WO2022231442A1 - Antipeptide immunoglobulins recognizing nonglycosylated epitopes within the amino-terminal extramembranous domain of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) membrane glycoprotein (m protein) and cognate oligopeptide- based immunogenic constructs, methods for producing and utilizing the same, and systems relating thereto for the detection of antigens comprising said epitopes - Google Patents

Abstract

The invention provides: antipeptide immunoglobulins recognizing epitopes within amino-terminal extramembranous domain sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) membrane glycoprotein (M protein); cognate oligopeptide-based immunogenic constructs comprising said epitopes; methods for producing said immunoglobulins and said constructs; methods and systems for detecting antigens comprising said epitopes; use of said constructs for producing said0 immunoglobulins; and use of said immunoglobulins for detecting said antigens.

Description

ANTIPEPTIDE IMMUNOGLOBULINS RECOGNIZING NONGLYCOSYLATED EPITOPES WITHIN THE AMINO-TERMINAL EXTRAMEMBRANOUS DOMAIN OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-CoV-2) MEMBRANE GLYCOPROTEIN (M PROTEIN) AND COGNATE OLIGOPEPTIDE-

BASED IMMUNOGENIC CONSTRUCTS, METHODS FOR PRODUCING AND UTILIZING THE SAME, AND SYSTEMS RELATING THERETO FOR THE DETECTION OF ANTIGENS COMPRISING

SAID EPITOPES

Field of the Invention

The present invention relates to the field of antipeptide immunoglobulins, including their production and biomedical use, particularly in relation to such immunoglobulins that recognize epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Background of the Invention Pathogen (e.g., virus) genomes code for pathogen- associated proteins (e.g., forming structural components of pathogens themselves) that are produced in the course of pathogen propagation (e.g., viral replication). Said proteins may be recognized by host immune-system components such as immunoglobulins (e.g., antibodies), which are thus useful for various biomedical applications such as immunodiagnostics and immunization for the control and prevention of infectious diseases.

Consequent to infection with a pathogen, the immune system of a vertebrate host typically mounts an immune response that comprises an antibody response, whereby antibodies are produced that recognize pathogen-associated proteins. Said antibody response is typically initiated by the binding of surface immunoglobulins to said proteins, said immunoglobulins being physically associated with the plasma membranes of B cells (i.e., B lymphocytes) of the host immune system. Said B cells may thus be activated to proliferate and subsequently differentiate into plasma cells, which secrete said antibodies (i.e., soluble immunoglobulins). Said antibodies are thus analytes of interest from the standpoint of immunodiagnostics, as their presence may indicate current or past infection by said pathogen, and also insofar as they may be used to detect antigens of said pathogen in biological or clinical samples. Furthermore, said antibodies may also be of therapeutic and/or prophylactic value, as they may be useful for immunization that enables the treatment and/or prevention of infection by said pathogen.

Said immunization may be either active where said antibodies are endogenously produced or passive where they are exogenously supplied. Active immunization may thus occur as the result of infection with a pathogen, such that the infected host mounts an immune response against the pathogen and/or pathogen biomolecules. Alternatively, active immunization may be attained via vaccination, with administration of a vaccine that stimulates the host immune system to mount an immune response, possibly even before the host ever encounters the pathogen. In contrast, passive immunization may be achieved by administering preformed antibodies (e.g., produced via active immunization) to a recipient host, for the treatment and/or prevention of infection by the pathogen . Pathogen-associated proteins may thus be used for immunodiagnostics to detect antibodies and/or for immunization (e.g., for vaccination to attain active immunization and thus produce antibodies, which may be used for immunodiagnostics to detect pathogens and also for passive immunization to prevent or treat infectious disease caused by pathogens). However, production of said proteins for such applications is problematic in that this entails safety issues related to the handling of potentially biohazardous materials (e.g., pathogens themselves and/or their associated biomolecules) and more generally to the use of biotechnology (e.g., genetic manipulation and microbial propagation), which pose the problem of dual use (i.e., utility for both peaceful and military aims).

For detection and production of antibodies, synthetic chemistry provides a safer alternative to biotechnology. In particular, synthetic chemistry enables the production of relatively short synthetic peptides that comprise amino-acid residue sequences of pathogen- associated proteins, said sequences being recognized by immunoglobulins that bind to said proteins. This can obviate the use of biotechnology while still enabling detection and even production of antibodies, using peptide-based constructs instead of said proteins for immunodiagnostics and vaccines. Yet, such a peptide-based approach is applicable only in cases where said immunoglobulins still recognize said sequences when removed from the structural context of said proteins, which may assume conformations that are difficult to mimic among synthetic peptides (e.g., due to excessive conformational flexibility of such peptides relative to natively folded protein segments of identical amino-acid residue sequence). Hence, binding of said immunoglobulins to said sequences may be abrogated or otherwise too weak to be of practical use.

In principle, synthetic peptides can be incorporated into immunogenic constructs that can be used to immunize suitable vertebrate species and thereby elicit production of cognate antipeptide immunoglobulins, which recognize said peptides. Said constructs are typically produced by covalently linking said peptides to immunogenic carriers (e.g., proteins), which enable immunoglobulin production in said species (e.g., by recruiting T-cell help for B cells, which are thus activated to proliferate and differentiate into either antibody-secreting plasma cells or memory B cells that form the basis for more rapid and intense antibody responses upon subsequent booster immunization with the same immunogenic construct) . Said immunoglobulins may recognize proteins in addition to said peptides, for example, in cases where a common amino-acid residue sequence is shared by said proteins and said peptides.

Antipeptide immunoglobulins can thus afford highly specific binding to relatively short (e.g., 20-mer or shorter) amino-acid residue sequences that occur as parts of proteins, especially if said immunoglobulins are affinity purified using peptides comprising said sequences; but because said immunoglobulins are so structurally well adapted for binding to said sequences, even seemingly minor modifications of said sequences may abrogate said binding, notably where steric hindrance is thus introduced. This is exemplified by an amino-acid residue substitution that replaces one (e.g., glycine) residue with a larger residue, such that steric clashes due to said larger residue preclude binding of an immunoglobulin. Among the 20 proteogenic amino acids represented in standard codon tables, the smallest one is glycine It is also the only one that is achiral, as its side chain is a hydrogen atom. In contrast, each of the other said amino acids is chiral, as its side chain comprises at least one non-hydrogen atom. Hence, immunoglobulin binding may be abrogated by steric hindrance resulting from replacement of glycine by a chiral residue in a sequence in an antigen. Such steric hindrance is the physical basis for the ability of pathogen escape mutants (e.g., viral variants) to evade antibody-mediated immune mechanisms where glycine is replaced by a chiral residue in a pathogen-associated protein antigen.

Antipeptide immunoglobulins may thus be produced by immunization with a synthetic peptide comprising an amino-acid residue sequence, said immunoglobulins recognizing proteins that likewise comprise said sequence. Yet, occurrence of said sequence in said proteins may be insufficient as basis for said immunoglobulins to recognize said proteins, particularly where key features other than sequence differ between the peptide and protein structural contexts. Such features include conformational state (e.g., relatively flexible in a short peptide versus relatively rigid in a natively folded protein), sequence location in the primary structure (e.g., at the amino terminus of a peptide versus within an internal segment of a protein) and posttranslational modification (e.g., glycosylation absent in a peptide but present in a protein comprising the sequence of said peptide, as an antipeptide immunoglobulin produced via immunization with said peptide may fail to recognize said protein due to glycosylation of said protein and consequent steric hindrance arising from steric clashes between said immunoglobulin and a carbohydrate moiety thus linked to said protein). Hence, such features must be carefully considered in the selection of sequences from proteins for use in peptides to produce antipeptide immunoglobulins, in order to avoid or minimize differences in structural context that tend to prevent said immunoglobulins from recognizing said proteins.

Antibodies are known to be produced against the membrane glycoprotein (hereafter referred to as M protein) of severe acute respiratory syndrome coronavirus (SARS-CoV, now also known as and hereafter referred to as SARS-CoV-1) and also against M protein of severe acute respiratory syndrome coronavirus 2 (hereafter referred to as SARS-CoV-2). For example, such antibody production against M protein can occur in a suitable vertebrate (e.g., mammalian) host subsequent to infection of said host by either SARS-CoV-1 or SARS-CoV-2.

In both SARS-CoV-1 and SARS-CoV-2, M protein is largely embedded in the viral envelope (i.e., viral membrane), being a transmembrane glycoprotein comprising an extramembranous amino-terminal domain that is oriented outward from the outer surface of said envelope. Typically, said domain comprises an amino-acid residue sequence less than 20 residues long, said sequence comprising an additional residue in SARS-CoV-2 relative to SARS-CoV-1, said additional residue being a serine residue located between an aspartate residue and an asparagine residue within said sequence, which further comprises a glycine residue downstream of said asparagine residue, said glycine residue being present in both SARS-CoV-1 and SARS-CoV-2.

Accordingly, said glycine residue shall hereafter be referred to as the consensus glycine residue, which typically occurs as the middle residue Xaa in a classic tripeptide N-linked glycosylation motif of the form Asn-Xaa-Thr, where Asn (i.e., said asparagine residue) is the glycosyl acceptor site and Xaa is a residue other than proline. Hence, said motif shall hereafter be referred to as the consensus glycosylation motif.

After infection of vertebrate (e.g., human) hosts by either SARS-CoV-1 or SARS-CoV-2, antibodies may be obtained from said hosts, with immunoassays subsequently revealing case-to-case variability among said hosts in the ability of said antibodies to recognize amino-terminal extramembranous domain sequences of M protein, said sequences being used in the form of synthetic oligopeptides (e.g., shorter than 20 amino-acid residues in length) as immunological probes to detect said antibodies in said immunoassays. In some cases, said immunoassays fail to detect said antibodies even where said oligopeptides comprise said sequences. In other cases, said immunoassays may detect said antibodies where said oligopeptides comprise said sequences or even only parts of said sequences, with said oligopeptides consistently comprising either the consensus glycine residue or an even smaller chemical moiety (e.g., an acetyl group) rather than a larger chemical moiety (e.g., a chiral amino-acid residue) in place of said glycine residue, thereby avoiding steric clashes between said oligopeptides and said antibodies, thus enabling recognition of said oligopeptides by said antibodies in said immunoassays . A problem is thus posed by the above-described inconsistency of antibody responses resulting from immunization of vertebrate hosts using antigens comprising whole M protein, for example, in the course of infection wherein said hosts are immunized via exposure to whole virus particles (e.g., of SARS- CoV-1 or SARS-CoV-2), which comprise whole M protein. Said inconsistency thus hampers attempts to deliberately and consistently produce antibodies that recognize predefined short (e.g., less than 20- mer) sequences of M protein, particularly within its amino-terminal extr amembranous domain. Such antibodies are, by virtue of the ability to recognize and thus bind such sequences, potentially useful as immunodiagnostic reagents (e.g., for detecting antigens that comprise M protein or fragments thereof) and possibly also as prophylactic or even therapeutic agents against infection.

In order to address the problem of inconsistency in the production of antibodies against amino- terminal extramembranous domain sequences of M protein, one possible approach is to still proceed with immunization using antigens that comprise whole M protein, to attempt production of monoclonal antibodies that recognize said sequences. This might afford the advantage of obtaining a clonal cell population (e.g., of a hybridoma) that, in principle, could be maintained and used indefinitely to produce antibodies of well defined molecular structure and, consequently, well defined binding specificity. However, the initial process of immunization and antibody screening would still be subject to said problem of inconsistency, and the immunization strategy itself would thus be inadequate for applications such as active immunization to elicit production of antibodies that mediate protective immunity, for example, by specifically targeting said sequences.

Alternatively, said problem of inconsistency can be more comprehensively addressed by producing antipeptide antibodies that recognize said sequences, for example, by immunizing a suitable vertebrate (e.g., mammalian or avian) species using oligopeptide-based immunogenic constructs for active immunization, said constructs comprising oligopeptide moieties that in turn comprise said sequences. Antipeptide antibodies can thus be consistently produced and even affinity purified using oligopeptides comprising said sequences, thereby obtaining monospecific, albeit polyclonal, antipeptide antibodies that recognize said sequences. Such an approach is suggested by published data relating to production of rabbit antipeptide antibodies via immunization with a 31-mer nonglycosylated synthetic peptide identical in sequence to the 31-mer amino-terminal segment of SARS-CoV-1 M protein, demonstrating that said antibodies are thus produced and bind to (i.e., recognize) said synthetic peptide; but native M protein is known to be glycosylated, with glycosylation expected at the consensus glycosylation motif comprising the consensus glycine residue. In order to avoid an outcome wherein said antibodies would be unable to recognize native M protein (e.g., in whole virus particles) due to steric clashes between said antibodies and the carbohydrate component of said protein, faithful structural mimicry of said amino-terminal segment could be attempted by covalently coupling a suitable carbohydrate moiety to said peptide prior to use for immunization; but this would be more difficult and thus more expensive than simple peptide synthesis for producing a nonglycosylated product. Presented with the dilemma of more expensive synthetic peptide chemistry to faithfully mimic protein glycosylation versus likely failure to produce antipeptide immunoglobulins that recognize SARS-CoV-2 M-protein amino-terminal extramembranous- domain sequences protruding outward from the viral envelope, a person skilled in the art would be motivated to search for and ultimately select an alternative SARS-CoV-2 protein sequence that obviously satisfies known criteria for avoiding and minimizing structural differences between peptides and proteins for the purpose of producing antipeptide immunoglobulins that also recognize proteins.

However, such aversion to selecting glycosylated protein sequences risks failure to realize production of antipeptide immunoglobulins that bind downstream of the typical glycosyl acceptor site in said amino-terminal sequences, without any effort to mimic protein glycosylation at the level of synthetic peptide chemistry, which is of practical relevance to antibody-based applications such as antigen detection that address SARS-CoV-2 infection. Furthermore, even if said person were to use nonglycosylated synthetic peptide analogs of SARS- CoV-2 M-protein amino-terminal extramembranous- domain sequences for producing antipeptide immunoglobulins, said person would conceivably fail to anticipate the possibility that said immunoglobulins would also recognize homologs of said sequences wherein the consensus glycine residue is replaced by a chiral residue, said homologs being useful as immunological probes to detect said immunoglobulins.

In view of the preceding considerations, a need therefore exists to explore the possibility of using nonglycosylated amino-terminal peptide sequences in order to produce antipeptide immunoglobulins that recognize subsequences of the SARS-CoV-2 M-protein amino-terminal extramembranous domain, particularly downstream of the glycosyl acceptor site of the consensus glycosylation motif comprising the consensus glycine residue, while also exploring the possibility that said immunoglobulins also recognize homologs of said subsequences wherein the consensus glycine residue is replaced by a chiral amino-acid residue (e.g., a cysteine residue) or, more generally, wherein a residue of said subsequences is replaced by a bulkier residue in said homologs, which may thus be useful as immunological probes to detect said immunoglobulins. This need is fulfilled by the present invention as described below.

Summary of the Invention

In order to obtain and use antipeptide immunoglobulins that bind downstream of the glycosyl acceptor site of the consensus glycosylation motif in SARS-CoV-2 M-protein amino-terminal extramembranous -domain sequences, without any effort to mimic protein glycosylation at the level of synthetic peptide chemistry, the present invention provides: antipeptide immunoglobulins recognizing epitopes within amino-terminal extramembranous domain sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) membrane glycoprotein (M protein); cognate oligopeptide-based immunogenic constructs comprising said epitopes; methods for producing said immunoglobulins and said constructs; methods and systems for detecting antigens comprising said epitopes; use of said constructs for producing said immunoglobulins; and use of said immunoglobulins for detecting said antigens .

Accordingly, the present invention provides antipeptide immunoglobulins, each being suitable for binding an oligopeptide sequence (e.g., to elicit production of said immunoglobulins, affinity purify them and use them to detect antigens) and, in addition, at least two target sequences (e.g., to detect said immunoglobulins), said target sequences hereafter referred to as the first and second target sequences, said oligopeptide and target sequences all being distinct from one another, yet each comprising a core subsequence thus shared by all said sequences that is devoid of N-linked glycosylation motifs, thereby avoiding N-linked glycosylation of said subsequence during protein biosynthesis (e.g., in eukaryotes, as in the course of viral infection).

Said core subsequence perfectly matches part of a SARS-CoV-2 sequence, which is a membrane- glycoprotein amino-terminal extramembranous -domain sequence. Furthermore, said SARS-CoV-2 sequence and first target sequence each comprise a glycine residue located immediately amino- terminal to said core subsequence. However, said second target sequence comprises a chiral amino-acid residue (e.g., a cysteine residue) located immediately amino-terminal to said core subsequence. Hence, said immunoglobulins recognize one or more epitopes in said SARS-CoV-2 sequence that are devoid of N-linked glycosylation motifs (and thus tend to remain unglycosylated even in vivo), while also tolerating substitution of said glycine residue with chiral (e.g., cysteine) residues in the production of synthetic peptide-based constructs (e.g., oxidation- polymerized cysteine-containing peptide antigens) comprising epitopes of said SARS-CoV-2 sequence.

Brief Description of the Drawings For a more complete understanding of the invention, reference is made to the following description and accompanying drawings, in which:

FIG. 1 shows a liquid chromatography profile wherein the dominant peak, exhibiting an elution time of 18.375 minutes and comprising 90.285% of the integrated peak area, corresponds to artificial oligopeptide of sequence MADSNGTITVEELKKLLEQC with unblocked amino-terminus and amidated carboxy- terminus ;

FIG. 2 shows a mass-to-charge ratio (m/z) spectrum wherein the dominant ions correspond to artificial oligopeptide of sequence MADSNGTITVEELKKLLEQC with unblocked amino-terminus and amidated carboxy- terminus ;

FIG. 3 shows a set of indirect ELISA results for detection of rabbit antipeptide antibodies using their cognate artificial oligopeptide (of sequence

MADSNGTITVEELKKLLEQC with unblocked amino-terminus and amidated carboxy-terminus) as coating antigen;

FIG. 4 shows a set of indirect ELISA results for detection of rabbit antipeptide antibodies using polymeric peptides pMl and pMls (of sequences

CADSNGTITVEELKKLLEQC and CTITVEELKKLLEQC, with unblocked and acetylated amino-terminus, respectively, each with amidated carboxy-terminus) as coating antigens; and FIG. 5 shows a set of indirect ELISA results for detection of antibodies using polymeric peptides pMls and pTlC+ (of sequences CTITVEELKKLLEQC and CFIGITELKKLESKINKVFC , respectively, each with acetylated amino-terminus and amidated carboxy- terminus) as coating antigens, in conjunction with either rabbit antipeptide antibodies (A) or human anti-tetanus antibodies (B). Definition

The term "chiral" describes an amino acid residue whose side chain is a chemical group larger than a hydrogen atom (i.e., the side chain of glycine). The term "peptide" refers to a plurality of amino- acid residues covalently linked via the main chain (as opposed to side chain) by a peptide amide bond between consecutive amino-acid residues along a linear molecular sequence of such residues, with unblocked or blocked amino-terminus and/or carboxy- terminus (such that the amino-terminus is unblocked with a free main-chain amino group or blocked, whereas the amino-terminus may be either unblocked with a free main-chain carboxyl group or blocked with carboxy-terminal main-chain carbonyl group forming part of a carboxamide or other non-carboxyl chemical group) .

The term "oligopeptide" refers to a peptide consisting of relatively few amino-acid residues, more specifically at most 20 such residues per peptide chain for the purpose of describing the present invention herein.

The term "moiety" refers to a part of a molecule (e.g., an oligopeptide), said part itself occurring as part of another structure (e.g., a macromolecular entity comprising an oligopeptide moiety covalently linked to a protein). The term "resin" refers to an insoluble solid material that can support the artificial solid-phase chemical synthesis of a peptide, such that the peptide becomes incorporated into the physical structure of said material.

The term "antigen" refers to a substance recognized by a vertebrate immune-system component such as an immunoglobulin.

The term "immunogen" refers to an antigen that elicits a specific immune response directed toward itself, for example, production of a vertebrate immune-system component such as an immunoglobulin that recognizes said antigen.

The term "immunoglobulin" refers to a protein produced by a vertebrate immune system and capable of recognizing an antigen via binding thereto. An immunoglobulin may be either surface immunoglobulin, which is physically bound to the plasma membrane (typically on B lymphocytes), or antibody, which is secreted (typically by plasma cells).

The term "immunoglobulin G" (hereafter abbreviated as "IgG") refers to a class of immunoglobulin which typically constitutes the majority of circulating antibody in mammalian blood plasma.

The term "epitope" refers to a structural feature (e.g., amino-acid residue sequence) bound by an immunoglobulin .

The term "paratope" refers to an epitope-binding portion of an immunoglobulin.

The term "immunogenic construct" refers to an artificially produced immunogen.

The term "immunogenic carrier" refers to an immunogen (e.g., a protein) that can be covalently linked to another antigen (e.g., an oligopeptide), thus yielding an immunogenic construct that elicits production of immunoglobulins that recognize the latter antigen.

The term "conjugation chemistry" refers to chemical means whereby one material entity (e.g., an oligopeptide) is covalently linked to another (e.g., a protein).

The term "glycosylation motif " refers to an amino-acid residue sequence that tends to be posttranslationally modified by glycosylation (i.e., enzyme-catalyzed covalent linkage of one or more carbohydrate moieties) during protein biosynthesis

(typically in eukaryotic cells).

The term "N-linked glycosylation motif" refers to a glycosylation motif wherein covalent linkage to a carbohydrate moiety occurs via a nitrogen atom (typically of the sidechain amide group of an asparagine [Asn] residue).

The term "glycosyl acceptor site" refers to an amino-acid residue that forms part of a glycosylation motif and participates directly in the covalent linkage to a carbohydrate moiety. Detailed Description of the Preferred Embodiments

The present invention provides antipeptide immunoglobulins (e.g., antibodies) suitable for binding a SARS-CoV-2 sequence, which is a membrane- glycoprotein extramembranous-domain sequence comprising a glycine- containing N-linked glycosylation motif of the form Asn-Gly-Thr, said immunoglobulins thus being suitable for binding an oligopeptide sequence as well as a first target sequence and a second target sequence. Said oligopeptide, first target and second target sequences are all distinct from one another yet each comprise a core subsequence that perfectly matches part of said SARS-CoV-2 sequence and is devoid of N- linked glycosylation motifs. Moreover, said SARS- CoV-2 and first target sequences each comprise a glycine residue located immediately amino-terminal to said core subsequence, whereas said second target sequence comprises a chiral amino-acid residue (e.g., a cysteine residue) located immediately amino- terminal to said core subsequence. Such tolerance for substitution of glycine by chiral amino-acid residues supports practical applications exemplified by use of peptide-based antigens for detection of said immunoglobulins, said antigens consisting of said core subsequence flanked by cysteine residues.

The present invention also provides immunogenic constructs suitable for generating said immunoglobulins, free oligopeptides suitable for producing said constructs, and oligopeptide precursor resins suitable for producing said oligopeptides. Each of said constructs comprises at least one oligopeptide moiety comprising said oligopeptide sequence and covalently linked to an immunogenic carrier component (e.g., a protein such as keyhole limpet hemocyanin [KLH]). Each of said oligopeptides comprises said oligopeptide sequence; and each of said resins comprises said oligopeptide sequence covalently linked to a solid support matrix, such that said oligopeptide sequence may be cleaved (e.g., by suitable chemical means) from said matrix, said resin being produced via well-established means (e.g., solid-phase peptide synthesis). Methods for producing said resin and cleaving said oligopeptide sequence from said matrix are well known to a person skilled in the art (e.g., in the context of commercial production of synthetic peptides according to customer specifications of amino-acid residue sequence and chemical modifications such as acetylation and/or amidation of amino- and carboxy- termini, respectively).

Accordingly, the present invention provides methods for producing said immunogenic constructs by covalently linking said free oligopeptides to said immunogenic carrier components, said linking being accomplished via suitable conjugation chemistry well known to a person skilled in the art. This is exemplified by covalent linkage between a thiol group (e.g., of a cysteine residue of a peptide) and a primary amine group (e.g., of a lysine residue of a protein) using a suitable heterobifunctional crosslinker (e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester [MBS]) . Furthermore, the present invention also provides methods for producing said immunoglobulins via immunization of suitable vertebrate species using said immunogenic constructs, as exemplified by immunization of mammalian species (e.g., rabbits) to produce antibodies that can be affinity purified using said oligopeptide sequences as affinity ligands. Additionally, the present invention provides methods for detecting antigens using said immunoglobulins, as well as systems comprising said immunoglobulins for detecting said antigens, for example, in the context of enzyme- linked immunosorbent assay (ELISA). EXAMPLES

The following examples are provided for illustrative purposes, to set forth the best mode contemplated for reducing the present invention to practice, and therefore shall be interpreted as illustrative and not in a limiting sense.

EXAMPLE 1

A previously filed patent application (Intellectual Property Office of the Philippines [IPO PHL] patent application no. 12020050482, filed 19

November 2020 and hereafter referred to as Document Dl) details sequence analysis of SARS-CoV-2 membrane glycoprotein and production of polymeric peptides comprising oligopeptide analogs of the amino- terminal sequence of said glycoprotein, in part enabling the present invention.

As disclosed in Document Dl, the 20-residue amino-terminal sequence of SARS-CoV-2 membrane glycoprotein, with sequence MADSNGT ITVEELKKLLEQW, was identified and selected for modification to produce artificial homologs. As further disclosed in Document Dl, such an artificial homolog was designed with sequence CADSNGTITVEELKKLLEQC, synthesized as an nonglycosylated oligopeptide with unblocked amino-terminus and amidated carboxy-terminus, and oxidized to yield an antigen hereafter referred to as pMl. In accordance with methods disclosed in Document Dl, another artificial homolog was designed with the shorter sequence CTITVEELKKLLEQC, synthesized as a nonglycosylated oligopeptide with acetylated amino-terminus and amidated carboxy- terminus, and oxidized to yield an antigen hereafter referred to as pMls. The classic N-linked glycosylation motif NGT (Asn-Gly-Thr) of the glycoprotein amino- terminal sequence

(MADSNGTITVEELKKLLEQW) was thus entirely present in pMl, whereas the glycosyl acceptor site (Asn) and middle residue (Gly) of said motif were replaced by the amino-terminal (blocking) acetyl group and a cysteine residue, respectively, in pMls.

Yet another artificial homolog was designed having an artificial sequence of

MADSNGTITVEELKKLLEQC . Subsequently, a commercial service provider (GenScript) synthesized said artificial sequence as an oligopeptide, prepared an immunogenic construct using said oligopeptide, immunized animals using said construct, prepared pooled serum from said animals, and prepared antipeptide antibodies from said serum using affinity purification with said oligopeptide as affinity ligand, ultimately yielding said antibodies in lyophilized form. Said provider performed said procedures under their Custom Rabbit Polyclonal Antibody Services commercial-service package (GenScript catalog number SC2046), according to standard methods that are well known to one skilled in the art.

In particular, said artificial oligopeptide was synthesized using solid-phase chemistry, with unblocked amino-terminus and amidated carboxy- terminus, and without glycosylation. The unblocked amino-terminus served to mimic the corresponding unblocked amino-terminus of the glycoprotein, whereas the amidated carboxy-terminus served to mimic the uncharged backbone of internal (versus amino- or carboxy-terminal ) residues in the glycoprotein. Said artificial oligopeptide was analyzed by liquid chromatography (LC) on an Inertsil ODS-3 4.6 x 250 mm column using a mobile phase consisting of 0.065% trifluoroacetic acid (TFA) in 100% water (v/v) for solvent A and 0.05% TFA in 100% acetonitrile (v/v) for solvent B with a total flow of 1 ml per minute and time program set with 5% solvent B initially and 65% solvent B at 25 minutes, with continuous UV absorbance monitoring of the eluate at 220 nm. A single dominant peak corresponding to said artificial oligopeptide was thus observed exhibiting an elution time of 18.375 minutes and comprising 90.285% of the integrated peak area, as depicted in FIG. 1. Mass spectrometry was performed on said peak, yielding a mass-to-charge ratio (m/z) spectrum that showed dominant ions at m/z 741.4 [M+3H]3+ and m/z 1111.6

[M+2H]2+, as depicted in FIG. 2. Analysis of said mass spectrum thus gave an estimated mass of 2221.1 Da, which closely approximates the theoretical mass of 2221.56 Da calculated for said artificial oligopeptide .

Furthermore, the carboxy- terminal cysteine residue served to enable covalent linkage of said artificial oligopeptide to an immunogenic carrier in the form of a protein, namely keyhole limpet hemocyanin (KLH). Accordingly, the free thiol (i.e., sulfhydryl) group of the oligopeptide cysteine residue was coupled to a free amino group of KLH using conjugation chemistry with the heterobifunctional crosslinker m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS). An immunogenic construct in the form of peptide-KLH conjugate was thus prepared and used to immunize two New Zealand rabbits, with subcutaneous injection of 0.2 mg of said peptide-KLH conjugate per rabbit, using either Freund's complete adjuvant for the first injection or Freund's incomplete adjuvant for subsequent injections, thus injecting each rabbit thrice at two- week intervals. Each rabbit was bled one week after the third and final injection, with serum then being prepared from the blood of each rabbit. Rabbit sera thus obtained were analyzed by indirect ELISA. Toward this end, said artificial oligopeptide was thus used in the form of free peptide as coating antigen at a concentration of 4 μg/ml in phosphate buffered saline (pH 7.4), using 100 mΐ of coating antigen solution per well, with anti-rabbit IgG Fc monoclonal secondary antibody horseradish peroxidase conjugate (GenScript catalog number A01856) as detecting molecule. Antipeptide antibody titers thus observed were at least 512,000 for both rabbit sera, as depicted in FIG. 3. Said sera were then pooled. From the resulting pooled serum, antipeptide antibodies were affinity purified using said artificial oligopeptide as affinity ligand Approximately 7.2 mg of affinity-purified antibodies was obtained by lyophilizing 8.1 ml of antibody solution, and the lyophilized antibodies were stored at -20°C prior to use.

EXAMPLE 2

The lyophilized antibodies obtained above in EXAMPLE 1 were dissolved in sterile water for injection, thereby producing an antibody stock solution with antibody concentration of 0.89 mg/ml. Commercial anti-tetanus antibodies (Ig TETANO human tetanus immunoglobulin; Kedrion S.p.A., Naples, Italy) produced prior to the pandemic spread of SARS- CoV-2 were used as a control antibody stock solution with anti-tetanus antibody concentration of 250 IU/ml (noting that each IU corresponds to approximately 0.03384 mg of anti-tetanus antibodies).

The antibodies were then analyzed using indirect ELISA with high-binding polystyrene microtiter plates (Costar 3590, Corning Inc., NY, USA) according to methods disclosed in Document Dl, using pMl and pMls, each at a concentration of 20 μg/ml in 0.05 M carbonate-bicarbonate buffer pH 9.6 (coating buffer), as coating antigens. In place of pMl or pMls, pTlC+ (a structural analog of pMls wherein the M-protein sequence [TITVEELKKLLEQ] is replaced by a tetanus toxin sequence [FIGITELKKLESKINKVF]) was used as a control peptide. Coating buffer containing no peptide was used as a negative control. A washing step, that is, removal of well contents and washing of wells thrice with 0.5% Tween 20 in phosphate buffered saline, pH 7.4 (wash buffer), was performed between successive incubation steps. For blocking (37°C, 30 minutes), wells were loaded (100 μL/well) with blocking buffer (5% skim milk in wash buffer). For antibody binding (room temperature, 1 hour), the antibody stock solution was diluted in dilution buffer (blocking buffer diluted 10-fold in wash buffer), and serial two-fold dilutions of the resulting solution were prepared. Wells were loaded (100 μL/well) with either the affinity-purified antibodies obtained above in EXAMPLE 1 or the above- described human anti-tetanus antibodies in serial two-fold dilutions with dilution buffer (0.5% skim milk in wash buffer). For conjugate binding (room temperature, 1 hour), wells were loaded (50 μL/well) with protein A-peroxidase (0.5 mg/mL in dilution buffer) . Fresh chromogenic substrate solution (CSS) was prepared by dissolving 1 mg of 3,3’,5,5’- tetramethylbenz idine in DMSO (200 μL), diluting with phosphate-citrate buffer (9.8 mL) and adding 30% hydrogen peroxide (2 mE). Wells were incubated (room temperature, 30 minutes) with CSS (50 μL/well), after which 1 M H2S04 (50 μL/well) was added to stop the enzymatic reaction. Well absorbance values at 450 nm were then obtained using a Bio-Rad Model 680 microplate reader.

ELISA results revealed intense signals in all wells containing antipeptide antibodies where either pMl or pMls was used as coating antigen, as depicted in FIG. 4; whereas no signal was observed for said antibodies where pTlC+ was used as coating antigen, as depicted in FIG. 5. Said results are consistent with the production of antipeptide antibodies that bind to one or more epitopes downstream of the asparagine (Asn) glycosyl acceptor site within the SARS-CoV-2 membrane-glycoprotein amino-terminal extramembranous -domain sequence. This demonstrates that immunoglobulins produced in response to immunization with a nonglycosylated peptide analog of said sequence are capable of recognizing nonglycosylated epitopes of said sequence.

Hence, the objects set forth above, among those made apparent from the preceding description, are efficiently attained and, because certain changes may be made in carrying out the above methods and in the constructions set forth without departing from the spirit and scope of the invention, all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. Furthermore, the following claims are intended to cover all of the generic and specific features of the invention herein described and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.

Claims

What is claimed:

1. An antipeptide immunoglobulin (AI); AI being suitable for binding an oligopeptide sequence (OS), a first target sequence (Tl) and a second target sequence (T2); OS, Tl and T2 all being distinct from one another; OS, Tl and T2 each comprising a core subsequence (CS); characterized by CS perfectly matching part of a SARS-CoV-2 membrane-glycoprotein amino-terminal extramembranous-domain sequence (SS); SS and Tl each comprising a glycine residue located immediately amino-terminal to CS; T2 comprising a chiral amino-acid residue located immediately amino- terminal to CS.

2. The immunoglobulin of claim 1, further characterized by said oligopeptide sequence comprising a cysteine residue.

3. The immunoglobulin of claim 2, further characterized by said cysteine residue being at a carboxy-terminal position.

4. The immunoglobulin of claim 1, 2 or 3, further characterized by said core subsequence being TITVEELKKLLEQ .

5. The immunoglobulin of claim 4, further characterized by said oligopeptide sequence being MADSNGTITVEELKKLLEQC .

6. An immunogenic construct (IC), IC comprising at least one oligopeptide moiety (OM), OM comprising an oligopeptide sequence (OS), IC further comprising an immunogenic carrier component (CC), OM being covalently linked to CC, IC being suitable for generating an antipeptide immunoglobulin (AI); AI being suitable for binding OS, a first target sequence (Tl) and a second target sequence (T2); OS, T1 and T2 all being distinct from one another; OS, Tl and T2 each comprising a core subsequence (CS); characterized by CS perfectly matching part of a SARS-CoV-2 membrane-glycoprotein amino-terminal extramembranous -domain sequence (SS); SS and Tl each comprising a glycine residue located immediately amino-terminal to CS; T2 comprising a chiral amino- acid residue located immediately amino-terminal to CS.

7. The construct of claim 6, further characterized by said oligopeptide sequence comprising a cysteine residue .

8. The construct of claim 7, further characterized by said cysteine residue being at a carboxy-terminal position .

9. The construct of claim 6, 7 or 8, further characterized by said core subsequence being TITVEELKKLLEQ .

10. The construct of claim 9, further characterized by said oligopeptide sequence being

MADSNGTITVEELKKLLEQC .

11. A free oligopeptide (FO), FO comprising an oligopeptide sequence (OS), FO being suitable for producing an immunogenic construct (IC), IC comprising at least one oligopeptide moiety (OM), OM comprising OS, IC further comprising an immunogenic carrier component (CC), OM being covalently linked to CC, IC being suitable for generating an antipeptide immunoglobulin (AI); AI being suitable for binding OS, a first target sequence (T1) and a second target sequence (T2); OS, Tl and T2 all being distinct from one another; OS, Tl and T2 each comprising a core subsequence (CS); characterized by CS perfectly matching part of a SARS-CoV-2 membrane- glycoprotein amino-terminal extramembranous -domain sequence (SS); SS and Tl each comprising a glycine residue located immediately amino-terminal to CS; T2 comprising a chiral amino-acid residue located immediately amino-terminal to CS.

12. The oligopeptide of claim 11, further characterized by said oligopeptide sequence comprising a cysteine residue.

13. The oligopeptide of claim 12, further characterized by said cysteine residue being at a carboxy-terminal position.

14. The oligopeptide of claim 11, 12 or 13, further characterized by said core subsequence being

TITVEELKKLLEQ .

15. The oligopeptide of claim 14, further characterized by said oligopeptide sequence being MADSNGTITVEELKKLLEQC .

16. An oligopeptide precursor resin (PR), PR comprising an oligopeptide sequence (OS) and a solid support matrix (SM), OS being covalently linked to SM, OS being cleavable from SM, PR being suitable for producing a free oligopeptide (FO), FO comprising OS, FO being suitable for producing an immunogenic construct (IC), IC comprising at least one oligopeptide moiety (OM), OM comprising OS, IC further comprising an immunogenic carrier component (CC), OM being covalently linked to CC, IC being suitable for generating an antipeptide immunoglobulin (AI); AI being suitable for binding OS, a first target sequence (Tl) and a second target sequence (T2); OS, Tl and T2 all being distinct from one another; OS, Tl and T2 each comprising a core subsequence (CS); characterized by CS perfectly matching part of a SARS-CoV-2 membrane-glycoprotein amino-terminal extramembranous-domain sequence (SS); SS and Tl each comprising a glycine residue located immediately amino-terminal to CS; T2 comprising a chiral amino-acid residue located immediately amino- terminal to CS.

17. The resin of claim 16, further characterized by said oligopeptide sequence comprising a cysteine residue .

18. The resin of claim 17, further characterized by said cysteine residue being at a carboxy-terminal position .

19. The resin of claim 16, 17 or 18, further characterized by said core subsequence being TITVEELKKLLEQ .

20. The resin of claim 19, further characterized by said oligopeptide sequence being MADSNGTITVEELKKLLEQC .

21. A method for producing an immunogenic construct

(IC), said method comprising the steps of: (i) providing a suitable free oligopeptide (FO); and (ii) covalently linking FO to an immunogenic carrier component (CC), said linking being accomplished via suitable conjugation chemistry, FO comprising an oligopeptide sequence (OS), FO being suitable for producing IC, IC comprising at least one oligopeptide moiety (OM), OM comprising OS, IC further comprising CC, OM being covalently linked to CC, IC being suitable for generating an antipeptide immunoglobulin (AI); AI being suitable for binding OS, a first target sequence (Tl) and a second target sequence (T2); OS, Tl and T2 all being distinct from one another; OS, Tl and T2 each comprising a core subsequence (CS); characterized by CS perfectly matching part of a SARS-CoV-2 membrane-glycoprotein amino-terminal extramembranous-domain sequence (SS); SS and T1 each comprising a glycine residue located immediately amino-terminal to CS; T2 comprising a chiral amino-acid residue located immediately amino- terminal to CS.

22. The method of claim 21, further characterized by said oligopeptide sequence comprising a cysteine residue .

23. The method of claim 22, further characterized by said cysteine residue being at a carboxy-terminal position .

24. The method of claim 21, 22 or 23, further characterized by said core subsequence being TITVEELKKLLEQ .

25. The method of claim 24, further characterized by said oligopeptide sequence being

MADSNGTITVEELKKLLEQC .

26. A method for producing an antipeptide immunoglobulin (AI), said method comprising the steps of: (i) providing a composition (X) for inducing immunoglobulin production in a suitable vertebrate species; and (ii) using X to immunize a member of said vertebrate species according to a suitable immunization schedule, X comprising an immunogenic construct (IC), IC comprising at least one oligopeptide moiety (OM), OM comprising an oligopeptide sequence (OS), IC further comprising an immunogenic carrier component (CC), OM being covalently linked to CC, IC being suitable for generating AI; AI being suitable for binding OS, a first target sequence (Tl) and a second target sequence (T2); OS, Tl and T2 all being distinct from one another; OS, Tl and T2 each comprising a core subsequence (CS); characterized by CS perfectly matching part of a SARS-CoV-2 membrane-glycoprotein amino-terminal extramembranous-domain sequence (SS); SS and Tl each comprising a glycine residue located immediately amino-terminal to CS; T2 comprising a chiral amino-acid residue located immediately amino- terminal to CS.

27. The method of claim 26, further characterized by said oligopeptide sequence comprising a cysteine residue .

28. The method of claim 27, further characterized by said cysteine residue being at a carboxy-terminal position .

29. The method of claim 26, 27 or 28, further characterized by said core subsequence being TITVEELKKLLEQ .

30. The method of claim 29, further characterized by said oligopeptide sequence being MADSNGTITVEELKKLLEQC .

31. A method for detecting an antigen (Y), said method comprising the steps of: (i) contacting Y with an antipeptide immunoglobulin (AI); and (ii) detecting AI bound to Y, Y comprising at least one epitope of SARS-CoV-2; AI being suitable for binding an oligopeptide sequence (OS), a first target sequence (Tl) and a second target sequence (T2); OS, T1 and T2 all being distinct from one another; OS, Tl and T2 each comprising a core subsequence (CS); characterized by CS perfectly matching part of a SARS-CoV-2 membrane-glycoprotein amino-terminal extramembranous -domain sequence (SS); SS and Tl each comprising a glycine residue located immediately amino-terminal to CS; T2 comprising a chiral amino- acid residue located immediately amino-terminal to CS.

32. The method of claim 31, further characterized by said oligopeptide sequence comprising a cysteine residue .

33. The method of claim 32, further characterized by said cysteine residue being at a carboxy-terminal position.

34. The method of claim 31, 32 or 33, further characterized by said core subsequence being TITVEELKKLLEQ .

35. The method of claim 34, further characterized by said oligopeptide sequence being

MADSNGTITVEELKKLLEQC .

36. A system for detecting an antigen (Y), Y comprising at least one epitope of SARS-CoV-2, said system comprising an antipeptide immunoglobulin (AI); AI being suitable for binding an oligopeptide sequence (OS), a first target sequence (Tl) and a second target sequence (T2); OS, Tl and T2 all being distinct from one another; OS, Tl and T2 each comprising a core subsequence (CS); characterized by CS perfectly matching part of a SARS-CoV-2 membrane- glycoprotein amino-terminal extramembranous -domain sequence (SS); SS and Tl each comprising a glycine residue located immediately amino-terminal to CS; T2 comprising a chiral amino-acid residue located immediately amino-terminal to CS.

37. The system of claim 36, further characterized by said oligopeptide sequence comprising a cysteine residue .

38. The system of claim 37, further characterized by said cysteine residue being at a carboxy-terminal position .

39. The system of claim 36, 37 or 38, further characterized by said core subsequence being TITVEELKKLLEQ .

40. The system of claim 39, further characterized by said oligopeptide sequence being MADSNGTITVEELKKLLEQC .

41. Use of the immunoglobulin of claim 1, 2 or 3 in a method for detecting an antigen.

42. Use of the immunoglobulin of claim 4 in a method for detecting an antigen.

43. Use of the immunoglobulin of claim 5 in a method for detecting an antigen.

44. Use of the construct of claim 6, 7 or 8 in a method for producing an antipeptide immunoglobulin.

45. Use of the construct of claim 9 in a method for producing an antipeptide immunoglobulin.

46. Use of the construct of claim 10 in a method for producing an antipeptide immunoglobulin.

47. Use of the oligopeptide of claim 11, 12, or 13 in a method for producing an immunogenic construct.

48. Use of the oligopeptide of claim 14 in a method for producing an immunogenic construct.

49. Use of the oligopeptide of claim 15 in a method for producing an immunogenic construct.

50. Use of the resin of claim 16, 17 or 18 in a method for producing an immunogenic construct.

51. Use of the resin of claim 19 in a method for producing an immunogenic construct.

52. Use of the resin of claim 20 in a method for producing an immunogenic construct.