HIV gp41 variants for immunoassay diagnostics

Abstract

The present invention relates to a novel HIV gp41 antigen composition suitable for detecting antibodies against HIV in an isolated biological sample that gives highly specific immunoassay results. The present invention further relates to a method for detecting HIV antibodies, the use of the novel HIV gp41 antigen composition in an immunoassay, and a reagent kit comprising the novel HIV gp41 antigen composition.

Description

Technical Field

[0001] The present invention relates to an HIV gp41 antigen composition, a reagent kit containing the HIV gp41 antigen composition, and a method for producing the HIV gp41 antigen composition. A method for detecting anti-HIV antibodies in an isolated sample using the HIV gp41 antigen composition is also included.

Background Art

[0002] The envelope protein of human immunodeficiency virus (HIV) is essential for the cell infection process. In the first stage of HIV infection, the viral membrane undergoes a fusion process with the target cell membrane. Here, the viral envelope proteins, namely gp41 and gp120, are involved, and both of them are derived from the precursor protein gp160 that is proteolytically cleaved into these two fragments. The larger subunit, gp120, is the surface-binding receptor-binding subunit, and gp41 forms a transmembrane subunit that is involved in membrane fusion during viral entry into the target cell. Regarding the assumed binding mechanism between the virus and its target cell, the contact of gp120 / gp41 with the host cell membrane protein CD4 and other co-receptors induces a series of conformational changes, resulting in the formation of a hairpin trimer structure in gp41 (Root et al. Science 2001, 291, 884-888).

[0003] Patients / animals infected with HIV usually generate antibodies against gp41 and other HIV proteins. Therefore, for at least the past 20 years, gp41 has been an important component for in vitro diagnostic immunoassays for detecting antibodies against HIV. Immunoassays using the wild-type sequence of HIV gp41 already show high specificity. This means that samples containing HIV antibodies are usually correctly identified as positive.

[0004] However, in reality, there are still a significant number of false positive samples that, even though the sample is actually negative and does not contain HIV antibodies, the assay results indicate the presence of antibodies against HIV. These results can cause false alarms, thorough retesting, and confirmatory testing procedures, so these false positives can, of course, be a serious problem even in the normal, everyday laboratory diagnostic situation. Furthermore, false positive results should be particularly avoided in the blood bank situation. Here, thousands of samples obtained from blood donations are screened daily with high-throughput diagnostic analyzers, and a positive result means that the entire blood donation from the patient may be discarded.

[0005] Scholz et al. (J. Mol. Biol. 2005, 345, 1229 - 1241) described gp41 polypeptide sequences derived from HIV-1 and the corresponding gp36 from HIV-2 that were engineered so that the polypeptides that tend to aggregate can be expressed in a soluble form. However, these polypeptides do not completely avoid false positive results when used as antigens in in vitro diagnostic immunoassays for the detection of HIV antibodies.

[0006] International Publication No. WO 2001 / 044286 discloses an artificially designed five-helix protein having a gp41 element that can be used to inhibit HIV infection in human cells. This inhibitor comprises three segments derived from the N-terminal helix domain from gp41 and two segments of the C-terminal helix domain from this molecule. However, this genetically engineered construct (also described by Root et al. supra) lacks many domains and many antigenic epitopes of the native molecule and, in particular, does not contain the so-called loop motifs which are known to possess immunogenic epitopes in particular. The five-helix protein folds into a stable structure and binds to a peptide corresponding to the C-peptide region of HIV gp41, and as a result, inhibits HIV infection of human cells. It is also disclosed that the five-helix protein can be used as a drug screening or antibody screening tool. Further, a six-helix protein containing the gp41 sequence is disclosed. This six-helix protein comprising three N-helices and three C-helices of HIV gp41 linked by a linker can be used as a negative control in screening for drugs that inhibit membrane fusion.

[0007] gp41 variants have been widely described in the prior art, but the publications do not describe the identification of gp41 antigens that avoid false positive results in in vitro diagnostic immunoassays for detecting HIV antibodies.

[0008] The technical problem underlying the present invention can be found in providing means and methods that avoid the recognized problems to the extent possible in accordance with the aforementioned needs. The technical problem is characterized in the claims and is solved by the embodiments described hereinafter in this specification. SUMMARY OF THE INVENTION

[0009] In a first aspect, the present invention relates to a composition suitable for detecting antibodies against HIV gp41 in an isolated sample, wherein the composition comprises at least two individual HIV gp41 antigens, the first HIV antigen comprises SEQ ID NO: 1, and the second HIV gp41 antigen comprises at least one of SEQ ID NO: 2 or 3. In particular, the antigen does not contain additional HIV-specific amino acid sequences.

[0010] In a second aspect, the present invention relates to a method for producing a composition of HIV gp41 antigens, wherein for each of the antigens, a) culturing a host cell transformed with an expression vector containing a recombinant DNA molecule encoding one of the antigens of the first aspect of the present invention, operably linked, in particular an E. coli cell; b) expressing the antigen; c) purifying the antigen; d) mixing the HIV gp41 antigen containing SEQ ID NO: 1 obtained by steps a) to c) with at least one HIV gp41 antigen containing at least one of SEQ ID NO: 2 or 3 obtained by steps a) to c) to form a composition of HIV gp41 antigens and relates to a method.

[0011] In a third aspect, the present invention relates to a method for detecting antibodies specific for HIV in an isolated sample, wherein the composition according to the first aspect of the present invention, or the HIV gp41 antigen composition obtained by the method according to the second aspect of the present invention, is used as a capture reagent and / or a binding partner for the anti-HIV antibody.

[0012] In a fourth aspect, the present invention relates to a method for detecting antibodies specific for HIV in an isolated sample, wherein a) forming an immunoreaction mixture by mixing a body fluid sample with the HIV gp41 antigen composition of the first aspect of the present invention or the HIV gp41 antigen composition obtained by the method of the second aspect of the present invention; b) maintaining the immunoreaction mixture for a time sufficient to allow antibodies in the body fluid sample against the HIV gp41 antigen composition to immunoreact with the HIV gp41 antigen as part of the HIV gp41 antigen composition to form an immunoreaction product; c) detecting the presence and / or concentration of any of the immunoreaction products; relates to a method comprising.

[0013] In a fifth aspect, the present invention is a method for identifying whether a patient / animal has been previously exposed to HIV infection, comprising: a) forming an immunoreaction mixture by mixing a body fluid sample of the patient / animal with the HIV gp41 antigen composition of the first aspect of the present invention or the HIV gp41 antigen composition obtained by the method of the second aspect of the present invention; b) maintaining the immunoreaction mixture for a time sufficient to allow antibodies in the body fluid sample against the HIV gp41 antigen composition to immunoreact with the HIV gp41 antigen as part of the HIV gp41 antigen composition to form an immunoreaction product; c) detecting the presence and / or absence of any of the immunoreaction products; comprising, wherein the presence of an immunoreaction product indicates that the patient / animal has been previously exposed to HIV infection.

[0014] In a sixth aspect, the present invention relates to the use of the HIV gp41 antigen composition of the first aspect of the present invention or the HIV gp41 antigen composition obtained by the method of the second aspect of the present invention in a high-throughput in vitro diagnostic test for the detection of anti-HIV antibodies.

[0015] In a seventh aspect, the present invention relates to a reagent kit for the detection of anti-HIV virus antibodies, comprising the HIV gp41 antigen composition of the first aspect of the present invention or the HIV gp41 antigen composition obtained by the method of the second aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016]

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[0017] List of sequences

Chemical formula

Chemical formula

Chemical formula

Mode for Carrying Out the Invention

[0018] Based on the known structure of the single peptide chain six-helix (6hel) construct of gp41 (Root et al., supra), various mutations were introduced into the molecule. As a starting point, positions on the outside of the single helix exposed to the solvent, i.e., positions that are potential binding sites for non-specific antibodies, were mutated by exchanging the original amino acids with glycine residues. These point mutations have already led to an improvement in specificity in binding gp41 antibodies in the sample. However, the improvement was not ultimately satisfactory. As a next step, the inventors exchanged 21 positions in the C-terminal 7-amino acid repeat (CHR, Figure 1). After each position was exchanged for 12 representative amino acids (arginine, lysine, aspartic acid, serine, asparagine, alanine, valine, isoleucine, phenylalanine, tyrosine, and glycine), small-scale expression, purification, modification for designing appropriately labeled antigens, and screening for antibody binding were performed. The best variants were then expressed and purified on a large scale, labeled, and tested. Furthermore, combinations of point mutations in the six-helix (6hel) were introduced, expressed, purified, labeled, and also tested for antibody binding. Additionally, since certain HIV-specific antibodies are known to bind to particularly immunogenic loop structures that are not part of the six-helix, several point mutations were also introduced into the gp41 variants. In total, the inventors of the present application designed 242 HIV gp41 mutant antigens (Figure 3). However, contrary to the inventors' expectations, among these 242 produced variants, only a few antigens showed satisfactory performance in immunoassays for detecting the appropriate gp41 sequence. No regular pattern or consistent logic for identifying appropriate HIV gp41 variants could be discerned.

[0019] Surprisingly, among this large number of variants, the inventors have identified gp41-derived polypeptides and corresponding compositions of peptides that overcome false positive results in IVD immunoassays for detecting HIV antibodies to a significant extent, and as a result, were able to provide immunological antibody detection with high specificity while maintaining high sensitivity.

[0020] Before the present invention is described in detail below, it is to be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, and these may vary. It should also be understood that the terms used herein are for the purpose of describing only particular embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0021] Throughout this specification, several documents have been cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In case of any conflict between the definitions or teachings of the references so incorporated and the definitions or teachings cited herein, the text of this specification shall prevail.

[0022] The elements of the present invention will be described below. Although these elements are listed with specific embodiments, it should be understood that these elements can be combined in any way and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the explicitly described embodiments. This specification should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Further, any permutation and combination of all the elements described in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0023] Definitions The term "comprise", as well as variations such as "comprises" and "comprising", are meant to include the specified integer or step, or group of integers or steps, but not to exclude any other integer or step, or group of integers or steps.

[0024] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0025] Concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a "range". It is understood that such a range format is merely used for convenience and brevity, and thus should be interpreted flexibly to include not only the numerical values explicitly listed as the boundaries of the range, but also all individual numerical values or sub-ranges subsumed within that range as if each numerical value and sub-range were explicitly listed. By way of illustration, the numerical range of "150 mg to 600 mg" should be interpreted to include not only the explicitly listed values of 150 mg to 600 mg, but also the individual values and sub-ranges within the indicated range. Thus, this numerical range includes individual values such as 150, 160, 170, 180, 190, ··· 580, 590, 600 mg, and sub-ranges such as 150 - 200, 150 - 250, 250 - 300, 350 - 600. This same principle applies to ranges that enumerate only one numerical value. Further, such an interpretation should apply regardless of the width of the range or the property being described.

[0026] The term "about", when used in connection with a numerical value, means a value within a range that has a lower limit 5% less than the indicated numerical value and an upper limit 5% greater than the indicated numerical value.

[0027] The term "HIV gp41" refers to a polypeptide derived from the surface protein gp41 of human immunodeficiency virus type 1. HIV gp41 mediates both cell attachment and membrane fusion of HIV to host cells. The wild-type sequence can be found under UniProt ID P03375. Positions 535 - 681 of the HIV envelope polyprotein define the gp41 wild-type polypeptide. Soluble variants of gp41 are described, for example, in WO 2003 / 000877.

[0028] As used herein, "patient / animal subject" means any mammal, fish, reptile or bird that can benefit from the diagnosis, prognosis or treatment described herein. In particular, "patient / animal subject" is selected from the group consisting of laboratory animals (e.g., mice, rats, rabbits or zebrafish), domestic animals (e.g., guinea pigs, rabbits, horses, donkeys, cows, sheep, goats, pigs, chickens, camels, cats, dogs, turtles, tortoises, snakes, lizards or goldfish), or primates including chimpanzees, bonobos, gorillas and humans. It is particularly preferred that the "patient / animal subject" is a human.

[0029] The terms "sample", "isolated sample", "isolated biological sample" or "sample of interest" are used interchangeably herein and refer to a tissue, organ or part or piece of an individual, and is usually smaller than such a tissue, organ or individual that is intended to represent the whole of the tissue, organ or individual. In the course of analysis, the sample provides information regarding the state of the tissue, or the health or disease state of the organ or individual. Examples of samples include, but are not limited to, fluid samples such as blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid, or solid samples such as tissue extracts, cartilage, bone, synovium, and connective tissue. Analysis of the sample can be achieved visually or chemically. Visual analysis includes, but is not limited to, microscopic imaging or radiation scanning of the tissue, organ or individual that enables morphological evaluation of the sample. Chemical analysis includes, but is not limited to, detection of the presence or absence of specific indicators or changes in their amounts, concentrations or levels. The sample is an in vitro sample isolated from the body and will be analyzed in vitro and will not be returned to the body.

[0030] A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it can affect the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is arranged to facilitate translation.

[0031] The term "array comparison" refers to a process in which one array acts as a reference array against which a test array is compared. When using an array comparison algorithm, the test array and the reference array are input into a computer program, subsequence coordinates are specified as needed, and array algorithm program parameters are specified. Default program parameters are commonly used, or alternative parameters can be specified. The array comparison algorithm then calculates the percent sequence identity or similarity of the test array to the reference array based on the program parameters. In sequence alignment, the term "comparison window" refers to a stretch of contiguous positions in an array that is compared to a reference stretch of contiguous positions in an array having the same number of positions. The number of contiguous positions selected may range from 10 to 1000, i.e., may include 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 contiguous positions. Typically, the number of contiguous positions ranges from about 20 to 800 contiguous positions, from about 20 to 600 contiguous positions, from about 50 to 400 contiguous positions, from about 50 to about 200 contiguous positions, from about 100 to about 150 contiguous positions. Methods of aligning arrays for comparison are well known in the art.Optimal alignment of arrays for comparison can be performed, for example, by the local algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)). Algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977), and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra).These initial neighboring word hits function as seed values to initiate a search to find longer HSPs that contain them. The word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for pairs of matching residues; always greater than 0) and N (penalty score for mismatched residues; always less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word hit in each direction stops when the cumulative alignment score decreases by an amount X from its maximum achieved value, when the cumulative score becomes zero or less due to the accumulation of one or more negatively scored residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses, by default, a word length of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989), an alignment (B) of 50, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87, 1993). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences could occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence when the minimum total probability upon comparison of a test nucleic acid and a reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001.

[0032] The term "recombinant DNA molecule" refers to a molecule created by the combination of two originally separated segments of a DNA sequence achieved by the isolation of a polynucleotide segment by genetic engineering techniques or by the artificial manipulation of chemically synthesized polynucleotides. By doing so, polynucleotide segments of desired functions can be ligated together to generate a combination of desired functions. Recombinant DNA techniques for the expression of proteins in prokaryotic host cells or lower eukaryotic host cells or higher eukaryotic host cells are well known in the art. They are described, for example, by Sambrook et al., (1989, Molecular Cloning: A Laboratory Manual).

[0033] The terms "vector" and "plasmid" are used interchangeably herein and refer to a protein or polynucleotide or a mixture thereof that can be introduced into a cell or that contains proteins and / or nucleic acids that can be introduced into a cell. Examples of plasmids include, but are not limited to, plasmids, cosmids, phages, viruses or artificial chromosomes.

[0034] The term "amino acid" generally refers to any monomer unit that includes a substituted or unsubstituted amino group, a substituted or unsubstituted carboxy group, and one or more side chains or side chain groups, or analogs of any of these groups. Exemplary side chains include, for example, thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azide, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioic acid, hydroxylamine, or any combination of these groups. Other representative amino acids include amino acids containing photoactivatable crosslinkers, metal-binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids having novel functional groups, amino acids that interact covalently or noncovalently with other molecules, photocaged and / or photoisomerizable amino acids, radioactive amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids containing polyethylene glycol or polyethers, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, carbon-linked sugar-containing amino acids, redox-active amino acids, amino thio acid-containing amino acids, and amino acids containing one or more toxic moieties, but are not limited thereto. As used herein, the term "amino acid" includes the following 20 natural or genetically encoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0035] The terms "measurement", "measuring", "detecting", or "detection" preferably include qualitative, semi-quantitative, or quantitative measurements. The term "detect the presence of" refers to a qualitative measurement, indicating the presence or absence without a description of the quantity (e.g., a yes or no description). The term "detect the quantity of" refers to a quantitative measurement in which an absolute number is detected (ng). The term "detect the concentration of" refers to a quantitative measurement in which the quantity is determined with respect to a given volume (e.g., ng / ml).

[0036] The term "immunoglobulin (Ig)", as used herein, refers to glycoproteins of the immunoglobulin superfamily that confer immunity. "Surface immunoglobulin" is attached to the membrane of effector cells by their transmembrane regions and includes, but is not limited to, molecules such as B cell receptors, T cell receptors, class I and II major histocompatibility complex (MHC) proteins, β2-microglobulin (β2M), CD3, CD4, and CD8.

[0037] Typically, the term "antibody" as used herein refers to a secreted immunoglobulin that lacks a transmembrane region and can thus be released into the bloodstream and body cavities. Human antibodies are classified into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains denoted by Greek letters: α, γ, δ, ε, and μ. The type of heavy chain present defines the class of antibody, each playing a different role and directing an appropriate immune response against different types of antigens, i.e., these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. The different heavy chains vary in size and composition and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is found in mucosal areas such as the gastrointestinal tract, respiratory tract, and urogenital tract, as well as in saliva, tears, and breast milk, and it prevents colonization by pathogens (Underdown & Schiff (1986) Annu. Rev. Immunol. 4:389 - 417). IgD mainly functions as an antigen receptor on B cells that have not been exposed to antigens and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118:429 - 437; Chen et al. (2009) Nat. Immunol. 10:889 - 898). IgE is involved in allergic reactions through binding to allergens that cause histamine release from mast cells and basophils. IgE is also involved in protection against parasites (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides most of the antibody - based immunity against invading pathogens and is the only antibody isotype that can pass through the placenta to confer passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). In humans, there are four different IgG subclasses (IgG1, 2, 3, and 4), named in order of their abundance in serum, with IgG1 being the most abundant (about 66%), followed by IgG2 (about 23%), IgG3 (about 7%), and IgG4 (about 4%).The biological profiles of different IgG classes are determined by the structures of their respective hinge regions. IgM is expressed on the surface of B cells in monomeric form and in a secreted pentameric form with very high avidity. IgM is involved in the elimination of pathogens at the initial stage of B cell-mediated (humoral) immunity before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118:429-437).

[0038] Typically, in the process of detecting antibodies against HIV antigens in an in vitro diagnostic setting, differential diagnosis between early IgM antibodies and later IgG antibodies is not performed.

[0039] The term "binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including but not limited to assays based on surface plasmon resonance (e.g., BIAcore assays as described in PCT Application Publication WO2005 / 012359); enzyme-linked immunosorbent assay (ELISA); and competitive assays (e.g., RIA). Low-affinity antibodies generally tend to bind antigens slowly and dissociate easily, while high-affinity antibodies generally tend to bind antigens more rapidly and maintain the bound state for a longer time. Various methods for measuring binding affinity are known in the art, and any of these can be used for the purposes of the present invention.

[0040] The term "antigen (Ag)" refers to a molecule or molecular structure that is bound by an antigen-specific antibody (Ab) or B cell antigen receptor (BCR). The presence of an antigen in the body typically elicits an immune response. In the body, each antibody is specifically produced to match the antigen after immune system cells come into contact with the antigen, thereby enabling the accurate identification or matching of the antigen and the initiation of an individualized response. In most cases, an antibody can react and bind to only one specific antigen. However, in some instances, antibodies can cross-react and bind to more than one antigen. Antigens are typically proteins, peptides (amino acid chains), and polysaccharides (chains of monosaccharides / single sugars) or combinations thereof. In the present invention, an antigen that is present in the sample to be analyzed and specifically binds to an antibody that binds to the antigen is used as a specific component in an immunoassay. The terms "antigen" and "polypeptide" may be used interchangeably.

[0041] In diagnostic tests, antigens are often used in serological tests to evaluate whether a patient / animal has been exposed to a specific pathogen (e.g., a virus or bacterium) and has produced antibodies against such a pathogen. Typically, these antigens are recombinantly produced and can be linear peptides or more complex folded molecules designed to present native antigens.

[0042] Antigens can be generated by polymerizing monomeric antigens by chemical cross-linking in order to better mimic natural antigens and to obtain a high epitope density. There are numerous homo- and hetero-bifunctional cross-linking agents that can be used very advantageously and are well known in the art. However, there are several significant drawbacks to the chemically induced polymerization of antigens for use as specifiers in serological assays. For example, the insertion of cross-linker moieties into the antigen can impair antigenicity by interfering with the native-like conformation or by shielding important epitopes. Furthermore, the introduction of non-native tertiary contacts can interfere with the reversibility of protein folding / unfolding and can further cause interference problems that must be overcome by anti-interference strategies in immunoassay mixtures.

[0043] More recent techniques involve fusing the antigen of interest to an oligomeric chaperone, thereby conferring a high epitope density on the antigen. The advantages of this technique lie in its high reproducibility and the triple function of the oligomeric chaperone fusion partner. First, the chaperone improves the expression rate of the fusion polypeptide in the host cell (e.g., in E. coli), second, the chaperone promotes the refolding process of the target antigen and improves its overall solubility, and third, the chaperone reproducibly assembles the target antigen into a regular oligomeric structure.

[0044] The term "chaperone" is well known in the art and refers to protein folding helpers that assist in the folding and maintenance of the structural integrity of other proteins. Examples of folding helpers are described in detail in WO 2003 / 000877. By way of example, chaperones of the peptidylprolyl isomerase class, such as chaperones of the FKBP family, can be used for fusion to antigen variants. Examples of FKBP chaperones suitable as fusion partners are FkpA (aa 26-270, UniProt ID P45523), SlyD (1-165, UniProt ID P0A9K9) and SlpA (2-149, UniProt ID P0AEM0). A further chaperone suitable as a fusion partner is Skp (21-161, UniProt ID P0AEU7), a trimeric chaperone derived from the periplasm of Escherichia coli (E. coli) that does not belong to the FKBP family. It is not always necessary to use the complete sequence of the chaperone. Functional fragments of the chaperone (so-called binding-competent modules) that still have the required capabilities and functions can also be used (see WO 98 / 13496).

[0045] The term "not comprising additional HIV-specific amino acid sequences" means that the HIV gp41 antigen is designed such that antibodies against other HIV antigens, such as gp120, p24 or the protease or reverse transcriptase, which are HIV enzymes, do not bind to the HIV gp41 antigen. Amino acid sequences derived from other HIV proteins are not part of any portion of the HIV gp41 antigen. In addition, this term means that no more than 15, in one embodiment no more than 10, in one embodiment no more than 5, and in yet another embodiment no more than 2 consecutive amino acids of a known gp41 polypeptide, such as a part of UniProt P03375 or SEQ ID NO: 11, are fused to the C-terminus or N-terminus of the HIV gp41 antigen according to the invention.

[0046] The antigen may further comprise an "effector group" such as, for example, a "tag" or a "label". The term "tag" refers to an effector group that confers on the antigen the ability to bind to other molecules or to be bound by other molecules. Examples of tags include, but are not limited to, His tags attached to an antigen sequence to enable purification of the antigen sequence. A tag may also include a partner of a bioaffine binding pair that enables the antigen to be bound by the second partner of the binding pair. The term "bioaffine binding pair" refers to two partner molecules that have a strong affinity for binding to each other (i.e., the two partners in a pair). Examples of partners of a bioaffine binding pair are: a) biotin or a biotin analog / avidin or streptavidin; b) hapten / anti-hapten antibody or antibody fragment (e.g., digoxin / anti-digoxin antibody); c) saccharide / lectin; d) complementary oligonucleotide sequences (e.g., complementary LNA sequences), and generally e) ligand / receptor.

[0047] The term "label" refers to an effector group that enables detection of the antigen. Labels include, but are not limited to, spectroscopic, photochemical, biochemical, immunochemical or chemical labels. Exemplary suitable labels include fluorescent dyes, luminescent or electrochemiluminescent complexes (e.g., ruthenium or iridium complexes), high electron density reagents, and enzyme labels.

[0048] As used herein, "particle" means a small localized object to which physical properties such as volume, mass or average size can be ascribed. Thus, the particle may have a symmetrical, spherical, essentially spherical or globular shape, or may have an irregular, asymmetrical shape or form. The size of the particle can vary. The term "fine particle" refers to particles having diameters in the nanometer and micrometer ranges.

[0049] The microparticles defined in the above specification may include, or may consist of, any suitable material known to those skilled in the art. For example, they may include, or may consist of, inorganic or organic materials, or may consist essentially of them. Typically, the microparticles may include, or may consist of, or may consist essentially of metals or metal alloys, or organic materials, or may include, or may consist of, or may consist essentially of carbohydrate moieties. Examples of materials contemplated for the microparticles include agarose, polystyrene, latex, polyvinyl alcohol, silica, and ferromagnetic metals, alloys or composite materials. In one embodiment, the microparticles are magnetic or ferromagnetic metals, alloys or compositions. In a further embodiment, the material may have certain properties, such as being hydrophobic or hydrophilic. Such microparticles are typically dispersed in an aqueous solution and retain a small negative surface charge to keep the microparticles separated and avoid non-specific cluster formation.

[0050] In one embodiment of the present invention, the microparticles are paramagnetic microparticles, and the separation of such particles in the measurement method according to the present disclosure is facilitated by magnetic force. To remove paramagnetic or magnetic particles from a solution / suspension and to hold the paramagnetic or magnetic particles as desired, a magnetic force is applied, during which the liquid of the solution / suspension can be removed and the particles can be washed, for example.

[0051] In a diagnostic test, it is necessary to determine whether the measured value is classified as "negative" (or "normal" or "non-reactive") or "positive" (or "pathological" or "reactive"). If the measured signal is in a range below a predetermined threshold, the sample is considered non-reactive or negative. If the measured parameter is in a range above the threshold, the sample is classified as reactive or positive. Such a threshold is a dividing point on the measurement scale set for the test procedure to distinguish between positive and negative values. The threshold can be selected such that the test still provides a predetermined high sensitivity (high true positive rate), while at the same time ensuring a predetermined high specificity (high true negative rate) to avoid false positive and false negative results. Depending on the test design and to avoid false positive results, the cut-off value can be defined as a multiple of the background signal or as a multiple of the result of a normal (negative) sample. The result of the test can be the ratio of the result signal obtained for the sample divided by a predetermined cut-off value, and can be provided in the form of a "Cut-off Index" (COI) which gives a signal sample / cut-off ratio. In particular, in HIV diagnosis, the cut-off and the calculated COI can be selected such that a high sensitivity and high specificity of the assay are achieved, i.e., ideally all positives should be detected and there should be no, or at least as few as possible, false positives among them. In many cases, the sensitivity and specificity for the most highly regulated infectious disease tests are at least 98% (e.g., in the range of 98 - 99.99%). For HIV diagnosis, a minimum sensitivity of 100% and a specificity of more than 99.8% are required.

[0052] A "kit" or "reagent kit" is any product (e.g., a package or container) that includes at least one reagent, such as a medicament for treating a disorder, or a probe for specifically detecting a biomarker gene or protein of the present invention. The kit is preferably promoted, distributed, or sold as a unit for carrying out the methods of the present invention. Usually, the kit may further comprise carrier means compartmentalized to securely house one or more container means, such as vials, tubes, etc. In particular, each of the container means contains one of the distinct elements used in the method of the first aspect. The kit may further comprise one or more other containers containing additional materials, including but not limited to buffers, diluents, filters, needles, syringes, and accompanying documents with instructions for use. Labels may be presented on the containers to indicate that the composition is for a particular use, and the labels may also indicate instructions regarding either in vivo or in vitro use. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disc), or directly to a computer or data processing device, or made available via a data cloud setting. Further, the kit may contain a standard amount for the biomarker for calibration purposes.

[0053] "Accompanying document" is used to refer to the instructions customarily included in the commercial package of a diagnostic product that contains information regarding the intended use of the product, such as instructions on how to use the product (a "method sheet") that is present on a diagnostic analyzer, the range of expected results, disorders observed during development or the registration process, etc.

[0054] Embodiments As further described above, currently available immunoassays for detecting anti-HIV antibodies using antigens derived from gp41 show a significant number of false positive results, i.e., they may lack specificity. Surprisingly, by restricting the antigen to the HIV gp41 antigen as further described below, the number of samples that react erroneously can be reduced while maintaining the high sensitivity of the assay.

[0055] In a first aspect, the present invention is a composition suitable for detecting antibodies against HIV gp41 in an isolated sample, said composition comprising at least two, preferably three, individual HIV gp41 antigens, a first HIV antigen comprising SEQ ID NO: 1 and other HIV gp41 antigens comprising at least one of SEQ ID NO: 2 and / or 3.

[0056] In a plurality of embodiments, each of the antigens does not contain additional HIV-specific amino acid sequences.

[0057] In a plurality of embodiments, each of the HIV gp41 antigens is immunoreactive, i.e., antibodies present in a biological sample bind to the antigen. Thus, any peptides derived from HIV gp41 that are not bound by the antibody are not included.

[0058] In a plurality of embodiments, each of the HIV gp41 antigens is soluble and suitable for use in in vitro assays aimed at detecting antibodies against said antigens in an isolated biological sample.

[0059] Therefore, each of the composition and its HIV gp41 antigen is suitable for use in in vitro assays aimed at detecting anti-HIV antibodies with high sensitivity and specificity. In multiple embodiments, the sensitivity is >95%, >96%, >97%, >98%, >99%, >99.5%, >99.8%. In certain embodiments, the sensitivity is >99.5% or >99.8%. In certain embodiments, the sensitivity is 100%. In multiple embodiments, the specificity is >95%, >96%, >97%, >98%, >99%, >99.5%. In certain embodiments, the specificity is >99% or >99.5%. In certain embodiments, the specificity is >99.9%. In certain embodiments, the sensitivity is 100% and the specificity is >99.9%.

[0060] In multiple embodiments, the composition of the HIV gp41 antigen is suitable for detecting antibodies against HIV in a fluid sample or detects antibodies against HIV in a fluid sample. In certain embodiments, the sample is a human sample, particularly a human body fluid sample. In certain embodiments, the sample is a human blood or urine sample. In certain embodiments, the sample is a human whole blood, plasma or serum sample.

[0061] In multiple embodiments, each of the HIV gp41 antigens is in its native state. In certain embodiments, the HIV gp41 specific amino acid sequences contained in each of the HIV gp41 antigens are folded in their native state.

[0062] In multiple embodiments, variants of the HIV gp41 specific amino acid sequences of SEQ ID NOs: 1, 2, and 3 are included. These variants are readily made by those skilled in the art by conservative or homologous substitutions of the disclosed amino acid sequences (e.g., substitution of cysteine by alanine or serine, etc.). In multiple embodiments, the variant exhibits a modification to its amino acid sequence, particularly selected from the group consisting of amino acid exchanges, deletions or insertions as compared to the amino acid sequences of SEQ ID NOs: 1, 2, and 3.

[0063] In multiple embodiments, the amino acid has a deleted C-terminus or N-terminus, or 1 to 10 amino acids, in one embodiment 1 to 5 amino acids, are inserted at one or both ends. However, no additional HIV-specific amino acid sequences need to be added. In particular, the variant can be an isoform that represents the most common protein isoform. In one embodiment, such substantially similar proteins have at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% sequence homology to SEQ ID NO: 1, 2, or 3.

[0064] In multiple embodiments, the variant includes a post-translational modification selected particularly from the group consisting of glycosylation or phosphorylation.

[0065] It is understood that such variants are classified as HIV gp41 antigen variants, i.e., they can bind and detect anti-HIV gp41 antibodies present in an isolated sample.

[0066] In multiple embodiments, since the overall three-dimensional structure of each of the HIV gp41 antigens remains unchanged, epitopes that were previously (i.e., in the wild type) accessible for binding to antibodies are still accessible in the variant.

[0067] In multiple embodiments, at least one of the HIV gp41 antigens further includes at least one chaperone. Thus, the HIV gp41 antigen includes the HIV gp41-specific amino acid sequence of SEQ ID NO: 1, 2, or 3 above or below, and the amino acid sequence of the chaperone.

[0068] In a preferred embodiment, only the HIV gp41 antigen of SEQ ID NO: 1 contains at least one chaperone. In a more preferred embodiment, only the HIV gp41 antigen of SEQ ID NO: 2 contains at least one chaperone. In a further more preferred embodiment, only the HIV gp41 antigen of SEQ ID NO: 3 contains at least one chaperone.

[0069] In a plurality of embodiments, each of the HIV gp41 antigens further contains at least one chaperone. Thus, the HIV gp41 antigen contains the HIV gp41-specific amino acid sequence of SEQ ID NO: 1, 2 or 3 above or below, and the amino acid sequence of the chaperone.

[0070] In a particular embodiment, the HIV gp41 antigen contains two chaperones. In a plurality of embodiments, the chaperone is selected from the group consisting of SlyD, SlpA, FkpA and Skp. In a particular embodiment, the chaperone is SlyD having the amino acid sequence given in particular by accession number UniProt ID P0A9K9.

[0071] In a particular embodiment, the HIV gp41 antigen contains the HIV gp41-specific amino acid sequence described in SEQ ID NO: 1, 2 or 3 and one SlyD chaperone. In a particular embodiment, the HIV gp41 antigen contains the HIV gp41-specific amino acid sequence described in SEQ ID NO: 1, 2 or 3 and two SlyD chaperones. The fusion of the two chaperones results in a higher solubility of the resulting antigen. In a particular embodiment, SEQ ID NO: 1 is fused to two SlyD chaperone molecules.

[0072] In multiple embodiments, the chaperone is fused to an HIV gp41-specific amino acid sequence at the N-terminus and / or C-terminus of the HIV gp41 antigen, and in particular is fused to the N-terminus of the HIV gp41 antigen. Thus, in certain embodiments, the HIV gp41 antigen comprises one SlyD chaperone attached to the N-terminus of the HIV gp41-specific amino acid sequence. In certain embodiments, the HIV gp41 antigen comprises two SlyD chaperones attached to the N-terminus of the HIV gp41-specific amino acid sequence. In multiple embodiments, the HIV gp41 antigen comprises one SlyD chaperone attached to the N-terminus of the HIV gp41-specific amino acid sequence and one SlyD chaperone attached to the C-terminus of the HIV gp41-specific amino acid sequence.

[0073] In multiple embodiments, the HIV gp41 antigen or antigen further comprises a linker sequence. These sequences are not specific to anti-HIV gp41 viral antibodies and are not recognized in in vitro diagnostic immunoassays. In particular, the HIV gp41 antigen comprises a linker sequence between the sequence of HIV gp41 and one or more chaperones. In certain embodiments, the linker is a Gly-rich linker. In certain embodiments, the linker has a sequence shown in any of SEQ ID NOs: 14, 15, and 16.

[0074] In certain embodiments, the HIV gp41 antigen comprises the amino acid sequence set forth in SEQ ID NO: 5. In multiple embodiments, the HIV gp41 antigen does not include any additional amino acid sequences. In certain embodiments, the HIV gp41 antigen consists of the amino acid sequence set forth in SEQ ID NO: 5.

[0075] In certain embodiments, the HIV gp41 antigen comprises the amino acid sequence set forth in SEQ ID NO: 6. In multiple embodiments, the HIV gp41 antigen does not include any additional amino acid sequences. In certain embodiments, the HIV gp41 antigen consists of SEQ ID NO: 6.

[0076] In certain embodiments, the HIV gp41 antigen comprises the amino acid sequence set forth in SEQ ID NO: 7. In a plurality of embodiments, the HIV gp41 antigen does not include any additional amino acid sequences. In certain embodiments, the HIV gp41 antigen consists of SEQ ID NO: 7.

[0077] In certain embodiments, the HIV gp41 antigen comprises the amino acid sequence set forth in SEQ ID NO: 8. In a plurality of embodiments, the HIV gp41 antigen does not include any additional amino acid sequences. In certain embodiments, the HIV gp41 antigen consists of SEQ ID NO: 8.

[0078] It is understood that the HIV gp41 antigen consisting of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 does not include any additional amino acid sequences, but may still include other chemical molecules such as labels and / or tags.

[0079] In a plurality of embodiments, a composition suitable for detecting antibodies against HIV gp41 in an isolated sample comprises at least two, preferably three, individual HIV gp41 antigens, wherein the first HIV antigen comprises SEQ ID NO: 1 and the second HIV gp41 antigen comprises at least one of SEQ ID NO: 2 or 3. In a plurality of embodiments, the composition comprises the HIV gp41 antigens set forth in SEQ ID NOs: 1, 2, and 3.

[0080] In a plurality of embodiments, the composition comprises SEQ ID NOs: 5, 6, 7, and 8.

[0081] In a plurality of embodiments, each HIV gp41 antigen further comprises a tag or a label. Thus, the HIV gp41 antigen comprises the HIV gp41-specific amino acid sequence at any of SEQ ID NOs: 1, 2, 3, 6, 7, 8, or 9 above or below, and a tag or a label, and optionally comprises the amino acid sequence of one or more chaperones.

[0082] In certain embodiments, the tag enables direct or indirect binding of the HIV gp41 antigen to a solid phase. In certain embodiments, the tag is a partner of a bioaffinity binding pair. In certain embodiments, the tag is selected from the group consisting of biotin, digoxigenin, hapten, or a complementary oligonucleotide sequence (particularly a complementary LNA sequence). In certain embodiments, the tag is biotin.

[0083] In certain embodiments, the label enables detection of the HIV gp41 antigen. In certain embodiments, the HIV gp41-specific sequence is labeled. In embodiments where at least one chaperone is present in the antigen, either the HIV gp41-specific sequence is labeled, or at least one chaperone is labeled, or both are labeled. In certain embodiments, the label is an electrochemiluminescent ruthenium or iridium complex. In certain embodiments, the electrochemiluminescent ruthenium complex is a negatively charged electrochemiluminescent ruthenium complex. In certain embodiments, the label is a negatively charged electrochemiluminescent ruthenium complex present in the antigen in a stoichiometry of 1:1 to 15:1. In certain embodiments, the stoichiometry is 2:1, 2.5:1, 3:1, 5:1, 10:1, or 15:1.

[0084] In multiple embodiments, the composition comprises one or more additional HIV antigens. In certain embodiments, the composition comprises the HIV gp120 antigen, the HIV reverse transcriptase antigen, or the HIV p24 antigen, or any combination thereof. In certain embodiments, the composition comprises the HIV reverse transcriptase antigen as an additional antigen.

[0085] In multiple embodiments, the additional HIV antigens are immunoreactive, i.e., antibodies present in a biological sample bind to said antigens. Thus, any peptides derived from HIV that are not bound by anti-HIV antibodies are not included.

[0086] In multiple embodiments, the additional HIV gp41 antigen is soluble. Thus, the antigen is suitable for use in in vitro assays aimed at detecting antibodies against said antigen in an isolated biological sample.

[0087] In a second aspect, the present invention is a method for producing a composition of HIV gp41 antigen, comprising: a) culturing a host cell, particularly an E. coli cell, transformed with an expression vector containing a recombinant DNA molecule encoding the antigen of the first aspect of the present invention, operably linked; b) expressing said antigen; c) purifying said antigen; d) mixing each of the HIV gp41 antigens obtained by steps a) to c) to form a composition of HIV gp41 antigen; and relates to a method comprising.

[0088] Optionally, as an additional step e), functional solubilization is required to render each HIV gp41 antigen into a soluble and immunoreactive conformation by refolding techniques known in the art.

[0089] In certain embodiments, the host cell is an E. coli cell, a CHO cell or a HEK cell. In certain embodiments, the host cell is an E. coli cell.

[0090] In embodiments where the antigen comprises an HIV gp41 sequence and one or more chaperones, the recombinant DNA molecule according to the invention may also contain a sequence encoding a linker peptide of 5 to 100 amino acid residues between the HIV gp41 antigens. Such linker sequences may have, for example, proteolytic cleavage sites. In one embodiment, the addition of non-HIV gp41-specific linkers or peptidic fusion amino acid sequences to the HIV gp41 antigen is possible because these sequences are not specific for anti-HIV antibodies and are not recognized in in vitro diagnostic immunoassays.

[0091] In a third aspect, the present invention relates to a method for detecting antibodies specific for HIV in an isolated sample, wherein the composition of the first aspect of the present invention, or the HIV gp41 antigen obtained by the method of the second aspect of the present invention, is used as a capture reagent for the anti-HIV antibody and / or as a binding partner.

[0092] In a fourth aspect, the present invention relates to a method for detecting antibodies specific for HIV in an isolated sample, comprising: a) forming an immunoreaction mixture by mixing a body fluid sample with the HIV gp41 antigen composition of the first aspect of the present invention or the HIV gp41 antigen composition obtained by the method of the second aspect of the present invention; b) maintaining the immunoreaction mixture for a time sufficient to allow antibodies in the body fluid sample against the HIV gp41 antigen composition to immunoreact with the HIV gp41 antigen as part of the composition to form an immunoreaction product; c) detecting the presence and / or concentration of any of the immunoreaction products. The present invention also relates to a method comprising the above steps.

[0093] In a plurality of embodiments, the method is an in vitro method. In a plurality of embodiments, the method exhibits high sensitivity and specificity. In a plurality of embodiments, the sensitivity is >95%, >96%, >97%, >98%, >99%, >99.5%. In certain embodiments, the sensitivity is >99% or >99.5%. In certain embodiments, the sensitivity is 100%. In a plurality of embodiments, the specificity is >95%, >96%, >97%, >98%, >99%, >99.5%. In certain embodiments, the specificity is >99% or >99.5%. In certain embodiments, the specificity is 99.8%. In certain embodiments, the sensitivity is 100% and the specificity is >99.9%.

[0094] In multiple embodiments, the antibodies detected by the method of the present invention are anti-HIV virus antibodies of the IgG, IgM, or IgA subclass, or all three subclasses, in the same immunoassay.

[0095] In multiple embodiments, the detected antibodies are made against gp41 of the human immunodeficiency virus (HIV), particularly gp41 of HIV-1.

[0096] In multiple embodiments, the isolated biological sample in which HIV-specific antibodies are detected is a human sample, particularly a human body fluid sample. In certain embodiments, the sample is a human blood or urine sample. In certain embodiments, the sample is a human whole blood, plasma, or serum sample. In certain embodiments, the sample is a human whole blood, plasma, or serum sample from a vein or capillary.

[0097] In multiple embodiments, in step a), the HIV gp41 antigen mixed with the isolated biological sample comprises at least one HIV gp41-specific amino acid sequence described in SEQ ID NO: 1, 2, or 3 or a variant thereof. In multiple embodiments, the HIV gp41 antigen does not contain additional HIV gp41 virus-specific amino acid sequences.

[0098] In multiple embodiments, the composition applied in the method for detecting antibodies specific to HIV in an isolated sample comprises the HIV gp41 antigens described in SEQ ID NO: 5, 6, 7, and 8. In certain embodiments, the HIV-specific sequences of the HIV gp41 antigen consist of SEQ ID NO: 5, 6, 7, and 8.

[0099] In multiple embodiments, the HIV gp41 antigen is immunoreactive, i.e., the antibodies present in the biological sample bind to the antigen. Accordingly, any peptides derived from HIV gp41 that are not bound by the antibodies are not included.

[0100] In multiple embodiments, the HIV gp41 antigen is soluble. Thus, the HIV gp41 antigen is suitable for use in in vitro assays aimed at detecting antibodies against said antigen in an isolated biological sample.

[0101] In multiple embodiments, the method includes the further step of adding a solid phase to the immunoreaction mixture. In multiple embodiments, the solid phase is a solid phase extraction (SPE) cartridge or beads. In certain embodiments, the solid phase comprises or consists of particles. In multiple embodiments, the particles are non-magnetic, magnetic or paramagnetic. In multiple embodiments, the particles are coated. The coating can vary depending on the intended use, i.e., the intended capture molecule. Which coating is suitable for which analyte is well known to those skilled in the art. The particles can be made of a variety of different materials. The beads may have various sizes and may have a porous or non-porous surface.

[0102] In certain embodiments, the particles are microparticles. In multiple embodiments, the microparticles have a diameter of 50 nanometers to 20 micrometers. In multiple embodiments, the microparticles have a diameter of 100 nm to 10 μm. In multiple embodiments, the microparticles have a diameter of 200 nm to 5 μm, particularly 750 nm to 5 μm, particularly 750 nm to 2 μm. In certain embodiments, the microparticles are magnetic or paramagnetic. In particular, the microparticles are paramagnetic.

[0103] In multiple embodiments, the solid phase is added either before adding the sample to the antigen or after the immunoreaction mixture has been formed. Thus, the addition of the solid phase can be carried out in step a) of the method, in step b) of the method, or after step b) of the method.

[0104] In multiple embodiments, the method implemented is an immunoassay for detecting anti-HIV antibodies in an isolated biological sample. Immunoassays for detecting antibodies are well known in the art, as are methods for performing such assays and actual applications and procedures. The HIV gp41 antigen according to the present invention can be used to improve assays for detecting anti-HIV antibodies regardless of the label used and regardless of the detection format (e.g., radioisotope assay, enzyme immunoassay, electrochemiluminescence assay, etc.) or assay principle (e.g., test strip assay, sandwich assay, indirect test concept or homogeneous assay, etc.).

[0105] In multiple embodiments, the method implemented is an immunoassay for detecting anti-HIV antibodies in an isolated sample according to the so-called dual antigen sandwich concept (DAGS). Since two antigens are cross-linked by the antibody analyte, this assay concept is sometimes referred to as the dual antigen cross-linking concept. In such an assay, the ability of the antibody to bind at least two different molecules of a given antigen with its two (IgG, IgE), four (IgA) or ten (IgM) paratopes is utilized.

[0106] In multiple embodiments, the immunoassay for determination of anti-HIV gp41 antibodies in DAGS format is performed by incubating a sample containing anti-HIV gp41 antibodies with two different HIV gp41 antigens, namely a first ("capture") HIV gp41 antigen and a second HIV gp41 virus ("detection") antigen, each of the two antigens being specifically bound by the anti-HIV gp41 antibody.

[0107] In multiple embodiments, the structures of the "capture antigen" and the "detection antigen" are immunologically cross-reactive. An essential requirement for implementing this method is that one or more related epitopes are present on both antigens. Thus, both antigens contain the HIV gp41-specific amino acid sequences described above or below. In multiple embodiments, the two antigens contain the same or different fusion moieties (e.g., SlyD fused to an HIV gp41-specific antigen tagged to be bound by a solid phase, and FkpA fused to an HIV gp41-specific antigen labeled to be detected, for example), and such variations significantly alleviate the problem of non-specific binding and thus reduce the risk of false positive results.

[0108] In multiple embodiments, the first antigen can be directly or indirectly bound to a solid phase and typically has an effector group that is part of a bioaffinity binding pair. In certain embodiments, the first antigen is conjugated to biotin and the complementary solid phase is coated with either avidin or streptavidin. In multiple embodiments, the second antigen has a label that alone or forms a complex with another molecule to confer specific detectability to this antigen molecule. In certain embodiments, the second antigen has a ruthenium complex label.

[0109] Thus, in step b) of the method, an immunoreaction mixture is formed that includes the first antigen, the sample antibody, and the second antigen.

[0110] This ternary complex consisting of the analyte antibody sandwiched between two antigen molecules is called an immune complex or immunoreaction product.

[0111] In multiple embodiments, the method may include a further step of separating the liquid phase from the solid phase.

[0112] Thus, in multiple embodiments, a method for detecting antibodies specific for the HIV gp41 virus in an isolated sample is a) adding to the sample a first HIV gp41 antigen having an effector group that can be directly or indirectly bound to a solid phase and is part of a bioaffinity binding pair, and a second HIV gp41 antigen having a detectable label, wherein the first and second HIV gp41 antigens specifically bind to the anti-HIV gp41 antibody; b) forming an immunoreaction mixture comprising the first antigen, the sample antibody, and the second antigen, wherein a solid phase having the corresponding effector group of the bioaffinity binding pair is added before, during, or after the formation of the immunoreaction mixture; c) maintaining the immunoreaction mixture for a time sufficient to allow anti-HIV gp41 antibodies in the body fluid sample against the HIV gp41 antigen to immunoreact with the HIV gp41 antigen to form an immunoreaction product; d) separating the liquid phase from the solid phase; e) detecting the presence of any of the immunoreaction products in the solid phase, the liquid phase, or both; and comprising.

[0113] Finally, the presence of any of the immunoreaction products is detected in the solid phase, the liquid phase, or both.

[0114] In a plurality of embodiments, the maximum total duration of the method for detecting HIV gp41 antibody is less than 1 hour, i.e., less than 60 minutes, in one embodiment less than 30 minutes, in a further embodiment less than 20 minutes, in one embodiment 15 - 30 minutes, and in one embodiment 15 - 20 minutes. The duration includes pipetting the sample and the reagents necessary to perform the assay, as well as the incubation time, any optional washing steps, the detection step, and the final output of the results.

[0115] In a fifth aspect, the present invention is a method for identifying whether a patient / animal has been previously exposed to HIV infection, comprising: a) Forming an immunoreaction mixture by mixing the body fluid sample of the patient / animal with the HIV gp41 antigen composition of the first aspect of the present invention or the HIV gp41 antigen obtained by the method of the second aspect of the present invention; b) Maintaining the immunoreaction mixture for a time sufficient to allow antibodies in the body fluid sample against the HIV gp41 antigen composition to immunoreact with the HIV gp41 antigen as part of the HIV gp41 antigen composition to form an immunoreaction product; c) Detecting the presence and / or absence of any of the immunoreaction products; comprising; The presence of the immunoreaction product relates to a method indicating that the patient / animal has been exposed to HIV infection in the past.

[0116] In a plurality of embodiments, the patient / animal has been exposed to HIV infection prior to the implementation of the method. In particular, the patient / animal has been exposed to HIV infection at least 5 days prior to the implementation of the method. In particular, the patient / animal has been exposed to HIV infection at least 10 days prior to the implementation of the method. In particular, the patient / animal has been exposed to HIV infection at least 14 days prior to the implementation of the method.

[0117] In a sixth aspect, the present invention relates to the use of the HIV gp41 antigen composition of the first aspect of the present invention or the HIV gp41 antigen composition obtained by the method of the second aspect of the present invention in a high-throughput in vitro diagnostic test for the detection of anti-HIV antibodies.

[0118] In a seventh aspect, the present invention relates to a reagent kit for the detection of anti-HIV virus antibodies, comprising the HIV gp41 antigen composition of the first aspect of the present invention or the HIV gp41 antigen composition obtained by the method of the second aspect of the present invention.

[0119] In a plurality of embodiments, the reagent kit contains the HIV gp41 antigen composition of the first aspect of the present invention or the HIV gp41 antigen obtained by the method of the second aspect of the present invention in separate containers or in separate compartments of a single container unit. In certain embodiments, the included HIV gp41 antigen is covalently bound to biotin.

[0120] In a plurality of embodiments, the reagent kit further contains microparticles, particularly microparticles coated with avidin or streptavidin, in separate containers or in separate compartments of a single container unit.

[0121] All definitions provided for the HIV gp41 antigen as part of the composition provided in the first to third aspects of the present invention are also applicable mutatis mutandis to the fourth, fifth, sixth and seventh aspects of the present invention.

[0122] In a further embodiment, the present invention relates to the following items.

[0123] Item 1: A composition suitable for detecting antibodies against HIV gp41 in an isolated sample, said composition containing at least two individual HIV gp41 antigens, the first HIV antigen containing SEQ ID NO: 1, the second HIV gp41 antigen containing SEQ ID NO: 2 and / or 3, and each of said individual HIV gp41 antigens not containing a further HIV-specific amino acid sequence.

[0124] Item 2: The composition according to item 1, wherein at least one of said HIV gp41 antigens is fused to at least one chaperone, and in one embodiment, two chaperones.

[0125] Item 3: The composition according to item 2, wherein said chaperone is selected from the group consisting of SlyD, SlpA, FkpA and Skp.

[0126] Item 4: The composition according to item 1 or 2, wherein the chaperone is fused to an HIV gp41-specific amino acid sequence at the N-terminus and / or C-terminus of the HIV gp41 antigen.

[0127] Item 5: The composition according to any one of items 1 to 4, wherein each of the antigens is soluble and immunoreactive.

[0128] Item 6: The composition according to any one of items 1 to 5, wherein the HIV gp41 antigen comprises SEQ ID NO: 1 and 2 or SEQ ID NO: 1 and 3.

[0129] Item 7: The composition according to any one of items 1 to 6, wherein the HIV gp41 antigen comprises SEQ ID NO: 1, 2, and 3.

[0130] Item 8: The composition according to any one of items 1 to 7, wherein the HIV gp41 antigen comprises SEQ ID NO: 5, 6, 7, and 8.

[0131] Item 9: The composition according to any one of items 1 to 8, wherein the HIV-specific sequence of the HIV gp41 antigen consists of SEQ ID NO: 5, 6, 7, and 8.

[0132] Item 10: An HIV gp41 antigen comprising SEQ ID NO: 1, suitable for detecting antibodies against HIV in an isolated biological sample, wherein the antigen does not contain any additional HIV-specific amino acid sequences.

[0133] Item 11: An HIV gp41 antigen comprising SEQ ID NO: 2, suitable for detecting antibodies against HIV in an isolated biological sample, wherein the antigen does not contain any additional HIV-specific amino acid sequences.

[0134] Item 12: An HIV gp41 antigen comprising SEQ ID NO: 3, suitable for detecting antibodies against HIV in an isolated biological sample, wherein the antigen does not contain any additional HIV-specific amino acid sequences.

[0135] Item 13: The HIV gp41 antigen according to any one of Items 10 to 12, further comprising a transglutaminase peptide, wherein in one embodiment, the antigen comprises the amino acid sequence YRYRQ (SEQ ID NO: 13).

[0136] Item 14: The HIV gp41 antigen according to any one of Items 10 to 13, further comprising a sortase peptide, wherein in one embodiment, the antigen comprises the amino acid sequence LPETG (SEQ ID NO: 12).

[0137] Item 15: The HIV gp41 antigen according to any one of Items 10 to 14, further comprising a linker peptide, wherein in one embodiment, the antigen comprises two or three glycine residues, in one embodiment GGGS (SEQ ID NO: 14), in another embodiment GGGSGGGSGGGSGGG (SEQ ID NO: 15), and in another embodiment SGGG (SEQ ID NO: 16).

[0138] Item 16: The HIV gp41 antigen according to any one of Items 10 to 15, further comprising a histidine peptide HHHHHH (SEQ ID NO: 17).

[0139] Item 17: A method for producing a composition of the HIV gp41 antigen according to any one of Items 1 to 9, wherein for each of the antigens, a) culturing a host cell transformed with an expression vector containing a recombinant DNA molecule operably linked to encode one of each of the antigens; b) expressing each of the antigens; c) purifying each of the antigens; d) mixing the HIV gp41 antigen containing SEQ ID NO: 1 obtained by steps a) to c) with at least one HIV gp41 antigen containing at least one of SEQ ID NO: 2 and / or 3 obtained by steps a) to c) to form a composition of the HIV gp41 antigen comprising a method.

[0140] Item 18: The method according to item 17, wherein in step d), the mixed HIV gp41 antigen consists of SEQ ID NO: 5, 6, 7 and 8.

[0141] Item 19: A method for detecting an antibody specific for HIV gp41 in an isolated sample, wherein the composition of the HIV gp41 antigen according to any one of items 1 to 9 is used as a capture reagent for the anti-HIV antibody and / or as a binding partner.

[0142] Item 20: A method for detecting an antibody specific for HIV gp41 in an isolated sample, comprising: a) forming an immunoreaction mixture by mixing a body fluid sample with the HIV gp41 antigen composition according to any one of items 1 to 9; b) maintaining the immunoreaction mixture for a time sufficient to allow antibodies against HIV gp41 present in the body fluid sample to immunoreact with the HIV gp41 antigen composition to form an immunoreaction product; c) detecting the presence and / or concentration of any of the immunoreaction products. The method comprising the above steps.

[0143] Item 21: A method for detecting an antibody specific for HIV in an isolated sample according to item 20, wherein the immunoreaction is: a) adding to the sample a first HIV gp41 antigen according to items 10 to 16 or a first antigen composition according to items 1 to 9 that can be directly or indirectly bound to a solid phase and has an effector group that is part of a bioaffinity binding pair, and a second HIV gp41 antigen according to items 10 to 16 or a second antigen composition according to items 1 to 9 having a detectable label, wherein the first and second HIV gp41 antigens specifically bind to the anti-HIV antibody. b) forming an immunoreaction mixture comprising the first antigen, the sample antibody, and the second antigen, wherein a solid phase having the corresponding effector group of the bioaffinity binding pair is added before, during, or after the formation of the immunoreaction mixture; c) maintaining the immunoreaction mixture for a time sufficient to allow an anti-HIV antibody in the body fluid sample against the HIV gp41 antigen to immunoreact with the HIV gp41 antigen to form an immunoreaction product; d) separating the liquid phase from the solid phase; e) detecting the presence of any of the immunoreaction products in the solid phase, the liquid phase, or both; A method implemented in a double antigen sandwich format, comprising:

[0144] Item 22: A method for identifying whether a patient / animal has been exposed to HIV infection in the past, comprising: a) forming an immunoreaction mixture by mixing a body fluid sample of the patient / animal with the HIV gp41 antigen composition according to any one of Items 1-9; b) maintaining the immunoreaction mixture for a time sufficient to allow an antibody against the HIV gp41 antigen composition present in the body fluid sample to immunoreact with the HIV gp41 antigen composition to form an immunoreaction product; c) detecting the presence and / or absence of any of the immunoreaction products; comprising: A method, wherein the presence of the immunoreaction product indicates that the patient / animal has been exposed to HIV infection in the past.

[0145] Item 23: Use of the HIV gp41 antigen composition according to any one of Items 1-9 for detecting anti-HIV gp41 antibodies in an isolated sample.

[0146] Item 24: Use of the HIV gp41 antigen composition according to Item 23 in a high-throughput in vitro diagnostic test for detecting anti-HIV antibodies in an isolated sample.

[0147] Item 25: A reagent kit for detecting anti-HIV antibodies, comprising the HIV gp41 antigen composition according to any one of Items 1 to 9.

[0148] Item 26: A reagent kit for detecting anti-HIV antibodies, comprising the composition according to any one of Items 1 to 9, or the HIV gp41 antigen composition obtained by the method according to Item 17.

[0149] Item 27: A reagent kit according to Item 23, comprising at least microparticles coated with avidin or streptavidin and the HIV gp41 antigen composition according to any one of Items 1 to 9 or the HIV gp41 antigen composition obtained by the method according to Item 17, wherein each of the HIV gp41 antigens is covalently bound to biotin, in separate containers or in separate compartments of a single container unit.

[0150] Item 28: A reagent kit according to Item 27, comprising the HIV gp41 antigen composition according to any one of Items 1 to 9 or the HIV gp41 antigen composition obtained by the method according to Item 17 in an additional separate container or in an additional separate compartment of a single container, wherein each of the HIV gp41 antigens in the additional separate container or additional separate compartment is covalently bound to a detectable label, in one embodiment an electrochemiluminescent ruthenium complex.

[0151] The following examples and figures are provided to assist in the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications may be made to the procedures described without departing from the spirit of the present invention.

Examples

[0152] Example 1: Expression and purification of recombinant HIV1 gp41 and 6hel antigens Small-scale preparation of recombinant 6hel antigen for high-throughput screening Plasmids containing the gp41-6hel gene with different point mutations and a C-terminal hexahistidine tag were synthesized by Twist Bioscience and cloned into pET29a via NdeI (5'-end) and XhoI (3'-end) restriction sites.

[0153] Small-scale expression of recombinant gp41-6hel protein After diluting the plasmid to 5 ng / μl - 10 ng / μl in 10 mM Tris-HCl buffer (pH 8.5), 1 μl of DNA was added to HT96 BL21(DE3) competent cell plates (Novagen). Transformation was performed according to the manufacturer's protocol, and 20 microliters of the transformation reaction was plated onto 48-well LB-kanamycin (50 μg / ml) agar plates (Teknova).

[0154] Culturing and expression of all variants were performed in 96-well plate format. For preculture, one colony per variant was picked into 96-well flat-bottom microtiter plates (Corning) filled with 200 μl of 4× yeast-kanamycin (50 μg / ml) medium per well. Additionally, each plate contained at least one wild-type gp41 antigen as a reference. Cells were grown overnight at 37 °C without shaking. For long-term storage, 50 μl of 50% (v / v) glycerol was added before freezing. Expression was carried out in 96 deep-well plates containing 1000 μl of 4× yeast-kanamycin (50 μg / ml) medium per well with 0.1 mM IPTG. Variants were expressed at 30 °C and 800 rpm (Microplate Shaker TiMix; Edmund Buhler GmbH) and harvested after 16 hours by centrifugation at 4700 rpm for 10 minutes.

[0155] Small-scale purification of recombinant gp41-6hel protein For small-scale purification of the 6hel antigen, bacterial cell pellets from 1 ml of E. coli culture were resuspended in 125 μl of 100% BugBuster according to the manufacturer's protocol (登録商標)(Merck Millipore) was dissolved. 125 μl of 2× equilibration buffer (0.1 M NaH 2 PO 4 pH 8.0; 1% (v / v) Tween-20; 1 M NaCl; 40 mM imidazole) was added, and after clarifying the cell lysate by centrifugation (4700 rpm, 10 min), the lysate was transferred to a 96-well V-bottom plate (Corning) using a pipetting robot (Biomek). Small-scale purification was performed using a robotic pipette tip called a PhyTip (PhyNexus) pre-packed with Ni-NTA resin. First, the Phytips were equilibrated with equilibration buffer (0.05 M NaH 2 PO 4 pH 8.0; 0.5% (v / v) Tween-20; 0.5 M NaCl; 20 mM imidazole). Then they were transferred to the sample for protein binding. To remove non-specifically bound proteins, the Phytips were washed twice with wash buffer 1 (0.05 M NaH 2 PO 4 pH 8.0; 0.5% (v / v) Tween-20; 0.5 M NaCl; 20 mM imidazole), followed by two washing steps with wash buffer 2 (0.05 M NaH 2 PO 4 pH 8.0; 0.5% (v / v) Tween-20; 0.15 M NaCl; 20 mM imidazole). Finally, the 6hel antigen was eluted in 100 μl of elution buffer (0.05 M NaH 2 PO 4 pH 8.0; 0.5% (v / v) Tween-20; 0.15 M NaCl; 200 mM imidazole). The protein samples were analyzed by SDS-PAGE gel. After Ni-NTA purification, buffer exchange into conjugation buffer (0.15 M KH TM 96-well microdialysis plate was performed using the instructions provided by Pierce Biotechnology. 2 PO 4 pH 8.0; 0.1 M KCl; 0.5 mM EDTA).

[0156] Small-scale ruthenation and biotinylation of recombinant gp41-6hel protein Conjugation was performed in a black 96-well half-area plate (Corning) using NHS-chemistry. Prior to conjugation, the protein concentration in each well in the microtiter plate was determined by a BCA assay using the Pierce TM BCA Protein Assay Kit (ThermoFisher). To 20 μl of purified antigen, 180 μl of BCA solution was added per well and measured at 562 nm using a Tecan sunrise TM microplate reader.

[0157] For ruthenium conjugation, antigen (approx. 1 mg / ml) and label were rapidly mixed to a final antigen-to-label ratio of 1 to 4 and for biotin conjugation 1 to 5 and a DMSO concentration of 10% (v / v). The plate was incubated at 600 rpm for 30 min at room temperature. The labeling reaction was stopped by adding L-lysine to a final concentration of 10 μM. For small-scale preparations, free unbound ruthenium label was not removed, but free unbound biotin label was removed by using a PD MultiTrap TM G-25 96-well plate (GE Healthcare). The concentrations of ruthenated and biotinylated antigens were determined by using the BCA assay as described above. The ruthenated and biotinylated gp41-6hel variants were stored at 4 °C until assayed by the Elecsys test system.

[0158] In summary, 171 out of 242 6hel mutants (71%) were successfully purified, labeled, and further assayed by immunoassays. The 71 lost variants failed in DNA synthesis, could not be expressed, or had too low a yield of purified protein to perform the labeling reaction.

[0159] Large-scale preparation of recombinant HIV1 gp41 and 6hel antigens for complete screening A plasmid containing recombinant HIV-1 gp41 (amino acids 536 - 681), the 6hel gene with different point mutations, and a C-terminal hexahistidine tag was synthesized by Eurofins Genomics GmbH and cloned into pET24a(+) via NdeI (5'-end) and XhoI (3'-end) restriction sites.

[0160] Furthermore, the N-terminus of recombinant gp41 (aa536 - 681) was fused to two SlyD chaperones from Escherichia coli (E. coli) via a glycine - serine rich linker (Scholz, C. et al., J. Mol. Biol. (2005) 345, 1229 - 1241) to obtain the EcSlyD-EcSlyD-gp41 fusion protein, which is simply referred to as gp41 hereinafter.

[0161] Using standard LB medium and IPTG induction, the expression of gp41 and the 6hel construct was carried out in BLR(DE3) E. coli cells at 37 °C for 3 hours. Cells were harvested by centrifugation (20 minutes, 5000g) and stored at -20 °C for further processing.

[0162] Large-scale purification of recombinant HIV-1 gp41 and 6hel antigens Recombinant HIV-1 gp41 and 6Hel antigens were purified under denaturing conditions followed by on-column refolding. Specifically, bacterial pellets from 700 ml of E. coli cultures were resuspended in chaotropic lysis buffer (50 mM sodium phosphate pH 8.0; 4 M guanidinium chloride; 5 mM imidazole) and stirred at room temperature for 90 minutes. For clarification, the cell lysate was centrifuged and filtered through a filter (5 / 0.8 / 0.2 μm). The clarified supernatant was applied to a Roche cOmplete His-tag purification column equilibrated with lysis buffer. Nonspecifically bound proteins were removed from the column by thorough washing to the baseline with lysis buffer. On-column refolding of the antigen was performed by on-column refolding using refolding buffer (50 mM sodium phosphate pH 8.0; 100 mM NaCl). The refolded target protein was eluted from the column using an elution buffer containing imidazole (50 mM sodium phosphate pH 8.0; 50 mM imidazole; 100 mM NaCl). For buffer exchange and final purification, the protein was applied to a Superdex 200 column equilibrated with SEC-buffer 1 (50 mM Tris-HCl pH 8.0; 150 mM KCl) for site-specific labeling or SEC-buffer 2 (150 mM potassium phosphate pH 8.9; 100 mM KCl; 0.5 mM EDTA) for labeling using NHS chemistry. Gp41 eluted in three peaks, and one prominent peak corresponded to the oligomeric arrangement. The oligomeric fraction was concentrated and proceeded to biotinylation and ruthenation. The 6Hel eluate eluted in one peak, which was concentrated and proceeded to biotinylation and ruthenation.

[0163] Example 2: Ruthenation and biotinylation of recombinant gp41 protein Large-scale ruthenation and biotinylation of recombinant HIV-1 gp41 and 6Hel antigens using NHS chemistry For the conjugation of the antigen with biotin or ruthenium, the protein concentration should ideally be 10 mg / ml in SEC buffer 2. The conjugation was carried out using NSH chemistry at a 1:4 molar concentration antigen-to-label ratio and a 5% (v / v) DMSO concentration. The label and the antigen were rapidly mixed and stirred at room temperature for 30 minutes. The labeling reaction was stopped by adding L-lysine to a final concentration of 10 mM. For large-scale preparations, free unbound label was removed from the reaction by size-exclusion chromatography using a Superdex 200 Increase (GE Healthcare) column equilibrated with storage buffer (50 mM sodium phosphate pH 7.5; 100 mM KCl; 0.5 mM EDTA). The concentration of the ruthenium-conjugated antigen was determined by use of the BCA assay, and the concentration of the biotinylated antigen was determined by absorbance measurement at 280 nm.

[0164] Large-scale rutheniumylation and biotinylation of recombinant HIV1 gp41 and 6hel using transglutaminase Using recombinant transglutaminase (KalbTG) derived from Kutzneria albida, antigens can be site-specifically labeled by forming Gln-Lys isopeptide bonds between Q-tag-containing antigens and their respective K-tag-containing labels (Steffen, W. et al. J. Mol. Biol. (2017) 292, 15622-1563). For the conjugation of HIV1 antigens with biotin or ruthenium, the protein concentration should ideally be 10 mg / ml in SEC-buffer 2. Conjugation was carried out with a 1:5 molar Q-tag to label ratio and a 1:300 enzyme to antigen dearth. The antigen, label, and activated enzyme were mixed and incubated at 37 °C for 20 h with gentle mixing. After 20 h of incubation, the reaction was stopped by adding 10 mM ammonium sulfate. Finally, free unbound label and KalbTG were removed from the labeled antigen by size exclusion chromatography using a Superdex 200 Increase (GE Healthcare) column equilibrated with storage buffer (50 mM sodium phosphate pH 7.5; 100 mM KCl; 0.5 mM EDTA). The concentration of rutheniumylated antigen was determined by use of a BCA assay and the concentration of biotinylated antigen was determined by absorbance measurement at 280 nm.

[0165] Large-scale biotinylation of recombinant HIV1 gp41 and 6hel using sortase Using a recombinant sortase, antigens can be site-specifically labeled by forming a peptide bond between the threonine of the C-terminal sortase recognition site (LPETG) and the glycine residue in each label. For the conjugation of the HIV1 antigen with biotin by sortase, the protein concentration should ideally be 10 mg / ml in phosphate-free SEC-buffer 1. Conjugation was carried out in the presence of 10 mM calcium chloride at an antigen-to-label ratio of 1:50 and an enzyme input of 50 U / μmol antigen. The antigen, label, and activated enzyme were mixed and incubated at 37 °C for 1 hour with gentle mixing. After 1 hour of incubation, the reaction was loaded onto Roche cOmplete His-tag resin to remove sortase and unlabeled antigen from the reaction mixture. Finally, free unbound label was removed by size-exclusion chromatography using a Superdex 200 Increase (GE Healthcare) column equilibrated with storage buffer (50 mM sodium phosphate pH 7.5; 100 mM KCl; 0.5 mM EDTA). The concentration of the biotinylated antigen was determined by absorbance measurement at 280 nm.

[0166] Example 3: Biochemical analysis of recombinant HIV1 gp41 and 6hel antigens Spectroscopic measurements of recombinant HIV1 gp41 and 6hel antigens Protein concentration was measured using a NanoDrop One® Micro-UV / Vis spectrophotometer (Thermo Scientific). The molar extinction coefficient (ε 280nm ) of the antigen was calculated using the equation reported by Pace et al. (Protein Sci. 1995 Nov;4(11):2411-23).

Table 1

[0167] Circular dichroism (CD) spectrum of recombinant HIV1 6hel antigen The far-UV CD spectrum (190 - 250 nm) of the 6hel antigen was recorded using a Jasco-720 spectropolarimeter and finally converted to mean residue ellipticity (Θ mrw、λ ). All samples were diluted in 50 mM potassium phosphate pH 7.5, 100 mM KCl, 0.5 mM EDTA to a concentration of 0.21 mg / ml. The spectrometer settings during the measurement were as follows: 0.2 cm path length, scan range of 190 nm - 330 nm, scan speed of 20 nm / min, bandwidth of 2.0 nm, resolution of 0.5 nm and response of 1 second. All spectra were measured 9 times and averaged.

[0168] In the far-UV region, since the absorption in this UV region is mainly caused by peptide bonds, CD spectroscopy can be used to analyze secondary structure proteins. Therefore, the far-UV CD spectra of all helical 6hel antigens compared to the mutated variants provide reliable insights into the structure of the antigen and the effect of point mutations on protein folding.

[0169] HPLC analysis of recombinant HIV1 6hel antigen HPLC analysis was performed to analyze the purity and aggregation tendency of the mutated antigens and also to estimate the molecular weight of the purified 6hel antigen. Therefore, at least 25 μg of recombinant protein was loaded onto a Superdex 200 column using 50 mM potassium phosphate pH 7.5, 100 mM KCl and 0.5 mM EDTA as the mobile phase. An internal HPLC standard was also analyzed as a reference. HPLC analysis enables the assessment of the aggregation behavior of the mutated 6hel antigens compared to the wild-type construct.

[0170] Example 4: Immunoreactivity of different recombinant HIV1 gp41 and 6hel antigens in an anti-HIV immunoassay An automated Elecsys was used with a double antigen sandwich (DAGS) format (登録商標)The cobas analyzer (Roche Diagnostics GmbH) was used to assay the immunoreactivity (antigenicity) of HIV-1 gp41 and 6Hel variants. The automated Elecsys (登録商標) Signal detection in the cobas analyzer is based on electrochemiluminescence. In the case of the DAGS assay format, the biotinylated capture antigen is immobilized on the surface of streptavidin-coated magnetic beads, while the same detection antigen is conjugated to a ruthenium complex. Upon activation, the ruthenium complex switches between redox states 2+ and 3+ and generates a light signal. In the presence of a specific immunoglobulin, in this case anti-HIV IgG antibodies in human serum, the ruthenium complex is cross-linked to the solid phase and luminescence at 620 nm is induced at the electrode by adding tripropylamine.

[0171] To evaluate the binding ability to anti-HIV-1 IgG antibodies, in this study, all 171 mutated variants of recombinant 6Hel from small-scale expression and labeling (Figure 3) were examined.

[0172] Immunoreactivity of different recombinant HIV-1 gp41 and 6Hel antigens in the DAGS assay setup For a more complete analysis, approximately 20 of the best mutations in the 6Hel antigen, identified as having improved immunological specificity in the initial screening, were expressed on a large scale, labeled, and comprehensively analyzed in the DAGS assay setup. Furthermore, the same selected mutations were also introduced into the HIV-1 gp41 antigen (International Publication No. WO 03 / 000877) and their specificity was evaluated. In addition to these constructs, 6Hel and gp41 antigens containing the most promising combinations of mutations were generated and assayed.

[0173] Specifically, different gp41-biotin or 6hel-biotin and gp41-ruthenium or 6hel-ruthenium antigens were used in reagent buffer 1 (R1) and R2, respectively. Recombinant gp41 antigens labeled at a concentration of 30 ng / ml to 300 ng / ml in R1 and R2 were used. The concentrations of various labeled 6hel antigens were 2 ng / ml to 130 ng / ml in R1 and R2, depending on the mutation.

[0174] To avoid immunological cross-reaction via the chaperone fusion unit of recombinant HIV1 gp41 antigen, unlabeled EcSkp-EcSlyD-EcSlyD (EP2893021 (B1)) or chemically polymerized EcSlyD-EcSlyD was added in large excess (5 - 30 μg / ml) to the reaction buffer as an anti-interference substance.

[0175] To assess the specificity and sensitivity of different recombinant HIV1 gp41 and 6hel antigens, Elecsys measurements using HIV-negative and -positive samples and seroconversion samples were analyzed.

[0176] The results of three of the most excellent antigens (SEQ ID NOs: 1, 2, and 3) are shown in Figure 6. Specifically, A) The cut-off index (COI) of 10 highly positive HIV samples from patients infected with different HIV-1 subtypes. All samples tested with the improved anti-HIV module (AHIVII) were positive, as they were already positive with the standard AHIVI module. A COI value < 1 is shown as non-reactive, while samples with COI > 1 indicate the presence of anti-HIV antibodies. B) Comparison of the performance of the standard Elecsys HIV Duo assay (black HIV Duo I) and the optimized Elecsys HIV Duo II assay (gray HIV Duo II), as well as separate comparisons of the anti-HIV modules AHIV I and AHIV II in gray and black, respectively. The comparison was made with five commercially available seroconversion panels (1 - 5) by serial blood sampling. The optimized anti-HIV II module shows higher sensitivity compared to the AHIV I module. The higher sensitivity of the AHIV II module is particularly effective in seroconversion panels 2 and 3. In these two panels, blood sampling 9 or 5 (highlighted in gray) was negative with the AHIV I module and clearly positive with the optimized AHIV II module. This higher sensitivity reduces the risk of the second window period after infection and significantly decreases the risk of false-negative HIV results. C) Graphical representation and D) numerical representation of the specificity of the current and optimized anti-HIV modules shown in black and gray, respectively. The specificity of both modules was determined using 6046 HIV-negative daily samples from different suppliers. When the HIV-positive threshold was set at COI > 1 (highlighted in bold), the standard AHIV I module showed four false-positive samples, resulting in a specificity of 99.92. On the other hand, the optimized AHIV II module showed no signal with COI > 1, resulting in a specificity of 100. In summary, the experiments conducted here showed a significant optimization in the sensitivity and specificity of the AHIV II module compared to the AHIV module.

[0177] Example 5: Data from an external specificity study To fully evaluate the specificity of the mutated and thereby optimized gp41 and 6hel antigens, 15,242 routine blood samples were analyzed in an external study (Figure 7a). The assays were performed by independent laboratories using both the AHIV module of the Elecsys HIV Duo and the AHIV module of the optimized Elecsys HIV Duo II assay (containing SEQ ID NOs: 1, 2, and 3).

[0178] Within this study, 44 samples gave false positive signals in the Elecsys HIV Duo I assay (specificity 99.71%), whereas using the optimized HIV Duo II assay, only 7 false positive samples were detected (specificity 99.95%) (Figure 7A). Twenty-one false positive samples of the Elecsys HIV Duo I assay and four false positive samples of the HIV Duo II were caused by the gp41 and 6hel antigens within the anti-HIV module of the HIV Duo Elecsys assay (Figure 7b). Thus, even within an independent external study, significant specificity improvements of the mutated and optimized gp41 and 6hel antigens could be shown, and the potential for interference of the gp41 antigen decreased to less than 20% after optimization.

[0179] Example 6: Improvement of the sensitivity and specificity of an HIV immunoassay by a combination of mutated and optimized HIV gp41 antigens Compared with the HIV immunoassay containing the HIV gp41 antigen containing SEQ ID NO: 10, the combination of SEQ ID NOs: 1, 2 and 3 not only shows significantly improved antigenicity, but also two combinations of the already optimized HIV gp41 antigens (SEQ ID NO: 1 and 2 or SEQ ID NO: 1 and 3) show significantly improved immunological reactivity (Figure 8). By analyzing 103 HIV-negative samples, the specificity of different antigen combinations was assayed (Figure 8a). Furthermore, when using the non-optimized gp41 antigen, only 91 out of 103 negative samples (88.35%) showed a COI of 0.02 - 0.05, whereas when using different combinations of the mutated gp41 and 6hel antigens, at least 98% of the negative samples were within this COI range (Figure 8a, columns 2 - 3). Therefore, when using the optimized antigen, the scattering of the samples is significantly lower. Furthermore, not only are the specificity and scattering improved, but the sensitivity of the different combined optimized gp41 and 6hel antigens is also significantly better (Figure 8b). For example, sample Sero01 clearly shows that the sensitivity is much higher in all combinations using the mutated antigens, and SEQ ID NOs: 1, 2 and 3 are the best combination.

Claims

1. A composition suitable for detecting antibodies against HIV gp41 in an isolated sample, said composition comprising at least two individual HIV gp41 antigens, a first HIV antigen comprising SEQ ID NO: 1, and a second HIV gp41 antigen comprising SEQ ID NO: 2 and / or 3, each of said individual HIV gp41 antigens not comprising additional HIV-specific amino acid sequences.

2. The composition according to claim 1, wherein at least one of said HIV gp41 antigens is fused to at least one chaperone.

3. The composition according to claim 2, wherein said chaperone is selected from the group consisting of SlyD, SlpA, FkpA and Skp.

4. The composition according to any one of claims 1 to 3, wherein each of said antigens is soluble and immunoreactive.

5. The composition according to any one of claims 1 to 4, wherein said HIV gp41 antigens comprise SEQ ID NO: 1 and 2 or SEQ ID NO: 1 and 3.

6. The composition according to any one of claims 1 to 5, wherein said HIV gp41 antigens comprise SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3.

7. A method for producing a composition of HIV gp41 antigens according to any one of claims 1 to 6, for each of said antigens, a) culturing a host cell transformed with an expression vector comprising a recombinant DNA molecule encoding one of each of said antigens operably linked; b) expressing each of said antigens; c) purifying each of said antigens; d) mixing the HIV gp41 antigen comprising SEQ ID NO: 1 obtained by steps a) to c) with at least one HIV gp41 antigen comprising at least one of SEQ ID NO: 2 or 3 obtained by steps a) to c) to form a composition of HIV gp41 antigens comprising.

8. A method for detecting antibodies specific for HIV gp41 in an isolated sample, wherein the composition of HIV gp41 antigens according to any one of claims 1 to 6 is used as a capture reagent for anti-HIV antibodies and / or as a binding partner.

9. A method for detecting antibodies specific for HIV gp41 in an isolated sample, a) forming an immunoreaction mixture by mixing a body fluid sample with the HIV gp41 antigen composition according to any one of claims 1 to 6; b) maintaining the immunoreaction mixture for a time sufficient for an antibody to HIV gp41 present in the body fluid sample to immunoreact with the HIV gp41 antigen as part of the HIV gp41 antigen composition to form an immunoreaction product; c) detecting the presence and / or concentration of any of the immunoreaction products; A method comprising the steps of: **Claim 10** A method for identifying whether a patient / animal has been previously exposed to HIV infection, comprising: a) forming an immunoreaction mixture by mixing a body fluid sample of the patient / animal with the HIV gp41 antigen composition according to any one of claims 1 to 6; b) maintaining the immunoreaction mixture for a time sufficient for an antibody to the HIV gp41 antigen composition present in the body fluid sample to immunoreact with the HIV gp41 antigen as part of the HIV gp41 antigen composition to form an immunoreaction product; c) detecting the presence and / or absence of any of the immunoreaction products; wherein the presence of an immunoreaction product indicates that the patient / animal has been previously exposed to HIV infection. A method as claimed. **Claim 11** Use of the HIV gp41 antigen composition according to any one of claims 1 to 6 for detecting anti-HIV gp41 antibodies in an isolated sample. **Claim 12** A reagent kit for detecting anti-HIV antibodies, comprising the HIV gp41 antigen composition according to any one of claims 1 to 6. **Claim 13** The reagent kit according to claim 12, comprising at least microparticles coated with avidin or streptavidin and the HIV gp41 antigen composition according to claims 1 to 5 in separate containers or in separate compartments of a single container unit, wherein each of the individual HIV gp41 antigens is covalently bound to biotin. **Claim 14** The reagent kit according to claim 13, comprising the HIV gp41 antigen composition according to any one of claims 1 to 5 in an additional separate container or in an additional separate compartment of a single container, wherein each of the individual HIV gp41 antigens in the additional separate container or additional separate compartment is covalently bound to a detectable label.

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