is responsible for the binding with its receptor angiotensin- converting enzyme 2 (ACE2) on host cells.
  • S2 domain is the transmembrane subunit that facilitates viral and cellular membrane fusion for entry into host cell. Therefore, the SARS-CoV-2 uses its spike glycoprotein for entry into host cells by binding to ACE2 receptor.
  • the spike glycoprotein is the main antigenic component in the SARS-CoV which is an important target of host defense system for producing antibodies, and neutralizing the virus. Hence, the spike glycoprotein is a common target for vaccine and therapeutic development.
  • the nucleocapsid protein is the most abundant protein in SARS-CoV-2 and is required for coronavirus RNA synthesis, and has RNA chaperone activity that may be involved in template switch. It is a highly immunogenic phosphoprotein and rarely changes/mutates.
  • the nucleocapsid protein of SARS-CoV-2 is often used as a marker in diagnostic assays.
  • RT-PCR Reverse transcriptase polymerase chain reaction
  • qRT-PCR Quantitative RT-PCR
  • the test can be done on respiratory samples obtained by various methods, including a nasopharyngeal swab or sputum sample, and on saliva. Testing using qRT-PCR approaches for detection of the SARS-CoV-2usually takes only 4-6 hours. However, the main limitation is, the typical turnaround time for screening and diagnosing patients with suspected SARS-CoV-2 being>24h including the time needed to ship samples overnight to reference laboratories.
  • Another major limitation of this method is its dependency on the location from which the sample is collected from the patients’ body. It is seen that in the cases of SARS- CoV-2 infection, in the first week of the infection the virus is detected in the upper respiratory tract, saliva etc. However, after one week, the virus may disappear from the upper tract and reach lungs. This disappearance can result in negative results if the sample is collected from upper respiratory tract, though the patient is still infected and is spreading the disease. In such cases further confirmatory tests are required.
  • Another diagnostic approach is the antigen testing, wherein the presence of viral antigen in the biological samples (nasal swabs, saliva, blood etc.) is tested in a quantitative, semi-quantitative, or qualitative assay.
  • the antigen testing requires monoclonal antibodies specific for the viral antigens.
  • An assay using a combination of different monoclonal antibodies against more than one viral antigen may result in more effective diagnosis.
  • Easy antigen testing assays which can be visualized with naked eye can be developed.
  • antigen testing in early stages may be difficult because it requires high viral count to work effectively and the results have poor accuracy, as this testing does not amplify the viral antigen.
  • the nasal and throat swabs of asymptomatic people may not have enough antigen particles to be detected as they have very little if any nasal or saliva discharge; even though these asymptomatic people can still spread the virus. Hence if test results are negative, it may require further confirmation by RT-PCR testing.
  • Another diagnostic approach would be to devise blood tests for antibodies against the SARS-CoV-2 virus, the serological tests.
  • Part of the immune response to infection is the production of antibodies including IgM and IgG antibodies.
  • Dual ELISA tests can be used to detect specific IgM and IgG against the virus in the blood of infected patients.
  • a fluorescent immunoassay system can measure quantitatively or semi-quantitatively the concentration of the IgM/IgG.
  • ELISA tests can be used to detect specific IgM, IgA and IgG against the virus in the blood of infected patients.
  • the quantity of circulating antibodies detectable by these assays will typically require 4-6 days for IgM antibodies, >6 days for IgA antibodies and 5-10 days for IgG post infection.
  • serological testing is one of the fastest and best methods to identify people who were recently infected or exposed, even if they are no longer shedding the virus. Individuals will typically have detectable antibodies for weeks to months after infection regardless of severity, mild or no clinical signs were present. Serological tests can be used in a situation like COVID-19 pandemic for large population testing, enabling identification followed by subsequent isolation of potential virus spreaders. Low-cost, rapid assays upon exit/ entry of any person in a location is more reliable than existing thermal assays, especially useful at airports assisting international travel.
  • the major limitation in the serological assays for SARS-CoV-2 is that few infected people develop more antibodies towards viral spike glycoprotein, whereas others develop more antibodies towards nucleocapsid proteins.
  • the present invention provides a rapid serological diagnostic method for detecting total antibodies (IgM/IgG/IgA) against SARS-CoV-2.
  • the present invention takes into account the drawbacks of the prior art and provides an invention with the main object of the invention providing a novel recombinant nucleospike fusion protein for rapid detection of antibodies against SARS-CoV2,a method for rapid detection of antibodies against SARS- CoV-2 in a biological sample, and a diagnostic kit for performing the same.
  • Another object of the invention is to provide a novel recombinant nucleospike fusion protein to develop biosensors, diagnostic assays, vaccines, therapeutic agents etc. for SARS-CoV-2.
  • Yet another object of the invention to provide an in vitro method for rapid detection of antibodies against SARS-CoV-2 in a biological sample by bilayer interferometry enabling rapid and high throughput screening, and detection within 120 seconds, more specifically detection of antibodies against SARS-CoV- 2 within 60 seconds.
  • Yet another object of the invention to provide an in vitro method for rapid detection of antibodies against SARS-CoV-2 using recombinant nucleospike fusion protein which is highly sensitive because it detects total antibodies including IgM, IgG, and IgA against SARS-CoV-2 in biological samples such as serum, saliva, sputum etc.
  • Yet another object of the invention to provide an in vitro method for rapid detection of antibodies against SARS-CoV-2 using recombinant nucleospike fusion protein enabling population level testing or seroepidemiology of countries, races, etc.
  • the invention provides a novel recombinant nucleospike fusion protein of SARS-CoV-2of Seq. ID 1 encoded by DNA of Seq. ID 2, capable of binding to antibodies against nucleocapsid protein, and spike glycoproteins of SARS-CoV-2, which has application in development of biosensors, diagnostics assays, vaccines, and therapeutic compositions.
  • the detection of antibodies against SARS-CoV-2 in a biological sample indicates infection of patient with SARS-CoV-2 from whom the biological sample is derived.
  • the invention provides an in vitro method for rapid detection of antibodies against SARS-Cov-2 in a biological sample by biolayer interferometry comprising the steps of
  • step (a) immobilizing a bio-layer interferometry biosensor’ tip with a purified recombinant ligand to detect antibodies against SARS-Cov-2; (b) washing the biosensor obtained in step (a) to remove excess unbound molecules of recombinant ligand;
  • the recombinant ligand is recombinant nucleospike fusion protein of SARS-CoV- 2 represented by Seq. ID 1 which has affinity for antibodies against SARS-CoV-2;
  • Seq. ID 1 is encoded by Seq. ID 2.
  • the invention provides a bio-layer interferometry assay for detecting antibodies againstSARS-CoV-2 in a biological sample, which is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized ligand/protein on the biosensor tip, and an internal reference layer.
  • the immobilized ligand on the biosensor tip binds to an analyte for which the ligand has affinity, it causes a shift in the interference pattern. This shift is proportional to number of molecules of ligand bound to analyte, hence more the binding of ligand and analyte, the more is the shift.
  • Seq. ID 1 is immobilized on a biolayer interferometry biosensors’ tip.
  • Seq. ID 1 is a 1405 amino acid long fusion protein comprising of spike glycoprotein sequence of SARS-CoV2 from amino acid 1-970, a linker sequence from amino acids 971- 974, nucleocapsid protein sequence SARS-CoV2 from amino acid 975-1392; and a His-tag tail sequence from amino acids 1393-1405.
  • the biosensors’ tip with immobilized Seq. ID 1 is exposed to a biological sample; if the biological sample has antibodies against SARS-CoV2, the subsequent biomolecular interactions between the Seq.
  • ID 1 on biosensors’ tip and antibodies in the biological sample will result in shift of interference pattern thus enabling in vitro detection of antibodies against SARS-CoV2 in the biological sample. Further, the quantification of the amount of antibodies against SARS-CoV2by analysis of the shift of interference will enable determination of stage and progression of the infection by SARS-CoV2.
  • the invention provides an in vitro method which is more sensitive for detection of total antibodies against SARS-CoV2.
  • Seq. ID 1 is fusion of nucleocapsid protein and spike glycoprotein of SARS-CoV2 which enables detection of antibodies against both the proteins.
  • Known serological assays use either one of the proteins to detect one kind of antibodies only which makes the assay less sensitive.
  • the present invention enables detection of total antibodies against SARS- CoV-2 with high specificity, compared to known tests such as ELISA tests. Further, the method for detection of antibodies against SARS-CoV-2 in a biological sample using bio-layer interferometry biosensor with immobilized Seq. ID 1 is rapid and can be achieved within 120 seconds, more specifically, the detection of antibodies against SARS-CoV-2 is achieved within 60 seconds. This makes the method useful for high throughput screening which is highly desirable for handling situations of epidemics and pandemics like that of Covid-19.
  • FIG. 1A is a schematic elucidation of basic principle of bio-layer interferometry assay and its analysis
  • Figure IB is a schematic elucidation of analysis of bio-layer interferometry sensogram using biosensor with ligands at different stages such as equilibration, ligand binding, washing, and analyte binding
  • Figure 2 is a graphical representation of binding rates of ligands -nucleocapsid ligand (N), S1 subunit spike protein ligand of (S1) - 16-681 amino acid stretch, Equimolar mixture of nucleocapsid ligand and S1 subunit spike protein ligand in 1:1 ratio (N+Sl), S1 and part of S2 subunit of spike protein (S-16-986), and Seq.
  • N nucleocapsid ligand
  • S1 subunit spike protein ligand of (S1) - 16-681 amino acid stretch Equimolar mixture of nucleocapsid ligand and S1 subunit spike protein ligand in 1:1 ratio (N+Sl), S1 and part of S2 subunit of spike protein (S-16
  • FIG. 3A is a bio-layer interferometry sensorogram depicting ligand-analyte binding rates of bio-layer interferometry biosensor immobilized with Seq. ID 1 (ligand) with antibodies (analyte) against SARS-CoV-2 in SARS-CoV-2 RT-PCR +ve serum sample with dilutions of RT-PCR +ve serum sample ranging between 10 to 200 times;
  • Figure 3B is a graphical representation of ligand-analyte binding rates of bio- layer interferometry biosensor immobilized with Seq. ID 1 (ligand) and antibodies (analyte) against SARS-CoV-2 in SARS-CoV-2 RT-PCR +ve serum sample with dilutions of RT-PCR +ve serum sample ranging between 10 to 200 times;
  • Figure 4 is a bio-layer interferometry sensorogram depicting real-time ligand- analyte binding of bio-layer interferometry biosensor immobilized with Seq. ID 1 (ligand) and antibodies (analyte) against SARS-CoV-2 in SARS-CoV-2 RT-PCR +ve serum samples and RT-PCR -ve serum samples;
  • Figure 5A is a graphical representation comparing ligand-analyte binding rates of Seq. ID 1 (BLI-Seq.
  • Figure 5B is a graphical representation of receiver operating characteristic curves(ROC) area showing relation between sensitivity and specificity of each ligand-analyte binding assay - BLI-Seq. ID 1, ELISA IgG (Spike Sl-RBD), and ELISA IgM (Spike Sl-RBD), where BLI-Seq. ID 1 binding rates showed 1.000, confidence interval in between 1.000 tol.000, and p values ⁇ 0.0001, ELISA - IgG showed only 0.9023 and confidence interval in between 0.8100 to 0.9945, and ELISA - IgM showed only 0.9070 and confidence interval in between 0.8243 to 0.9898; Line indicates cut off point;
  • Figure 6A is a graphical representation comparing ligand-analyte binding rates of BLI-Seq. ID 1 to detect total antibodies, and ELISA assay using gamma irradiated whole viral particle of SARS-CoV-2 as antigen to detect IgG antibodies in RT-PCR +ve and RT-PCR -ve plasma samples;
  • Figure 7A is graphical representation showing binding rates rNS- Seq. ID 1 by bio-layer interferometry assay using 40 plasma samples collected beforeCOVID- 19 pandemic in India (Nov 2019) and 3 plasma samples (RT-PCR +ve) collected after COVID- 19 pandemic;
  • FIG. 7B depicts Receiver operating characteristic curve (ROC) analysis showing sensitivity versus specificity for discrimination of RT-PCR positive and plasma samples collected before COVID-19 pandemic showing 100% specificity (ROC area under the curve: 1.000, 95%confidence interval in between 1.000 to 1.000 and p values 0.004249);
  • ROC Receiver operating characteristic curve
  • Figure 8A is graphical representation showing binding rates rNS- Seq. ID 1 by bio-layer interferometry assay using 850 plasma samples collected from a community who were never diagnosed for SARS-CoV-2 (no COVID-19 symptoms) and 122 plasma samples collected from people diagnosed for SARS- CoV-2 using RT-PCR;
  • Figure 8B depicts Receiver operating characteristic curve (ROC) analysis showing sensitivity versus specificity showing curve area of 0.9818, at 95% confidence interval in between 0.9663 to 0.9973 and p ⁇ 0.0001.
  • ROC Receiver operating characteristic curve
  • Figure 9A depicts a binding curve (bio-layer interferometry sensogram) representing detection of total antibodies against SARS-CoV-2 in 20 ⁇ lblood sample collected from patients diagnosed for SARS-CoV-2 (2RT-PCR +ve) and healthy volunteers (5 RT-PCR -ve);
  • Figure 9B depicts a binding curve (bio-layer interferometry sensogram) representing detection of total antibodies against SARS-CoV-2 in 10 m ⁇ plasma sample collected from the same patients diagnosed for SARS-CoV-2 (2RT-PCR +ve) and same healthy volunteers (5 RT-PCR -ve); and
  • Figure 9C is a graphical representation comparing binding rates of ligand-analyte using bio-layer interferometry assay with RT-PCR +ve blood and plasma samples using the following ligands-nucleocapsid ligand (N), S1 subunit spike protein ligand of (S1) - 16-681 amino acid stretch, Equimolar mixture of nucleocapsid ligand and S1 subunit spike protein ligand in 1:1 ratio (N+Sl), and Seq. ID 1 (rNS).
  • N ligands-nucleocapsid ligand
  • S1 subunit spike protein ligand of (S1) - 16-681 amino acid stretch Equimolar mixture of nucleocapsid ligand and S1 subunit spike protein ligand in 1:1 ratio (N+Sl)
  • Seq. ID 1 (rNS) Seq. ID 1
  • the invention provides a novel recombinant nucleospike fusion protein of SARS-CoV-2 of amino acid sequence of Seq. ID l.
  • the invention also provides the nucleotide sequence of Seq. ID 2 as which encodes Seq. ID 1.
  • Seq. ID 1 is a 1405 amino acid long fusion protein comprising of spike glycoprotein sequence from amino acid 1-970, a linker sequence from amino acids 971-974, nucleocapsid protein sequence from amino acid 975-1392; and a His-tag tail sequence from amino acids 1393-1405.
  • the spike glycoprotein sequence in the Seq. ID 1 corresponds to amino acids 16-985 of Chain A spike glycoprotein of SARS-CoV-2 (NCBI accession no: QHD43416.1) corresponding to subunit 1 and part of subunit 2 of spike protein; and nucleocapsid protein sequence in Seq.
  • ID 1 corresponds to amino acids 2-419 of nucleocapsid protein of SARS-CoV-2 (NCBI accession no: QHD43417.1).C-terminal His-tag of recombinant nucleospike fusion protein enables purification, and immobilization onto affinity based biosensors, chips etc.
  • the invention provides application of Seq. ID 1 to detect SARS-CoV-2 infection in a patient.
  • Seq. ID 1 comprises of highly immunogenic domains of spike glycoprotein and nucleocapsid protein of SARS-CoV-2.
  • Seq. ID 1 is capable to bind to total antibodies produced against SARS-CoV-2, thus enabling indirect detection of SARS-CoV-2 infection in patients from whom the biological samples were obtained.
  • Systemic immune response to infection results in the production of antibodies including IgM, IgA and IgG antibodies.
  • ELISA tests can be used to detect only specific IgM, IgA and IgG against the virus in the blood of infected patients.
  • the quantity of circulating antibodies detectable by these assays will typically require 4-6 days for IgM antibodies, >6 days for IgA antibodies and 5-10 days for IgG post infection.
  • the invention provides an in vitro rapid antibody detection method based on bio-layer interferometry total antibody assay.
  • BLI-TAA is a label-free optical analytical technology for measuring bio-molecular interactions by analyzing the interference pattern of white light reflected from two surfaces.
  • the binding between a ligand immobilized on the biosensor and an analyte in solution produces an increase in optical thickness at the biosensor tip, which results in absorption properties with a wavelength shift, which is a direct measure of the change in thickness of the biological layer.
  • the invention provides an in vitro method for rapid detection of total antibodies against SARS-CoV-2 in a biological sample of SARS-CoV-2 of Seq. ID 1 by biolayer interferometry.
  • the sensitivity of the biolayer interferometry assay using Seq. ID 1 as ligand is highly enhanced as Seq. ID 1 acts as antigen which would be having affinity to all kinds of antibodies IgG, IgM, and IgA, and the assay does not only bind to IgG or IgM alone antibodies as in the case of ELISA tests.
  • virus-specific ligand - Seq. ID 1 is immobilized on the sensor tip.
  • ID 1 comes in contact with a patient’s plasma or whole blood containing the antibodies against SARS-CoV-2 (analyte), a shift in wavelength is observed indicating presence of antibodies against SARS-CoV-2.
  • This testing method takes less than a minute to detect the presence of SARS-CoV-2 antibodies during and after infection. If there is no shift in wavelength it indicates absence of SARS- CoV-2 antibodies, hence no infection by SARS-CoV-2.
  • the present invention relates to a novel recombinant nucleospike fusion protein of SARS-CoV-2 represented by Seq. ID 1 which has affinity for antibodies against SARS-CoV-2 and its use in rapid detection of total antibodies against SARS-CoV-2 by bio-layer interferometry assay.
  • Seq. ID 1 a novel recombinant nucleospike fusion protein of SARS-CoV-2 represented by Seq. ID 1 which has affinity for antibodies against SARS-CoV-2 and its use in rapid detection of total antibodies against SARS-CoV-2 by bio-layer interferometry assay.
  • Seq. ID 1 ligand recombinant nucleospike fusion protein-rNS
  • Ligands corresponding to antigens of SARS-CoV-2 viral particles used for experiments are provided below: i.
  • ACE2 toangiotensin-converting enzyme 2
  • Spike protein plays an important role in the induction of neutralizing- antibodies and T-cell responses, as well as protective immunity.
  • S1 subunit (S1) corresponding to 16-681 amino acid stretch of NCBI accession no. QHD43416 was used for experimental studies as a purified ligand.
  • the S2 subunit (686-1273 residues) of spike protein plays a key role in mediating virus-cell fusion and its integration into host cells.
  • a portion of S2 subunit 682- 985 amino acid stretch was also a part of the rNS of Seq. ID 1, hence, the ligand
  • Nucleocapsid protein of SARS-CoV-2 is also known to act as an antigen efficient in eliciting a serological response in COVID-19 positive patients.
  • a study that used ELISA to measure only antibodies to the nucleocapsid protein found that patients become seropositive 10-18 days after the onset of symptoms (Guo et. al., 2020).
  • Another study showed that nucleocapsid antibodies emerge before spike antibodies (Burbelo et.ah, 2020).
  • Nucleocapsid protein of SARS-CoV-2 (N) corresponding to 2-418 amino acid stretch of NCBI accession no. QHD43417 was used for experimental studies as a purified ligand.
  • S1 subunit (S1) and Nucleocapsid protein (N) of SARS-CoV-2 were purified and mixed in a ratio of 1:1 and the mixture (N+Sl) was used for experimental studies as a purified ligands. Seq.
  • ID 1 ligand, rNS consisting of spike protein ranging from 16-986 amino acids (which includes Full length S1 (670 amino acids)subunit and Part of S2 subunit (300 amino acids)S2 Heptad region-1) of SARS-CoV-2, and Nucleocapsid protein (418 amino acids) inframe to S2 domain with Serine Glycine linker (SGSG).
  • S1 subunit (S1) of spike protein corresponding to 16-681 amino acid stretch of NCBI accession no. QHD43416 was cloned in into pcDNA3.1(+) with a secretory signal nucleotide sequence at the 5’ terminal C-terminal His-tag.
  • DNA encoding S1 subunit and part of S2 subunit(S (16-986)) of spike protein corresponding to 16-986 amino acid stretch of NCBI accession no. QHD43416 was cloned in into pcDNA3.1(+) with a secretory signal nucleotide sequence at the 5’ terminal C-terminal His-tag.
  • N Nucleocapsid protein corresponding to 2-418NCBI accession no. QHD43417 was cloned in pET28a(+), and pcDNA3.1(+) with a secretory signal nucleotide sequence at the 5’ terminal C-terminal His-tag.
  • Seq. ID 2 encoding Seq. ID 1, recombinant nucleospike fusion protein (rNS), was be cloned in into pcDNA3.1(+) in between Nhel and Xhol restriction sites with a secretory signal nucleotide sequence at the 5’ terminal, hence Seq. ID 1 is expressed with an N-terminal secretory signal peptide of sequence MGVKVLFALICIAVAEA which enables secretion of Seq. ID 1 outside host expressing cells, and C-terminal His-tag. This enables easy purification of Seq. ID 1 from the culturing media and purification.
  • FS-293 and CHO-S mammalian cells were used for expression of DNA vectors for ligands - S1, S (16-986), N, and rNS.
  • FS-293 cells were maintained in chemically defined FS-293 expression media and CHO-S maintained in chemically defined FS-CHO media respectively.
  • rNS phosphate buffered solution
  • the bio-layer interferometry biosensor used for this purpose has anti-His antibodies on its tip. His-tag of purified S1, S (16-986), N, and rNS of Seq. ID 1 were used for binding of Seq. ID 1 to the biosensors’ tip by antibody-antigen affinity. Purified S1 and N ligands were mixed in 1:1 ratio and used as ligand mixture for binding on to the biosensors’ tip.
  • C-terminal His tag of ligands were immobilized on bio-layer interferometry biosensor using inline protocol according manufactured instructions and then washed with PBS buffer. After removal of the excess unbound ligands from the biosensor, the biosensor was ready to be used for analysis of detection and quantification of SARS-CoV-2 in a biological sample.
  • a biological sample can be blood sample, serum sample taken from blood, sputum, saliva swab, nasal swab, throat swab etc. derived from patients diagnosed of SARS-CoV-2 infection by RT-PCR (RT-PCR +ve) and uninfected controls confirmed by RT-PCR tests (RT-PCR -ve).
  • the basic principle of bio-layer interferometry assay is based on affinity of ligand on the tip of biosensor and analyte in the biological sample.
  • a biosensor with only immobilized ligand will display a certain interference pattern for white light reflected from two surfaces: a layer of immobilized ligand on the biosensor tip, and an internal reference layer.
  • the ligand is bound to an analyte (biomolecular interactions between ligand and analyte)
  • it would increase the thickness of the biosensors’ tip which results in shift of interference pattern for white light.
  • This shift of interference pattern is analyzed as presence of analyte in the biological sample; and the extent of shift of interference pattern determines the quantity of analyte present in the biological sample.
  • the interference pattern of white light by the biosensor is taken in real time.
  • the bio- layer interferometry biosensor with anti-His was used for immobilization of C- terminal His-tag ligands - S1, S (16-986), N, N+Sl, and rNS of Seq. ID 1, and the bio-layer interferometry biosensor with immobilized ligands was exposed to the biological sample. If any antibodies against SARS-CoV-2 in the biological sample bind to the ligand on the biosensor tip it would result in a shift of interference pattern of white light.
  • Biological samples such as plasma or whole blood were obtained from infected patients who had tested positive for SAR-CoV2 by RT-PCR (RT-PCR +ve) and uninfected patients had tested negative for SAR-CoV2 by RT-PCR (RT-PCR - ve). i. Sample collections:
  • the ligands used for conducting the assay were one of the following: S1, S (16- 986), N, N+ S1 in 1:1 ratio, or rNS of Seq. ID 1.
  • the real-time data of interference pattern for all the steps of the method is provided within 300 seconds (6 mins) as depicted in Figure IB.
  • the real-time data of interference pattern to detect antibodies against SARS-CoV-2 in a biological sample is provided within 120 seconds. Shift in interference pattern due to presence of antibodies against
  • SARS-CoV-2 is detected within 60 seconds.
  • rNS of Seq. ID 1 has better sensitivity than spike protein (S1 or S-(16-986)), nucleocapsid protein, or even equimolar combination N+Sl.
  • the commercial kits which are available for serological tests for SAR-CoV2 generally comprise of either spike ligands or nucleocapsid ligands. It is clear from the above experiment that these ligands alone are not sensitive enough and may easily provide false negative results for detecting antibodies against SAR-CoV2. Even though simple combination of mixing nucleocapsid protein and spike protein showed better binding rate efficiency but still does not provide a good additive effect. Whereas, the recombinant nucleospike (rNS) of Seq. ID lshowed significantly better binding rate efficiency than nucleocapsid protein and spike protein combination. This showed the improved efficacy of the recombinant Seq. ID 1.
  • Plasma obtained from RT-PCR +ve patient was diluted using PBS in range of 10X to 200X and tested for ligand-analyte binding rates of bio-layer interferometry biosensor with rNS of Seq. ID 1 with antibodies (analytes) in diluted plasma sample.
  • Table 1 provides details of ligand-analyte binding rates with different dilutions of RT-PCR +ve serum sample ranging between 10 to 200 times.
  • a dilution oi plasma sample by 10 times provides sufficient binding efficiency between the biosensor with rNS of Seq. ID 1 (ligand) and the antibodies (analyte) against SARS-CoV-2 in the diluted plasma sample.
  • Higher dilutions show reduced binding constants indicating the reduction in the number of analytes, further confirming that the biosensor with rNS of Seq. ID 1 is capable to detect antibodies (analyte) against SARS-CoV-2 in a diluted plasma sample.
  • Figure 4 provides bio-layer interferometry sensorogram using biosensor with rNS of Seq. ID 1 comparing interference pattern of 20 times diluted plasma sample of RT-PCR +ve patient with20 times diluted plasma sample of seven negative control patients (RT-PCR -ve).
  • the data of interference pattern is acquired in real- time data during hydration of biosensor depicted in first 30 seconds in the graph, binding of rNS Seq. ID 1 (ligand) for 120 seconds depicted from 30-150 seconds in the graph, washing the biosensor to remove unbound rNS Seq. ID 1 for 30 seconds depicted from 150-180 seconds, and exposing biosensor with diluted plasma sample of patient depicted from 180-300 seconds.
  • bio-layer interferometry assay using the rNS of Seq. ID 1 as ligand for detecting total antibodies against SARS-CoV-2 was compared with commercially available ELISA kits (SARS-CoV-2 Spike Sl-RBD IgG & IgM ELISA Detection Kit from Genscript, China).
  • SARS-CoV-2 Spike Sl-RBD IgG & IgM ELISA Detection Kit from Genscript, China.
  • a total of 17 RT-PCR -Ve and 31 RT-PCR +Ve were analysed for total antibodies by ‘bio-layer interferometry assay using rNS of Seq. ID 1, and IgG & IgM ELISA using Spike Sl-RBD.
  • ID 1 as ligand for detecting total antibodies against SARS-CoV-2detected antibodies in all the 31 RT-PCR +Ve plasma samples, and the assay could’t detect any antibodies in RT-PCR -Ve plasma. Hence, there were no false positive or false negative results.
  • GenScript ELISA kit detected IgM in only 24 out of 31, and IgG in 29 out of 31 RT-PCR +ve samples thus giving around 25% false negative results in case of IgG and around 6.5% false negative results.
  • GenScript assay also showed positivity in one sample for IgM and in 4 samples for IgG in RT-PCR -Ve plasma, thus giving around 6% false positive in IgM detection, and 23.5% false positive in IgG detection. This shows that Bio-layer interferometry assay using the rNS of Seq. ID 1 as ligand is more sensitive and specific compared to the commercially available Covid-19or SARS-CoV-2 testing tests.
  • the assay was compared to a commercially available ELISA kit developed by National Institute of Virology, India using killed SARS-CoV-2 viral particles. Since the ELISA kit was developed using gamma irradiated whole viralparticle as antigen, it is supposed to detect antibodies against SARS-CoV-2 in total, i.e antibodies against all antigens, and reported to have 95% specificity and sensitivity against SARS-CoV-2. A total of 486 samples which never tested for SARS-CoV-2 were analyzed by both assays and sensitivity was compared.
  • bio-layer interferometry has more specificity and sensitivity in detecting antibodies.
  • a total of 4positive samples analysed by bio-layer interferometry assay using the rNS of Seq. ID 1 differed with said ELISA kit ELISA kit. This difference could be due to the fact that the ELISA kit detects only anti-IgG and not anti-IgM antibodies against SARS-CoV-2. Detection of IgMantibodies is of paramount importance to diagnose infection in early stages.
  • the bio-layer interferometry assay using the rNS of Seq. ID lkit shows greater sensitivity and specificity in detection of IgM, IgA, and IgG antibodies against SARS-CoV-2 infection.
  • Figure 7 A depicts a graph representing the binding rate of 40 plasma samples collected beforeCOVID-19 pandemic in India (Nov 2019) and 3 plasma samples (RT-PCR +ve) collected after COVID-19 pandemic.
  • Figure 7B depicts Receiver operating characteristic curve (ROC) analysis showing sensitivity versus specificity for discrimination of RT-PCR positive and plasma samples collected before COVID-19 pandemic showing 100% specificity (ROC area under the curve: 1.000, 95%confidence interval in between 1.000 to 1.000 and p values 0.004249).
  • ROC Receiver operating characteristic curve
  • Bio-layer interferometry assay using the rNS of Seq. ID 1 is suitable for rapid detection of total antibodies against SARS-CoV2 at a large scale with high specificity and sensitivity rates enabling population level testing or seroepidemiology of countries, races, etc. Such tests in large communities etc. are usefully to determine herd immunity etc.

Abstract

La présente invention concerne une nouvelle protéine de fusion nucléospicule recombinante de Seq. ID 1 pour la détection rapide d'anticorps totaux contre le SARS-CoV-2. L'invention concerne en outre un procédé de détection rapide d'anticorps totaux contre le SARS-CoV-2 dans un échantillon biologique par analyse d'interférométrie de bio-couche, avec des biocapteurs et avec un kit de diagnostic de ceux-ci.
PCT/IB2021/054637 2020-05-27 2021-05-27 Procédé de détection rapide d'anticorps contre sras-cov-2 à l'aide d'une protéine de fusion nucléospicule recombinante WO2021240425A1 (fr)

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WO2012012571A1 (fr) * 2010-07-21 2012-01-26 Hans Zassenhaus Mesure d'interférométrie à biocouche de cibles biologiques
CN111187354A (zh) * 2020-02-20 2020-05-22 北京新创生物工程有限公司 新型冠状病毒(SARS-CoV-2)IgM/IgG抗体检测试剂盒
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CN111196857A (zh) * 2020-02-05 2020-05-26 杭州贤至生物科技有限公司 一种新型冠状病毒多表位重组抗原及其制备方法
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