Modified HIV-1 group M cyclic gp41 peptides and their use in the detection of anti-HIV-1 antibodies
The invention involves a synthetic peptide derived from HIV-1 virus gp41 having sequence where X 615 is F or G, a method of preparing the synthetic peptide, a composition and a kit containing the synthetic peptide, and use of the synthetic peptide in immunoassays for the detection of infections caused by HIV-1 viruses.
1. An isolated cyclic peptide of human immunodeficiency virus type 1 (HIV-1) gp41 consisting of
wherein X is an amino acid selected from the group consisting of F and G, and
wherein (i) aa 1 and aa 2 are absent or (ii) aa 1 is alanine and aa 2 is serine.
2. An antigenic composition comprising a peptide according to claim 1 in combination with a carrier or diluent.
3. The peptide according to claim 1 , wherein the peptide consists Of
4. The peptide according to claim 1 , wherein the peptide consists of
5. The peptide according to claim 1 , wherein the peptide consists of
6. The peptide according to claim 1 , wherein the peptide consists of
7. An antigenic composition comprising a peptide according to claim 3 in combination with a carrier or diluent.
8. An antigenic composition comprising a peptide according to claim 4 in combination with a carrier or diluent.
9. An antigenic composition comprising apeptide according to claim 5 in combination with a carrier or diluent.
10. An antigenic composition comprising apeptide according to claim 6 in combination with a carrier or diluent.
11. A process for preparation of the cyclic peptide of claim 1 comprising the steps of:
a) reacting resin 9-fluorenylmethyloxycarbonyl (Fmoc) with reagents 2-(1H-b enzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and N-hydroxybenzotriazole (HOBt), in a ratio (moles/moles) reagents/substitutable groups on the resin equal to 5, to effect a resin-bound linear peptide;
b) contacting the resin-bound linear peptide with a trifluoroacetic acid solution, to separate the linear peptide from the resin;
c) contacting the linear peptide in solution with an ether solution, to precipitate the linear peptide;
d) purifying the precipitated linear peptide by high performance liquid chromatography (HPLC); and
e) oxidizing the purified linear peptide to effect the cyclic peptide.
12. A method for in vitro detection of anti-HIV-1 antibodies in a biological sample comprising the steps of:
a) bringing the biological sample into contact with at least one peptide of HIV-1 virus gp41 of sequence (III) as defined in claim 1 ;
b) incubating the sample-and-peptide mixture under conditions permitting the formation of antigen-antibody complexes between the at least one peptide of sequence (III) and anti-HIV-1 antibodies present in the biological sample;
c) washing the mixture; and
d) revealing and detecting the antigen-antibody complexes formed.
13. A method for the in vitro detection of anti-HIV-1 antibodies and anti-HIV-2 antibodies in a biological sample comprising the steps of:
a) bringing the biological sample into contact with (i) at least one peptide of gp41 of sequence (III) as defined in claim 1 and (ii) an HIV-2 antigen;
b) incubating the sample-peptide-antigen mixture under conditions permitting the formation of antigen-antibody complexes (i) between the at least one peptide of sequence (III) and anti-HIV-1 antibodies present in the biological sample and (ii) between the HIV-2 antigen and anti-HIV-2 antibodies present in the biological sample;
c) washing the mixture; and
d) revealing and detecting the antigen-antibody complexes formed.
14. A method for the in vitro detection of anti-HIV-1 antibodies and anti-HIV-1 O antibodies in a biological sample comprising the steps of:
a) bringing the biological sample into contact with at least one peptide of gp41 of sequence (III) as defined in claim 1 and an HIV-1 O antigen:
b) incubating the sample-peptide-antigen mixture under conditions permitting the formation of antigen-antibody complexes (i) between the at least one peptide of sequence (III) and anti-HIV-1 antibodies present in the biological sample and (ii) between the HIV-1 O antigen and anti-HIV-1 O antibodies present in the biological sample;
c) washing said mixture; and
d) revealing and detecting the antigen-antibody complexes formed.
15. A method for the in vitro detection of anti-HIV-1 antibodies anti-HIV-1 O antibodies, and of anti-HIV-2 antibodies in a biological sample comprising the steps of:
a) bringing the biological sample into contact with a first HIV-1 antigenic composition consisting of at least one peptide of gp41 of sequence (III), as defined in claim 1 , a second antigen consisting of an HIV-1 O antigen, and a third antigen consisting of an HIV-2 antigen;
b) incubating the mixture under conditions permitting the formation of antigen-antibody complexes between the peptide(s) of sequence (III) and anti-HIV-1 antibodies present in the biological sample, between the HIV-1 O antigen and anti-HIV-1 O antibodies present in the biological sample, and between the HIV-2 antigen and anti-HIV-2 antibodies present in the biological sample;
c) washing the mixture; and
d) revealing and detecting the antigen-antibody complexes formed.
16. A method for the in vitro detection of anti-HIV-1 antibodies in a biological sample comprising the steps of:
a) bringing the biological sample into contact with a composition according to claim 2 ;
b) incubating the sample-and-composition mixture under conditions permitting the formation of antigen-antibody complexes between the at least one peptide of sequence (III) and anti-HIV-1 antibodies present in the biological sample;
c) washing the mixture; and
d) revealing and detecting the antigen-antibody complexes formed.