Method for detecting HIV-1-specific antibodies using clade C env polypeptides

Abstract

The present invention relates to a method for detecting and/or quantifying human immunodeficiency virus (HIV) specific antibodies in a sample of a subject comprising the step of determining the presence and/or amount of antibodies binding to a) a peptide consisting of the amino acid sequence AIVCTRPNNNTRKSIRIGPGQVFYT (SEQ. ID No. 1), or b) a homolog having at least 70% identity with a peptide of a), or c) a fragment of a peptide of a) or b) consisting of 15 to 24 amino acid residues in said sample.

Claims

1. A method for detecting or quantifying human immunodeficiency virus type 1 (HIV-1) specific antibodies in a sample of a subject, the method comprising the steps of: a) immobilizing one or more peptides or polypeptides on a solid support, wherein said peptides consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5; b) contacting the solid support with the sample under conditions that permit antigen-antibody binding; and, c) detecting antigen-antibody binding through radioimmune assay (RIA), enzyme-linked immunosorbent assay (ELISA), Western blot assay, dot blot assay, bead assay, or peptide array assay.

2. The method according to claim 1, wherein a) is the peptide consisting of the amino acid sequence SEQ ID NO. 2.

3. The method according to claim 1, wherein a) is the peptide consisting of the amino acid sequence SEQ ID NO. 3.

4. The method according to claim 1, wherein a) is the peptide consisting of the amino acid sequence SEQ ID NO. 4.

5. The method according to claim 1, wherein a) is the peptide consisting of the amino acid sequence SEQ ID NO. 5.

6. The method according to claim 1, wherein further the presence or amount of antibodies binding to human immunodeficiency virus (HIV) capsid protein p24 in a sample of the individual is determined.

7. The method according to claim 1, wherein further the presence or amount of antibodies binding to at least one polypeptide selected from the group consisting of human immunodeficiency virus (HIV) integrase, HIV reverse transcriptase+RNAse H, HIV protease and HIV matrix protein p17 in a sample of the individual is determined.

8. The method according to claim 1, wherein the sample of the subject is a sample selected from the group consisting of blood sample, serum sample, plasma sample, saliva, tears, urine, nose secret, genital secretion, stool and breast milk.

9. The method according to claim 1, wherein the antibodies to be determined and/or quantified are selected from the group consisting of IgG, IgG1, IgG2, IgG3, IgG4, IgA and IgM, wherein IgG is particularly preferred.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.

(2) FIGS. 1A-C show antibody responses to HIV-1 clade C gp120 and gp120-derived peptides.

(3) FIG. 1A shows frequency (y-axes: number of reactive sera) of IgG, IgG subclass, IgA and IgM responses of African (left) and European (right) patients to recombinant gp120 and 24 overlapping gp120 peptides (x-axes: peptides 1-24).

(4) FIG. 1B shows position of overlapping gp120 peptides in gp120 of the HIV-1 clade C South African strain (clade C_ZA) and of the reference strain HXB2 (clade B_HXB2). Gaps (numbers of missing amino acids 20 are displayed) in gp120 from clade C and B required for optimal sequence alignment are indicated. Relevant protein domains de-scribed for gp120 clade B are indicated (SP: signal peptide, V1-V5: variable regions 1-5).

(5) FIG. 1C shows multiple sequence alignment of the amino acid sequence of peptide 120/15 (SEQ ID No.: 1) of HIV-1 clade C South African strain (Ref.C.ZA) with corresponding peptides from HIV-1 reference strains. Percentages of sequence identity with the Ref. C.ZA peptides are indicated on the right margin. Dots are conserved amino acids, dashes are gaps, N-linked glycosylation sites are underlined. The numbering system refers to HIV-1 strain HXB2 (Ref.B.FR) and “aa” stands for amino acid.

(6) FIGS. 2A-B show antibody responses to HIV-1 clade C gp41-derived peptides.

(7) FIG. 2A shows frequency (y-axes: number of reactive sera) of IgG, IgG subclass, IgA and IgM responses of African (left) and European (right) patients to 17 overlapping gp41 peptides (x-axes: peptides 1-17).

(8) FIG. 2B shows position of overlapping gp41 peptides in gp41 of the HIV-1 clade C South African strain (clade C_ZA) and of the reference strain HXB2 (clade B_HXB2). A gap (number of missing amino acids is displayed) in gp41 from clade B required for optimal sequence alignment is indicated. Relevant protein domains/epitopes described for gp41 clade B are indicated (F: fusion peptide, TM: transmembrane domain, ID: immunodominant region, IS: immunosuppressive region, MPER: membrane proximal external region).

(9) FIGS. 3A-B show purity and immunoreactivity of recombinant HIV-1 clade C structural, accessory and pol-derived proteins.

(10) FIG. 3A shows coomassie-stained SDS PAGE containing recombinant matrix (MA), capsid (CA), nucleocapsid (NC), NEF, TAT, VIF, protease (PR), reverse transcriptase+RNase H (RR), integrase (IN) and molecular weight markers (M). Molecular weights (kDa) are indicated on the left.

(11) FIG. 3B shows frequency (y-axes: number of reactive sera) of IgG, IgG subclass, IgA and IgM responses of African (left) and European (right) patients to structural, accessory and pol-derived proteins (x-axes).

(12) FIG. 4 shows IgG and IgM reactivity profiles to gp120-derived proteins and peptides of two individuals with negative results in established conventional HIV diagnostic tests. Shown are positive IgG and IgM antibody reactivities to HIV-1 clade C antigens (rgp120, MA: matrix; NEF, TAT, PR: protease, RR: reverse transcriptase+RNaseH, IN: integrase) and peptides. Negative test results obtained with the InnoLIA IgG immunoblot for HIV-1 antigens gp120, gp41, integrase (IN), capsid (CA), matrix (MA) and HIV-2 antigens gp105, gp36 are shown on the right margin.

(13) FIG. 5A shows an amino acid sequence alignment showing identical amino acids (*), substitutions (.Math.) and gaps (−). The peptide length is indicated on the right margin as number of amino acids. The peptides aligned in FIG. 5A are AIVCTRPNNNTRKSIRIGPGQVFYT (SEQ ID No. 1) and NTRKSIRIGPGQTFY (SEQ ID No. 42; see Casseb et al. Braz J Med Biol Res 3S (2002): 369-37S).

(14) FIG. 5B shows the medium IgG serum reactivity towards the two peptides of FIG. 5A in African and European HIV-infected patients, expressed as optical density (OD) subtracted of the reactivity to the negative control.

(15) FIG. 5C shows the frequency (y-axes: number of reactive sera) of IgG responses of African (left) and European (right) patients to peptides having SEQ ID No. 1 and 42.

DETAILED DESCRIPTION

EXAMPLES

Example 1

(16) Materials and Methods:

(17) Synthesis of HIV-1 Clade C Envelope Overlapping Peptides

(18) Twenty-four and 17 peptides covering the complete amino acid sequences of gp120 and gp41 from the South African HIV-1 clade C reference strain (isolate ZA.04.04ZASK146, Los Alamos HIV sequence database accession no. AY772699), were produced by solid phase synthesis on a CEM-Liberty (CEM, USA) or Applied Biosystems peptide synthesizer (Life technologies, USA). All peptides were 25 amino acids long, except the gp120 and gp41 C-terminal peptides accounting for 33 and 32 amino acids respectively, and had an overlap of 5 amino acids (see the following table).

(19) HIV-1 clade C Envelope Peptides:

(20) TABLE-US-00001 Peptides Amino acid sequence SEQ ID No. Solvent gp120 peptides 120/1 RVRGILRNWPQWWIWGILGFWMIII  6 10% DMF 120/2 WMIIICRGEENSWVTVYYGVPVWTE  7 2% DMSO, 1 mM DTE 120/3 PVWTEAKTTLFCASDAKAYEKEVHN  8 PBS, 1 mM DTE 120/4 KEVHNVWATHAVPTDPSPQELVLE  9 H20, 1 mM DTE 120/5 ELVLENVTESFNMWENDMVDQMHED 10 H20, 9 mM NaOH 120/6 QMHEDIIGLWDESLKPCVKTLPLCV 11 H20, 1 mM DTE 120/7 TPLCVTLNCNTTSHNNSSPSPMTNC 12 5% ACN, 1 mM DTE 120/8 PMTNSFNATTELRDKTQKVNALFY 13 H20, 1 mM DTE 120/9 NALFYRSDIVPLEKNSSEYILINCN  3 2% DMSO, 3 mM NaOH, 1 mM DTE 120/10 LINNTSTITQAPKVSFDPIPIHY 14 5% DMSO, 1 mM DTE 120/11 IPIHYCAPAGYAILKCNNKTFNGTG 15 H20, 1 mM DTE 120/12 FNGTGPSNVSTVQTHGIKPVVST 16 H20, 1 mM DTE 120/13 PVVSTQLLLNGSLAEGEIIIRSENL 17 3 mM NaOH 120/14 RSENLTDNAKTIIVHLNKSVAIVCT 18 H20, 1 mM DTE 120/15 AIVCTRPNNNTRKSIRIGPGQVFYT  1 H20, 1 mM DTE 120/16 QVFYTNEIIGNIRQAHCNISRELWN 19 5% DMF, 1 mM DTE 120/17 RELWNNTLEQVKKKLKEHFQNKTIE 20 H20 120/18 NKTIEFQPPAGGDLEVTTHSFNCRG 21 H20, 1 mM DTE 120/19 FNCRGEFFYCNTSNLFNITASNASD 22 PBS, 2 mM NaOH, 120/20 SNASDANNNTITLPCKIKQIINMWQ 23 H20 120/21 INMWQEVGRAMYAPPIAGNITCNSS 24 10% ACN, 1 mM DTE 120/22 TCNSSITGLLLTRDGGNNNDTGNNN 25 H20, 1 mM DTE 120/23 TGNNNDTEIFRPGGGNMKDNWRSEL 26 H20 120/24 WRSELYKYKVVEIKPLGIAPTKAKRRVVEREKR  5 H20 gp41 peptides 41/1 AVGLGAVLLGFLGTAGSTMGAASIT 27 5% ACN 41/2 AASITLTVQARQLLSGIVQQQSNLL 28 5% DMF, 1 mM Acetic acid 41/3 QSNLLRAIEAQQHMLQLTVWGIKQL 29 H20 41/4 GIKQLQARVLAIERYLKDQQLLGLW  4 H20 41/5 LLGLWGCSGKLICTTAVHWNSSWSN  2 5% DMSO, 1 mM DTE 41/6 SSWSNKSQDYIWGNMTWMQWDREIN 30 H20, 3 mM NaOH 41/7 DREINNYTDIIYTLLEESQSQQEKN 31 H20, 6 mM NaOH 41/8 QQEKNEKDLLALDSWNNLWNWFSIT 32 H20, 3 mM NaOH 41/9 WFSITKWLWYIKIFIMIVGGLIGLR 33 2% DMSO 41/10 LIGLRIILGVLSIVKRVRQGYSPLS 34 10% ACN 41/11 YSPLSFQTLPPNPRGPDRLRGIEEE 35 H20 41/12 GIEEEGGEQDKDRSIRLVSGFLALV 36 H20 41/13 FLALVWEDLRSLC-LFSYHRLRDFIL 37 5% DMF 41/14 RDFILIAGRAAELLGRSSLRGLQTG 38 H20 41/15 GLQTGWQALKYLGSLVQYWGLELKK 39 5% ACN 41/16 LELKKSAINLFDTTAIVVAEGTDRL 40 2% DMSO, 2 mM NaOH 41/17 GTDRLIEGLQGIGRAIYNIPRRIRQGFEAALL 41 2% DMF

(21) The synthesis was performed with the 9-fluorenyl-methoxy-carbonyl (Fmoc)-method, using PEG-PS preloaded resins. Synthesized peptides were washed with dichloromethane, cleaved from the resins in a mixture of 19 ml trifluoroacetic acid, 500 ul silane and 500 ul H20 and precipitated into tertbutylmethylether. Peptides were separated from by-products by reverse-phase HPLC in an acetonitrile gradient (UltiMate 3000 Pump, Dionex, USA) to a purity >90% and their identity was verified by mass spectrometry (Microflex MALDI-TOF, Bruker, USA). The peptides' chemical features were predicted by ProtParam on the Expasy proteomics server and were considered in the optimization of the solubilization conditions. Highly hydrophobic peptides were solubilized in dimethylformamide (DMF), dimethylsulfoxide (DMSO) or acetonitrile (ACN), while reducing agents such as dithioerythritol (DTE) were added to peptides rich in Cysteins and NaOH to strongly acidic peptides.

(22) Expression and Purification of Recombinant HIV-1 Clade C Proteins

(23) Recombinant matrix (MA), capsid (CA), nucleocapsid (NC) proteins as well as accessory NEF, TAT and VIF and the pol-derived protease (PR), reverse transcriptase+RNAseH (RR) and integrase (IN) were expressed in Escherichia coli (E. coli). Briefly, the cDNA sequences of the structural and accessory proteins as well as of the protease were derived from the HIV-1 clade C reference strain from South Africa (isolate ZA.04.04ZASK146, Los Alamos HIV sequence database accession no. AY772699). The reverse transcriptase-RNAseH and the integrase constructs were derived from the Ethiopian HIV-1 clade C isolate ET.86.ETH2220 (Los Alamos HIV sequence database accession no. U46016). The cDNA of the proteins, followed by a hexa-histidine tag, was codon-optimized for bacterial expression and cloned into a pET17b vector (ATG:biosynthetics, Germany). Expression of the recombinant proteins in E. coli BL21 (DE3) cells, grown to an OD600=0.4−0.6 in LB medium supplemented with l O Omg/l ampicillin, was induced by addition of 0.5-1.0 mM isopropyl-β-thiogalactopyranoside (IPTG) and proteins were purified by Nickel-affinity chromatography under native or denaturing conditions. For purification under native conditions of matrix, capsid and nucleocapsid proteins, cells were lysed in 50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole pH 8.0, PMSF (1 μl/ml), Lysozyme (1 mg/ml) by sequential cycles of freezing and thawing; DNA was cleaved by treatment with 5 μg/ml DNaseI (20 min at room temperature); and, after removal of cellular debris (by centrifugation at 18000 rpm, 20 min, 4° C.), the clear lysate was incubated with Nickel-agarose (QIAGEN, Germany) 2-4 h at 4° C. Elution of the His-tagged proteins was performed with an imidazole gradient (20, 50, 100, 250 mM Imidazole) in 50 mM NaH2PO4, 300 mM NaCl pH 8.0. For purification of NEF, TAT and protease under denaturing conditions, cells were lysed in 8M Urea or 6M Guanidinium chloride buffer for at least 2 h or overnight at room temperature. After removal of cellular debris (by centrifugation at 18000 rpm, 20 min, 4° C.) and incubation of the clear lysate with Nickel-agarose (QIAGEN, Germany) (2-4 h at room temperature or overnight at 4° C.), a pH gradient (pH 6.3, 5.6, 4.5) was used to elute the recombinant proteins in 8M Urea, 100 mM NaH2PO4, 10 mM Tris. For purification of recombinant integrase, the cells were first lysed under native conditions as described above, and, since after centrifugation (18000 rpm, 20 min, 4° C.) the protein was found in the insoluble fraction, this was resuspended in 8M Urea, 100 mM NaH2PO4, 10 mM Tris, pH 8.0 and incubated at room temperature overnight. After removal of cellular debris (by a second step of centrifugation at 18000 rpm, 20 min, 4° C.), the recombinant integrase was purified under denaturing conditions in 8M Urea as described above. Purification of VIF and reverse transcriptase+RNAse was achieved by using an inclusion body preparation protocol. Removal of urea and refolding of the recombinant proteins purified under denaturing conditions were achieved by sequential dialysis steps.

(24) The identity of the proteins was verified by SDS-PAGE and Coomassie staining (FIG. 3A) as well as by Westernblot and by mass spectrometry. Their secondary structure and thermal stability were analyzed by Circular dichroism spectroscopy on a Jasko J-810 spectropolarimeter (Japan Spectroscopic, Japan). The biochemical features of the recombinant HIV-1 clade C proteins, predicted with the ProtParam software on the Expasy server and assessed experimentally as described above, are outlined in the following table.

(25) Biochemical Features of Recombinant HIV-1 Clade C Proteins:

(26) TABLE-US-00002 Migration MW 2 SOS-PAGE Secondary Thermal Proteins 1 kDa kDa pI 4 structure4 Stability 5 MA 15.5 17.5 9.1 α-helical > Tm = 65° C. β-sheet CA 26.5 24.0 6.6 α-helical Tm = 66° C. NC 7.2 11.0 10.2 β-sheet < n.d. random coil NEF 24.6 30.0 6.2 α-helical & n.d. β-sheet TAT 12.2 17.0 9.0 Random coil n.d. VIF 23.7 24.0 10.5 β-sheet > Tm > 95° C. α-helical PR 11.7 12.0 8.7 β-sheet & Tm = 55° C. random coil RR 65.1 65.0 6.8 α-helical & Tm > 95° C. β-sheet IN 33.2 33.5 7.4 α-helical & n.d. β-sheet
.sub.1Protein abbreviations: MA: matrix, CA: capsid, NC: nucleocapsid, PR: protease, RR: reverse transcriptase+RNAseH, IN: integrase. .sub.2MW: Molecular weight in kilo Daltons (kDa), as predicted with ProtParam and verified by mass spectrometry. .sub.3pI: Isoelectrical point predicted with ProtParam. .sub.4Secondary structure: determined by circular dichroism spectroscopy, predominance of alphahelical or beta-sheet elements. .sub.5Thermal stability: determined by circular dichroism spectroscopy; Tm: melting temperature; n.d.: not done.

(27) Study Subjects and Routine Immunoassays

(28) Sera were taken from 14 African HIV-infected patients, from 2 highly exposed African individuals, from 15 European HIV-infected patients and from 10 uninfected individuals. HIV sero-positivity and -negativity was confirmed for each of the subjects by routine analyses. HIV-specific IgG immunoblots were carried out by Line Immuno Assay (InnoLIA, Innogenetics, Bel-gium) and asymptomatic subjects were additionally tested with the Abbott Murex HIV Ag/Ab combination (Abbott, USA).

(29) HIV-1 Clade C Specific IgG, IgA and IgM Determinations by ELISA

(30) ELISA plates (Nunc Maxisorp, Thermo Fisher Scientific, USA) were coated overnight at 4° C. with the HIV-1 clade C derived peptides and proteins diluted in 100 mM sodium bicarbonate buffer pH 9.6 (2 ug/ml). After washing in PBS, 0.05% v/v Tween20 and blocking in 2% w/v BSA, PBS, 0.05% v/v Tween20 for at least 4 h at room temperature, the plates were incubated with sera diluted 1:200 in 0.5% w/v BSA, PBS, 0.05% v/v Tween20 overnight at 4° C. Bound antibodies were detected with mouse anti-human IgG1, IgG2, IgG4, IgA, IgM (BD, 1:1000, 2 h at room temperature) or mouse anti-human IgG3 (1:5000, 2 h at room temperature, Sigma Aldrich, St. Louis, Mo.) and horseradish peroxidase (HRP)-coupled sheep anti-mouse IgG (1:2000, 1 h at room temperature, GE Healthcare, Waukesha, Wis.). Total IgG antibodies were detected with a directly labelled HRP-anti human IgG (1:5000, 1 h at room temperature, GE Healthcare, USA). The color reaction was induced with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) di-ammonium salt and the optical density (OD405 nm-OD490 nm) was measured on a Spectra Max spectrophotometer (Molecular Devices, USA). As control antigen for the analysis of envelope-specific reactivity, we used recombinant His-tagged gp120 from HIV-1 clade C, isolate CN54, expressed in 293 cells (#11233-VO SH, Sino Biological, China). For interplate normalization we analyzed on each plate a positive control serum specific for each antibody isotype/subclass. Additionally, reactivity of each serum sample to human serum albumin (HSA) was tested as negative control. Coating of the antigens was verified by detection of His-tagged proteins with a mouse anti-His-tag antibody (Dianova, Germany) and by detection of untagged proteins and peptides with a pool of HIV-infected sera. Unspecific binding of the detection antibodies to coated antigens was excluded by buffer control analyses. All determinations were carried out as duplicates and results are shown as normalized means of the raw data subtracted by the reactivity to the negative control antigen. Cut-off values were calculated for each antibody isotype/subclass and for each antigen as the mean+3SD of the results of the HIV uninfected subjects.

(31) Sequence Alignments

(32) For multiple sequence alignments, the amino acid sequence identity was calculated with ClustalW2 on the EMBL-EBI server and alignments were generated with the program Gene Doc. Sequences are numbered accordingly to the international HXB2 numbering scheme, with amino acid insertions carrying the number of the last corresponding HXB2 amino acid followed by sequential letters.

(33) Results

(34) Almost Identical IgG, IgA and IgM Responses Towards gp120 Epitopes from HIV-1 Clade C in HIV-Infected Patients from Africa and Europe

(35) For a detailed epitope mapping of gp120, sera from HIV-1-infected patients from Zimbabwe were tested, where clade C is the predominant HIV-1 subtype, and from European patients infected with HIV-1 strains from different clades for antibody reactivity with recombinant gp120 and 24 gp120-derived overlapping peptides. These peptides covered the complete amino acid sequence of gp120 derived from the South African HIV-1 subtype C reference strain (FIG. 1A, B). IgG, IgA, IgM and IgG1-4 subclass reactivities were measured.

(36) Interestingly, IgG, IgG subclass, IgA and IgM response pro-files towards the peptides were almost identical in the African (FIG. 1A, left panel) and in the European (FIG. 1A, right panel) HIV-infected patients. The recognition pattern of the peptides was similar for all antibody classes and the IgG subclasses. However, the analysis of antibody levels and frequencies of recognition showed that IgG and in particular IgG1 responses dominated in both populations. Peptide 120/15 was the major antibody-reactive peptide in terms of frequency and intensity of recognition for the African and European patients (FIG. 1A). Peptide 120/24 was also recognized by a majority of the individuals tested. The overview of gp120 (FIG. 1B) shows that peptide 120/15 resides in the V3 domain, which is supposed to contain a binding site for the co-receptor CCR5/CXCR4 on CD4 cells. Pep-tide 120/24 is located at the very C-terminal end of gp120.

(37) FIG. 1C contains a sequence alignment of peptide 120/15 with the corresponding peptides in various HIV-1 reference strains from different continents. A considerable degree of sequence variation of the peptide 120/15-corresponding regions was found among the strains, ranging from 88 to 72% sequence identity. De-pending on the strain, the peptide 120/15-defined region contains one or two N-linked glycosylation sites.

(38) African and European Patients Recognize Highly Similar Epitopes on gp41 from HIV-1 Clade C

(39) Next, the IgG, IgA and IgM responses of the African and European patients to 17 overlapping peptides derived from HIV-1 clade C gp41 was analyzed (FIGS. 2A, B). Again it was found that the African and European patients recognized similar peptides and that the immune response was dominated by IgG and in particular by IgG1 antibodies. Both the frequency and the intensity of recognition were lower towards the gp41 peptides than to the gp120-derived peptides. IgG and IgG1 antibodies were directed mainly to a region defined by peptides 41/4-8 (FIG. 2A). This region includes areas, which have been designated as “immunosuppressive” and “immunodominant” domains and contain the majority of the predicted N-linked glycosylation sites of gp41 (FIGS. 2A, B). Interestingly, the other antibody-reactive area defined by peptides 41/14-17 was located at the C-terminal portion of gp41, which is part of the so-called “cytoplasmic domain”.

(40) African and European HIV-Infected Patients React Primarily with Structural and Pol-Derived but not with Accessory Proteins

(41) In order to characterize the antibody response directed to viral proteins other than the surface antigens, HIV-1 clade C structural, pol-derived and accessory proteins were expressed in E. coli and purified and the protein-specific antibody levels were measured in African (FIG. 3B, left panel) and European sera (FIG. 3B, right panel). Similar as for the envelope proteins, the immune response was dominated by IgG and in particular by IgG1 antibodies. Again patients of both populations showed a similar recognition profile: The protease, reverse transcriptase+RNAseH, integrase, as well as the capsid protein and the matrix protein were the most frequently and strongly recognized antigens. Antibody responses towards the nucleocapsid protein and the accessory proteins were rare and low.

(42) IgG Subclass Recognition of HIV Proteins and Peptides is Indicative of Mixed Th1/Th2 Immune Response

(43) The measurements of IgG subclass responses towards the gp120 and gp41 peptides as well as against the structural, poi-derived and accessory proteins were performed using the same serum dilution. Since IgG1 represents the dominating IgG subclass, IgG1 responses were more intense and frequent than IgG2, IgG3 and IgG4 responses. However, the antigen and epitope recognition profile was similar for all subclasses in the African and European patients. The frequencies and intensities of IgG2 and IgG4 responses were comparable, which is indicative of a mixed Th1/Th2 immune response.

(44) High Sensitivity and Specificity of Diagnostic Tests Based on Recombinant Proteins and Peptides Assembling the Clade C Proteome

(45) The comprehensive analysis of antibody responses showed that the panel of HIV-1 clade C-derived antigens and peptides allowed the reliable detection of specific IgG antibodies in each of the HIV-infected patients from Africa and Europe. No false positive test results were obtained when sera from uninfected individuals were tested. Peptide 120/15 was as good as complete rgp120 for IgG-based diagnosis of HIV-infected patients because it allowed identifying 29 out of the 29 patients. Likewise, peptide 41/5 allowed identifying 29 out of 29 patients whereas peptide 120/24 was positive in 21 patients. Testing for IgG reactivity to the capsid protein identified 29/29, to reverse transcriptase+RNAseH 27/29, to integrase 27/29 and to protease 26/29 patients. Peptides 120/15 and 41/5 can therefore be used alone to diagnose HIV infections in individuals. Using a panel of peptides 120/15, 120/24, rgp120, capsid and pol-derived proteins each of the infected patients was diagnosed by IgG testing.

(46) Furthermore, it was found that HIV-highly exposed African individuals with an infection, who were negative in routine antigen/IgG+IgM determinations (Abbott Murex HIV Ag/Ab combination, Abbott, USA) as well as in Immunoblot-based assays (InnoLIA, Innogenetics, Belgium; shown in FIG. 4, right margin), could be diagnosed by IgG and IgM testing to the panel of HIV-1 clade C-derived antigens and peptides (FIG. 4).

Example 2

(47) In this example the reactivity of the peptides having the amino acid sequence AIVCTRPNNNTRKSIRIGPGQVFYT (SEQ ID No. 1; u120/15″) and NTRKSIRIGPGQTFY (SEQ ID No. 42; “Cons C”, see Casseb et al. Braz J Med Biol Res 35 (2002): 369-375) with serum of HIV infected individuals is examined. The results of this example are depicted in FIG. 5.

(48) FIG. 5A shows a comparison of the amino acid sequences of peptide 120/15 and the consensus peptide Cons C used by Casseb et al. 2002, which has been made to incorporate a Threonine in position 13 to resemble the sequences of most of the subtype C sequences, whereas 120/15 contains a Valine in this position. Despite the fact that the sequence of the consensus peptide was modified to resemble the amino acid sequence of most of the sub-type C sequences, 11 out of 29 HIV-positive serum samples did not react with this peptide and hence were false negative in the diagnostic test, whereas peptide 120/15 allowed to identify each of the 29 HIV-positive serum samples (see Fig. SC).

(49) Materials and Methods

(50) The peptide “Cons C” which was used by Casseb et al. 2002, was manufactured using solid phase peptide synthesis, as described for the gp120-derived peptides (see example 1). For ELISA experiments the peptides were solubilized in H20, and the immunological assays were performed as described for the gp120-derived peptides (see example 1).