WO2023022613A2

WO2023022613A2 - Antipeptide immunoglobulins to severe acute respiratory syndrome coronavirus 2 (sars-cov-2) spike glycoprotein (s protein) epitopes and cognate immunogens, method for producing and utilizing the same, and antigen detection systems relating thereto

Info

Publication number: WO2023022613A2
Application number: PCT/PH2022/050014
Authority: WO
Inventors: Salvador Eugenio CAOILI; Ruby Anne KING; Fresthel Monica CLIMACOSA; Riziel Hannah AGUIMATANG; Ayra Patrice ESPIRITU
Original assignee: University Of The Philippines Manila
Priority date: 2021-08-13
Filing date: 2022-08-13
Publication date: 2023-02-23
Also published as: WO2023022613A3

Abstract

The invention provides: antipeptide immunoglobulins recognizing epitopes within the subdomain SD2 major disulfide loop of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S 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.

Description

ANTIPEPTIDE IMMUNOGLOBULINS TO SEVERE ACUTE RESPIRATORY

SYNDROME CORONAVIRUS 2 (SARS-CoV-2) SPIKE GLYCOPROTEIN (S PROTEIN) EPITOPES AND COGNATE IMMUNOGENS, METHOD FOR PRODUCING AND UTILIZING THE SAME, AND ANTIGEN DETECTION

SYSTEMS RELATING THERETO

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 whereby an amino-acid residue (e.g., alanine) is replaced by a bulkier (i.e., larger) amino-acid residue, such that steric clashes due to said bulkier residue preclude binding of an immunoglobulin.

Among the 20 proteogenic amino acids represented in standard codon tables, alanine is the smallest chiral amino acid, its side chain being a methyl group, such that the other chiral amino acids each comprise a side chain bulkier than a methyl group. Hence, immunoglobulin binding may be abrogated by steric hindrance resulting from replacement of alanine by a bulkier 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 alanine is replaced by a bulkier 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 sequence 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 spike glycoprotein (hereafter referred to as S protein) of severe acute respiratory syndrome coronavirus (SARS-CoV, now also known as and hereafter referred to as SARS-CoV-1) and also against S protein of severe acute respiratory syndrome coronavirus 2 (hereafter referred to as SARS-CoV-2). For example, such antibody production against S 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, S protein comprises transmembrane sequences embedded in the viral envelope (i.e., viral membrane). Said protein is a homotrimeric transmembrane glycoprotein consisting of three identical monomeric units whose amino-terminal ends are oriented outward from the outer surface of said envelope. Each of said units comprises an amino-terminal SI subunit and a carboxy-terminal S2 subunit. Said subunits become physically separated from each other by proteolytic cleavage at an intervening S1/S2 cleavage site. Said SI subunit comprises an N-terminal domain (NTD), which is amino-terminal to a receptor binding domain (RBD), which is in turn amino-terminal to tandem subdomains SD1 and SD2, such that SD1 is amino-terminal to SD2. Furthermore, SD2 comprises two disulfide loops of unequal length, the longer one being amino-terminal to the shorter one and thus hereafter referred to as the SD2 major disulfide loop (SD2MDL). The canonical SARS-CoV-2 (Wuhan strain) SD2MDL reference sequence is CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGC, corresponding to amino-acid residue position numbers 617 through 649. Typically, the amino-terminal cysteine residue of SD2MDL is the middle residue Xaa in a classic tripeptide N-glycosylation motif of the form Asn- Xaa-Thr, such that SD2MDL is itself devoid of N-glycosylation sites. Moreover, the non-cysteine residues of SD2MDL are known to adopt conformations that vary among different experimentally obtained S-protein structures, said residues being mostly either annotated as “MISSING” or assigned to neither helical nor beta-strand secondary structures, all of which is consistent with dynamic conformational disorder (i.e., lack of persistently folded conformation) among said residues.

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 SD2MDL sequences of S 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. As a case in point, Immune Epitope Database (IEDB) assay record no. 12546810 (https://www.iedb.org/assay/12546810) relates to use of a peptide with sequence

EVPVAIHADQLTPTWRVYSTGSNVFQTRA (conceptually a truncated version of canonical SARS-CoV-2 SD2MDL) as an immunoassay antigen to detect antibodies in a series of 55 COVID-

19 convalescent plasma donors, such that antibodies were thus detected in only one of said donors. A problem is thus posed by the above-described inconsistency of antibody responses resulting from immunization of vertebrate hosts using antigens comprising whole S 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 S protein. Said inconsistency thus hampers attempts to deliberately and consistently produce antibodies that recognize predefined short (e.g., less than 20-mer) sequences of S protein, particularly within its SD2MDL. Such antibodies are, by virtue of their ability to recognize and thus bind such sequences, potentially useful as immunodiagnostic reagents (e.g., for detecting antigens that comprise S protein or fragments thereof) and possibly also as prophylactic or even therapeutic agents against infection, which is conceivable on the basis of dynamic conformational disorder of SD2MDL sequences in both whole S protein and synthetic peptide analogs thereof, as such disorder in proteins enables binding of cognate antipeptide antibodies via conformational selection.

In order to address the problem of inconsistency in the production of antibodies against sequences of S protein, one possible approach is to still proceed with immunization using antigens that comprise whole S 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 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 plausible in view of data accessed via IEDB assay record no. 1244220 (https://www.iedb.org/assay/1244220), which relates to use of a glutathione S-transferase (GST) fusion protein comprising recombinantly inserted sequence STAIHADQLTPAWRIY (conceptually a truncated version of SARS-CoV-1 SD2MDL) at an internal sequence location (i.e., comprising neither the amino-terminus nor the carboxyterminus of said fusion protein) to immunize BALB/c mice and thereby obtain mouse polyclonal antipeptide antibodies that bind to SARS-CoV-1 S protein. As sequence STAIHADQLTPAWRIY is only 16 residues long, it conceivably could be produced as a synthetic oligopeptide to replace said fusion protein, thus simplifying the process of antigen production. However, this would pose the problem of adequately mimicking the covalent backbone structure at an internal sequence location, as detailed below.

A linear peptide comprises an amino-terminal residue and a carboxy-terminal residue, both of which comprise covalent backbone structures that are sterically distinct from those of a residue at an internal sequence location, which is covalently linked to adjacent residues in the sequence by means of two peptide bonds. The covalent backbone structure of a linear peptide is thus truncated at both amino-terminal and carboxy-terminal ends, such that antipeptide antibodies recognizing said peptide may fail to recognize a longer peptide comprising the sequence of said linear peptide (i.e., wherein the sequence of said linear peptide is nested within the sequence of said longer peptide), due to steric clashes between the paratopes of said antibodies with structural components (e.g., additional amino- and/or carboxy-terminal residues) that are present only in said longer peptide. Furthermore, unblocked amino-terminal and carboxy-terminal residues comprise backbone amino and carboxyl groups, respectively, which tend to become formally charged (via protonation and deprotonation, respectively) at physiological (i.e., near neutral) pH, such that antipeptide antibodies that recognize peptides with such formally charged backbone structures may fail to recognize the sequences of said peptides where said sequences are at internal sequence locations (e.g., nested within the sequence of a longer peptide or protein) and thus lacking formally charged backbone structures. The problem posed by formal charges on unblocked amino- and carboxy-terminal residue backbone structures might be mitigated by covalently blocking said structures (e.g., via N-acetylation and amidation, respectively); but this would fail to fully mimic the steric features of residues at internal sequence positions.

Presented with the dilemma of biotechnological fusion-protein production to faithfully mimic protein backbone structure versus likely failure to produce antipeptide immunoglobulins that recognize SARS-CoV-2 SD2MDL sequences at internal sequence locations, a person skilled in the art would be motivated to opt for said fusion-protein production, let alone attempt use of linear peptides with unblocked amino- and carboxy-terminal residues for antipeptide-antibody production. However, such aversion to said use of linear peptides risks failure to realize production of antipeptide immunoglobulins that recognize SARS-CoV-2 SD2MDL sequences at internal sequence locations, without the complexity of said fusion-protein production or even covalent blocking of amino- and/or carboxy-terminal residues, which is of practical relevance to antibodybased applications such as antigen detection that address SARS-CoV-2 infection. Furthermore, even if said person were to use fusion proteins comprising SD2MDL sequences for producing immunoglobulins that recognize said sequences, said person would conceivably fail to anticipate the possibility that said immunoglobulins would also recognize homologs of said sequences wherein an alanine residue is replaced by a bulkier 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 linear peptide sequences in order to produce antipeptide immunoglobulins that recognize subsequences of SARS-CoV-2 S-protein SD2MDL, particularly where both amino- and carboxytermini are unblocked, while also exploring the possibility that said immunoglobulins also recognize homologs of said subsequences wherein an alanine residue is replaced by a bulkier (e.g., 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 recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-glycoprotein (S-protein) subdomain SD2 major disulfide loop (SD2MDL) sequences at internal sequence locations, without the complexity of biotechnological fusion-protein production or even covalent blocking of amino- and/or carboxy-terminal residues, the present invention provides: antipeptide immunoglobulins recognizing epitopes within SARS-CoV-2 S-protein SD2MDL; 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. Said core subsequence perfectly matches part of a SARS-CoV-2 sequence, which is a S-protein SD2MDL sequence.

Furthermore, said SARS-CoV-2 sequence and first target sequence each comprise a alanine residue located immediately amino-terminal to said core subsequence. However, said second target sequence comprises a bulkier 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 alanine residue with bulkier (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 16.097 minutes and comprising 92.193% of the integrated peak area, corresponds to artificial oligopeptide of sequence AIHADQLTPTWRVYC with unblocked amino-terminus and unblocked carboxy-terminus;

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

FIG. 3 shows a set of indirect ELISA results for detection of rabbit antipeptide antibodies using their cognate artificial oligopeptide (of sequence AIHADQLTPTWRVYC with unblocked amino-terminus and amidated carboxy-terminus) as coating antigen; and

FIG. 4 shows a set of indirect ELISA results for detection of rabbit antipeptide antibodies using polymeric peptides pSls and pSlc (of sequences CIHADQLTPTWRVYC and CPVAIHADQLTPTWRVYSTC, respectively, each with unblocked amino-terminus and amidated carboxy-terminus) as coating antigens, with coating buffer (CB) used as a negative control.

DEFINITIONS

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 aminoterminus 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 solidphase 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 which is a spike-gly coprotein (S-protein) subdomain SD2 major disulfide loop (SD2MDL) sequence, 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. Moreover, said SARS-CoV-2 sequence and said first target sequence each comprise a alanine residue located immediately amino-terminal to said core subsequence, whereas said second target sequence comprises a bulkier amino-acid residue (e.g., a cysteine residue) located immediately amino-terminal to said core subsequence. Such tolerance for substitution of alanine by bulkier 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 N- 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 DI) 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 by providing the basis for producing analogous polymeric peptides that comprise SARS-CoV-2 S-protein sequences.

The 20-residue sequence VPVAIHADQLTPTWRVYSTG, which is at an internal sequence location within SD2MDL of SARS-CoV-2 S protein, was identified and selected for modification to produce artificial homologs. In accordance with methods disclosed in Document DI, such an artificial homolog was designed with sequence CPVAIHADQLTPTWRVYSTC, synthesized as an oligopeptide with unblocked amino-terminus and amidated carboxy-terminus, and oxidized to yield an antigen hereafter referred to as pSlc. Also in accordance with methods disclosed in Document DI, another artificial homolog was designed with the shorter sequence CIHADQLTPTWRVYC, synthesized as an oligopeptide with unblocked amino-terminus and amidated carboxy-terminus, and oxidized to yield an antigen hereafter referred to as pS 1 s. For both pSlc and pSls, the unblocked amino-termini provided the option to covalently modify the polymeric peptides using suitable electrophilic agents (e.g., aldehydes and arylsulfonates) for cross-linking and/or haptenic labeling, although further covalent modification was ultimately deemed unnecessary for reducing the present invention to practice.

Yet another artificial homolog was designed having an artificial sequence of AIHADQLTPTWRVYC. 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 Complete Affinity-Purified Peptide Polyclonal Antibody Package commercial service (GenScript catalog number SCI 031), 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 unblocked carboxy -terminus. The unblocked amino- and carboxytermini obviated the added cost of covalent blocking (e.g., by N-acetylation and amidation, respectively), thereby minimizing the overall cost of antipeptide antibody production. 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 16.097 minutes and comprising 92.193% 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 887.8 [M+2H]2+, as depicted in FIG. 2. Analysis of said mass spectrum thus gave an estimated mass of 1773.6 Da, which closely approximates the theoretical mass of 1774.02 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 pooled and then 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 pg/ml in phosphate buffered saline (pH 7.4), using 100 pl 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 the pooled rabbit serum, as depicted in FIG. 3. From said pooled serum, antipeptide antibodies were affinity purified using said artificial oligopeptide as affinity ligand. Approximately 7.08 mg of affinity-purified antibodies was obtained by lyophilizing 15.4 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 0.46 mg/ml. The antibodies were then analyzed using indirect ELISA with polystyrene microtiter plates (Kartell S.p.A., Via delle Industrie, 1, 20082, Noviglio MI, Italy) using pSls and pSlc, each at a concentration of 20 pg/ml in 0.05 M carbonate-bicarbonate buffer pH 9.6 (coating buffer), as coating antigens. Coating buffer containing no peptide was used as a negative control. The coated plates were incubated for 24 hours at 4°C. 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, 1 hour), wells were loaded (100 pL/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 pL/well) with the affinity-purified antibodies obtained above in EXAMPLE 1. For conjugate binding (room temperature, 1 hour), wells were loaded (50 pL/well) with protein A-peroxidase (0.5 pg/mL in dilution buffer). Fresh chromogenic substrate solution (CSS) was prepared by dissolving 1 mg of 3,3’,5,5’-tetramethylbenzidine in DMSO (100 pL), diluting with phosphate-citrate buffer (9.9 mL) and adding 30% hydrogen peroxide (2 pL). Wells were incubated (room temperature, 30 minutes) with CSS (50 pL/well), after which 1 M H2SO4 (50 pL/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 detectable signals in wells containing antipeptide antibodies where either pSls or pSlc was used as coating antigen, as depicted in FIG. 4. Said results are consistent with the production of antipeptide antibodies that bind to one or more epitopes located between the cysteine residues of each monomeric oligopeptide unit of pSls, that is, within the core sequence IHADQLTPTWRVY. This demonstrates that immunoglobulins produced in response to immunization with a linear peptide analog of an internally located sequence of SARS-CoV-2 S- protein SD2MDL are capable of recognizing said sequence even where said analog has unblocked amino- and carboxy-termini such that the amino- and carboxy -terminal residues of said analog are structurally different from the correspondingly aligned residues of said internally located 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 (Al); Al 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 being identical to part of a SARS-CoV-2 surface-glycoprotein subdomain SD2 major disulfide loop sequence

CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGC (SS); SS and Tl each comprising an alanine residue located immediately amino-terminal to CS, T2 comprising a chiral aminoacid residue located immediately amino-terminal to CS, said chiral amino-acid residue being bulkier than an alanine residue. . The immunoglobulin of claim 1, further characterized by said oligopeptide sequence comprising a cysteine residue. . The immunoglobulin of claim 2, further characterized by said cysteine residue being at a carboxy-terminal position. . The immunoglobulin of claim 1, 2 or 3, further characterized by said core subsequence being IHADQLTPTWRVY. . The immunoglobulin of claim 4, further characterized by said oligopeptide sequence being AIHADQLTPTWRVYC. . 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 (Al); Al being suitable for binding OS, a first

22 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 being identical to part of a SARS-CoV-2 surface-glycoprotein subdomain SD2 major disulfide loop sequence CTEVPVAIHADQL1TTWRVYSTGSNVFQTRAGC (SS); SS and Tl each comprising an alanine residue located immediately amino-terminal to CS, T2 comprising a chiral amino-acid residue located immediately amino-terminal to CS, said chiral amino-acid residue being bulkier than an alanine residue.

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 carboxyterminal position.

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

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

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 (Al); Al 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 beingn identical to part of a SARS-CoV-2 surface-glycoprotein subdomain SD2 major disulfide loop sequence CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGC (SS); SS and Tl each comprising an alanine residue located immediately amino-terminal to CS, T2 comprising a chiral amino-acid residue located immediately amino-terminal to CS, said chiral amino-amino-terminal to CS, said chiral amino-acid residue being bulkier than an alanine residue. 2. The oligopeptide of claim 11, further characterized by said oligopeptide sequence comprising a cysteine residue. 3. 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 IHADQLTPTWRVY. 5. The oligopeptide of claim 14, further characterized by said oligopeptide sequence being AIHADQLTPTWRVYC. 6. 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 (Al); Al 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 being identical to part of a SARS-CoV-2 surface-glycoprotein subdomain SD2 major disulfide loop sequence Cl'EVPVAIHADQLlTTWRVYSTGSNVFQl'RAGC (SS); SS and T1 each comprising an alanine residue located immediately amino-terminal to CS, T2 comprising a chiral amino-acid residue located immediately amino-terminal to CS, said chiral amino-acid residue being bulkier than an alanine residue.

17. The resin of claim 16, further characterized by said oligopeptide sequence comprising a cysteine residue. 8. The resin of claim 17, further characterized by said cysteine residue being at a carboxyterminal position.

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

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

20. 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 (Al); Al 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 being identical to part of a SARS-CoV-2 surface-glycoprotein subdomain SD2 major disulfide loop sequence CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGC (SS);

25 SS and T1 each comprising an alanine residue located immediately amino-terminal to CS, T2 comprising a chiral amino-acid residue located immediately amino-terminal to CS, said chiral amino-acid residue being bulkier than an alanine residue.

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

22. 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 IHADQLTPTWRVY.

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

26. A method for producing an antipeptide immunoglobulin (Al), 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 Al; Al being suitable for binding OS, a first target sequence (T1 ) and a second target sequence (T2); OS, T1 and T2 all being distinct from one another; OS, T1 and T2 each comprising a core subsequence (CS); characterized by CS being identical to part of a SARS-CoV-2 surface- gly coprotein subdomain SD2 major disulfide loop sequence

CTEWVAIHADQLTPTWTtVYSTGSbATQTRAGC (SS); SS and T1 each comprising an alanine residue located immediately amino-terminal to CS, T2 comprising a chiral amino-

26 acid residue located immediately amino-terminal to CS, said chiral amino- acid residue being bulkier than an alanine residue.

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 carboxyterminal position.

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

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

31. A method for detecting an antigen (Y), said method comprising the steps of: (i) contacting

Y with an antipeptide immunoglobulin (Al); and (ii) detecting Al bound to Y, Y comprising at least one epitope of SARS-CoV-2; Al being suitable for binding an oligopeptide sequence (OS), a first target sequence (T1 ) and a second target sequence (T2); OS, T1 and T2 all being distinct from one another; OS, T1 and T2 each comprising a core subsequence (CS); characterized by CS being identical to part of a SARS-CoV-2 surface- glycoprotein subdomain SD2 major disulfide loop sequence

CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGC (SS); SS and T1 each comprising an alanine residue located immediately amino-terminal to CS, T2 comprising a chiral amino acid residue located immediately amino-terminal to CS, said chiral amino-acid residue being bulkier than an alanine residue.

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

27

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

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

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

36. A system for detecting an antigen (Y), Y comprising at least one epitope of SARS-CoV-2, said system comprising an antipeptide immunoglobulin (Al); Al 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 being identical to part of a SARS-CoV-2 surface-glycoprotein subdomain SD2 major disulfide loop sequence CTEVPV HADQLTPTWRVYSTGSNVFQTRAGC (SS); SS and Tl each comprising an alanine residue located immediately amino-terminal to CS, T2 comprising a chiral amino acid residue located immediately amino-terminal to CS, said chiral amino- acid residue being bulkier than an alanine residue.

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

IHADQLTPTWRVY.

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

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.

29