Synthetic peptides and mixtures thereof for detecting HIV...

Details Synthetic peptides and mixtures thereof for detecting HIV... Synthetic peptides and mixtures thereof for detecting HIV...

1988-01-27

2001-04-17

Budens, Robert D. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C530S317000, C530S324000

Reexamination Certificate

active

06218102

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to novel cyclic synthetic peptides and combination thereof with linear synthetic peptides for detecting HIV antibodies.
BACKGROUND OF THE INVENTION
The numbering system for amino acids used herein is that of Ratner et al., Nature, 313, 277-284, 1985 even though other numbering systems are used in the prior art referred to herein. The amino acids used herein in the peptides are given with the single letter code as follows: ala=A, arg=R, asn=N, asp=D, cys=C, gin=Q, glu=E, gly=G, his=H, ile=I, leu=L, lys=K, met=M, phe=F, pro=P, ser=S, thr=T, trp=W, tyr=Y and val=V.
The initial immunodiagnostic tests for the detection of antibodies in the serum of patients infected with HIV-1 utilized whole virus as the antigen. Second generation tests made use of polypeptide sequences obtained by the recombinant DNA methodology. Cabradilla et al. Bio/Technology 4, 128-133 (1986) and Chang et al. Bio/Technology 3, 905-909 (1985) succeeded in obtaining bacterially synthesized viral protein fragments of 82 and 102 amino acid residues respectively. Eur. Patent 86202314 and 86114243 describe recombinant polypeptides covering regions of the gp41 and gp120 that are immunoreactive alone or in mixtures. Shoeman et al. Anal. Biochem. 161, 370-379 (1987) also describe several polypeptides from gp41 that have immunoreactive properties with antibodies present in sera from patients infected with HIV. None of the above assay procedures is acceptable. Their lack of sensitivity is serious as it may permit blood-containing virus to escape detection and thereby potentially result in the infection of blood product receivers. The impurities present in these antigen preparations are also responsible for unacceptably high levels of false positive results which cause healthly individuals to suffer distress.
It then became apparent that a tendency of the prior art was the identification of shorter epitopes. This is because of the ease and lower cost with which they could be prepared and more importantly because of the reduced risk of obtaining falsely positive tests results due to the presence of shared epitopes with viral proteins not related to AIDS. In this regards, serum samples tested in each of these cases is very limited, specificity was found to be very high (95%-100%) with small synthetic peptides but the overall sensitivity varied between 80 and 100%. In the only example where 100% sensitivity was attained only ten samples had been tested.
Smith et al., (J. Clin. Microbiol. 25, 1498-1504, 1987) described two overlapping peptides, E32 and E34, that are highly immunoreactive. No false positive result, out of 240 seronegative specimens, were obtained but the test missed three seropositive samples out of 322 (sensitivity of 99.1%). Wang et al. (Proc. Natl. Acad. Sci. 83, 6159-6163, 1986) described a series of overlapping peptides (including amino acid residues of the E32 and E34 peptides discovered by Smith et al.) among which one 21-mer peptide showed 100% specificity and 98% sensitivity (out of 228 seropositive samples taken from patients with AIDS, 224 were found positive with this peptide).
In U.S. patent application Ser. No. 120,027 filed Nov. 13, 1987, there is disclosed a short synthetic peptide covering residues 606 to 620 (SGKLICTTAVPWNAS) of gp 41 (HIV-1), which is said to be immunoreactive with antibodies of patients infected by the AIDS viruses. In this example, specificity was also excellent (63/63) but six seropositive specimens out of 57 confirmed positive could not be detected (sensitivity of 89%).
Gnann et al. (J. Virol. 6, 2639-2641, 1987 and J. Infect. Dis. 156, 261-267, 1987) also reported a series of overlapping peptides from an immunodominant region of gp 41 (HIV-1). Of particular interest was their finding that one peptide having the sequence SGKLIC (606-611) was not immunoreactive with any of the 22 HIV-1 positive sera tested. The addition of a cysteine residue to the N-terminus restored some immunoreactivity, 21 of 44 sera reacted with the 7-mer peptide (48% sensitivity). Gnann et al. concluded that cys-605 was essential for the immunoreactivity of that segment of the gp 41 (HIV-1) protein.
Gnann et al. have also speculated that the cysteine residues at positions 605 and 611 (Ratner's numbering system) of gp 41 (HIV-1) might play a critical role in the antigenic conformation of this region of the protein possibly through the formation of a loop via disulfide bonding.
The 7-amino acid sequence containing two cysteine residues at position 605-611 also has been disclosed in other documents such as PCT/US 86/00831 published on Nov. 6, 1986 under International Publication No. WO 86/06414 where peptide X(39) which is encoded by the region from about bp 7516 through 7593 and peptide XIII(79) which is encoded by the region extending from about bp 7543 through bp 7593 both contain the 7-amino acid sequence (amino acids 605-611) discussed by Gnann et al. in the above noted publication. In PCT/US 86/0031 the peptides are reported as linear and there is no mention of any cyclic peptides or disulfide bridging between the 605 and 611 cysteines.
Although the references discussed above do provide peptides which are useful in identifying HIV-1 antibodies, they also present certain drawbacks such as inability to full detection (100%) of positive serum samples. For example, Gnann et al. in Journal of Virology, August 1987 P. 2639-2641 in their tests with their 600-611 amino sequence detected 22 out of 22 positive sera while they also states that similar tests carried out by another author at the Center for Disease Control, Atlanta, Ga. with the same 12-amino acid sequence (600-611) detected 78 out of 79 positive sera. The same authors in J. Infect. Dis., 156, 261-267, 1987 show that the same 12-amino acid sequence from gp 41 (HIV-1) was shown to be reactive with 131 out of 132 HIV-1-infected patients from the United States.
In the same article, it is also clearly shown that when the HIV-1 positive sera are diluted by a factor exceeding 500, some of these diluted, sera are found to be negative thus indicating a low sensitivity.
Another potential drawback of these prior art assays is their use of a poorly defined and unpredictable peptide mixture as the probe. This mixture comprises peptides having many oxidative forms of cysteine produced spontaneously during peptide preparation, processing and use. It would appear highly desirable to provide peptides or peptide mixtures which are resistant to spontaneous oxidation. Such peptides would, thus, have a well defined structure. Moreover, under normal test conditions, they detect all HIV-1 antibody-containing samples as positive even when extremely low levels of antibody are present.
SUMMARY OF THE INVENTION
In accordance with the present invention, there is now provided a novel series of peptides or amino acid sequences which are particularly adapted in detecting 100% of HIV-1 antibodies and which are still capable of fully detecting all the HIV-1 antibodies even when the sera are highly diluted.
More specifically, the novel peptides of the present invention comprise any amino acid sequence extending from 586 to 629 (gp41 HIV-1) wherein in any selected amino acid sequence there is always present the amino acid sequence which contains the cysteine residues at each terminus of the 605-611 amino acid sequence which are linked by a disulfide bond to provide the following partial sequence
Still more specifically, the novel cyclic peptides of the present invention are depicted in formula I and comprise therein the amino acid sequence 605-611 (gp 41 HIV-1):
a-x-CSGKLIC-y-b  (I)
wherein x represents if present one to nineteen amino acids corresponding to AA
604
to AA
586
-AA
604
of gp41 (HIV-1 ) or analogues thereof; y if present represents one to eighteen amino acids corresponding to AA
612
to AA
612
-AA
629
of gp41 (HIV-1 ) or analogues thereof; a represents th

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