FI99115B - Peptides which react with antibodies directed against an antigen which is bound at T4 receptors, and the diagnostic use thereof - Google Patents

Description

99115 Peptides Reactive with Tactors Targeting T-4 Receptor Binding Antigen and Their Diagnostic Use 5 Divided by application from FI 885630.

Brief Description of the Invention This invention relates to synthetically produced short peptide sequences that induce the production of antibodies to HTLV-III / 10 LAV, or HIV envelope protein. Consequently, these peptides and monoclonal antibodies raised against them could be used as diagnostic agents for the identification of the HIV virus. Therefore, peptides and antibodies directed against the peptides could have use in the preparation of test kits for the identification of HIV carriers or individuals with AIDS.

Background of the invention

Several researchers have reported the complete nucleic acid sequence of the AIDS (HIV) virus. (See Lee

Ratner et al., Nature 313, p. 277, January 1985; Muesing et al., Nature 313, p. 450, February 1985; and Wain-Habson. and others Cell 40, pp. 9-17, January 1985.) The envelope bed is specifically associated with antigenic cause and infectivity. The envelope region is also known to include regions that are highly variable. The HIV viral envelope glycoprotein has been shown to be covalently bound to human, rat and monkey brain membranes and immune system cells.

30 Understanding that viruses can show selectivity for cells and tissues by attaching to cells; highly specific regions of membrane surface receptors, has encouraged researchers to look for agents that: could bind to viral receptor sites on cell membranes and thus prevent a particular virus from binding to these cell membranes. Hin. It has previously been shown (Epstein et al., Nature 318, pp. 663-667) that infectivity mediated by specific receptors of the vaccinia virus can be shown to be ineffective with synthetic peptides.

The HIV virus has been shown to bind to a surface molecule known as the CD4 or T4 region, which is present in many different cells susceptible to HIV infection, including T lymphocytes and crude phage. (See Shaw et al. [Science 226, pp. 1165-1171] for a review of the selectivity of HTLV-10 lii).

In addition to the symptoms of immunodeficiency, AlDS patients show neuropsychological damage. The central nervous system and immune system share a large number of specific cell surface recognition molecules that act as receptors for intercellular communication mediated by neuropeptides. Neuropeptides and their receptors have been shown to be remarkably stable during development and have remained in almost unchanged form in unicellular organisms as well as in higher animals. In addition to Li-20, common DC4 (T4) cell surface recognition molecules that act as receptors for the HIV envelope glycoprotein (gp120) are detected in the central nervous system and immune system. Because the same largely conserved neuropeptide transporters combine immune and • · 25 brain function with receptors that are substantially similar to HIV receptors, it has been argued that the very similar amino acid sequence that] · * HIV glycoprotein 120: 11a with a short peptide previously identified elsewhere in the envelope region of Ep-Stein-Barr virus may indicate. the existence of a nuclear nucleus peptide that is essential; rus receptor binding. It was suggested that such peptides that bind to receptor sites would result in the production of antibodies directed against peptide sequences, and that these peptides could be used to provide imaging. an immunological basis for diagnosing AIDS.

»M

«· • · 3 99115

Object of the present invention was to provide diagnostic means for detecting antibodies to HIV or HIV envelope protein.

5 Detailed description of the invention

The octapeptide in the HIV envelope glycoprotein (gp120) was identified using computer-assisted analysis. This peptide, designated “peptide T” because of its high threonine content, has been shown to inhibit the binding of gp 10 120 to brain membranes.The amino acid sequence of the peptide is Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr. Subsequent Analysis revealed similar types of pentapeptides with similar binding properties.

The peptide of the present invention is characterized in that it has the formula I: X-R1-Ser-Thr-Thr-Thr-Asn-Tyr-Rb-X (I) in which R1 is an amino-terminal end group A- or D- Area-,

Rb is the carboxy-terminal end group -Thr or -Thr-amide,

And X is Cys or one or both Xs are absent, or have the formula II: 1 25 «X-R1-R2-R3-R4-R1-X (II) • 99 *: 1 wherein: T: R1 is an amino-terminal end group Thr, Ser, Asn, Leu, Ile, Arg or Glu, R2 is Thr, Ser or Asp, • · · R3 is Thr, Ser, Asn, Arg, Gln, Lys or Trp, «· R4 is Tyr, and • 1: R5 is the terminal amino acid group of the carboxy terminus, the corresponding * · · · ... 1 35 D-amino acid, the corresponding amide derivative or R5 is absent, and • · 9 • · • · * · · • »·, 99115 X is Cys or one or both Xs are absent, provided, however, that the peptide is not Ser-Thr-Asn-Tyr-Thr, or physiologically acceptable salts thereof.

R5 is preferably a carboxy-terminal end group -Thr, Arg or Gly or a derivative thereof. A peptide terminating in R4 (tyrosine) retains the binding properties of the group discussed herein. The active compounds of this invention may exist as physiologically acceptable salts of the peptides.

Peptides in this group have been found to bind to T4 viral receptors.

The most preferred peptides, as well as the peptide T described above, are the following octa-15 peptides of formula (I): D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr and D-Ala-Ser-Thr-Thr-Thr -Asn-Tyr-Thr-amide and the following pentapeptides of formula (II):

Thr-Asp-Asn-Tyr-Thr 20 Thr-Thr-Ser-Tyr-Thr

Thr-Thr-Asn-Tyr-Thr and analogs thereof having an amino-terminal D-Thr and / or a carboxy-terminal amide derivative.

. ' The peptides disclosed herein may also be modified to be spatially limited to improve stability.

The following abbreviations are used:. ’Amino acid Three-letter One-letter ··· code code» ·

30 arginine arg R

asparagine asn N

aspartic acid asp D

• · kystelini cys C

. ·. glycine Gly G

35 serine ser S

*

Γ threonine thr T

tyrosine tyr Y

5 99115

Unless otherwise indicated, the amino acids are, of course, in their natural L-stereoisomeric forms.

A comparison of the amino acid sequences of the twelve pentapeptides is shown in Table 1. Although historically, our initial computer search revealed peptide T (a peptide contained in ARV isolation) to be the relevant sequence, as new viral amino acid sequences became available, the bioactive sequence could be shorter. 10 consists of peptide T (4-8), or the sequence TTNYT. In the isolations we compared (Table 1), significant homology was observed only in this shorter range. Most of the changes are the exchange of serine (S) and threonine (T), two closely resembling amino acids, in one to 15 years. Tyrosine at position 7 of peptide T is an unchanged feature of all these forms, suggesting that it may be necessary for bioactivity. The substitution events occurring at position 5 include T, Gtn, R, and S. Positions 4 and 6 were first limited to (except for one exception) S, T, and N, all of which are amino acids with uncharged polar, closely related in space structure. element. The determination of the uniformity of the sequences between the five different AIDS virus isolation preparations reveals that the region adjacent to the peptide T sequence, to which it itself belongs, is highly variable. Such variability may indicate the development of sub-strains through an increase in the diversity of a strong activity (s) that is susceptible to selection events and located in this region.

Like opiate peptides, these peptide T analogs show *: *. occur in many forms, resembling met and% · Leu encephalins. These pentapeptide sequences, which are present in these various AIDS virus isolation preparations, are biologically active and capable of interacting with • · · · mm · «· 1 · * f · ·« · 6 99115

Been CD4 receptor - formerly more widely known as a T-helper cell surface "rougher" property - as an antagonist.

Table 1. Comparison of the ENV sequence from several AIDS virus isolation ^ 5 preparations

Isolation Section Section Peptide T TESTED Pert, C.B., and others PNAS (w / w) 10 * VALUE (195-199) Tested Willey, R.L. and others LAV TTSYT PNAS 83, p.5083, 1986

Z3 SSTYR

NY5 NTSYT

15 BIO (HTLV-III) TTSYT Starcich, B.R., and others WMJ-1 SSTYR Cell, 45, p. 637, 1986

HAT-3 NTSYG

Successive insulation products STNYR

20 WMJ-1 SSTYR Hahn, B.L. and others WMJ-2 SSRYR Science 232 p. 1548, WMJ-3 SSTYR 1986

· ': TTSYS

:: 25 1 The numbers refer to the relative positions of the amino acids in the ARV env sequence (9).

The seven amino acid peptide CYS-THR-.:. THR-ASN-TYR-THR-CYS is also active. Addition of cysteines ··. the core cycle does not significantly impair activity. Pep- • · 30 tids were synthesized as a service provided by Peninsula Laboratories under a confidentiality agreement between the inventors and the manufacturer. ‘Merrifield solid phase peptide synthesis was used. (See U.S. Patent No. 3,531,1258, which is incorporated herein by reference.) The peptides synthesized are in the primary position of • 7 · m ··· * ··· • · • · ···. Although peptide T and the pentapeptide included in it could be isolated from a virus, the peptides synthesized according to Merrifield do not contain residues from cells or viruses. As a result, reactions to site 5 contamination do not occur when synthesized peptides are used.

The peptides of this invention can be produced by conventional peptide synthesis methods. Both solid and liquid phase methods can be used. We have ha-10 assumed the solid phase of Merrifield to be particularly useful. In this method, the peptide is synthesized stepwise with the carboxy terminus of the chain covalently attached to an insoluble support. During the intermediate steps of the synthesis, the peptide remains in the solid phase and is thus easily handled. The solid support phase is a chloromethylated styrene-divinylbenzene copolymer.

The N-protected form of the terminal amino acid of the carboxy terminus, e.g., the t-butoxycarbonyl (Boc-) -protected amino acid, is reacted with the chloromethyl group of the chloromethylated styrene-divinylbenzene-20-copolymer resin as a bimethyl ester to give the protected aminoacyl. This is deprotected and reacted with the next protected form of the amino acid to give the protected dipeptide attached to the resin. The amino acid is usually used in its activated form, e.g. using a carbodiimide or an active ester. These steps are repeated and the peptide chain grows group by group by condensation at its amino terminus with the desired N-protected amino acids 30 until the desired peptide is assembled on a support. The peptide resin is then treated with anhydrous hydrofluoric acid, which. cleaves the ester bond connecting the assembled peptide to the carrier to release the desired peptide. At the same time, the functional groups of the amino acid side chains which have had to be protected during the synthesis steps can be liberated by conventional methods. The synthesis of a peptide containing an amide group at its carboxy terminus can be carried out conventionally using 4-methylbenzhydrylamine resin.

Another object of the present invention relates to test kits for detecting AIDS viruses, the antigen source of which comprises a peptide of the present invention. The assay kit of the invention for determining antibodies to an antigen that binds to the T4 receptor is characterized in that it comprises a peptide of the invention bound to a porous surface or solid support. For example, a peptide of this invention can be used in a test kit to detect AIDS infection and the diagnosis of AIDS and pre-AIDS conditions by using it as a test reagent in an enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunostaining assay. Such assay kits may include an insoluble porous surface or solid support to which the antigenic peptide has been pre-adsorbed or covalently attached, such surface or support being primarily in the form of a microtiter plate or wells; test sera; secondary antisera that specifically bind to and saturate the antigen or antibody adsorbed on the surface or carrier; several different diluents and buffer solutions; labeled conjugates to detect specifically associated antibodies; and mold signaling reagents such as enzyme substrates, cofactors, and chromogens.

: The present invention further relates to the use of a peptide of the invention in a diagnostic method for detecting human immunodeficiency virus (HIV) infection. a body fluid or tissue useful for diagnosing acquired immune deficiency syndrome (AIDS) or a precursor thereof, wherein 9 99115 a) reacting a sample with said antigenic peptide, b) forming an antigen-antibody complex, and c) detecting the formation of an antigen-antibody complex; antigen-antibody complex.

The peptide of this invention can also be used as an immunogen by conventional methods to induce the production of monoclonal antibodies that specifically bind to appropriate regions in the envelope region of the AIDS virus.

Experimental methods and test results

Radiolabeling of gp120, preparation of membranes from the brain, binding and cross-linking of gp120 to the receptor, and immunoprecipitation of T4 antigen 15 strains of HIV HTLV-IIIb were added in H9 cells and gp120 was isolated by immunoaffinity chromatography and pre-preparative NaD Using PAGE. Purified gp120 was labeled with 125 I by the chloramine-T method.

Fresh human, monkey, and rat cerebellum were rapidly homogenized (Polytron, Brinkmann Instruments) in 100 volumes of ice-cold Hepes (pH 7.4) buffer. The membranes collected by centrifugation (15,000 x g) were washed in a volume of the original buffer solution and used fresh or stored at -70 ° C. Brain membranes and highly purified T cells (Ref. 16; donation from Larry Wahl) were preincubated for 15-30 min in phosphate buffered saline (PBS) before use. A 20 mg aliquot (wet weight at the beginning) of brain-derived membranes (approximately 100 pg protein *) was incubated with a 28,000 cpm aliquot of 125 I-gp 120 for 30 hours at 37 ° C. 200 μl (final volume) of 50 mM Hepes buffer containing 0.1% bovine serum: 1; min albumin and the peptidase inhibitors bacitracin (0.005%), aprotinin (0.005%), leupeptin (0.001%) and ·· · chymostatin (0.001%). The incubation mixtures were quickly filtered under vacuum and the radioactivity was counted to determine the amount of material attached to the receptor.

Lymphocyte Activation

Immunoprecipitates were prepared by incubating (overnight at 4 ° C) with 0.5% Triton-X-100 / PBS solubilized lactoperoxidase / glucose oxidase / 125 I iodinated brain membranes or intact T cells against the indicated monoclonal antibodies. with the substances used at 10 μg per reaction. Solid phase immunoadsorbent 10 (Immunobeads, Bio-Rad) was used to precipitate immune complexes before separation with NaDod. Control incubations did not contain primary monoclonal antibodies or contained a subset of control monoclonal antibody (ΟΚΤβ).

15 Chemical Nerve Anatomy and Computer-Aided Density Measurement 25 μm cryostat sections from fresh frozen human, monkey, and rat brains were thawed and dried on gelatin-coated slides, and receptors were detected as described in reference (18). Incubations without antibodies to T4, T4A, T8 and T11 (10 μg / ml) or. these were performed overnight at 0 ° C in RPMI solution, cross-linked to their antigens and challenged with 125 I-labeled goat mouse antibody. Incubations of slide sections attached to slides to label antigen receptor 12SI-gp120 were performed in 5 ml slide wells with or without unlabeled gp120 (1 μM) or with 30 0KT4A monoclonal antibody (10 μg / ml). ) (Ort-. Ho Diagnostics) or without this.

; ·. Separation of T lymphocyte subgroups • T cell subgroups were obtained by treating peripheral circulating T cells purified in a Percoll density gradient with specific monoclonal antibodies (T4 or »· · • · 1 m · • ψ • · 11 99115 T8) with the concentration was 10 pg / ml. The treated cells were allowed to stand for 30 min at 4 ° C in a plastic petri dish fixed with immunoglobulin against goat [F (ab ') 2] mouse (Sero Lab, Eastbury, MA).

The remaining cells were then removed and washed and examined for reactivity with a flow cytometer. Separated T4 and T8 cell populations have other T cell subsets at <5% contamination level. The cells were then cultured in the presence of phytoheragglutinin (1 pg / ml) and exposed to HIV as described below. The nature of the appearance of HIV-bound cells was elucidated when cell toxicity assays were performed.

VIruss1toutuminen

The HTLV virus used for binding was isolated from a cultured T cell line requiring in-15 terleukin 2 (IL-2) isolated from fresh AIDS patient material and inoculated into HuT 78, a permissive cell line that is independent of IL-2. 2.

Description of the Drawings Figure IA shows the cross-linking of 125 I-gp120 to brain membranes and T cells. (a) 125 I-gp 120 alone; (b) monkeys; (c) rat; (d) the human brain; and (e) human T cells.

Figures IB and 1C show the immunoprecipitation of 125 I-labeled monkey brain membranes and human T cells, respectively; (f, i) a control without primary antibodies; (g, j) monoclonal antibody 0KT4; (h, k) 0KT8 monoclonal antibody.

: Figure 2A shows the displacement of specific 125 I-gp120 binding to fresh rat cerebellar membranes. Each assay was performed in triplicate; results. one experiment giving similar results three times is shown. The degree of specific binding that was displaceable by 10 pg / ml 0KT4 and 4A ranged from 27 to 85% of the total binding, which in the experiment shown was 2201 ± 74 cpm.

* 1 · * 12 99115

Figure 2B shows that peptide T and its synthetic analogs render viral binding ineffective. Each assay was performed in two parallel experiments. The results represent one experiment repeated three times with similar results.

Example 1 Binding of 125 I-gp120 specifically to skull monkey, rat or human brain membranes yields one radiolabeled, approximately 180 Kd cross-linked 10 product that is indistinguishable from the corresponding product bound to human T cells (Figure 1A). ). This result indicates that gp120 can be coupled to a protein of about 60 Kd; the unresponsive I25I-gp 120 travels next to the membrane-free control (channel a).

Immunoprecipitation of radiolabeled human brain membranes with 0KT4 and 0KT8 (10 pg / ml) (Figure IB) shows that the brain membranes contain T4 antigen of approximately 60 Kd in size and cannot be distinguished from human T-antigen. the corresponding 20 antigens identified from lymphocytes (Figure 1C); in contrast, immunoprecipitation with 0KT8 precipitates a low molecular weight (approximately 30 Kd) protein from T lymphocytes (Fig. 1C) that is not present in brain membranes (Fig. IB), suggesting that • · ·· '; that brain T4 is not derived from these localized • »* * 25 lymphocytes. Similar results (data not shown) were obtained using monkey and rat cerebellar membranes- * ,! by. These results indicate that the T4 antigen acts as a || * viral receptor and is a well-conserved 60 Kd molecule common to the immune system and central sensory 30.

y. The finding that Epstein-Barr and HTLV- »« · m-ym III / LAV have nearly identical octapeptide sequences led to the synthesis and study of "peptide T". Figure 2 shows, • ·; that the binding of 125 I-gp 120 to freshly prepared rat 35 brain membranes occurs with high affinity (0.1 »· •% * · 13 99115 nM range) and that this is saturable. Specificity (Figure 2B) is indicated by inhibition by 0KT4 and 0KT4A, but not by 0KT3 (0.1 pg / ml). Peptide T and two of its synthetic analogs significantly inhibited 125 I-gp120 binding in the 0.1 nM range (Figure 2C) (but this was not done by the non-associated octapeptide substance P (1-8)). D-threoninamide substitution at position 8 resulted in at least a 100-fold loss of receptor binding activity. The substitution of classical (L-domain) by (D-domain) 10 resulted in a permanently more potent, apparently more peptidase-resistant analog than peptide T; The C-terminal terminal threonine produces a permanently somewhat increased potency (Figure 3).

When the ability of synthetic peptides to prevent receptor binding in human T cells was tested, the experimenter was unaware of the results of the binding experiments. At level 10’7, the three peptides active in the binding assay reduce the detectable level of reverse transcriptase by almost 9-fold. The less active binding displacement (D-thre) peptide T also proved to be greatly impaired in inhibition of binding and was required 100-fold to achieve significant inhibition. higher concentration. Thus, not only the order between the classes of the four peptides ((D- (Alah peptide T-amide> (D-25 (Ala ^ peptide T> peptide T> (D- (Thr) 8-peptide T-amide),. but also the inhibition of their binding to the receptor is closely correlated with each other (Fig. 3).

Example 2 * A protein of about 60 Kd, similar if not identical to the T4 antigen of human T cells, was apparently present in preserved molecular form in membranes prepared from human *: brain; in addition, the radiolabeled HIV envelope glycoprotein (125I-gp120) can be covalently cross-linked to three mammalian molecules that can be distinguished by their size and in vitro precipitation properties. T4 antlgeenista. Using a method of detecting antibody-binding receptors in brain sections, a pattern formed by the neural anatomical distribution of brain T4 in the end network of the henr.coat of the ti-5 hay bark and presented analogously in all three mammals was presented.

Radiolabeled HIV envelope glycoprotein binding also produced a similar character in successive sections, suggesting that T4 was the 10 HIV receptor.

Example 3

Chemical nerve anatomy, computer-assisted ti-density measurement

25 micron cryostat sections of fresh frozen human, monkey, and rat brains were thawed and dried on gel-coated slides, and the receptors were detected as described in Herkenham and Pert, J. Neurosci., 2, pp. 1129-1149 (1982). Incubations with and without antibodies to T4, T4A, T8, and 20 T11 (10 pg / ml) were performed overnight at 0 ° C in RPM1, cross-linked to their antigens, and detected. ·. with an antibody directed against the IJ5I goat mouse.

Incubations of slide sections attached to the slide for antigen / receptor labeling with 1J5I-gp120 were performed in 5 ml slide vials using 10'6 unlabeled gp120 or OKT4A (10 μg / ml). ml) monoclonal antibody or omitting these *: as described above for the use of membranes.

30 A computer-aided conversion of autoradiographic film to quantitative color images was performed. Exposure in parallel with monkey brain sections using standards with known radioactivity enhancement resulted in a linear plot (4 => 0.99) of ·· * 35 log OD and cpm, of which the radioactivity ratio • · 15 99115 can be meaningfully extrapolated. Staining of alvolelkelt cells was performed by classical methods and the receptors were detected in the stained tissue.

5 Example 4

Experiments have been performed to determine the distribution of T4 antigen in a series extending from the anterior to posterior part of the skull monkey brain or in horizontal sections. These experiments show that the binding of T4 monoclonal antibody to areas relevant to the cellular architecture of the brainstem (e.g., substantia nigra) occurs at detectably high levels, but a striking cortex-enriching presence is evident at each level of the germ axis. 0KT8, a monoclonal antibody directed against T-lymphocyte from the same subgroup as 0KT4, does not form any recognizable character. The uppermost surface layers of the cerebral cortex generally contain the densest concentration of T4 antigen; the frontal lobe of the cerebral cortex and the portion of the cortical layer surrounding the limbis-20 system above the tonsil nucleus contain a particularly large number of receptors that are localized throughout the inner layers. The densest concentration of receptors targets the area of edema in the monkey, rat, and human brains. Dark field microscopy of skull monkey sections dipped in film development solution 25 revealed that the zone of densest labeling of receptors is located in the molecular layer of the tooth fold and in the cortex itself (which contains very few neurons). Thus, the receptors appear to be precisely located to cover the terminal network of neural causes (nerve extensions of nerve terminals and exporting branches) or to locate. to a specific subset of unstained star scaffold cells.

: Assays have been performed that demonstrate the specificity of the chemical neural anatomy and the results obtained * * 9999115 show that T4 and the viral envelope protein recognition molecule do not differ. Horizontal sections of rat brain revealed a very similar receptor distribution focused on the cerebral cortex / α1-5 of the large brain, regardless of whether 0KT4 or 12SI-gp120 was used for detection. In the presence of 0KT4A (10 pg / ml) or 0KT4 (10 pg / ml). No identifiable figure was obtained from rat brain with other murine monoclonal antibodies directed against other human T cell antigens, including 0KT8 and 0KT11, when detected with 1J5I goat anti-mouse XgG secondary antibody, as well as no reproducibly detectable antigen / receptor was found using the 15 secondary antibody alone.

• · • · • · 9 · 9 9 9 9 9 9 • · • · 9 • 999 9 9 99 ··· • · • * * · · ··· • · · · · I · • · · · · T · · · • · · • · • 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

Claims (10)

17 99115 A peptide, a known elite, not having the formula I: wherein X 1 is X-R 2 -Ser-Thr-Thr-Thr-Asn-Tyr-F 2 -X (I) in which R 1 is the aminopene end group Ala- or D-Ala-, Rb is the carboxy-pSSteryhmS -Thr or -Thr-amide, and X is Cye or one or both of the Xs are absent, or the peptide has the formula II: X-R1-R1-R1-R4-Re-X (II) in which R1 is an aminopene ether group Thr, Ser, Asn, Leu, Ile, Arg or Glu, R1 is Thr, Ser or Asp, R3 is Thr, Ser, Asn, Arg, Gln, Lys or Trp, R4 is Tyr, and R9 is the carboxypene pSAteamyl amino acid group A, corresponding .1. D-amino acid, corresponding amide derivative or R9 is absent , and • · · '' 25 X is Cys or one or both Xs are absent, provided that the peptide is not Ser-Thr- • · V Asn-Tyr-Thr, or a physiologically acceptable salt thereof. T: A peptide according to claim 1, characterized in that it is not a peptide of formula II, wherein *: ·. R1 is Thr, Ser or Asn, R3 is Thr, Ser, Asn or Arg, and R9 is -Thr, -Arg or -Gly. 999:: 999 9 999 • · ··· • · · • 99 9 · 99115 1β A peptide according to claim 1, characterized in that it is not a peptide of formula II, rivers a R * are absent. The peptide according to claim 1, characterized in that it is Cys-R1-R, -RJ-R4-R5-Cys. Peptide according to Claim 1, characterized in that it is Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, Thr-Thr-Asn-Tyr-Thr, 10 Ser-Ser-Thr-Tyr-Arg, Asn-Thr-Ser-Tyr-Thr, Thr-Thr-Ser-Tyr-Thr Asn-Thr-Ser-Tyx-Gly, Ser-Thr-Asn-Tyr-Arg, 15 Ser-Ser-Arg-Tyr-Arg, Thr-Thr-Ser-Tyr-Ser, or Cys-Thr-Thr-Asn-Tyr-Thr-Cys. A test kit for the determination of antibodies directed against an antigen which binds to the T4 receptor, characterized in that it comprises a peptide of claim 1 bound to a porous surface or fixed on a solid support. 7. A test kit according to claim 6, characterized in that the antigenic peptide is bound to the walls of a microtiter plate. Use of peptics according to any one of claims 1 to 5 in a diagnostic method for detecting human immunodeficiency virus (HIV) infection in a body fluid or tissue, which method is useful for diagnosing acquired immune deficiency syndrome (AIDS) or a precursor thereof, wherein; . a) reacting the sample with said antigenic * «peptide, l: b) forming an antigen-1 antibody complex, and * * 35 c) detecting the formation of an antigen-antibody complex. 19 99115 Use according to claim 8, characterized in that the peptide is used in a diagnostic method in which the antigen-antibody complex is determined by an immunoassay method. Use according to claim 9, characterized in that the peptide is used in a diagnostic method, wherein the antigen-antibody complex is determined by a radyl immunoassay method, an enzyme-linked immunoassay method or an immunoblot method. • · • · • · ··· • · · • »· * + · • · • · • · · · · · · · ··· 9 ···· 9 * 9 9 · · • ♦ 9 99 9 9 9 99 9 999 9 9 9 9 9 9 9 9 9 · 9 9 · 9 9 · 999 · 999 i: • · · • · »* · • * 999 9 9 9 9 9 9 99 9 9 20 99115