WO2022079001A2

WO2022079001A2 - Sars-cov-2 antigens, diagnostic and therapeutic uses

Info

Publication number: WO2022079001A2
Application number: PCT/EP2021/078138
Authority: WO
Inventors: Petra MORONI-ZENTGRAF; Christoph Keller; Egbert Siegfried MUNDT
Original assignee: Boehringer Ingelheim International Gmbh; Boehringer Ingelheim Veterinary Research Center Gmbh & Co. Kg
Priority date: 2020-10-14
Filing date: 2021-10-12
Publication date: 2022-04-21
Also published as: WO2022079001A3

Abstract

This invention relates to antigens of SARS-CoV-2, diagnostic and therapeutic modalities thereto, and related uses in the diagnosis, prophylactic and therapy of COVID-19. In particular, the present invention relates to a pharmacologically active compound which either elicits an immune response against the S2 subunit of SARS-CoV-2 spike protein, or neutralizes a biological activity of the S2 subunit of SARS-CoV-2 spike protein, for use in the prophylaxis or treatment of COVID-19.

Description

SARS-COV-2 ANTIGENS, DIAGNOSTIC AND THERAPEUTIC USES

BACKGROUND OF THE INVENTION

TECHNICAL FIELD

This invention relates to antigens of SARS-CoV-2, diagnostic and therapeutic modalities thereto, and related uses in the diagnosis, prophylactic and therapy of COVID-19.

BACKGROUND INFORMATION

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was first identified in December 2019 in Wuhan, Hubei, China, and has resulted in an ongoing pandemic. The first confirmed case has been traced back to 17 November 2019 in Hubei. As of 9 July 2020, more than 12.1 million cases have been reported across 188 countries and territories, resulting in more than 552,000 deaths (Guo et al., Frontiers in Cell and Developmental Biology 2020, 8:410).

Common symptoms include fever, cough, fatigue, shortness of breath, and loss of smell and taste. While the majority of cases result in mild symptoms, some progress to acute respiratory distress syndrome (ARDS) possibly precipitated by cytokine storm, multi-organ failure, septic shock, and blood clots. The time from exposure to onset of symptoms is typically around five days, but may range from two to fourteen days.

SARS-CoV-2 (GenBank accession number: MN908947, isolate Wuhan-Hu-1) is a strain of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). It is a positivesense single-stranded RNA (+ssRNA) virus, with a single linear viral RNA molecule. Each SARS-CoV-2 virion is 50-200 nanometres in diameter. Like other coronaviruses, SARS- CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; the N protein is associated with the viral RNA genome, while the S, E, and M proteins together build the viral envelope. The spike protein is the protein responsible for allowing the virus to attach to and fuse with the membrane of a host cell; specifically, its SI subunit attaches to the cellular receptor, the S2 subunit facilitates fusion with the cellular membrane fusion (Su et al., Vaccine 2020, 38: 5071-5075; Walls et al., Cell 2020, 180:281-292, Lan et al., Nature 2020, 581 :215-220, Perotta et al., Respiratory Medicine 2020, 168: 105996). It is suggested that the spike protein receptor binding domain (RBD) binds to the receptor angiotensin converting enzyme 2 (ACE2) on human cells.

The spike glycoprotein (in this application, the terms ’’spike glycoprotein” and “spike protein” are used synonymously) of SARS-CoV-2 is a membrane protein of 1273 amino acids (SEQ ID NO: 1, NCBI Reference Sequence: YP_009724390. Iv, isolate Wuhan-Hu- 1). The S2 subunit comprises amino acid 662-1270 of the spike protein sequence (SEQ ID NO.: 2). Sequence motifs within the S2 subunit comprise a fusion peptide region (FP, SEQ ID NO.: 3), and two heptad repeat regions (HR1, SEQ ID NO: 4, HR2, SEQ ID NO: 5).

Efforts to find prophylactic or therapeutic modalities for COVID-19, as well as improved diagnostic methods, are underway.

As one therapeutic option, the use of neutralizing antibodies targeting e.g. the spike protein or its subunits have been suggested, with a focus on antibodies targeting the RBD domain of the SI subunit (Ho M, Antibody Ther. 2020, 3(2): 109-114; Hansen et al., Science 2020, 10.1126/science.abd0827, Rogers et al., Science 2020, 10.1126/science.abc7520, .Elshabrawy, Vaccines 2020, 8:335, Jiang et al., Trends Immunol. 2020, 41(5): 355-359, Wang et al., Nature Communications 2020, 11 :2251).

Different approaches for vaccination have been suggested and some have been developed and approved by regulatory authorities. Also here, there is strong emphasis on vaccines comprising spike protein, or antigenic sequences thereof, in particular the RBD domain of the SI subunit. Proposed or approved vaccines are based on antigenic peptides, viral vectors or nucleic acids capable of expression of antigenic spike protein sequences, or inactivated virus (Ahmed et al, Viruses 2020, 12:254, Kalita et al., Microbial Pathogenesis 2020, 145: 10236, Bhattacharya e al., J Med Virol. 2020, 92:618-631, Smith et al., Nature Comm. 2020, 11 :2601; Zhu et al., Lancet 2020, 395: 1845-1854, Salvatori et al. J Transl Med (2020), 18:222, Wang et al., Med Sci Monit. 2020, 26:e924700, Doremalen et al., bioRxiv 2020.05.13.093195; Mulligan et al., medRxiv 2020.06.30.20142570, Jackson et al., New Engl J Med 2020, DOI: 10.1056/NEJMoa2022483). Neutralizing human monoclonal antibodies against highly conserved HR1 and HR2 domains of the spike protein S2 subunit, as well as vaccines comprising respective antigen, had been previously suggested as either therapeutic agents or prophylactic measures, respectively, against infection with related SARS-CoV-1 strain that led to an epidemic in 2002/2003 (Elshabrawy et al., PLOS One 2010, 7(11): e50366, Lip et al. 2006, J Virol. 80(2):841-950).

Immunoassays have been used to profile antibody responses post SARS-CoV-2 infection (Nisreen MAO et al., EID Journal 2020, 26(7): 1478-1488; Lee CY et al., Front. Immunol. 2020, 11 :879)

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a pharmacologically active compound which either elicits an immune response against the S2 subunit of SARS-CoV-2 spike protein, or neutralizes a biological activity of the S2 subunit of SARS-CoV-2 spike protein, for use in the prophylaxis or treatment of COVID-19.

In another aspect, the present invention relates to a method of prophylactic or therapeutic treatment of an individual being at risk of acquiring COVID-19, or having clinical signs of COVID-19, or having been tested positive for SARS-CoV-2 in a diagnostic test, comprising administering to the individual a therapeutically effective amount of a compound which either elicits an immune response against the S2 subunit of SARS-CoV- 2 spike protein, or neutralizes a biological activity of the S2 subunit of SARS-CoV-2 spike protein.

In another aspect, the present invention relates to an antibody which is specific for SARS- CoV-2 spike protein S2 subunit, but does not bind to the SI subunit, for use in the prophylaxis or treatment of COVID-19. Preferably, the antibody is a neutralizing antibody. Preferably, the antibody binds to an epitope within SEQ ID NO.: 2. In another aspect, the present invention relates to a vaccine against SARS-CoV-2 infection, comprising one or more antigenic peptides of spike protein S2 subunit, or a nucleic acid sequence coding for one or more antigenic peptides of spike protein S2 subunit, but no peptides of spike protein SI subunit, or respective nucleic acid sequence.

In another aspect, the present invention relates to a vaccine against SARS-CoV-2 infection, comprising one or more antigenic peptides of spike protein S2 subunit, or a nucleic acid sequence coding for one or more antigenic peptides of spike protein S2 subunit, and peptides of spike protein SI subunit, or respective nucleic acid sequence.

In another aspect, the present invention relates to diagnostic kit for the determination of the level of antibodies specific for SARS-CoV-2 spike protein S2 subunit in a sample, comprising a. isolated SARS-CoV-2 S2 subunit antigen, or fragment thereof b. means for detection whether antibodies from that sample have bound to said S2 subunit antigen.

In another aspect, the present invention relates to process for deciding on a therapeutic intervention for a patient suspected or known to be infected by SARS-CoV-2, with the steps a. Obtaining either a blood sample or a saliva sample or a tear fluid sample from said patient; b. Determining the level of antibodies against spike protein SI subunit and the level of antibodies against spike protein S2 subunit in said sample; and c. Deciding on the therapeutic intervention based on the results obtained in step.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Detectability of SARS-CoV-2 RNA and different antibodies against SARS- CoV-2 at different stages during the course of an infection and interpretation of combined diagnostic test results. This graphic is for illustrative purposes only and does not represent actual levels of RNA/each antibody. Figure 2. IgA against SI protein (Eurolmmun) over time. Panel A. Participants with COVID-19 history known before the program start (n=20); B. Participants with COVID-19 history identified during the program (n=10); C. Comparison of levels in all participants (n=141) over all four visits.

*An optical density [OD] ratio of serum sample/OD of calibrator below 0.8 was evaluated as negative, 0.8— <1.1 as borderline and >1.1 as positive. COVID-19ip: COVID-19 history unknown before program but identified as positive during the program; COVID-19kp: positive COVID-19 history known before program; COVID-19neg: COVID-19 history unknown before program and not identified during the program.

Figure 3. IgG against SI protein (Eurolmmun) over time. Panel A. Participants with COVID-19 history known before the program (n=20); B. Participants with COVID-19 history identified during the program (n=10); C. Comparison of levels in all participants (n=141) over all four visits.

*An optical density [OD] ratio of serum sample/OD of calibrator below 0.8 was evaluated as negative, 0.8-< 1.1 as borderline and >1.1 as positive. COVID-19ip: COVID-19 history unknown before program but identified as positive during the program; COVID-19kp: positive COVID-19 history known before program; COVID-19neg: COVID-19 history unknown before program and not identified during the program.

Figure 4. IgG against S1/S2 proteins (Diasorin CLIA) over time. Panel A. Participants with COVID-19 history known before the program start (n=20); B. Participants with COVID-19 history unknown before the program but identified during the program (n=10); C. Comparison of levels in all participants (n=141) over all four visits.

* A level of <12 IU was considered negative; 12— <15 as borderline and >15 as positive. COVID-19ip: COVID-19 history unknown before program but identified as positive during the program; COVID-19kp: positive COVID-19 history known before program; COVID-19neg: COVID-19 history unknown before program and not identified during the program. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the observation that a high S2 subunit specific IgG antibody titer after SARS-CoV-2 infection correlates with a mild or even subclinical course of the infection, or disease. On the other hand, a more severe course of the disease may be associated with high titer of SI subunit specific IgG antibodies. In another aspect, the present invention is based on the observation that S2 subunit specific IgG antibody titer increase in the course of healing COVID-19, whereas SI subunit specific IgG antibody titer may decrease rapidly.

The present invention relates to the S2 subunit of SARS-CoV-2 spike protein as a target for therapeutic intervention, and as source of antigen for an anti-SARS-CoV-2 vaccine. In a preferred embodiment, said S2 subunit comprises the amino acid sequence as represented in SEQ ID NO.: 2. In another embodiment, said S2 subunit consists of the amino acid sequence as represented in SEQ ID NO.: 2. It is understood that virus strains evolve over time and acquire mutations (substitutions, insertions, deletions). Hence, the amino acid sequence of the S2 subunit may differ from SEQ ID No.: 2 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more residues.

In one embodiment, the present invention relates to an antibody having specificity for the S2 subunit of the spike protein of SARS-CoV-2, for use in medicine. In particular, the present invention relates to an antibody having specificity for the S2 subunit of the spike protein of SARS-CoV-2, for use in the prophylactic or therapeutic treatment of COVID-19. Preferably, the epitope for the antibody is within SEQ ID NO.: 2, or a sequence which differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from SEQ ID NO.: 2.

“Having specificity”, or “specific for” in the context of the present invention means having a high binding affinity for the S2 subunit of the spike protein of SARS-CoV-2, but low or no measurable binding affinity to unrelated targets, for example human proteins. Importantly, antibodies specific for the S2 subunit of the spike protein in the context of the present invention must not bind, or bind only with very low affinity (Ko>100pM, see below) to isolated SI subunit, or discrete epitopes within the SI subunit of the spike protein. In other words, such antibodies shall discriminate between the SI and S2 subunits of the spike protein of SARS-CoV-2. Still, S2 specific antibodies in the context of the present invention may cross-react with S2 subunits of spike proteins of closely related corona virus strains. For example, the S2 subunit is highly conserved between SARS-CoV- 2 and SARS-CoV-1 that caused an outbreak in 2002/2003, and antibodies specific for the S2 subunit of the SARS-CoV-1 spike protein have been shown to be cross-reactive to SARS-CoV-2 (Zheng et al., Eurosurveillance 2020, 25(28): 19-28, Lv et al., Cell Reports 2020, 31(9): 1-6). Antibodies showing this type of cross-reactivity are therefore within the ambit of the present invention.

Affinity is the interaction between a single antigen-binding site on an antibody molecule and a single epitope. It is expressed by the association constant KA kass/kdiss, or the dissociation constant KD = kdiss/k_ass .

In one aspect , the antibody binds to the S2 subunit of the spike protein of SARS-.CoV-2 with an affinity, as determined e.g. by surface plasmon resonance analysis (Malmqvist M., "Surface plasmon resonance for detection and measurement of antibody-antigen affinity and kinetics.", Curr Opin Immunol. 1993 Apr;5(2):282-6.), with a KD value ranging from 1 pM to 100 pM, preferably 1 pM to 1 pM. Antibody affinity can also be measured using kinetic exclusion assay (KinExA) technology (Darling, R.J., and Brault P-A., “Kinetic exclusion assay technology: Characterization of Molecular Interactions.” ASSAY and Drug Development Technologies. 2004, Dec 2(6): 647-657).

Antibodies and methods of generating antibodies against defined antigens are well-known in the art. Antibodies (also known as immunoglobulins, abbreviated Ig) are gamma globulin proteins that can be found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. They are typically made of basic structural units - each with two large heavy chains and two small light chains - to form, for example, monomers with one unit, dimers with two units or pentamers with five units The basic unit is a heterotetrameric glycoprotein, typically of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is covalently linked to a heavy chain by one disulfide bond to form a heterodimer, and the heterotrimeric molecule is formed through a covalent disulfide linkage between the two identical heavy chains of the heterodimers. Although the light and heavy chains are linked together by one disulfide bond, the number of disulfide linkages between the two heavy chains varies by immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at the amino-terminus a variable domain (VH = variable heavy chain), followed by three or four constant domains (CHI, CH2, CH3, and CH4), as well as a hinge region between CHI and Cm. Each light chain has two domains, an amino-terminal variable domain (VL = variable light chain) and a carboxy-terminal constant domain (CL). The VL domain associates non-covalently with the VH domain, whereas the CL domain is commonly covalently linked to the CHI domain via a disulfide bond. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains. Antibodies can bind, by non-covalent interaction, to other molecules or structures known as antigens. This binding is specific in the sense that an antibody will only bind to a specific structure with high affinity. The unique part of the antigen recognized by an antibody is called an epitope, or antigenic determinant. The part of the antibody binding to the epitope is sometimes called paratope and resides in the so- called variable domain, or variable region (Fv) of the antibody. The variable domain comprises three so-called complementary-determining region (CDR’s) spaced apart by framework regions (FR’s).

Within the context of this invention, reference to CDR’s is based on the definition of Chothia (Chothia and Lesk, J. Mol. Biol. 1987, 196: 901-917), together with Kabat ( E.A. Kabat, T.T. Wu, H. Bilofsky, M. Reid-Miller and H. Perry, Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda (1983)).

The art has further developed antibodies and made them versatile tools in medicine and technology. Thus, in the context of the present invention the terms “antibody molecule” or “antibody” (used synonymously herein) do not only include antibodies as they may be found in nature, comprising e.g. two light chains and two or heavy chains, or just two heavy chains as in camelid species, but furthermore encompasses all molecules comprising at least one paratope with binding specificity to an antigen and structural similarity to a variable domain of an immunoglobulin. Thus, an antibody according to the invention may be a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a fragment of an antibody, in particular a Fv, Fab, Fab’, or F(ab’)2 fragment, a single chain antibody, in particular a single chain variable fragment (scFv), a domain antibody, a peptide aptamer, a nucleic acid aptamer or a nanobody.

Polyclonal antibodies represent a collection of antibody molecules with different amino acid sequences and may be obtained from the blood of vertebrates after immunization with the antigen by processes well-known in the art.

Monoclonal antibodies (mAb) are monospecific antibodies that are identical in amino acid sequence. They may be produced by hybridoma technology from a hybrid cell line (called hybridoma) representing a clone of a fusion of a specific antibody -producing B cell with a myeloma (B cell cancer) cell. Alternatively, monoclonal antibodies may be produced by recombinant expression in host cells.

For application in man, it is often desirable to reduce immunogenicity of antibodies originally derived from other species, like mouse. This can be done by construction of chimeric antibodies, or by a process called “humanization”. In this context, a “chimeric antibody” is understood to be antibody comprising a sequence part (e.g. a variable domain) derived from one species (e.g. mouse) fused to a sequence part (e.g. the constant domains) derived from a different species (e.g. human). A “humanized antibody” is an antibody comprising a variable domain originally derived from a non-human species, wherein certain amino acids have been mutated to make the overall sequence of that variable domain more closely resemble to a sequence of a human variable domain. Methods of chimerisation and humanization of antibodies are well-known in the art.

Furthermore, technologies have been developed for creating antibodies based on sequences derived from the human genome, for example by phage display or use of transgenic animals. Such antibodies are “human antibodies” in the context of the present invention.

Antibody molecules according to the present invention also include fragments of immunoglobulins which retain antigen binding properties, like Fab, Fab’, or F(ab’)2 fragments. Such fragments may be obtained by fragmentation of immunoglobulins e.g. by proteolytic digestion, or by recombinant expression of such fragments. For example, immunoglobulin digestion can be accomplished by means of routine techniques, e.g. using papain or pepsin. Papain digestion of antibodies typically produces two identical antigen binding fragments, so-called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields an F(ab')2. In Fab molecules, the variable domains are each fused to an immunoglobulin constant domain, preferably of human origin. Thus, the heavy chain variable domain may be fused to a CHI domain (a so-called Fd fragment), and the light chain variable domain may be fused to a CL domain. Fab molecules may be produced by recombinant expression of respective nucleic acids in host cells, see below.

Peptide Aptamers are artificial proteins selected or engineered to bind specific target molecules consisting of one or more peptide loops of variable sequences displayed by a protein scaffold. Aptamers are less immunogenic than antibodies and therefore provoke less undesirable reactions. Molecules like proteins reversibly bound to aptamers allow controlled release of biological molecules. On the other hand the bonds formed by aptamers are usually weak and they are easily digested by enzymes limiting their effectiveness.

Nucleic acid apatamers (from the Latin aptus - fit, and Greek meros - part) are oligonucleotide or peptide molecules that bind to a specific target molecule.

A number of technologies have been developed for placing variable domains of immunoglobulins, or molecules derived from such variable domains, in a different molecular context. Those should be also considered as “antibodies” in accordance with the present invention. In general, these antibody molecules are smaller in size compared to immunoglobulins, and may comprise a single amino acid chain or several amino acid chains. For example, a single-chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker, usually serine (S) or glycine (G). “Single domain antibodies” or „nanobodies” harbour an antigen-binding site in a single Ig-like domain. One or more single domain antibodies with binding specificity for the same or a different antigen may be linked together, resulting for example in bispecific antibodies.

The antibody molecule may be fused (as a fusion protein) or otherwise linked (by covalent or non-covalent bonds) to other molecular entities having a desired impact on the properties of the antibody molecule. For example, it may be desirable to improve pharmacokinetic properties of antibody molecules, stability e.g. in body fluids such as blood, in particular in the case of single chain antibodies or domain antibodies. A number of technologies have been developed in this regard, in particular to prolong the half-life of such antibody molecules in the circulation, such as pegylation, fusing or otherwise covalently attaching the antibody molecule to another antibody molecule having affinity to a serum protein like albumin, or expression of the antibody molecule as fusion protein with all or part of a serum protein like albumin or transferrin.

For the present invention, antibodies of the IgG subtype, e.g. IgGl, or IgG4, may be well suited. Alternatively, other IgG subtypes, e.g. IgG2 or IgG3, or IgA may be used.

Generation of antibodies having specificity for the S2 subunit of SARS-CoV-2 have been described in the prior art (Zheng et al., Eurosurveillance 2020, 25(28): 19-28, Lip et al. 2006, J Virol. 80(2):841-950).

In a preferred aspect of the invention, the antibody is a neutralizing antibody. A neutralizing antibody in the context of the present invention is capable, by its binding to the S2 subunit, of partly or completely inhibiting a biological activity of the spike protein and/or its S2 subunit, for example inhibiting or preventing fusion of the viral envelope and cellular membranes. Ideally, by this neutralizing activity, the infection of the cells and the subsequent pathogenic effects of SARS-CoV-2 can be diminished or prevented. Whether a given antibody having binding specificity for the S2 subunit of the spike protein has neutralizing activity can be determined by the expert with assay methods known in the art (Hansen et al., Science 2020, 10.1126/science.abd0827, Elshabrawy et al., PLOS One 2010, 7(11): e50366), Lip et al. 2006, J Virol. 80(2):841-950). Methods of manufacturing of an antibody in a quantity and quality that it can be used in medicine are well-known in the art.

In another aspect, the present invention provides a pharmaceutical composition comprising an antibody against the S2 subunit of the spike protein and a pharmaceutically acceptable carrier.

To be used in medicine, the antibody is included into pharmaceutical compositions appropriate to facilitate administration to animals or humans. Typical formulations of the antibody molecule can be prepared by mixing the antibody molecule with physiologically acceptable carriers, excipients or stabilizers, in the form of lyophilized or otherwise dried formulations or aqueous solutions or aqueous or non-aqueous suspensions. Carriers, excipients, modifiers or stabilizers are nontoxic at the dosages and concentrations employed. They include buffer systems such as phosphate, citrate, acetate and other anorganic or organic acids and their salts; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone or polyethylene glycol (PEG); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, oligosaccharides or polysaccharides and other carbohydrates including glucose, mannose, sucrose, trehalose, dextrins or dextrans; chelating agents such as EDTA; sugar alcohols such as, mannitol or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or ionic or non-ionic surfactants such as TWEEN™ (polysorbates), PLURONICS™ or fatty acid esters, fatty acid ethers or sugar esters. Also organic solvents can be contained in the antibody formulation such as ethanol or isopropanol. The excipients may also have a release-modifying or absorption-modifying function.

In one aspect, the pharmaceutical composition comprises the antibody in an aqueous, buffered solution at a concentration of 10, 20, 30, 40, or 50 mg/ml, or a lyophilisate made from such a solution. The antibody is administered by any suitable means, including, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

For the prevention or treatment of disease, the appropriate dosage of antibody will depend on a variety of factors such as the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

In another aspect, the present invention relates to a vaccine against SARS-CoV-2 infection, comprising one or more antigenic peptides of spike protein S2 subunit, but no peptides of spike protein S 1 subunit.

Methods of producing vaccines comprising peptides as antigens are known in the art. Such vaccines could comprise recombinantly produced S2 subunit protein of SEQ ID NO.: 2, or one or more fragments thereof.

In another aspect, the present invention relates to a vaccine against SARS-CoV-2 infection, comprising one or more nucleic acids encoding antigenic peptides of spike protein S2 subunit, but no nucleic acids encoding peptides of spike protein SI subunit.

Methods of producing vaccines comprising nucleic acids encoding antigenic peptides are known in the art.

For example, Doremalen et al., bioRxiv 2020.05.13.093195, describe production and testing of a COVID-19 vaccine based on an adenoviral vector (ChAdOxl nCov-19) that comprises a nucleic acid encoding amino acids 2-1273 of SARS-CoV-2 spike protein, i.e. including both SI and S2 subunits. One mode of carrying out the present invention would be to replace the spike sequence used by Doremalen by a shorter sequence comprising S2 epitopes only, e.g. amino acids 662-1273, or fragment thereof, like SEQ ID NO.: 2. A different way of carrying out the present invention is the production of an RNA-based vaccine as described in Mulligan et al. Nature 2020, https://doi.org/10.1038/s41586-02Q- 2639-4, or Jackson et al., New Engl J Med 2020, DOI: 10.1056/NEJMoa2022483, but replacing the full-length spike protein sequences, or the RBD domain sequences disclosed in these publications, by a sequence comprising S2 epitopes only, like SEQ ID NO.: 2, or fragment thereof.

EXAMPLES

METHODS

Study design and participants

Human volunteers aged between 20 and 63 years participated in the study. The study was performed as a two-group testing program according to COVID- 19 status: (i) known and recovered from COVID-19 and (ii) unknown COVID-19 history. This design enabled comparison of test results between a group known to have had COVID- 19 (COVID- 19 known positive [COVID- 19kp]) and a group with unknown history, but potentially infected unknowingly in the past or recently (COVID19uk). Testing schedule and subsequent actions were determined by participants’ status, which included not infected, acutely ill/infectious, or recovered/immune (Supplementary Figure SI and Table SI). Four regular visits were scheduled at approximately 2-week intervals (see Supplementary Appendix).

Baseline and disease characteristics

A comprehensive assessment of participants’ baseline and disease characteristics including medication use and influenza vaccination was performed.

Testing materials

All test kits were CE-marked and commercially available.

For detection of SARS-CoV-2 RNA by RT-PCR, a throat swab was collected by qualified healthcare personnel. Samples were assayed using the Kylt® SARS-CoV-2 Initial Screening/Confirmation (AniCon Labor GmbH, Holtinghausen, Germany). Confirmatory RT-PCR for positive initial RT-PCR results and IgM- (LFA) and IgA- (ELISA) positive participants were performed by an independent laboratory.

LFAs for IgM and IgG determination were used using fingertip blood or venous blood samples. The following test systems were evaluated: VivaDiag COVID-19 IgM/IgG Rapid Test (VivaChek Biotech Co Ltd, Hangzhou, China); 2019-nCoV IgG/IgM Rapid Test Cassette (MoLab GmbH, Langenfeld, Germany); SARS-CoV-2 rapid IgM/IgG antibody test (Nano Repro AG, Marburg, Germany); and PerGrande SARS-CoV-2 Antibody Detection Kit IgG (PerGrande BioTech Development Co Ltd, Beijing, China); all directed against the N protein of the virus.

Laboratory assays for specific SARS-CoV-2 IgA, IgG and total IgE (unspecific) were conducted using venous blood samples (IgG S 1/2: LIAISON SARS-CoV-2 S1/S2 IgG chemiluminescence immunoassay [CLIA; DiaSorin, Saluggia, Italy]; IgG SI: Anti-SARS-CoV-2 ELISA SI IgG [Euroimmun AG, Lubeck, Germany]; IgA (SI): anti-SARS-CoV-2 ELISA IgA [Euroimmun AG]; total IgE: Roche Elecsys Total IgE II assay [Roche Diagnostics, Rotkreuz, Switzerland]). All laboratory assays outlined above allow a quantitative readout. This feature was used to capture the temporal evolution of the antibody levels.

Digital self-monitoring

For real-time digital self-monitoring, participants were asked to record vital signs (body temperature and pulse) and any suspicious symptoms for COVID-19 daily.

Statistical analysis

Test results and demographic data were analyzed using descriptive statistics. Standard statistical parameters of the test performance, like sensitivity, specificity and positive and negative predictive values, were calculated. To that end, medical judgment and a post hoc classification of each participant’s Covid- 19 history — based on the full diagnostic information package accumulated during the testing program — was considered as gold standard. The (semi-)quantitative outcomes from the laboratory assay tests were plotted over time and evaluated by linear regression to quantify the dynamics of potential seroconversion. RESULTS

Participants and baseline characteristics

Baseline demographics and characteristics are shown in Table 1. In total, 141 participants aged between 20 and 63 years entered the program (61 female); amongst these, 20 participants were known positives for COVID-19 (COVID-19kp; confirmed by RT-PCR and clinical assessment). There were no incident infections during the testing phase. During the program, 10 participants (8.3%) out of the 121 COVID-19uk participants were medically judged to have had COVID-19 prior to testing, based on diagnostic results, evidence for risk of infection and medical history, indicating a mild course of infection with only minor clinical symptoms; this subset was subsequently denoted as COVID-19-identified-positive (COVID-19ip). Of these, nine participants were only positive by DiaSorin S1/S2 IgG CLIA and one participant was positive by IgA/IgG SI ELISA at Visit 1. The remaining 111 participants from the COVID- 19uk cohort were re-classified as CO VID-19 negative (COVID-19neg; Table 1 and Supplementary Figure S2). Participants from the COVID- 19kp or COVID-lOip groups were numerically less likely to report allergies compared with the COVID- 19neg group, including the most commonly reported allergy ‘allergic rhinitis due to pollen’, less likely to take antihistamines, to have baseline IgE values of >100 lU/mL and less likely to have had an influenza vaccination in 2019/2020 (Table 1 and Supplementary Figure S3).

Table 1. COVID infection by demographics and baseline characteristics

'Reported by at least 10 participants in any group. Detection of SARS-CoV-2 antibodies by LFA

Amongst COVID-19kp participants, the frequency of positive LFA IgG and IgM testing results generally decreased over time, except for the MoLab 2019-nCoV IgM LFA. In COVID-19ip participants, the frequency of positive LFA results (IgM and IgG) was much lower compared with COVID-19kp participants (0-10.8% vs. 33.8-75.7%, respectively).

Positive IgM results in the absence of additional indication of SARS-CoV-2 infection were found in 10 (8.3%) COVID-19neg participants, using at least one LFA and at least once over the observation period. In most cases, participants had an allergic disposition with symptoms of seasonal allergic reactions and/or potential infections during testing, with subacute symptoms (such as pain or increased body temperature). None of the participants were RT- PCR positive and no other SARS-CoV-2 antibodies were observed at any visit. It is therefore likely that these isolated IgM results were unspecific and probably caused by infection other than SARS-CoV-2.

Detection of SARS-CoV-2 antibodies by laboratory serology

A pronounced decline in S 1 IgA (Euroimmun) levels was observed over time, in particular amongst COVID-19kp participants (Figure 2A). In comparison, the overall assay levels of the COVID- 19ip group (Figure 2B) fell clearly below the COVID- 19kp group, with levels that were hardly sufficient to generate a positive test signal (Figure 2B).

A similar decline in levels was observed for the SI -specific IgG response (Euroimmun) in the COVID- 19kp group (Figure 3A). As observed with IgA, the overall assay levels of the COVID- 19ip group fell clearly below the “known positive” level (Figure 3B), and were therefore not sufficient to generate a positive test signal (Figure 3C).

In contrast to the Euroimmun ELISAs, the Diasorin CLIA, which detects IgG against both S 1 and S2, showed a sustained IgG response over time (Figure 4A) in the COVID-19kp group. In the COVID- 19ip group (Figure 4B), the observed immune response against S1/S2 remained stable and persistent, although notably below the COVID- 19kp group; however, the signal remained sufficiently large for a positive detection (Figure 4C).

SEQUENCES

Claims

WHAT WE CLAIM

1. Pharmacologically active compound which either elicits an immune response against the S2 subunit of SARS-CoV-2 spike protein or neutralizes a biological activity of the S2 subunit of SARS-CoV-2 spike protein for use in the prophylaxis or treatment of COVID-19.

2. Neutralizing antibody which is specific for SARS-CoV-2 spike protein S2 subunit, but does not bind to the SI subunit, for use in the prophylaxis or treatment of COVID-19.

3. Vaccine against SARS-CoV-2 infection and COVID-19, comprising one or more antigenic peptides of spike protein S2 subunit, or a nucleic acid sequence coding for one or more antigenic peptides of spike protein S2 subunit, but no peptides of spike protein SI subunit, or respective nucleic acid sequence.

4. Vaccine against SARS-CoV-2 infection and COVID-19, comprising one or more antigenic peptides of spike protein S2 subunit, or a nucleic acid sequence coding for one or more antigenic peptides of spike protein S2 subunit, and peptides of spike protein SI subunit, or respective nucleic acid sequence.

5. Diagnostic kit for the determination of the level of antibodies specific for SARS-CoV-2 spike protein S2 subunit in a sample, comprising a. isolated SARS-CoV-2 S2 subunit antigen, or fragment thereof b. means for detection whether antibodies from that sample have bound to said S2 subunit antigen.

6. Process for deciding on a therapeutic intervention for a patient suspected or known to be infected by SARS-CoV-2, with the steps a. Obtaining an either blood sample, or a saliva sample or a tear fluid sample from said patient; b. Determining the level of antibodies against spike protein SI subunit and the level of antibodies against spike protein S2 subunit in said sample; and c. Deciding on the therapeutic intervention based on the results obtained in step b. Method of prophylactic or therapeutic treatment of an individual being at risk of developing COVID-19, or having clinical signs of COVID-19, or having been tested positive for SARS-CoV-2 in a diagnostic test, comprising administering to the individual a therapeutically effective amount of a compound which either elicits an immune response against the S2 subunit of SARS-CoV-2 spike protein, or neutralizes a biological activity of the S2 subunit of SARS-CoV-2 spike protein.