GB2313376A - Polypeptide immunologically cross-reactive with NTM peptide and with vasoactive intestinal peptide (VIP) - Google Patents

Abstract

A polypeptide of the general formula: X.R1.R2.D.N.Y wherein: ```R1 is F or W; ```R2 is T or S; ```X and Y are either amino acid radicals or peptide sequences comprising from 2 to 15 amino acid radicals; (i) said polypeptide is immunologically cross-reactive with NTM peptide and with VIP; (ii) said polypeptide has, within the informational spectrum thereof, the dominant frequency component belonging to the frequency interval 0.180 to 0.220; provided that: said polypeptide is neither the VIP nor the NTM peptide, is described. Preferred polypeptides are selected from RSAHFTDNAKTPESVEIP, RSAHFTDNAKTHPESVEIH and HFTDNPHSVEIH. The polypetides, to which antibodies may be raised, may be of use in the detection of NTM.V antibodies, or in a pharmaceutical composition (especially for use in the treatment of AIDS). The polypeptides may be either (i) conjugated to a protein, especially human seum albumin, or (ii) expressed on the surface of a viral vector, particularly vaccinia virus.

Description

Peptides which react with Antibody representing the Prognostic Marker for HIV Disease Progression This invention relates to peptide sequences which react with human antibodies whose titre in the sera of individuals infected with Human Immunodeficiency Virus type 1 (HIV-1) correlates with Acquired Immune Deficiency Syndrome (AIDS) progression. Hence, these peptides are useful in preparing test kits for the prognostic evaluation of disease progression, as well as for monitoring the effect of antiviral therapy on HIV-1 infected patients, The peptides described in this invention also induce production of antibodies that influence HIV-1 infectivity and modulate the immune network. Hence, these peptides are also useful as vaccines to prevent the development of AIDS.

Monoclonal antibodies against the peptides from this invention may also be used as therapeutic agents to prevent AIDS progression.

Investigation of the difference in spectrum of antibodies directed against HIV-1 envelope glycoprotein gpl20 in two groups of HIV-infected persons, i.e. those who remained healthy for at least 10 years and those who developed AIDS within 5 years from the onset of infection, showed that the antibody recognising the peptide having the sequence, in the one-letter code, RSANFTDNAXTIIVQLNESVEINCTRP derived from the second conserved region of HIV-1 gp120, is significantly more prevalent in asymptomatic carriers than in AIDS patients [Neurath et al., AIDS Res. Hum. Retrovir.

6, 1183 (1990)]. This result indicates that declining levels or disappearance of this antibody (denoted as NTM.V Ab) may represent a possible factor contributing to the development of AIDS.

The origin of NTM.V Ab remains unknown since the region of HIV-1 gp120 encompassing the peptide pC2 is nonimmunogenic [Bradac and Mathieson, An epitope map to immunity to HIV-1: a roadmap for vaccine development, NIAID, NIH, Bethesda (1991)]. Based on the sequence and spectral homology between HIV-1 gpl20-derived peptide RSANFTDNAKTIIVQLNESVEIN (hereinafter "peptide NTM") and human vasoactive intestinal peptide (VIP), it has been suggested that NTM.V Ab probably represents the natural anti-VIP autoantibody [Veljkovic et al., Biochem. Biophys. Res. Commun. 189, 7005 (1992)]. This assumption was confirmed by the strong reactivity of the sera collected from HIV-negative asthma patients, containing high titres of natural anti-VIP antibodies, with theXpeptide NTM (the main end-point titre for hyperimmune sera was 1/10000) [Veljkovic et al., Biochem. Biophys. Res. Commun.

196, 1019 (1993)1 The putative protective role of NTM.V Ab could be ascribed to the following two possibilities: (1) This antibody blocks the "secondary interaction" between HIV-1 gp120 and CD4 receptor [Veljkovic and Metlas, Cancer Biochem. Biophys. 10, p. 191 (1988); Pollard et al., Proc. Natl. Acad Sci. USA 88, 11320 (1991)], playing an important role in the infection process [Stamatos and Cheng-Mayer, J. Virol. 67, 5635 (1993); Brynmor et al., J.

Virol. 67, 7493(1993)]. This conclusion is in accordance with the fact that jacalin a chain-derived peptide VVVRSLTFKTNRKT, which is highly homologous with the peptide NTM, effectively blocks viral infection in vivo without impairing lymphocyte function [Favero et al., Eur. J.

Immunol. 23, 179 (1993)].

(2) An unusual set of local homologies between the C-terminus of the second conserved region of HIV-1 gp120 and some human proteins (Figure 1) indicates that this region contains one of the epitopes around which the human immune network is organised [Veljkovic et al., Cancer J. 8, 308 (1995)]. This means that NTM-crossreactive antibodies could represent important components of the immune network and that their decrease negatively influences the immune network dynamic [Veljkovic and Metlas, Immunol. Today 1i, 38 (1992); Metlas and Veljkovic, Vaccine 13, 355 (1995)].

WO 93/17108 discloses analogous peptides of the internal image of a HIV-1 gp120. This reference also relies on the protein informational analysis technique originally developed by the present applicants (Veljkovic V. et al., Phys. Rev. Lett. 29, 105, 1972) and using a set -of the HIV-1 gp160, determined the frequency 0.1855 as a main frequency component that correlates with binding of gp120 to CD4 receptor. Based on this frequency, artificial peptides, expressing immunological cross-reactivity with the peptide derived from the C-terminus of HIV-1 gp120, have been designed. The frequency component 0.1855 correlates however with the gp120/gp41 interaction, not with the gp120/CD4 interaction. The above mentioned peptides are, therefore, entirely different from those according to the present invention.

In a first aspect, the present invention provides a peptide having the general formula I: X - Rl - R2 - D - N - Y (I) wherein: X is an amino terminal sequence of arbitrary length and arbitrary amino acid composition; R1 is phenylalanine or trypthophan; R2 is threonine or serine; D is aspartic acid; N is asparagine; the amino terminal group X is the residue of an amino acid or a peptide sequence having from 2 to 15 amino acids; the carboxy terminal group Y is the residue of an amino acid or a peptide sequence having from 2 to 15 amino acids, said peptide I immunologically cross-reacting with NTM peptide and with vasoactive intestinal peptide and having in its informational spectrum the dominant frequency component belonging to the frequency interval 0.180 - 0.220 with the proviso that said peptide is not either the vasoactive intestinal or the NTM peptide.

Advantages of certain of the embodiments of the present invention are as follows: peptides useful as a component of test kit identifying the presence of NTM.V Ab in sera of HIV-1-infected patients are provided - it has been shown that the titre of NTM.V Ab correlates with disease progression so that this antibody is useful as a prognostic marker; peptides of the invention are useful as vaccines to give rise to NTM.V Ab to hinder development of AIDS in HIV-1 infected subjects; peptides of the invention are also useful as immunogens to elicit monoclonal NTM.V Ab which can be used as a therapeutic agent ot prevent development of AIDS or slow its progression; and antibodies elicited by or reactive with a peptide according to claim 1 or 2 may be useful for preventing AIDS or slowing down its progression.

The informational spectrum represents the amplitude spectrum obtained by Fourier transformation of the numerical sequence generated by representation of amino acids in the peptide primary structure with corresponding values of the electron-ion interaction potential (hereinafter EIIP) [Veljkovic et al., IEEE Trans. Biomed.

Eng. 32, 337 (1985)]. The values of EIIP for amino acids, calculated according to the General-Model-Pseudopotential [Veljkovic V. and Slavic I., Phys. Rev. Lett. 29, 105 (1972); Veljkovic V. Phys. Lett. 45A, 41 (1973)] and the quasi valence number of amino acids [Veljkovic V. A Theoretical Approach in Preselection of Carcinogens and Chemical Carcinogenesis, Gordon & Breach Sci. Pub., New York (1980)] are given in Table 1.

Table 1 Amino acid EIIP [Ry]* L 0.0000 I 0.0000 N 0.0036 G 0.0050 V 0.0057 E 0.0058 P 0.0198 H 0.0242 K 0.0371 A 0.0373 Y 0.0516 W 0.0548 Q 0.0761 M 0.0823 S 0.0829 C 0.0829 T 0.0941 F 0.0946 R 0.0959 D 0.1263 * Ry = Rydberg unit A strong correlation between reactivity of the peptides of he invention with the sera of HIV-1-infected patients and their stage of disease has been found.

The peptides of the invention may be used as a test reagent in an enzyme-linked immunosorbent assay (ELISA) or an enzyme immunodot assay for the detection of NTM.V Ab.

This test may be used for prognosis of disease progression and for monitoring the effect of antiviral and immuno therapy in HIV-l-infected patients. Such test kits may include a porous surface or solid substrate to which the antigenic peptide has been preabsorbed or covalently bound, said surface substrate being preferably in the form of microtitre plates or wells; test sera; various diluents and buffers; labelled conjugates for the detection of specifically bound antibodies and other signal-generating reagents such as enzyme substrates, cofactors and chromogens.

A further aspect of the invention relates to vaccine preparations containing a peptide according to the invention, for protecting against development of AIDS or slowing down its progression in HIV-1 infected patients.

Such vaccines contain an effective immunogenic amount of peptide, e.g. 1 pg to 20 mg/kg body weight, optionally conjugated to a protein such as human serum albumin, or expressed on the surface of a suitable viral vector, such as vaccinia virus, in a suitable vehicle, e.g. sterile water, saline or buffered saline. Adjuvants may be employed, such as aluminium hydroxide gel.

The peptides of the invention may be administered by injection, e.g. intradermal, intramuscularly, intraperitoneally, subcutaneously or intravenously. Administration may take place once or at a plurality of times, e.g. at 1 4 week intervals.

Antigenic sequences from the crab, for example, as well as proteins from other invertebrates can also be added to the peptides of the invention to promote antigenicity, if so desired.

The peptides according to the invention may be used as immunogens to elicit monoclonal NTM.V Ab using conventional techniques. Such monoclonal antibodies may be used as therapeutic agents, for the prevention of development of AIDS or slowing down its progression.

EXAMPLES Methods Studv population.

Serum specimens were collected from 194 HIV-l-infected patients divided into three groups: asymptomatic (AS) patients - 87, patients with AIDS related complex (ARC) - 85, and patients who had developed AIDS - 22.

According to the route of infection, patients belonged to the following three groups: intravenous drug users, homosexuals and heterosexuals. Approximately half of the studied patients received antiretroviral therapy (AZT, DDC, DDI, D4T).

As a control, the sera collected from healthy HIVnegative blood donors was used.

Peptides.

The following three artificial peptides of the invention were designed and synthesised according to conventional methods: P 32 RSAHFTDNAKTPESVE I P P3 3 RSAHFTDNAKTHPESVEIH P34 HFTDNHPSVEIH The natural peptides VIP and NTM were also chemically synthesised.

Enzvme-linked immunosorbent assav.

Antigen coating - Peptides were diluted in coating buffer and microtitre plates were coated with 100 pl of peptide solution (1 g/well). Plates were incubated at 40C overnight and washed 3x with phosphate-buffered saline (PBS)/Tween. For each wash, all wells are filled with PBS/Tween prior aspirating and poured on paper towels.

Blocking - Remaining binding sites were blocked with 200 Cil/well of blocking buffer for 1 h at room temperature and washed 2x with PBS/Tween.

Primary antibody reaction - Sera were diluted in 0.18 Bovine Serum Albumin (BSA) in PBS and 100 Zl per well were added. Plates were incubated for 1 h at room temperature, then washed 3x with PBS/Tween.

Application of secondary antibody - Peroxidase labelled goat anti-human IgG antibody, Fc fragment specific diluted 1:5000 in 0.1% BSA in PBS was added at 100 Zl per well.

After 1 h of incubation at room temperature, the plates were washed 5x with PBS/Tween, the last one with PBS.

Substrate - Colour was developed with ABTS substrates supplemented with H2O2. The reaction was stopped after 30 min of incubation by adding 150 zl of 0.1 M citric acid with 0.01% NaN3, pH 1.8. Absorbance was measured at 450 nm in a microplate reader.

Reagents Coating buffer: 0.1M NaHCO3 and 0.1M Na2CO3, pH 9.6; PBS solution: 0.2 g KH2PO4, 1.2 g Na2HPO4, 8.0 g NaCl and 0.2 g KCl in 1I milliQ water; PBS/Tween: 1I PBS solution with 0.5 ml Tween 20 and 0.1 g NaN; Blocking buffer: 1 % BSA in PBS; ABTS substrate; 5 mg ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) in 0.1 ml 0.1 M citric acid pH 4.35 adjusted with NaOH. Prior to use, 4 pl 30% H202 was added; peptide stock solution: 1 mg peptide per 1 ml milliQ water, stored in aliquots at - 200C.

The present invention will be further illustrated with respect to the accompanying drawings, in which: Figure 1 shows the homology between the peptide Cterminus of the second conserved region of HIV-1 gp120 and some human proteins; Figure 2 shows the comparison of reactivity of the natural peptides NTM and VIP with sera collected from AIDS, ARC and AS patients; Figure 3 shows the comparison of the reactivity profiles between the peptides NTM and P34 and sera of the HIV-1-infected patients; Figures 4, 5 and 6 show the relationship between the CD4 count and reactivity of the peptide P34 with sera collected from AIDS, ARC and AS patients, respectively; Figure 7 shows the relationship between the CD4 count and ratio between number of sera above and below the cut-off for ARC and AS patients; and Figures 8, 9, 10 and 11 show the influence of the antiretroviral therapy on reactivity of the peptide P34 with sera collected from ARC and AS patients.

Example 1.

Experiments have been carried out to compare the reactivity of artificial peptides designed according to the criteria described in this invention with that of VIP and NTM natural peptides with sera of the HIV-l-infected patients.

All three artificial peptides P32, P33 and P34 showed a profile of reactivity with HIV-positive sera similar to that of the naturally peptides NTM and VIP. None of the tested peptides has shown a significant reactivity with sera of healthy HIV-negative blood donors. The comparison of the reactivity between VIP, NTM and P34 is reported in Figure 2.

Example 2.

Experiments have been effected to establish the relationship between the reactivity of the peptide P34 with sera of the HIV-1-infected patients and their disease stage. The results of this study are given in Figures 3-6.

The cut-off value of absorbance (O.D.) separating the hyperimmune NTM.V Ab serum from nonhyperimmune serum selected as O.D. cut-off > 0.7, the values were calculated as 3 x O.D.Nmem of normal serum [Ritchie et al., Methods in Immunology, Academic Press, New York (1983)]. The O.D.Nem obtained as the mean value of 200 normal sera was 0.233.

Figures 3 - 6 show that most analysed sera with O.D.

r 0.7 correspond to HIV patients with CD4 values between 200 and 400 cells/mm3. It is important to note that in this interval of CD4 count, the ratio of hyperimmune to nonhyperirmrtune HIV-positive sera is 3.7 times greater for AS than for ARC patients. In the region of CD4 count < 200 the number of patients with the hyperimmune NTM.V Ab sera is remarkably smaller than in patients with nonhyperimmune sera (Figure 3 and 4). The same picture corresponds to the region of CD4 count > 400 (Figure 5).

Based on the above results, the following general pattern for disease evolution could be proposed in terms of NTM.V Ab production in HIV-l-infected patients. At the beginning of infection (CD4 500) the NTM.V Ab titre is low and similar to that of healthy HIV-negative individuals. Near to CD4 count of 500, this antibody increases. In prognostic terms it may be concluded that this increase represents the beginning of disease progression. In the interval of CD4 count 200 - 400, the NTM.V Ab titre remains high for some time and then decreases with further disease progression from AS to ARC and AIDS. Finally, most of ARC and AIDS patients having a low titre of NTM.V Ab have a CD4 count < 200.

The above results show that NTM.V Ab together with CD4 count are useful prognostic markers for monitoring disease progression in HIV-l-infected patients. The peptides according to the invention may be therefore used as antigenic components in a prognostic ELISA test kit, for example.

Example 3 In order to evaluate the effect of the antiretroviral therapy on the NTM.V Ab titre, sera collected from treated and untreated patients were separately analysed. The results reported in Figures 8 11 show a positive trend supporting the correlation of therapy in AS patients (CD4 count > 400) with the decrease in NTM.V Ab titre, and with the increase in this antibody titre in ARC patients with CD4 count 200 - 400. On the contrary, in ARC patients with CD4 count < 200, antiretroviral therapy correlates with the decrease in the NTM.V Ab titre. These results are in agreement with the reported effect of the deferred antiretroviral therapy in HIV patients [O'Brien et al., New Engl. J. Med. 334, 426 (1996)].

Claims (15)

CLAIMS:

1. A peptide having the general formula I: X - R1 - R2 - D - N - Y (I) wherein: X is an amino terminal sequence of arbitrary length and arbitrary amino acid composition; R1 is phenylalanine or trypthophan; R2 is threonine or serine; D is aspartic acid; N is asparagine; the amino terminal group X is the residue of an amino acid or a peptide sequence having from 2 to 15 amino acids; the carboxy terminal group Y is the residue of an amino acid or a peptide sequence having from 2 to 15 amino acids, said peptide immunologically cross-reacting with NTM peptide and with vasoactive intestinal peptide and having in its informational spectrum the dominant frequency component belonging to the frequency interval 0.180 - 0.220 with the proviso that said peptide is neither the vasoactive intestinal nor the NTM peptide.

2. A peptide having the sequence selected from: RSAHFTDNAKTPESVE I P RSAHFTDNAKTHPESVEIH HFTDNHPSVEIH.

3. A test kit for the detection of NTM.V antibodies recognising the peptide NTM (RSANFTDNA}rIIVQLNESvEIN) derived from the second conserved region of HIV-1 and human VIP, containing an antigenic peptide according to Claim 1 or 2, bound to a porous surface or a solid substrate.

4. A test kit according to Claim 3, wherein the antigen peptide is bound to wells of a microtitre plate.

5. A pharmaceutical composition containing as the active ingredient a peptide as claimed in Claim 1 or 2 together with a pharmaceutical carrier therefor.

6. A composition of matter comprising a peptide according to Claim 1 or 2 conjugated to a protein.

7. A composition according to Claim 6 wherein the protein is human serum albumin.

8. A composition of matter comprising a peptide according to Claim 1 or 2 expressed on the surface of a viral vector.

9. A composition according to claim 8 wherein the viral vector is vaccinia virus.

10. Use of a peptide according to Claim 1 or 2, for the manufacture of a medicament for preventing AIDS or slowing down its progression.

11. Use of the antibodies elicited by or reactive with a peptide according to Claim 1 or 2, for preventing AIDS or slowing down its progression.

12. Use of a peptide according to claim 1 or 2 in anti AIDS therapy.

13. A test kit comprising a peptide as defined in claim 1 or 2, substantially as described herein.

14. A peptide as defined in claim 1 or 2, for use in anti AIDS therapy.

15. An antibody capable of recognising a peptide as defined in claim 1 or 2, for use in anti-AIDS therapy.