HIV GP41 variants for immunodiagnostic assays

Abstract

The present invention relates to novel HIV gp41 antigen compositions suitable for the detection of antibodies against HIV in isolated biological samples, thereby providing highly specific immunoassay results. The invention further relates to a method for detecting an HIV antibody, to the use of the novel HIV gp41 antigen composition in an immunoassay, and to a kit comprising the novel HIV gp41 antigen composition.

Description

[0001] The present invention relates to HIV gp41 antigen composition and a kit containing the same and a method for producing the same. The present invention also covers a method for detecting anti-HIV antibodies in a separated sample using the HIV gp41 antigen composition. 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, it is related to viral envelope proteins, i.e. gp41 and gp120, both of which are derived from the precursor protein gp160 that is cut into these two fragments by proteolysis. Larger subunit gp120 is a surface-related receptor binding subunit, and gp41 forms a transmembrane subunit, which participates in membrane fusion during the virus enters the target cell. About the hypothetical binding mechanism of the virus and its target cell, the contact of gp120 / gp41 with host cell membrane protein CD4 and other co-receptors triggers 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 infected with HIV often produce antibodies against gp41 and other HIV proteins, so for at least the past two decades, gp41 has been an important component of in vitro diagnostic immunoassays used to detect antibodies against HIV. Immunoassays using the wild-type sequence of HIV gp41 have been shown to be highly specific. This means that samples containing HIV antibodies are often correctly identified as positive.

[0004] However, there are still a large number of false-positive samples, meaning that the assay result indicates the presence of antibodies against HIV, although in fact the sample is negative and does not contain HIV antibodies. These false positives can become critical not only in routine laboratory diagnostic settings, where these results lead to false alarms, intensive retesting, and confirmatory testing procedures. In addition, false-positive results should be particularly avoided in the blood bank setting. Here, thousands of samples from blood donations are screened every day on high-throughput diagnostic analyzers, and a positive result means that the whole blood donation from the patient may be discarded.

[0005] Scholz et al. (J. Mol. Biol. 2005, 345, 1229-1241) describe gp41 polypeptide sequences from HIV-1 and the corresponding gp36 from HIV-2 that have been engineered so that aggregation-prone polypeptides can be expressed in soluble form. However, when these polypeptides are used as antigens in in vitro diagnostic immunoassays for detecting HIV antibodies, false positive results cannot be completely avoided.

[0006] WO2001 / 044286 discloses a kind of artificially designed five helical protein with gp41 element, which can be used to inhibit HIV infection in human cells. The inhibitor comprises three sections of the N-terminal helical domain derived from gp41 and two sections of the C-terminal helical domain of the molecule. However, this genetically engineered construct (also described by Root et al., supra) lacks many domains and many antigenic epitopes of the natural molecule, and it is particularly free of so-called loop motifs, which are known to contain specific immunogenic epitopes. The five helical protein folds into a stable structure and binds to a peptide corresponding to the C-peptide region of HIV gp41, thereby inhibiting HIV infection of human cells. It is also disclosed that the five helical protein can be used as a drug screening or antibody screening tool. In addition, a six helical protein comprising a gp41 sequence is also disclosed. This six helical protein comprises three N helices and three C helices of HIVgp41 connected by a joint, which can be used as a negative control for screening drugs that inhibit membrane fusion.

[0007] Although gp41 variants have been extensively described in the prior art, these publications do not address the identification of gp41 antigens that avoid false positive results in in vitro diagnostic immunoassays for the detection of HIV antibodies.

[0008] The technical problem underlying the present invention can be seen as providing means and methods that meet the aforementioned needs, avoiding as much as possible the identified problems. This technical problem is solved by the embodiments characterized in the claims and described below. 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, said composition comprising at least two separate HIV gp41 antigens, wherein the first HIV antigen comprises SEQ ID NO: 1 and wherein the second HIV gp41 antigen comprises at least one of SEQ ID NO: 2 or 3. In particular, said antigens do not comprise 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, said method comprising the following steps for each of said antigens:

[0011] a) culturing a host cell, in particular an E. coli cell, transformed with an expression vector comprising an operably linked recombinant DNA molecule encoding one of the antigens of the first aspect of the present invention,

[0012] b) expressing said antigen, and

[0013] c) purifying the antigen, and

[0014] 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.

[0015] 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 invention, or the HIV gp41 antigen composition obtained by the method of the second aspect of the invention is used as a capture reagent and / or binding partner for the anti-HIV antibodies.

[0016] In a fourth aspect, the present invention relates to a method for detecting antibodies specific for HIV in an isolated sample, the method comprising

[0017] a) forming an immune reaction 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

[0018] b) maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the HIV gp41 antigen composition present in the body fluid sample to immunologically react with the HIV gp41 antigen as part of the HIV gp41 antigen composition to form an immune reaction product; and

[0019] c) detecting the presence and / or concentration of any said immunoreaction product.

[0020] In a fifth aspect, the present invention relates to a method for identifying whether a patient has been exposed to HIV infection in the past, the method comprising

[0021] a) forming an immune reaction mixture by mixing a body fluid sample of a patient 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

[0022] b) maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the HIV gp41 antigen composition present in the body fluid sample to immunologically react with HIV gp41 antigens as part of the HIV gp41 antigen composition to form an immune reaction product; and

[0023] c) detecting the presence and / or absence of any of said immune reaction products,

[0024] The presence of immune response products indicates that the patient has been exposed to HIV infection in the past.

[0025] In a sixth aspect, the present invention relates to 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 detecting anti-HIV antibodies.

[0026] In a seventh aspect, the present invention relates to a kit for detecting 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

[0027] Figure 1 : Sequence alignment of wild-type HIV gp41 (P03375, positions 512 to 868 as shown in SEQ ID NO: 11) with the N-terminal (aa543-581; SEQ ID NO: 18) and C-terminal (aa625-662; SEQ ID NO: 19) heptad repeat sequences used in the 6he1 (six-helix) construct. Highlighted are the positions that were mutated to optimize specificity against HIV antigens (light grey: N636, dark grey N637, black H643). The bottom line of each of the three upper sections shows the position of the gp41 of WO03 / 0000877 (SEQ ID NO: 10) relative to the entire gp41 sequence.

[0028] Figure 2: Amino acid sequence of the 6hel construct depicting the corresponding amino acid positions in gp41 and the positions in the heptad repeat sequence (a to g). The N-terminal and C-terminal heptad repeat sequences are shown in light grey and dark grey, respectively. Positions marked with an x ​​were mutated during the optimization process. The optimal position is underlined.

[0029] Figures 3a to 3f : Table lists all 242 designed HIV gp41 variants. Mutants highlighted in grey could not be synthesized or expressed and therefore could not be tested in anti-HIV immunoassays.

[0030] Figure 4: CD data of wild-type recombinant 6hel (6hel_wt) antigen (SEQ ID NO: 4: black) and two mutated 6hel variants containing N636D / H643Y mutations (SEQ ID NO: 3: light grey (6hel_N636D / H643Y, 3mut) and SEQ ID NO: 2: dark grey (6hel_N636D / H643Y, 2mut)). The CD spectra of all three antigen variants show two minima characteristic of α-helices at 208 nm and 222 nm in the far UV range, which strongly suggests that the introduction of point mutations in the 6hel construct does not interfere with protein folding and therefore does not impair the immunoreactivity of the mutant antigen.

[0031] Figure 5 : HPLC analysis of 6hel_wt antigen (SEQ ID NO:4, A) compared with two different 6hel mutant antigens; B): SEQ ID NO:2 (6hel_N636D / H643Y, 2mut); C) SEQ ID NO:3 (6hel_N636D / H643Y, 3mut). The analysis was performed on a Superdex20010 / 300 (A) or on a Superdex 5 / 150 (B and C) column. For quality control and for molecular weight estimation, HPLC standards (grey) were plotted in the chromatogram together with the 6hel antigen (black). The HPLC chromatogram strongly suggests that the introduction of point mutations in the 6hel antigen does not interfere with protein folding and therefore does not impair the immunoreactivity of the mutant antigen.

[0032] Figure 6 : Performance of the improved anti-HIV module (AHIVII) comprising antigens with SEQ ID NO: 1, 2 and 3 compared to the standard AHIV module of the Elecsys HIV Duo assay (AHIVI) comprising only the antigen with SEQ ID NO: 10. Detailed analysis is shown in the Examples section.

[0033] Figure 7: Performance of the improved HIV Duo II assay containing antigens with SEQ ID NO: 1, 2 and 3 compared to the standard Elecsys HIV Duo assay containing only antigen with SEQ ID NO: 10. A) Shows the number of negative samples analyzed in an independent external study and the number of false positive results in this study and the specificity generated by the HIV Duo I assay compared to the HIV Duo II assay. B) Shows the detailed extraction of all false negative samples interfering with the different gp41 and 6hel antigens used in HIV Duo I (SEQ ID NO: 10) or HIV Duo II (SEQ ID NO: 1, 2 and 3). All samples with COI>0.8 were considered positive and highlighted in gray. Please refer to the Examples section for more details.

[0034] Figure 8: Specificity and sensitivity of different combinations of gp41 and 6hel antigens. A) Specificity of HIV immunoassays containing antigens with SEQ ID NO: 10, SEQ ID NO: 1 and 2, or SEQ ID NO: 1 and 3. B) Sensitivity of the same antigen combination was assessed using 10 different seroconversion samples close to the COI cutoff from light grey to dark grey. See the Examples section for more details.

[0035] Sequence Listing

[0036] SEQ ID NO: 1: gp41 variant (N637E / H643Y) mutations are printed in bold and underlined

[0037] SEQ ID NO:2:6hel(N636D / H643Y,2mut), mutations are printed in bold and underlined

[0038] SEQ ID NO:3:6hel(N636D / H643Y,3mut), mutations are printed in bold and underlined

[0039]

[0040] SEQ ID NO:4: 6hel-N(543-581)x3 / C(625-662)x3-Srt wild type

[0041] MQLLSGIVQQ QNNLLRAIEA QQHLLQLTVW GIKQLQARIL GGSGGHTTWM EWDREINNYT

[0042] SLIHSLIEES QNQQEKNEQE LLEGSSGGQL LSGIVQQQNN LLRAIEAQQH LLQLTVWGIK

[0043] QLQARILGGSGGHTTWMEWD REINNYTSLI HSLIEESQNQ QEKNEQELLE GSSGGQLLSG

[0044] IVQQQNNLLR AIEAQQHLLQ LTVWGIKQLQ ARILGGRGGH TTWMEWDREI NNYTSLIHSL

[0045] IEESQNQQEK NEQELLGGLP ETGHHHHHH

[0046] SEQ ID NO:5: 6hel-N(543-581)x3 / C(625-662,N636D / H643Y)x2 / C(625-662)x1-Srt, mutations are printed in bold and underlined

[0047]

[0048] SEQ ID NO:6:6hel-(N(543-581)x3 / C(625-662,N636D / H643Y)x3-Q-tag, mutations are printed in bold and underlined

[0049]

[0050] SEQ ID NO:7: EcSlyD-EcSlyD-gp41(536-681; N637E / H643Y)-Q-tag, mutations are printed in bold and underlined

[0051]

[0052] SEQ ID NO:8: EcSlyD-EcSlyD-Q tag-gp41(536-681; N637E / H643Y), mutations are printed in bold and underlined

[0053]

[0054] SEQ ID NO: 9: Hexahelix according to WO2001 / 044286

[0055] MQLLSGIVQQ QNNLLRAIEA QQHLLQLTVW GIKQLQARIL AGGSGGHTTW MEWDREINNY

[0056] TSLIHSLIEE SQNQQEKNEQ ELLEGSSGGQ LLSGIVQQQN NLLRAIEAQQ HLLQLTVWGI

[0057] KQLQARILAG GSGGHTTWME WDREINNYTS LIHSLIEESQ NQQEKNEQEL LEGSSGGQLL

[0058] SGIVQQQNNL LRAIEAQQHL LQLTVWGIKQ LQARILAGGR GGHTTWMEWD REINNYTSLI

[0059] HSLIEESQNQ QEKNEQELLG GHHHHHH

[0060] SEQ ID NO:10: HIV gp41 according to WO03 / 0000877

[0061] TLTVQARQLL SGIVQQQNNE LRAIEAQQHL LQLTVWGTKQ LQARELAVER YLKDQQLLGI

[0062] WGASGKLIAT TAVPWNASWS NKSLEQIWNN MTWMEWDREI NNYTSLIHSL IEESQNQQEK

[0063] NEQELLELDK WASLWNWFNI TNWLWY

[0064] SEQ ID No:11: UniprotP03375 HIV 1gp41 (positions 512 to 856)

[0065] AVGIGALFLG FLGAAGSTMG AASMTLTVQA RQLLSGIVQQ QNNLLRAIEA QQHLLQLTVW

[0066] GIKQLQARIL AVERYLKDQQ LLGIWGCSGK LICTTAVPWN ASWSNKSLEQ IWNNMTWMEW

[0067] DREINNYTSL IHSLIEESQN QQEKNEQELL ELDKWASLWN WFNITNWLWY IKLFIMIVGG

[0068] LVGLRIVFAV LSVVNRVRQG YSPLSFQTHL PIPRGPDRPE GIEEEGGERD RDRSIRLVNG

[0069] SLALIWDDLR SLCLFSYHRL RDLLLIVTRI VELLGRRGWE ALKYWWNLLQ YWSQELKNSA

[0070] VSLLNATAIA VAEGTDRVIE VVQGAYRAIR HIPRRIRQGL ERILL

[0071] SEQ ID NO: 12: Sortase sequence

[0072] LPETG

[0073] SEQ ID NO: 13: Transglutaminase sequence

[0074] YQ

[0075] SEQ ID NO: 14: Linker

[0076] GGGS

[0077] SEQ ID NO: 15: Linker

[0078] GGGSGGGSGGGSGGG

[0079] SEQ ID NO: 16: Linker

[0080] SGGG

[0081] SEQ ID NO: 17: His-tag

[0082] HHHHHH

[0083] SEQ ID NO:18: 6he1NHR (543-581; 6-helical N-heptad repeat sequence)

[0084] QLLSGIVQQQ NNLLRAIEAQ QHLLQLTVWG IKQLQARIL

[0085] SEQ ID NO:19: 6helCHR (625-662; 6-helical C-heptad repeat sequence)

[0086] HTTWMEWDRE INNYTSLIHS LIEESQNQQE KNEQELLE DETAILED DESCRIPTION

[0087] Based on the known structure of the single peptide chain six helical (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 serve as potential binding sites for nonspecific antibodies) were mutated by replacing the original amino acid with a glycine residue. These point mutations have indeed led to improved specificity of gp41 binding antibodies in the sample. However, this improvement was ultimately unsatisfactory. As a next step, the inventors exchanged 21 positions in the C-terminal heptad repeat sequence (CHR, Figure 1 ). Each position is exchanged with 12 representative amino acids (arginine, lysine, aspartic acid, serine, asparagine, alanine, valine, isoleucine, phenylalanine, tyrosine and glycine), then expressed on a small scale, purified, modified to design appropriately labeled antigens, and screened for antibody binding. Then, the best variants are expressed and purified, labeled and tested on a large scale. In addition, a combination of point mutations in the six-helix (6hel) is introduced, expressed, purified, labeled, and antibody binding is also tested. In addition, and because it is known that some HIV-specific antibodies bind to specific immunogenic loop structures that are not part of the six-helix, some point mutations are also introduced into the gp41 variants. The inventors of the present application have designed a total of 242 HIV gp41 mutant antigens (Fig. 3). However, contrary to the inventors' expectations, only a few of the 242 variants produced showed satisfactory performance in immunoassays for detecting suitable gp41 sequences. No regular pattern or consistent logic was found to identify suitable HIV gp41 variants.

[0088] Surprisingly, among such a large number of variants, the inventors were able to identify gp41-derived polypeptides and corresponding peptide compositions that largely overcome false positive results in IVD immunoassays for detecting HIV antibodies, thereby providing immunological antibody detection with high specificity while maintaining high sensitivity.

[0089] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise indicated, all scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0090] This specification text quotes several documents throughout. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions for use, etc.), whether cited above or below, is incorporated herein by reference in its entirety. In the event that the definition or teaching of such incorporated references conflicts with the definition or teaching cited in this specification, the text of this specification shall prevail.

[0091] Elements of the present invention will be described below. These elements are listed together with specific embodiments, however, it should be understood that they can be combined in any manner and in any number to create other embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the embodiments clearly described. This description should be understood to support and encompass embodiments that combine the embodiments clearly described with any number of disclosed and / or preferred elements. In addition, unless the context otherwise indicates, any arrangement and combination of all described elements in this application should be considered to be disclosed by the specification sheet of the application.

[0092] definition

[0093] The word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps.

[0094] As used in this specification and the appended claims, the singular forms "a," "an," "the," and "said" include plural referents unless the content clearly dictates otherwise.

[0095] Concentration, amount and other numerical data can be expressed or presented in "range" format in this article.It should be understood that such range format is used only for convenience and simplicity, and therefore should be flexibly interpreted as not only including the numerical value clearly listed as range limit, but also including all individual numerical values ​​or sub-ranges covered by the range, just as each numerical value and sub-range are clearly listed.As an illustration, the numerical range of "150mg to 600mg" should be interpreted as not only including the value 150mg to 600mg clearly listed, but also including individual values ​​and sub-ranges within the indicated range.Therefore, individual values ​​are included in this numerical range, such as 150, 160, 170, 180, 190, ..., 580, 590, 600mg and sub-ranges, such as from 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc.This same principle applies to the scope of only quoting one numerical value.In addition, no matter how wide the range or feature is, such explanation is applicable.

[0096] When used in connection with a numerical value, the term "about" is meant to encompass the numerical value within a range having a lower limit that is 5% less than the indicated numerical value and an upper limit that is 5% greater than the indicated numerical value.

[0097] The term "HIV gp41" refers to a polypeptide derived from the surface protein gp41 of the human immunodeficiency virus 1. HIV gp41 mediates both cell attachment and membrane fusion with host cells of HIV. The wild-type sequence can be found under UniProt ID P03375. Positions 535 to 681 of the HIV envelope polyprotein define the gp41 wild-type polypeptide. Soluble variants of gp41 have been described, for example, in WO2003 / 000877.

[0098] As used herein, "patient" means any mammal, fish, reptile or bird that can benefit from the diagnosis, prognosis or treatment described herein. In particular, the "patient" is selected from the group consisting of laboratory animals (e.g., mice, rats, rabbits or zebrafish), livestock (including, for example, guinea pigs, rabbits, horses, donkeys, cattle, sheep, goats, pigs, chickens, camels, cats, dogs, turtles, tortoises, snakes, lizards or goldfish), or primates, including chimpanzees, bonobos, gorillas and humans. Particularly preferred "patients" are humans.

[0099] The terms "sample", "isolated sample", "isolated biological sample" or "target sample" are used interchangeably herein and refer to a portion or slice of a tissue, organ or individual, usually smaller than such tissue, organ or individual, intended to represent the entire tissue, organ or individual. When analyzed, the sample provides information about the state of the tissue or the healthy or diseased state of the organ or individual. Examples of samples include, but are not limited to, liquid samples such as blood, serum, plasma, synovial fluid, urine, saliva and lymph; or solid samples such as tissue extracts, cartilage, bone, synovium and connective tissue. Analysis of samples can be completed on a visual or chemical basis. Visual analysis includes, but is not limited to, microscopic imaging or radiographic scanning of tissues, organs or individuals to allow morphological assessment of the sample. Chemical analysis includes, but is not limited to, detecting the presence or absence of a specific indicator or changes in its amount, concentration or level. A sample is an in vitro sample isolated from the body that will be analyzed in vitro and will not be moved back into the body.

[0100] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0101] For the term "sequence comparison", it refers to a process in which one sequence is used as a reference sequence and is compared to a test sequence. When a sequence comparison algorithm is used, the test and reference sequences are input into a computer program, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters are usually used, or alternative parameters can be specified. Then based on the program parameters, the sequence comparison algorithm calculates the sequence identity or similarity percentage of the test sequence relative to the reference sequence. In sequence alignment, the term "comparison window" refers to those segments of the continuous positions of the sequence compared with the reference segments of the continuous positions of the sequence with the same number of positions. The number of selected continuous positions can be in the range of 10 to 1000, that is, 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 continuous positions can be included. Typically, the number of contiguous positions ranges from about 20 to 800 contiguous positions, about 20 to 600 contiguous positions, about 50 to 400 contiguous positions, about 50 to about 200 contiguous positions, about 100 to about 150 contiguous positions. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences 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 search for similarity method of Pearson and Lipman, (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerized 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)). Suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are 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 analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short fields of length W in the query sequence that match or meet a certain positive threshold score T when aligned with the same length field of the database sequence. T is called the neighboring field score threshold (Altschul et al., supra). These initial neighboring field hits serve as seeds for initiating searches to find longer HSPs containing them. Field hits are extended bidirectionally along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (a reward score for a pair of matching residues; always>0) and N (a penalty score for mismatched residues; always<0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. When the cumulative alignment score decreases by a number X from its maximum achieved value; when the cumulative score reaches or falls below zero due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached, extension of the field hits in each direction stops. BLAST algorithm parameters W, T and X determine the sensitivity and speed of comparison. BLASTN program (for nucleotide sequences) uses default word length (W) 11, expected value (E) 10, M = 5, N = -4, and compares two chains. For amino acid sequences, BLASTP program defaults to word length 3 and expected value (E) 10, and BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915, 1989) comparison (B) 50, expected value (E) 10, M = 5, N = -4, and comparison of two chains. BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-87, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001.

[0102] The term "recombinant DNA molecule" refers to a molecule made by combining two originally separate segments of a DNA sequence that is artificially manipulated by genetic engineering techniques or chemical synthesis of separated polynucleotide segments. In doing so, polynucleotide segments of desired functions can be linked together to produce the desired functional combination. Recombinant DNA techniques for expressing proteins in prokaryotic or lower or higher eukaryotic host cells are well known in the art. They have been described, for example, by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual).

[0103] The terms "vector" and "plasmid" are used interchangeably herein and refer to proteins or polynucleotides or mixtures thereof that can be introduced into or introduce the proteins and / or nucleic acids contained therein into cells. Examples of plasmids include, but are not limited to, plasmids, cosmids, phages, viruses or artificial chromosomes.

[0104] The term "amino acid" generally refers to a monomeric unit comprising a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group and one or more side chains or groups or analogs of any of these groups. Exemplary side chains include, for example, thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, borate, borate, phospho, phosphino, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising photoactivated cross-linkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, amino acids comprising metals, amino acids with novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photosensitive photocaged and / or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids comprising carbon-linked sugars, redox-active amino acids, amino thioacid-containing amino acids, and amino acids comprising one or more toxic moieties. As used herein, the term "amino acid" includes the following twenty naturally occurring or genetically encoded α-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).

[0105] The terms "measurement", "measuring", "detecting" or "detection" preferably include qualitative, semi-quantitative or quantitative measurements. The term "detecting the presence" refers to a qualitative measurement, which indicates the presence or absence without making any statement about the amount (e.g., a yes or no statement). The term "detecting the amount" refers to a quantitative measurement in which an absolute number (ng) is detected. The term "detecting the concentration" refers to a quantitative measurement in which the amount is determined relative to a given volume (e.g., ng / ml).

[0106] As used herein, the term "immunoglobulin (Ig)" refers to the immunity-conferring glycoprotein of the immunoglobulin superfamily. "Surface immunoglobulins" are attached to the membrane of effector cells via their transmembrane regions and encompass molecules such as, but not limited to, B cell receptors, T cell receptors, class I and class II major histocompatibility complex (MHC) proteins, beta-2 microglobulin (about 2M), CD3, CD4, and CDS.

[0107] Generally, as used herein, the term "antibody" refers to a secretory immunoglobulin that lacks a transmembrane region and can therefore be released into the bloodstream and body cavity. Human antibodies are divided into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains, represented by Greek letters: α, γ, δ, ε and μ. The type of heavy chain present defines the category of the antibody, i.e., these chains are present in IgA, IgD, IgE, IgG and IgM antibodies, respectively, each playing a different role, and directing the appropriate immune response for different types of antigens. Different heavy chains differ in size and composition; and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is present in mucosal areas, such as the intestinal tract, respiratory tract and urogenital tract, as well as in saliva, tears and breast milk, to prevent pathogen colonization (Underdown & Schiff (1986) Annu. Rev. Immunol. 4: 389-417). IgD acts primarily as an antigen receptor on B cells that are not 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 participates in allergic reactions by binding to allergens, triggering mast cells and basophils to release histamine. IgE is also involved in preventing 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 cross the placenta to provide 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 according to their abundance order in serum, with IgG1 being the most abundant (about 66%), followed by IgG2 (about 23%), IgG3 (about 7%) and IgG (about 4%). The biological properties of different IgG classes are determined by the structure of the corresponding hinge region. IgM is expressed on the surface of B cells in monomeric and secretory pentamer forms with very high avidity. Before sufficient IgG is produced, IgM participates in the elimination of pathogens in the early stages of B cell-mediated (humoral) immunity (Geisberger et al. (2006) Immunology 118: 429-437).

[0108] Typically, during the detection of antibodies to HIV antigens in an in vitro diagnostic setting, a differential diagnosis between early IgM antibodies and late IgG antibodies is not performed.

[0109] The term "binding affinity" generally refers to the strength of the sum 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 intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including but not limited to surface plasmon resonance-based assays (e.g., BIAcore assays described in PCT Application Publication No. WO2005 / 012359); enzyme-linked immunosorbent assays (ELISAs); and competition assays (e.g., RIAs). Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen quickly and tend to remain bound for longer periods of time. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0110] The term "antigen (Ag)" is a molecule or molecular structure that binds to an antigen-specific antibody (Ab) or B cell antigen receptor (BCR). The presence of an antigen in the body typically triggers an immune response. In the body, each antibody is specifically produced to match the antigen after the cells of the immune system come into contact with the antigen; this allows accurate identification or matching of the antigen and initiates a specialized response. In most cases, antibodies can only react and bind to one specific antigen; however, in some cases, antibodies may cross-react and bind to more than one antigen. Antigens are typically proteins, peptides (amino acid chains) and polysaccharides (monosaccharide / simple sugar chains) or a combination thereof. For the present invention, antigens are used as specific components in immunoassays that specifically bind to antibodies present in the analysis sample and bound to the antigen. The terms "antigen" and "polypeptide" can be used interchangeably.

[0111] In diagnostic testing, antigens are often used in serological tests to assess whether a patient has been exposed to a pathogen (such as a virus or bacteria) and has produced antibodies against it. Typically, these antigens are recombinantly produced and can be linear peptides or more complex folded molecules designed to represent natural antigens.

[0112] In order to more closely resemble the native antigen and obtain a high epitope density, antigens can be generated by polymerizing monomeric antigens by means of chemical cross-linking. There are a large number of homobifunctional and heterobifunctional cross-linkers that have great advantages when used and are well known in the art. However, there are some serious disadvantages to chemically induced antigen polymerization for use as specific markers in serological assays. For example, the insertion of cross-linker moieties into the antigen may impair antigenicity by interfering with the native-like conformation or by masking critical epitopes. In addition, the introduction of non-native tertiary contacts may interfere with the reversibility of protein folding / unfolding and, in addition, it may be a source of interference problems that must be overcome by anti-interference strategies in the immunoassay mixture.

[0113] A more recent technique is to fuse the target antigen to an oligomeric chaperone protein, thereby delivering a high epitope density to the antigen. The advantage of this technique lies in its high reproducibility and the triple function of the oligomeric chaperone protein fusion partner: first, the chaperone protein enhances the expression rate of the fusion polypeptide in the host cell (e.g., in E. coli), second, the chaperone protein promotes the refolding process of the target antigen and enhances its overall solubility, and third, it reproducibly assembles the target antigen into an ordered oligomeric structure.

[0114] The term "chaperone protein" is well known in the art and refers to a protein folding aid that assists the folding of other proteins and maintains structural integrity. Examples of folding aids are described in detail in WO 2003 / 000877. For example, chaperone proteins of the peptidyl prolyl isomerase class such as chaperone proteins of the FKBP family can be used for fusion with antigenic variants. Examples of FKBP chaperone proteins 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). Another chaperone protein suitable as a fusion partner is Skp (21-161, UniProt ID P0AEU7), a trimeric chaperone protein from the periplasm of Escherichia coli, which does not belong to the FKBP family. It is not always necessary to use the complete sequence of the chaperone protein. It is also possible to use functional fragments of chaperone proteins which still possess the desired capabilities and functions (so-called modules with binding capacity) (cf. WO 98 / 13496).

[0115] The term "does not comprise further HIV-specific amino acid sequences" means that the HIV gp41 antigen is designed in such a way that antibodies against other HIV antigens, such as, for example, gp120, p24 or the HIV enzymes protease or reverse transcriptase do not bind to the HIV gp41 antigen. Amino acid sequences derived from other HIV proteins are not part of any HIV gp41 antigen. Additionally, the term means that no more than 15, in one embodiment no more than 10, in one embodiment no more than 5, in a further embodiment no more than 2 consecutive amino acids of a known gp41 polypeptide, which is, for example, part of UniProt P03375 or SEQ ID NO: 11, are fused to the C- or N-terminus of the HIV gp41 antigen according to the invention.

[0116] The antigen may further comprise an "effector group", such as, for example, a "tag" or a "label". The term "label" refers to those effector groups that provide the antigen with the ability to bind to or be bound to other molecules. Examples of labels include, but are not limited to, for example, a His tag, which is attached to the antigen sequence to allow its purification. Tags may also include partners of a bioaffine binding pair, which allows the antigen to be bound by the second partner of the binding pair. The term "bioaffine binding pair" refers to two partner molecules (i.e., the two partners in a pair) that have a strong affinity to bind to each other. Examples of partners of a bioaffine binding pair are a) biotin or a biotin analogue / avidin or streptavidin; b) hapten / anti-hapten antibody or antibody fragment (e.g. digoxin / anti-digoxin antibody); c) sugar / lectin; d) complementary oligonucleotide sequences (e.g. complementary LNA sequences), and generally e) ligand / receptor.

[0117] The term "label" refers to those effector groups that allow detection of an antigen. Labels include, but are not limited to, spectroscopic labels, photochemical labels, biochemical labels, immunochemical labels, or chemical labels. For example, suitable labels include fluorescent dyes, luminescent or electrochemiluminescent complexes (e.g., ruthenium or iridium complexes), electron-dense reagents, and enzymatic labels.

[0118] As used herein, "particle" means a small, localized object to which physical properties (such as volume, mass, or average size) can be attributed. A particle can thus be symmetrical, spherical, substantially spherical or spherical, or of an irregular, asymmetrical shape or form. The size of the particle can vary. The term "microparticle" refers to a particle having a diameter in the nanometer and micrometer range.

[0119] Microparticles as defined above may comprise or consist of any suitable material known to those skilled in the art, for example they may comprise or consist of inorganic or organic materials or consist of or consist essentially of. Typically, they may comprise or consist of metals or metal alloys, or organic materials or consist of or consist essentially of, or comprise carbohydrate elements or consist of or consist essentially of. Examples of materials envisioned for microparticles include agarose, polystyrene, latex, polyvinyl alcohol, silicon dioxide and ferromagnetic metals, alloys or composite materials. In one embodiment, the microparticle is magnetic or ferromagnetic metal, alloy or composition. In further embodiments, the material may have specific characteristics, and for example, be hydrophobic or hydrophilic. Such microparticles are usually dispersed in aqueous solution and retain a small negative surface charge, thereby keeping the microparticles separated and avoiding nonspecific aggregation.

[0120] In one embodiment of the invention, the microparticles are paramagnetic microparticles, and the separation of such microparticles in the measurement method according to the present disclosure is facilitated by magnetic forces. Magnetic forces are applied to pull the paramagnetic or magnetic particles out of the solution / suspension and retain them as desired, while the liquid of the solution / suspension can be removed and the particles can, for example, be washed.

[0121] In diagnostic tests, it is necessary to determine whether a measurement is classified as "negative" (or "normal" or "non-reactive") or "positive" (or "pathological" or "reactive"). If the measured signal range is below a predefined threshold, the sample is considered non-reactive or negative. If the measured parameter range is above the threshold, the sample is classified as reactive or positive. This 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 so that the test still provides a predefined high sensitivity (high true positive rate), but also ensures a predefined high specificity (high true negative rate), thereby avoiding false positive and false negative results. Depending on the test design, and in order to avoid false positive results, the cutoff value can be defined as a multiple of the background signal or a multiple of the normal (negative) sample result. The test result can be provided in the form of a "cutoff index" (COI), which can be the ratio of the result signal obtained for the sample divided by the predefined cutoff value, thereby obtaining a signal sample / cutoff ratio. Particularly in HIV diagnostics, the cutoff values ​​and the calculated COI can be selected in such a way that a high sensitivity and a high specificity of the assay are achieved, i.e., ideally all positives must be detected, and among these there should not be any false positives, or at least as few false positives as possible. In many cases, most highly regulated infectious disease tests have a sensitivity and specificity of at least 98% (e.g., ranging from 98% to 99.99%). For HIV diagnostics, a minimum sensitivity of 100% and a specificity of >99.8% are required.

[0122] A "kit (kit / reagent kit)" is any article (e.g., package or container) comprising at least one reagent of the present invention (e.g., a drug for treating a disease, or a probe for specifically detecting a biomarker gene or protein). The kit is preferably marketed, distributed or sold as a unit for performing the method of the present invention. Typically, the kit may further include a separated carrier device to receive one or more container devices, such as vials, tubes, etc., in a tightly defined space. In particular, each container is meant to contain one of the individual elements to be used in the method of the first aspect. The kit may further include one or more other containers containing other materials, including but not limited to buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. Markings may be present on the container to indicate that the composition is used for a specific application, and may also indicate instructions for 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 disk), or directly provided on a computer or data processing device, or may be obtained via a data cloud setting. In addition, the kit may include a standard amount of a biomarker for calibration purposes.

[0123] “Package insert” is used to refer to the instructions customarily included in the commercial package of a diagnostic product that contains information about the intended use of the product, instructions on how to use the product, such as a diagnostic analyzer (“method sheet”), the expected range of results, interferences observed during development or during the registration process, etc.

[0124] Implementation

[0125] As further explained above, currently available immunoassays for detecting anti-HIV antibodies using gp41-derived antigens show considerable false positive results, i.e., they sometimes lack specificity. Surprisingly, by limiting the antigen to HIV gp41 antigens as further explained below, the number of falsely reactive samples can be reduced while maintaining a high sensitivity of the assay.

[0126] In a first aspect, the present invention relates to a composition suitable for detecting antibodies against HIV gp41 in an isolated sample, the composition comprising at least two, preferably three separate HIV gp41 antigens, wherein the first HIV antigen comprises SEQ ID NO.1 and wherein the other HIV gp41 antigens comprise at least one of SEQ ID NO:2 or and / or 3.

[0127] In an embodiment, each of said antigens does not comprise additional HIV-specific amino acid sequences.

[0128] In an embodiment, each of the HIV gp41 antigens is immunoreactive, ie, antibodies present in the biological sample bind to said antigen. Thus, any peptide derived from HIV gp41 that does not bind to an antibody is not contemplated.

[0129] In embodiments, each of the HIV gp41 antigens is soluble and suitable for use in an in vitro assay designed to detect antibodies against said antigen in an isolated biological sample.

[0130] Therefore, each of the composition and its HIV gp41 antigen is suitable for in vitro assays intended to detect anti-HIV antibodies with high sensitivity and specificity. In embodiments, the sensitivity is >95%, >96%, >97%, >98%, >99%, >99.5%, >99.8%. In specific embodiments, the sensitivity is >99.5% or >99.8%. In specific embodiments, the sensitivity is 100%. In embodiments, the specificity is >95%, >96%, >97%, >98%, >99%, >99.5%. In specific embodiments, the specificity is >99% or >99.5%. In specific embodiments, the specificity is >99.9%. In specific embodiments, the sensitivity is 100% and the specificity is >99.9%.

[0131] In embodiments, the composition of HIV gp41 antigen is suitable for detecting or detecting antibodies against HIV in a fluid sample. In particular embodiments, the sample is a human sample, particularly a human body fluid sample. In particular embodiments, the sample is a human blood or urine sample. In particular embodiments, the sample is a human whole blood, plasma or serum sample.

[0132] In embodiments, each of the HIV gp41 antigens is in its native state. In specific embodiments, the HIV gp41 specific amino acid sequence contained in each of the HIV gp41 antigens is folded in its native state.

[0133] In an embodiment, variants of the HIV gp41 specific amino acid sequences of SEQ ID NOs: 1, 2 and 3 are encompassed. Those skilled in the art can easily generate these variants by conservative or homologous substitutions of the disclosed amino acid sequences (such as, for example, substitutions of cysteine ​​with alanine or serine). In an embodiment, the variant exhibits a modification of its amino acid sequence, in particular selected from the group consisting of amino acid exchanges, deletions or insertions compared to the amino acid sequences of SEQ ID NOs: 1, 2 and 3.

[0134] In an embodiment, the amino acids are deleted or inserted at one or both ends, C-terminally or N-terminally, by 1 to 10 amino acids, in one embodiment by 1 to 5 amino acids. However, no further HIV-specific amino acid sequence can be added. In particular, the variant may be an isoform that exhibits the most common protein isoform. In one embodiment, such a substantially similar protein has a sequence homology of at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% to SEQ ID NO: 1, 2 or 3.

[0135] In an embodiment the variant comprises a post-translational modification, in particular selected from the group consisting of glycosylation or phosphorylation.

[0136] It will be appreciated that such variants are classified as HIV gp41 antigenic variants, ie, are capable of binding and detecting anti-HIV gp41 antibodies present in an isolated sample.

[0137] In embodiments, the overall three-dimensional structure of each of the HIV gp41 antigens remains unchanged, such that epitopes previously (ie, in the wild type) accessible to antibody binding remain accessible in the variant.

[0138] In an embodiment, at least one of the HIV gp41 antigens further comprises at least one chaperone protein. Thus, the HIV gp41 antigen comprises the HIV gp41 specific amino acid sequence of SEQ ID NO: 1, 2 or 3 as described above or below, and the amino acid sequence of a chaperone protein.

[0139] In a preferred embodiment, only the HIV gp41 antigen of SEQ ID NO: 1 comprises at least one chaperone protein. In a further preferred embodiment, only the HIV gp41 antigen of SEQ ID NO: 2 comprises at least one chaperone protein. In a further preferred embodiment, only the HIV gp41 antigen of SEQ ID NO: 3 comprises at least one chaperone protein.

[0140] In an embodiment, each of the HIV gp41 antigens further comprises at least one chaperone protein. Thus, the HIV gp41 antigen comprises the HIV gp41 specific amino acid sequence of SEQ ID NO: 1, 2 or 3 as described above or below, and the amino acid sequence of a chaperone protein.

[0141] In a particular embodiment, the HIV gp41 antigen comprises two chaperone proteins. In an embodiment, the chaperone proteins are selected from the group consisting of SlyD, SlpA, FkpA and Skp. In a particular embodiment, the chaperone protein is SlyD, in particular having the amino acid sequence given in Accession No.: UniProt ID P0A9K9.

[0142] In a specific embodiment, the HIV gp41 antigen comprises an HIV gp41 specific amino acid sequence according to SEQ ID NO: 1, 2 or 3 and a SlyD chaperone. In a specific embodiment, the HIV gp41 antigen comprises an HIV gp41 specific amino acid sequence according to 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 specific embodiment, SEQ ID NO: 1 is fused to two SlyD chaperone molecules.

[0143] In an embodiment, 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, in particular to the N-terminus of the HIV gp41 antigen. Thus, in a specific embodiment, the HIV gp41 antigen comprises a SlyD chaperone attached to an HIV gp41-specific amino acid sequence at the N-terminus and / or C-terminus. In a specific embodiment, the HIV gp41 antigen comprises two SlyD chaperones attached to an HIV gp41-specific amino acid sequence at the N-terminus. In an embodiment, the HIV gp41 antigen comprises a SlyD chaperone attached to an HIV gp41-specific amino acid sequence at the N-terminus and a SlyD chaperone attached to an HIV gp41-specific amino acid sequence at the C-terminus.

[0144] In embodiments, the HIV gp41 antigen or antigens further comprise a linker sequence. These sequences are not specific for 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 HIV gp41 sequence and one or more chaperone proteins. In specific embodiments, the linker is a Gly-rich linker. In specific embodiments, the linker has a sequence as shown in any one of SEQ ID NOs: 14, 15, and 16.

[0145] In certain embodiments, the HIV gp41 antigen comprises the amino acid sequence according to SEQ ID NO: 5. In embodiments, the HIV gp41 antigen does not comprise any additional amino acid sequence. In certain embodiments, the HIV gp41 antigen consists of the amino acid sequence according to SEQ ID NO: 5.

[0146] In certain embodiments, the HIV gp41 antigen comprises the amino acid sequence according to SEQ ID NO: 6. In embodiments, the HIV gp41 antigen does not comprise any additional amino acid sequence. In certain embodiments, the HIV gp41 antigen consists of SEQ ID NO: 6.

[0147] In certain embodiments, the HIV gp41 antigen comprises the amino acid sequence according to SEQ ID NO: 7. In certain embodiments, the HIV gp41 antigen does not comprise any additional amino acid sequence. In certain embodiments, the HIV gp41 antigen consists of SEQ ID NO: 7.

[0148] In certain embodiments, the HIV gp41 antigen comprises the amino acid sequence according to SEQ ID NO: 8. In certain embodiments, the HIV gp41 antigen does not comprise any additional amino acid sequence. In certain embodiments, the HIV gp41 antigen consists of SEQ ID NO: 8.

[0149] It should be 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 contain any additional amino acid sequence, but may still contain other chemical molecules, such as for example markers and / or tags.

[0150] In an embodiment, a composition suitable for detecting antibodies against HIV gp41 in an isolated sample comprises at least two, preferably three, separate HIV gp41 antigens, wherein the first HIV antigen comprises SEQ ID NO: 1 and wherein the second HIV gp41 antigen comprises at least one of SEQ ID NO: 2 or 3. In an embodiment, the composition comprises HIV gp41 antigens according to SEQ ID NO: 1, 2 and 3.

[0151] In an embodiment, the composition comprises SEQ ID NOs: 5, 6, 7 and 8.

[0152] In an embodiment, each HIV gp41 antigen further comprises a tag or label. Thus, the HIV gp41 antigen comprises an HIV gp41 specific amino acid sequence as in any one of SEQ ID NO: 1, 2, 3, 6, 7, 8 or 9 as described above or below, and a tag or label, and optionally an amino acid sequence of one or more chaperone proteins.

[0153] In a particular embodiment, the tag allows direct or indirect binding of the HIV gp41 antigen to a solid phase. In a particular embodiment, the tag is a partner of a bioaffinity binding pair. In a particular embodiment, the tag is selected from the group consisting of: biotin, digoxigenin, a hapten or a complementary oligonucleotide sequence (particularly a complementary LNA sequence). In a particular embodiment, the tag is biotin.

[0154] In certain embodiments, the label allows detection of HIV gp41 antigen. In certain embodiments, HIV gp41 specific sequences are labeled. In embodiments in which at least one chaperone protein is present in the antigen, the HIV gp41 specific sequence is labeled or at least one chaperone protein 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 that is present in the antigen in a stoichiometric ratio of 1:1 to 15:1. In certain embodiments, the stoichiometric ratio is 2:1, 2.5:1, 3:1, 5:1, 10:1 or 15:1.

[0155] In an embodiment, the composition comprises one or more additional HIV antigens. In a specific embodiment, the composition comprises HIV gp120 antigen, HIV reverse transcriptase antigen or HIV a p24 antigen or any combination thereof. In a specific embodiment, the composition comprises HIV reverse transcriptase antigen as an additional antigen.

[0156] In an embodiment, the additional HIV antigen is immunoreactive, ie antibodies present in the biological sample bind to said antigen. Thus, any HIV-derived peptide that does not bind to anti-HIV antibodies is not contemplated.

[0157] In an embodiment, the additional HIV gp41 antigen is soluble. Thus, the antigen is suitable for use in an in vitro assay aimed at detecting antibodies against the antigen in an isolated biological sample.

[0158] In a second aspect, the present invention relates to a method for producing a composition of HIV gp41 antigen, the method comprising the following steps:

[0159] a) culturing a host cell, in particular an E. coli cell, transformed with an expression vector comprising an operably linked recombinant DNA molecule encoding the antigen of the first aspect of the present invention,

[0160] b) expressing said antigen, and

[0161] c) purifying the antigen, and

[0162] d) mixing each of the HIV gp41 antigens obtained through steps a) to c) to form a composition of HIV gp41 antigens.

[0163] Optionally, as an additional step e), functional solubilization may be performed to render each HIV gp41 antigen in a soluble and immunoreactive conformation by refolding techniques known in the art.

[0164] 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.

[0165] In embodiments where the antigen comprises an HIV gp41 sequence and one or more chaperone proteins, the recombinant DNA molecule according to the invention may further contain a sequence encoding a linker peptide having 5 to 100 amino acid residues between the HIV gp41 antigens. This linker sequence may, for example, contain a proteolytic cleavage site. In embodiments, it is possible to add non-HIV gp41 specific linker or peptide fusion amino acid sequences to the HIV gp41 antigen, since these sequences are not specific for anti-HIV antibodies and will not be recognized in in vitro diagnostic immunoassays.

[0166] In a third aspect, the present invention relates to a method for detecting antibodies specific to HIV in an isolated sample, wherein the composition of the first aspect of the invention, or the HIVgp41 antigen obtained by the method of the second aspect of the invention is used as a capture reagent and / or binding partner for the anti-HIV antibodies.

[0167] In a fourth aspect, the present invention relates to a method for detecting antibodies specific for HIV in an isolated sample, the method comprising

[0168] a) forming an immune reaction 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

[0169] b) maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the HIV gp41 antigen composition present in the body fluid sample to immunologically react with the HIV gp41 antigen as part of the composition to form an immune reaction product; and

[0170] c) detecting the presence and / or concentration of any said immunoreaction product.

[0171] In embodiments, the method is an in vitro method. In embodiments, the method exhibits high sensitivity and specificity. In embodiments, the sensitivity is >95%, >96%, >97%, >98%, >99%, >99.5%. In specific embodiments, the sensitivity is >99% or >99.5%. In specific embodiments, the sensitivity is 100%. In embodiments, the specificity is >95%, >96%, >97%, >98%, >99%, >99.5%. In specific embodiments, the specificity is >99% or >99.5%. In specific embodiments, the specificity is 99.8%. In specific embodiments, the sensitivity is 100% and the specificity is >99.9%.

[0172] In an embodiment, the antibodies detected by the method of the invention are anti-HIV virus antibodies of the IgG, IgM or IgA subclass, or all three subclasses in the same immunoassay.

[0173] In an embodiment, the antibodies detected are directed against gp41 of human immunodeficiency virus (HIV), in particular against gp41 of HIV-1.

[0174] In embodiments, the isolated biological sample in which HIV-specific antibodies are detected is a human sample, particularly a human body fluid sample. In specific embodiments, the sample is a human blood or urine sample. In specific embodiments, the sample is a human whole blood, plasma or serum sample. In specific embodiments, the sample is a venous or capillary human whole blood, plasma or serum sample.

[0175] In an embodiment, the HIV gp41 antigen mixed with the isolated biological sample in step a) comprises at least one HIV gp41 specific amino acid sequence according to SEQ ID NO: 1, 2 or 3 or a variant thereof. In an embodiment, the HIV gp41 antigen does not comprise additional HIV gp41 virus specific amino acid sequences.

[0176] In an embodiment, the composition used in the method for detecting antibodies specific for HIV in an isolated sample comprises HIV gp41 antigen according to SEQ ID NO: 5, 6, 7 and 8. In a specific embodiment, the HIV-specific sequence of the HIV gp41 antigen consists of SEQ ID NO 5, 6, 7 and 8.

[0177] In an embodiment, the HIV gp41 antigen is immunoreactive, ie, antibodies present in the biological sample bind to the antigen. Thus, any peptide derived from HIV gp41 that does not bind to antibodies is not contemplated.

[0178] In an embodiment, the HIV gp41 antigen is soluble. Thus, the HIV gp41 antigen is suitable for use in an in vitro assay aimed at detecting antibodies against the antigen in an isolated biological sample.

[0179] In embodiments, the method includes the additional step of adding a solid phase to the immunoreaction mixture. In embodiments, the solid phase is a solid phase extraction (SPE) column or a bead. In specific embodiments, the solid phase comprises or consists of particles. In embodiments, the particles are nonmagnetic, magnetic or paramagnetic. In embodiments, the particles are coated. Depending on the intended use, i.e., depending on the intended capture molecule, the coating can be different. Which coating is suitable for which analyte is well known to the technician. The particles can be made of a variety of different materials. The beads can have various sizes and include surfaces with or without holes.

[0180] In a particular embodiment, the particle is a microparticle. In an embodiment, the microparticle has a diameter of 50 nanometers to 20 micrometers. In an embodiment, the microparticle has a diameter between 100 nm and 10 μm. In an embodiment, the microparticle has a diameter of 200 nm to 5 μm, in particular 750 nm to 5 μm, in particular 750 nm to 2 μm. In a particular embodiment, the microparticle is magnetic or paramagnetic. In particular, the microparticle is paramagnetic.

[0181] In an embodiment, the solid phase is added before adding the sample to the antigen or after forming the immunoreaction mixture. Thus, the addition of the solid phase may occur in step a) of the method, in step b) of the method or after step b) of the method.

[0182] In an embodiment, the method performed 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 practical applications and procedures. The HIV gp41 antigen according to the present invention can be used to improve assays for detecting anti-HIV antibodies, which are independent of the label used and independent of the detection mode (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.).

[0183] In an embodiment, the method performed is an immunoassay for detecting anti-HIV antibodies in separated samples according to the so-called double antigen sandwich concept (DAGS). Sometimes, this assay concept is also called the double antigen bridge concept, because the two antigens are bridged by the antibody analyte. In this assay, the ability of an antibody to bind to at least two different molecules of a given antigen and its two (IgG, IgE), four (IgA) or ten (IgM) paratopes is utilized.

[0184] In an embodiment, an immunoassay for determining anti-HIV gp41 antibodies according to a DAGS format is performed by incubating a sample containing anti-HIV gp41 antibodies with two different HIV gp41 antigens, a first ("capture") HIV gp41 antigen and a second HIV gp41 viral ("detection") antigen, wherein each of the two antigens is specifically bound by the anti-HIV gp41 antibodies.

[0185] In embodiments, the structures of the "capture antigen" and the "detection antigen" are immunologically cross-reactive. The basic requirement for performing the method is that the relevant epitope or multiple relevant epitopes are present on both antigens. Therefore, both antigens comprise HIV gp41-specific amino acid sequences as described above or below. In embodiments, the two antigens comprise the same or different fusion parts (e.g., SlyD fused to an HIV gp41-specific antigen labeled to be solid-phase bound, and, for example, FkpA fused to an HIV gp41-specific antigen labeled to be detected), because this change significantly alleviates the problem of non-specific binding and thus reduces the risk of false positive results.

[0186] In embodiments, the first antigen may be directly or indirectly bound to a solid phase and typically carries an effector group as part of a bioaffinity binding pair. In specific embodiments, the first antigen is conjugated to biotin and the complementary solid phase is coated with avidin or streptavidin. In embodiments, the second antigen carries a label that confers specific detectability to the antigen molecule alone or in complex with other molecules. In specific embodiments, the second antigen carries a ruthenium complex label.

[0187] Thus, in step b) of the present method, an immunoreaction mixture is formed comprising the first antigen, the sample antibody and the second antigen.

[0188] This ternary complex consisting of the analyte antibody sandwiched between two antigen molecules is called an immune complex or immune reaction product.

[0189] In embodiments, the method may include the additional step of separating the liquid phase from the solid phase.

[0190] Thus, in an embodiment, a method for detecting antibodies specific for HIV gp41 virus in an isolated sample comprises

[0191] a) adding to the sample a first HIV gp41 antigen and a second HIV gp41 antigen, the first HIV gp41 antigen being directly or indirectly bound to a solid phase and carrying an effector group as part of a bioaffinity binding pair, the second HIV gp41 antigen carrying a detectable label, wherein the first HIV gp41 antigen and the second HIV gp41 antigen specifically bind to the anti-HIV gp41 antibody

[0192] b) forming an immunoreaction mixture comprising the first antigen, the sample antibody and the second antigen, wherein a solid phase carrying the corresponding effector groups of the bioaffinity binding pair is added before, during or after forming the immunoreaction mixture,

[0193] c) maintaining the immune reaction mixture for a period of time sufficient to allow the anti-HIV gp41 antibodies against the HIV gp41 antigen in the body fluid sample to react with the HIV gp41 antigen to form an immune reaction product,

[0194] d) Separation of liquid and solid phases

[0195] e) detecting the presence of any said immunoreaction product in solid phase or liquid phase or both.

[0196] Finally, the presence of any such immunoreaction products is detected in solid phase or liquid phase, or both.

[0197] In an embodiment, the maximum total duration of the method for detecting HIV gp41 antibodies is less than one hour, i.e., less than 60 minutes, in one embodiment less than 30 minutes, in another embodiment less than 20 minutes, in one embodiment between 15 and 30 minutes, in one embodiment between 15 and 20 minutes. The duration includes pipetting the sample and the reagents required to perform the assay as well as the incubation time, optional washing steps, detection steps, and the final output of the results.

[0198] In a fifth aspect, the present invention relates to a method for identifying whether a patient has been exposed to HIV infection in the past, the method comprising

[0199] a) forming an immune reaction mixture by mixing a body fluid sample of a patient 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

[0200] b) maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the HIV gp41 antigen composition present in the body fluid sample to immunologically react with HIV gp41 antigens as part of the HIV gp41 antigen composition to form an immune reaction product; and

[0201] c) detecting the presence and / or absence of any of said immune reaction products,

[0202] The presence of immune response products indicates that the patient has been exposed to HIV infection in the past.

[0203] In an embodiment, the patient is exposed to HIV infection before performing the method. In particular, the patient is exposed to HIV infection at least 5 days before performing the method. In particular, the patient is exposed to HIV infection at least 10 days before performing the method. In particular, the patient is exposed to HIV infection at least 14 days before performing the method.

[0204] In a sixth aspect, the present invention relates to 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 detecting anti-HIV antibodies.

[0205] In a seventh aspect, the present invention relates to a kit for detecting 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.

[0206] In an embodiment, the kit comprises the HIV gp41 antigen composition of the first aspect of the invention, or the HIV gp41 antigen obtained by the method of the second aspect of the invention, in separate containers or in separate compartments of a single container unit. In a specific embodiment, the HIV gp41 antigen comprised is covalently coupled to biotin.

[0207] In an embodiment, the kit further comprises microparticles, in particular microparticles coated with avidin or streptavidin, in separate containers or in separate compartments of a single container unit.

[0208] All definitions provided for the HIV gp41 antigen as part of the composition provided in the first to third aspects of the invention also apply mutatis mutandis to the fourth, fifth, sixth and seventh aspects of the invention.

[0209] In a further embodiment, the present invention relates to the following items:

[0210] Item 1: A composition suitable for detecting antibodies against HIV gp41 in an isolated sample, the composition comprising at least two separate HIV gp41 antigens, wherein the first HIV antigen comprises SEQ ID NO.1, and wherein the second HIV gp41 antigen comprises SEQ ID NO:2 and / or 3, and wherein each of the separate HIV gp41 antigens does not contain additional HIV-specific amino acid sequences.

[0211] Item 2: A composition according to Item 1, wherein at least one of the HIV gp41 antigens is fused to at least one chaperone protein, in one embodiment to two chaperone proteins.

[0212] Item 3: The composition according to Item 2, wherein the chaperone protein is selected from the group consisting of SlyD, SlpA, FkpA and Skp.

[0213] Item 4: A composition according to Item 1 or 2, wherein the chaperone protein is fused to an HIV gp41-specific amino acid sequence at the N and / or C terminus of the HIV gp41 antigen.

[0214] Item 5: A composition according to any of the preceding items, wherein each of the antigens is soluble and immunoreactive.

[0215] Item 6: A composition according to any one of the preceding items, wherein the HIV gp41 antigen comprises SEQ ID NOs 1 and 2 or SEQ ID NOs 1 and 3.

[0216] Item 7: A composition according to any of the preceding items, wherein the HIV gp41 antigen comprises SEQ ID NOs 1, 2 and 3.

[0217] Item 8: A composition according to any of the preceding items, wherein the HIV gp41 antigen comprises SEQ ID NOs 5, 6, 7 and 8.

[0218] Item 9: A composition according to any one of the preceding items, wherein the HIV-specific sequence of the HIV gp41 antigen consists of SEQ ID NOs 5, 6, 7 and 8.

[0219] Item 10: An HIV gp41 antigen suitable for detecting anti-HIV antibodies in an isolated biological sample, comprising SEQ ID NO: 1,

[0220] The antigen does not contain additional HIV-specific amino acid sequences.

[0221] Item 11: An HIV gp41 antigen suitable for detecting anti-HIV antibodies in an isolated biological sample, comprising SEQ ID NO: 2,

[0222] The antigen does not contain additional HIV-specific amino acid sequences.

[0223] Item 12: An HIV gp41 antigen suitable for detecting antibodies against HIV in an isolated biological sample, comprising SEQ ID NO: 3, wherein the antigen does not contain additional HIV-specific amino acid sequences.

[0224] Item 13: HIV gp41 antigen according to any one of items 10 to 12, wherein the antigen further comprises a transglutaminase peptide, in one embodiment comprising the amino acid sequence YRYRQ (SEQ ID NO: 13).

[0225] Item 14: HIV gp41 antigen according to any one of items 10 to 13, wherein the antigen further comprises a sortase peptide, in one embodiment comprising the amino acid sequence LPETG (SEQ ID NO: 12).

[0226] Item 15: HIV gp41 antigen according to any one of items 10 to 14, wherein the antigen further comprises a linker peptide, in one embodiment comprising two or three glycine residues, in one embodiment comprising GGGS (SEQ ID NO: 14), in another embodiment comprising GGGSGGGSGGGSGGG (SEQ ID No: 15), and in another embodiment comprising SGGG (SEQ ID NO: 16).

[0227] Item 16: HIV gp41 antigen according to any one of Items 10 to 15, wherein the antigen further comprises the histidine peptide HHHHHH (SEQ ID NO: 17).

[0228] Item 17: A method for producing a composition of HIV gp41 antigens according to any one of Items 1 to 9, the method comprising the following steps for each of the antigens

[0229] a) culturing a host cell transformed with an expression vector comprising an operably linked recombinant DNA molecule encoding one of each of said antigens,

[0230] b) expressing each of said antigens,

[0231] c) purifying each of said antigens, and

[0232] 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 and / or 3 obtained by steps a) to c) to form a composition of HIV gp41 antigens.

[0233] Item 18: The method according to Item 17, wherein in step d), the mixed HIV gp41 antigen consists of SEQ ID NOs 5, 6, 7 and 8.

[0234] Item 19: A method for detecting antibodies specific for HIV gp41 in an isolated sample, wherein a composition of HIV gp41 antigen according to any one of items 1 to 9 is used as a capture reagent and / or binding partner for the anti-HIV antibodies.

[0235] Item 20: A method for detecting antibodies specific for HIV gp41 in an isolated sample, the method comprising

[0236] a) forming an immune reaction mixture by mixing a body fluid sample with the HIV gp41 antigen composition according to any one of items 1 to 9,

[0237] b) maintaining the immune reaction mixture for a period of time sufficient to allow the antibodies against HIV gp41 present in the body fluid sample to react immunologically with the HIV gp41 antigen composition to form an immune reaction product; and

[0238] c) detecting the presence and / or concentration of any said immunoreaction product.

[0239] Item 21: A method for detecting antibodies specific for HIV in an isolated sample according to Item 20, wherein the immune reaction is performed in a double antigen sandwich format, the method comprising 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, which can be directly or indirectly bound to a solid phase and carries an effector group as 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, which carries a detectable label, wherein the first HIV gp41 and the second HIV gp41 antigen specifically bind to the anti-HIV antibody,

[0240] b) forming an immunoreaction mixture comprising the first antigen, the sample antibody and the second antigen, wherein a solid phase carrying the corresponding effector groups of the bioaffinity binding pair is added before, during or after forming the immunoreaction mixture,

[0241] c) maintaining the immune reaction mixture for a period of time sufficient to allow the anti-HIV antibodies against the HIV gp41 antigen in the body fluid sample to react with the HIV gp41 antigen to form an immune reaction product,

[0242] d) Separation of liquid and solid phases

[0243] e) detecting the presence of any said immunoreaction product in solid phase or liquid phase or both.

[0244] Item 22: A method for identifying whether a patient has been exposed to HIV infection in the past, the method comprising

[0245] a) forming an immune reaction mixture by mixing a body fluid sample of the patient with the HIV gp41 antigen composition according to any one of items 1 to 9,

[0246] b) maintaining the immune reaction mixture for a period of time sufficient to allow the antibodies against the HIV gp41 antigen composition present in the body fluid sample to immunologically react with the HIV gp41 antigen composition to form an immune reaction product; and

[0247] c) detecting the presence and / or absence of any of said immune reaction products,

[0248] The presence of the immune reaction product indicates that the patient has been exposed to HIV infection in the past.

[0249] Item 23: Use of the HIV gp41 antigen composition according to any one of Items 1 to 9 for detecting anti-HIV gp41 antibodies in an isolated sample.

[0250] 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 isolated samples.

[0251] Item 25: A kit for detecting anti-HIV antibodies, comprising the HIVgp41 antigen composition according to any one of Items 1 to 9.

[0252] Item 26: A kit for detecting anti-HIV antibodies, comprising the composition described in any one of Items 1 to 9, or the HIV gp41 antigen composition obtained by the method described in Item 17.

[0253] Item 27: A kit according to Item 23, which comprises microparticles coated with at least avidin or streptavidin in separate containers or in separate compartments of a single container unit, and an HIV gp41 antigen composition according to any one of Items 1 to 9 or an HIV gp41 antigen composition obtained by the method according to Item 17, wherein each of the HIV gp41 antigens is covalently coupled to biotin.

[0254] Item 28: A kit according to Item 27, comprising an HIV gp41 antigen composition according to any one of Items 1 to 9 or an 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 coupled to a detectable label, in one embodiment to an electrochemiluminescent ruthenium complex.

[0255] The following examples and drawings are provided to aid the understanding of the present invention, the true scope of the invention being set forth in the appended claims.It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention.

[0256] Examples

[0257] Example 1: Expression and purification of recombinant HIV1gp41 and 6hel antigens

[0258] Small-scale preparation of recombinant 6hel antigen for high-throughput screening

[0259] 242 plasmids containing the gp41-6hel gene with different point mutations and a C-terminal hexahistidine tag were synthesized at Twist Bioscience and cloned into pET29a via NdeI (5' end) and XhoI (3' end) restriction sites.

[0260] Small-scale expression of recombinant gp41-6hel protein

[0261] After plasmid was diluted to 5 to 10 ng / μl in 10 mM Tris-HCl buffer (pH 8.5), 1 μl 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 were plated on 48-well LB-kanamycin (50 μg / ml) agar plates (Teknova).

[0262] Cultivation and expression of all variants were performed in a 96-well plate format. For pre-culture, one colony of each mutant was selected in a 96-well flat-bottom microtiter plate (Corning) containing 200 μl 4x yeast-kanamycin (50 μg / ml) medium in each well. In addition, 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 50% (v / v) glycerol was added before freezing. Expression was performed in 96 deep-well plates, each well including 1000 μl 4x yeast-kanamycin (50 μg / ml) medium containing 0.1 mM IPTG. Mutants were expressed at 30°C and 800 rpm (Microplate Shaker TiMix; Edmund Bühler GmbH) and harvested after 16 hours by centrifugation at 4700 rpm for 10 minutes.

[0263] Small-scale purification of recombinant gp41-6hel protein

[0264] For small-scale purification of 6hel antigen, 125 μl of 100% (Merck Millipore) lysed bacterial cell pellets from 1 ml of E. coli culture. After adding 125 μl of 2x equilibration buffer (0.1M NaH2PO4 pH 8.0; 1% (v / v) Tween-20; 1M NaCl; 40mM imidazole) and clearing the cell lysate by centrifugation (4700rpm, 10 minutes), the lysate was transferred to a 96-well V-bottom plate (Corning) using a pipetting robot (Biomek). Small-scale purification was performed using robotic pipette tips called PhyTips (PhyNexus) pre-loaded with Ni-NTA resin. First, Phytips were equilibrated with equilibration buffer (0.05M NaH2PO4 pH8.0; 0.5% (v / v) Tween-20; 0.5M NaCl; 20mM imidazole). They were then transferred to the sample for protein binding. To remove non-specifically bound proteins, Phytips were washed twice with wash buffer 1 (0.05 M NaH2PO4 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 NaH2PO4 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 elution buffer (0.05 M NaH2PO4 pH 8.0; 0.5% (v / v) Tween-20; 0.15 M NaCl; 200 mM imidazole). Protein samples were analyzed by SDS-PAGE gel. After Ni-NTA purification, the 6hel antigen was purified using Pierce Biotechnology according to the instructions provided by Pierce Biotechnology. TM 96-well microdialysis plates were buffer exchanged into conjugation buffer (0.15 M KH2PO4 pH 8.0; 0.1 M KCl; 0.5 mM EDTA).

[0265] Small-scale ruthenylation and biotinylation of recombinant gp41-6Hel protein

[0266] Conjugation was performed using NHS chemistry in black 96-well half-area plates (Corning). Prior to conjugation, the conjugate was purified using Pierce TM The protein concentration in each well was determined by BCA assay in a microtiter plate using a BCA protein assay kit (ThermoFisher). 180 μl of BCA solution was added to 20 μl of purified antigen per well and quantified using a Tecan sunrise TM The microplate reader was used for measurement at 562 nm.

[0267] Antigen (approximately 1 mg / ml) and label were quickly mixed to a final antigen to label ratio of 1:4 (ruthenium conjugated) and 1:5 (biotin conjugated) at a DMSO concentration of 10% (v / v). The plate was incubated at 600 rpm for 30 minutes at room temperature. The labeling reaction was terminated by adding L-lysine to a final concentration of 10 μM. For small-scale preparations, free, unbound ruthenium label was not removed but was removed by using PD MultiTrap TM G-2596 well plates (GE Healthcare) were used to remove free, unbound biotin label. The concentrations of ruthenylated and biotinylated antigens were determined by using the BCA assay as described above. The ruthenylated and biotinylated gp41-6hel mutants were stored at 4°C until assessed by the Elecsys test system.

[0268] In total, 171 of the 242 6hel mutations (71%) could be successfully purified, labeled, and further assessed via immunoassay. The 71 missing variants either failed to express due to DNA synthesis failure or the yield of purified protein was too low to allow labeling reactions.

[0269] Large-scale preparation of recombinant HIV1 gp41 and 6hel antigens for thorough screening

[0270] Plasmids containing recombinant HIV1 gp41 (aa536-681) and 6hel genes (with different point mutations and a C-terminal hexahistidine tag) were synthesized at Eurofins Genomics GmbH and cloned into pET24a(+) via NdeI (5' end) and XhoI (3' end) restriction sites.

[0271] In addition, recombinant gp41 (aa536-681) was fused to two SlyD chaperone proteins from Escherichia coli at the N-terminus via a glycine-serine-rich linker (Scholz, C. et al., J. Mol. Biol. (2005) 345, 1229-1241) to produce EcSlyD-EcSlyD-gp41 fusion protein, hereinafter simply referred to as gp41.

[0272] gp41 and 6hel constructs were expressed in BLR(DE3) E. coli cells using standard LB medium and IPTG induction for three hours at 37° C. Cells were harvested by centrifugation (20 min, 5000 g) and stored at −20° C. before further processing.

[0273] Large-scale purification of recombinant HIV1 gp41 and 6hel antigens

[0274] Recombinant HIV1 gp41 and 6hel antigens were purified under denaturing conditions and subsequently refolded on the column. Specifically, bacterial pellets from 700 ml of E. coli culture were resuspended in a chaotropic lysis buffer (50 mM sodium phosphate pH 8.0; 4 M guanidine chloride; 5 mM imidazole) and stirred at room temperature for 90 minutes. For removal, the cell lysate was centrifuged and filtered (5 / 0.8 / 0.2 μm). The clarified supernatant was added to a Roche cOmplete His-tag purification column balanced with lysis buffer. Non-specifically bound proteins were removed from the column by thorough washing to the baseline with lysis buffer. Refolding of the antigen was performed by refolding on the column using a refolding buffer (50 mM sodium phosphate pH 8.0; 100 mM NaCl). The refolded target protein was eluted from the column with an elution buffer containing imidazole (50 mM sodium phosphate pH 8.0; 50 mM imidazole; 100 mM NaCl). For buffer exchange and polishing, 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 with one major peak representing the oligomeric arrangement. The oligomeric fraction was concentrated and biotinylated and ruthenylated. 6hel eluted in one peak, which was concentrated and biotinylated and ruthenylated.

[0275] Example 2: Ruthenylation and biotinylation of recombinant gp41 protein

[0276] Large-Scale Ruthenylation and Biotinylation of Recombinant HIV1gp41 and 6HEL Antigens Using NHS Chemistry

[0277] For the conjugation of antigen to biotin or ruthenium, the protein concentration in SEC buffer 2 should ideally be 10mg / ml. Using NSH chemistry, conjugation is performed with a molar ratio of 1:4 antigen to label and a DMSO concentration of 5% (v / v). The label and antigen are quickly mixed and stirred at room temperature for 30 minutes. The labeling reaction is terminated by adding L-lysine to a final concentration of 10mM. For large-scale preparation, a Superdex 200Increase (GE Healthcare) column balanced with a storage buffer (50mM sodium phosphate pH 7.5; 100mM KCl; 0.5mM EDTA) is used to remove free unbound labels from the reaction by size exclusion chromatography. The concentration of ruthenylated antigen is determined by using BCA assay, and the concentration of biotinylated antigen is determined by absorption measurement at 280nm.

[0278] Large-scale ruthenylation and biotinylation of recombinant HIV1 gp41 and 6hel using transglutaminase

[0279] Recombinant transglutaminase from Kutzneria albida (KalbTG) can be used to site-specifically label antigens by forming a Gln-Lys isopeptide bond between a Q-tagged antigen and a corresponding K-tagged antigen (Steffen, W. et al., J. Mol. Biol. (2017) 292, 15622-1563). For conjugation of HIV1 antigens with biotin or ruthenium, the protein concentration in SEC buffer 2 should ideally be 10 mg / ml. Conjugation was performed with a molar ratio of Q tag to label of 1:5 and a lack ratio of enzyme to antigen of 1:300. The antigen, label and activated enzyme were mixed and incubated at 37°C for 20 hours while gently mixing. After 20 hours of incubation, the reaction was terminated 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 ruthenylated antigen was determined by using a BCA assay, and the concentration of biotinylated antigen was determined by absorbance measurements at 280 nm.

[0280] Large-scale biotinylation of recombinant HIV1 gp41 and 6hel using sortase

[0281] Recombinant sortase can be used to site-specifically label antigens by forming a peptide bond between the threonine of the C-terminal sortase recognition site (LPETG) and the glycine residue in the corresponding tag. For conjugation of HIV1 antigens to biotin by sortase, the protein concentration in phosphate-free SEC buffer 1 should ideally be 10 mg / ml. Conjugation was performed in the presence of 10 mM calcium chloride, with an antigen to tag ratio of 1:50 and an enzyme input of 50 U / μmol antigen. Antigen, tag, and activated enzyme were mixed and incubated at 37°C for 1 hour with gentle mixing. After incubation for 1 hour, the reaction was loaded onto Roche cOmplete His-tag resin to remove sortase and unlabeled antigen from the reaction mixture. Finally, free, unbound tag was removed by size exclusion chromatography using a Superdex200 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 biotinylated antigen was determined by absorbance measurement at 280 nm.

[0282] Example 3: Biochemical analysis of recombinant HIV1 gp41 and 6hel antigens

[0283] Spectroscopic Measurement of Recombinant HIV1gp41 and 6Hel Antigens

[0284] Using NanoDrop Protein concentration was measured using a micro UV / visible spectrophotometer (Thermo Scientific). The molar extinction coefficient (ε) of the antigen was calculated using the equation reported by Pace et al. (Protein Sci. 1995 Nov; 4(11): 2411-23). 280nm ).

[0285] Table 1: Protein parameters of the five best recombinant HIV1 gp41 and 6hel antigens

[0286]

[0287] Circular dichroism (CD) spectrum of recombinant HIV16 hel antigen

[0288] The far-UV CD spectra (190-250 nm) of the 6hel antigen were recorded using a Jasco-720 spectropolarimeter and finally converted to average residue ellipticity (θ mrw,λ ). All samples were diluted to 0.21 mg / ml in 50 mM potassium phosphate pH 7.5, 100 mM KCl, 0.5 mM EDTA. The spectrometer was adjusted during the measurements as follows: 0.2 cm pathlength, scan range 190 to 330 nm, scan speed 20 nm / min, bandwidth 2.0 nm, resolution 0.5 nm, response time 1 s. All spectra were measured nine times and averaged.

[0289] In the far-UV range, CD spectroscopy allows the analysis of secondary structured proteins, since the absorption in this UV range is mainly caused by peptide bonds. Therefore, the far-UV CD spectrum of the full-helical 6hel antigen compared to the mutant variants provides reliable insights into the antigen structure and the effects of point mutations on protein folding.

[0290] HPLC Analysis of Recombinant HIV16 Hel Antigen

[0291] In order to analyze the purity and aggregation tendency of the mutant antigens and to estimate the molecular weight of the purified 6hel antigen, HPLC analysis was performed. 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 mobile phase. As a reference, an internal HPLC standard was also analyzed. HPLC analysis allows the assessment of the aggregation behavior of the mutant 6hel antigen compared to the wild-type construct.

[0292] Example 4: Immunoreactivity of different recombinant HIV1 gp41 and 6hel antigens in anti-HIV immunoassay

[0293] Immunoreactivity (antigenicity) of HIV1 gp41 and 6HEL variants in automated The assay was performed using a double antigen sandwich (DAGS) format on the cobas analyzer (Roche Diagnostics GmbH). 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. After activation, the ruthenium complex switches between the redox states 2+ and 3+, generating a light signal. In the presence of specific immunoglobulins (in this case anti-HIV IgG antibodies in human serum), the ruthenium complex is bridged to the solid phase and light emission at 620 nm is triggered at the electrode by the addition of tripropylamine.

[0294] In this study, all 171 mutant variants of recombinant 6hel expressed and labeled on a small scale (Figure 3) were investigated to assess their binding potential to anti-HIV1 IgG antibodies.

[0295] Immunoreactivity of different recombinant HIV1gp41 and 6Hel antigens in the DAGS assay setting

[0296] For a more thorough analysis, approximately 20 best mutations in the 6hel antigen, identified in the initial screening as having improved immunospecificity, were expressed and labeled on a large scale and fully analyzed in a DAGS assay setting. In addition, the same selected mutations were also transferred to the HIV1 gp41 antigen (WO03 / 000877) and their specificity was assessed. In addition to these constructs, 6hel and gp41 antigens containing the most promising mutation combinations were generated and evaluated.

[0297] In detail, different gp41-biotin or 6hel-biotin and gp41-ruthenium or 6hel-ruthenium antigens were used in reagent buffer 1 (R1) and R2, respectively. Labeled recombinant gp41 antigens were used at concentrations between 30 ng / ml and 300 ng / ml in R1 and R2. The concentrations of various labeled 6hel antigens in R1 and R2 were between 2 ng / ml and 130 ng / ml, depending on the mutation.

[0298] To avoid immunological cross-reaction via the chaperone fusion unit of the recombinant HIV1 gp41 antigen, a large excess (5 to 30 μg / ml) of unlabeled EcSkp-EcSlyD-EcSlyD (EP2893021 (B1)) or chemically polymerized EcSlyD-EcSlyD was added to the reaction buffer as an anti-interference substance.

[0299] To assess the specificity and sensitivity of different recombinant HIV1 gp41 and 6hel antigens, Elecsys measurements performed with HIV negative and positive and seroconverted samples were analyzed.

[0300] The results of the three best antigens (SEQ ID No 1, 2 and 3) are as follows Figure 6 As shown. In detail, A) cut-off index (COI) of 10 high positive HIV samples from patients infected with different HIV-1 subtypes. All samples tested with improved anti-HIV module (AHIVII) were positive, as was the standard AHIVI module. COI value <1 was indicated as non-reactive, while COI>1 sample indicated the presence of anti-HIV antibodies. B) Performance comparison of standard Elecsys HIV Duo assay (black HIV Duo I) and optimized Elecsys HIV Duo II assay (gray HIV Duo II), and separate comparison of anti-HIV modules AHIV I and AHIV II, which are gray and black, respectively. This comparison was carried out on five commercially available seroconversion plates (1-5), and blood was drawn in sequence. Compared with AHIV I module, optimized anti-HIV II module showed higher sensitivity. The higher sensitivity of AHIV II module plays a role especially in seroconversion plates 2 and 3. In both panels, nine or five blood draws (highlighted in grey) were negative in the AHIV I module and clearly positive in the optimized AHIV II module. This higher sensitivity reduces the risk of a second window period after infection and significantly reduces the risk of false negative HIV results. C) Graphics and D) numbers showing the specificity of the current and optimized anti-HIV modules in black and grey, respectively. The specificity of the two modules was determined using 6046 HIV-negative routine samples from different suppliers. Setting the threshold for HIV positivity to COI>1 (highlighted in bold), the standard AHIV I module showed four false positive samples, resulting in a specificity of 99.92. The optimized AHIV II module, on the other hand, does not show any COI>1 signals, resulting in a specificity of 100. In summary, the experiments performed here show that the sensitivity and specificity of the AHIV II module are significantly optimized compared to the AHIV module.

[0301] Example 5: Data from external specificity studies

[0302] To thoroughly evaluate the specificity of the mutated and thus optimized gp41 and 6HEL antigens, 15,242 routine blood samples were analyzed in an external study ( Figure 7a). The assessment was performed by an independent laboratory using the AHIV module of Elecsys HIV Duo and the optimized Elecsys HIV Duo II assay (containing SEQ ID NOs 1, 2 and 3).

[0303] In this study, 44 samples generated false-positive signals in the Elecsys HIVDuo I assay (specificity 99.71%), whereas only 7 false-positive samples were detected using the optimized HIVDuo II assay (specificity 99.95%) (Figure 7A). The 21 false-positive samples for the Elecsys HIV Duo I assay and the 4 false-positive samples for HIV Duo II were caused by the gp41 and 6hel antigens within the anti-HIV module of the HIV DuoElecsys assay ( Figure 7b ). Thus, a significant specificity improvement of the mutated and optimized gp41 and 6hel antigens could also be shown in independent external studies, and the interference potential of the gp41 antigen was reduced to <20% after optimization. .

[0304] Example 6: Improving the sensitivity and specificity of HIV immunoassays by combining mutated and optimized HIV gp41 antigens sex

[0305] Compared to the HIV immunoassay containing the HIV gp41 antigen comprising SEQ ID NO. 10, not only the combination of SEQ ID NO. 1, 2 and 3 showed significantly improved antigenicity, but also the combination of two optimized HIV gp41 antigens (SEQ ID NO. 1 and 2 or SEQ ID NO. 1 and 3) has shown significantly improved immunoreactivity (Figure 8). The specificity of different antigen combinations was evaluated by analyzing 103 HIV negative samples ( Figure 8a When the gp41 antigen without further optimization was used, only 91 of 103 negative samples (88.35%) showed a COI between 0.02 and 0.05, whereas when different combinations of mutated gp41 and 6hel antigens were used, at least 98% of the negative samples were within this COI range ( Figure 8a Columns 2 to 3). Thus, the scatter of the samples was significantly reduced when the optimized antigens were used. In addition, not only the specificity and scatter were improved, but also the sensitivity was significantly better for the different combinations of optimized gp41 and 6hel antigens ( Figure 8b ). For example, sample Sero01 clearly showed much higher sensitivity in all combinations using mutant antigens, with SEQ ID NO. 1, 2 and 3 being the best combination.

Claims

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

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

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

4. A composition according to any one of the preceding claims, wherein each of the antigens is soluble and immunoreactive.

5. The composition according to any one of the preceding claims, wherein the HIV gp41 antigen comprises SEQ ID NOs 1 and 2 or SEQ ID NOs 1 and 3.

6. The composition of any one of the preceding claims, wherein the HIV gp41 antigen comprises 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, said method comprising the following steps for each of said antigens a) culturing a host cell transformed with an expression vector comprising an operably linked recombinant DNA molecule encoding one of each of said antigens, b) expressing each of said antigens, c) purifying each of said antigens, and 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.

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

9. A method for detecting antibodies specific for HIV gp41 in an isolated sample, the method comprising a) forming an immune reaction 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 immune reaction mixture for a period of time sufficient to allow antibodies against HIV gp41 present in the body fluid sample to immunologically react with HIV gp41 antigens as part of the HIV gp41 antigen composition to form immune reaction products; and c) detecting the presence and / or concentration of any said immunoreaction product.

10. A method for identifying whether a patient has been exposed to HIV infection in the past, the method comprising a) forming an immune reaction mixture by mixing a body fluid sample of the patient with the HIV gp41 antigen composition according to any one of claims 1 to 6, b) maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the HIV gp41 antigen composition present in the body fluid sample to immunologically react with HIV gp41 antigens as part of the HIV gp41 antigen composition to form an immune reaction product; and c) detecting the presence and / or absence of any of said immune reaction products, The presence of the immune reaction product indicates that the patient has been exposed to HIV infection in the past.

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.

12. A kit for detecting anti-HIV antibodies, comprising the HIVgp41 antigen composition according to any one of claims 1 to 6.

13. The 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 separate HIV gp41 antigens is covalently coupled to biotin.

14. The kit according to claim 13, comprising the HIV gp41 antigen composition according to any one of claims 1 to 5 in a further individual container or in a further separate compartment of a single container, wherein each of the individual HIV gp41 antigens in the further individual container or further separate compartment is covalently coupled to a detectable label.

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