Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1994-06-30
2001-11-27
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C530S300000, C530S324000, C530S325000, C530S326000, C530S327000, C424S188100
Reexamination Certificate
active
06322964
ABSTRACT:
1. FIELD OF INVENTION
A novel human retrovirus, designated lymphadenopathy virus, type 2 (“LAV-2”), has recently been isolated from patients with Acquired Immune Deficiency Syndrome (AIDS) from West Africa. Although this virus is related to HIV-1 (also known as LAV/HTLV-III), sera from patients infected with LAV-2 are repeatedly negative or react poorly in screening assays for antibodies to HIV-1. Due to the serious threat to public health posed by a new virus associated with AIDS which is not sufficiently detected with the presently available tests for HIV-1, there has arisen an immediate need for a new antibody screening test. Desirably the test would be able to distinguish between individuals with antibodies to HIV-1 versus LAV-2.
To develop an immunodiagnostic test to identify individuals previously exposed to LAV-2, it is necessary to have LAV-2 proteins and/or antibodies to such proteins. Because of the hazards to laboratory workers associated with growing LAV-2, as well as the technical difficulties and expense of large scale culture and purification of virus, there is considerable interest in developing alternative means for obtaining the viral proteins or their immunologic equivalents. In choosing alternatives, one must be concerned with the fact that the HIV family of retroviruses has been reported to be highly polymorphic, as well as the possibility that additional members of the HIV family may be discovered.
2. BRIEF DESCRIPTION OF THE BACKGROUND ART
The isolation and partial characterization of LAV-2 from two AIDS patients from West Africa was initially described in Clavel et al.,
C.r. Acad. Sci.
(Paris), 302:485-488 (1986) and Clavel et al.,
Science
233:343 (1986). This virus is related to HIV-1, the causative agent of AIDS, by both its morphology and its biological properties, the latter including its T4+ lymphotropism and its cytopathic effect on T4 cell lines and lymphocytes. However, DNA hybridization studies suggest that there are regions of extensive homology between HIV-1 and LAV-2 particularly in the gag gene, the reverse transcriptase domain in pol, the Q genes, the F gene, and the 3′ LTR. The envelope gene, the tat gene, and part of the pol gene seem very divergent. Clavel et al. (ibid.)
Thus, even though HIV-1 and LAV-2 may be related, the heterogeneity in the genes which encode the envelope proteins may explain why serological cross-reactivity between the two species appears to be limited to epitopes of the gag and pol proteins. This may also account for the difficulties of the commercially available HIV-1 screening tests to adequately detect antibodies to LAV-2. Clavel et al. (ibid.) have predicted that, despite the wide divergence between the env genes of HIV-1 and LAV-2, there may exist homologous domains in the envelope glycoproteins of the two viruses which bind them to T4 receptors on the target cells.
It appears that LAV-2 may also be related to a virus known as Simian T-lymphotropic Virus Type 3 (“STLV-3”), a retrovirus first isolated from captive macaques with an AIDS-like disease. Clavel et al. (
Nature
324:691-695 (1986)) used radioimmunoprecipitation assays to show that sera from patients infected with LAV-2 reacted with all the STLV-3 proteins, although the proteins of LAV-2 and STLV-3 were distinguishable by differences in molecular weights. The nucleotide sequence of LAV-2 may also be very similar to STLV-3, as LAV-2 probes bound STLV-3 gag and pol regions under stringent conditions, whereas HIV-1 probes did not react with the STLV-3 genome. Southern blots of STLV-3 DNA showed a restriction pattern that was clearly different, however, from LAV-2 DNA.
Another human retrovirus, designated HTLV-IV, has also been isolated from West African patients but has not been associated with an AIDS-like disease. This virus also appears related to STLV-3 and HIV-1 (Kanki et al.,
Science (
1986) 232:238-243). Comparisons of HTLV-IV and LAV-2 have not been reported. Serological evidence for another new retrovirus related to STLV-3 has been reported in residents of West Africa. Barin et al.,
Lancet,
2:1387-89 (1985).
Hopp and Woods,
Proc. Nat. Acad. Sci. USA
(1981) 78:3824, describe criteria for selecting peptides as potential epitopes of polypeptides based on their relative hydrophilicity. In one study employing these criteria, a 12-amino acid peptide was synthesized that bound 9% of antibodies elicited by the native protein (Hopp,
Molec Immunol.
(1981) 18:869). In general Hopp/Woods criteria have not been shown to have a high predictive value, particularly for those regions not associated with the highest peak of hydrophilicity for a particular protein. Furthermore, epitopes have been demonstrated which are not hydrophilic (Kazim et al.,
Biochem. J.
(1982) 203:201). Other studies of polypeptide antigenicity include Green et al.,
Cell
(1982) 28:477, where peptides were employed which elicited antibodies, which antibodies were capable of binding to the native protein, while conversely antibodies which were elicited by the native protein failed to bind to the peptides; and Trainer et al.,
Nature
(1984) 312:127, whose results with myohaemerythrin paralleled those of Green et al.
Synthetic peptides have been generated which immunologically mimic or have been predicted to mimic proteins encoded by HIV-1. See U.S. Pat. No. 4,629,783 and Pat. Publication No. WO 86/06414, which are incorporated herein by reference; NTIS Publication No. PB 86-131562 (U.S. patent application Ser. No. 779,431) and Chang et al.,
Proc. Natl. Acad. Sci. USA
83:6159-63 (1986). Pauletti et al.,
Analyt. Biochem.
151:540-546 (1985) used a computer program to predict possible antigenic determinants for the envelope glycoprotein (gp160) of HIV-1. Starcich et al.,
Cell
45:637-648 (1986) analyzed the entire envelope gene of isolates of HIV-1 and, based on predicted secondary protein structure and hydrophilicity, postulated that certain hypervariable regions represented antigenic sites.
3. SUMMARY OF THE INVENTION
Peptide sequences capable of immunologically mimicking proteins encoded in the env and gag regions of the LAV-2 retrovirus are provided as reagents for use in the screening of blood and blood products for prior exposure to the retrovirus. The peptides are at least five amino acids in length and can be used in various specific binding assays for the detection of antibodies to LAV-2 virus, for the detection of LAV-2 antigens, or as immunogens. Peptides described in the examples enable one to distinguish the presence of antibodies in a body fluid to LAV-2 from antibodies to HIV-1.
One peptide (41-2-1) described herein has the following amino acid sequence:
Y
n
-Arg-Val-Thr-Ala-Ile-Glu-Lys-Tyr-Leu-Gln-Asp-Gln-Ala-Arg-Leu-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-Y′
n′
in which
Y and Y′ each comprises one or more amino acid residues; and
n and n′ each comprises an integer of at least 0.
Another peptide (41-2-2) described herein has the following amino acid sequence:
Y
n
-Ser-Lys-Ser-Leu-Glu-Gln-Ala-Gln-Ile-Gln-Gln-Glu-Lys-Asn-Nor-Tyr-Glu-Leu-Gln-Lys-Leu-Asn-Ser-Trp-Asp-Y′
n′
in which Y and Y′ each comprises one or more amino acid residues, and n and n′ each comprises an integer of at least 0.
Another peptide (25-2-2) from the gag region described herein has the following amino acid sequence:
Y
n
-Asp-Cys-Lys-Leu-Val-Leu-Lys-Gly-Leu-Gly-Nor-Asn-Pro-Thr-Leu-Glu-Gln-Nor-Leu-Thr-Ala-Cys-Y′
n′
in which Y and Y′, each represents one or more amino acid residues; and n and n′ each are integers of at least 0.
3.1. DEFINITIONS
As used herein, “nor” is meant to refer to norleucine and is intended as a substitution for certain methionine residues.
As used herein, HIV is meant to refer to a human immunodeficiency virus characterized as a human retrovirus which is tropic for cells expressing the CD4 antigen and cytopathic to the host cell which it infects. This group includes but is not limited to HIV-1 (LAV-1/HTLV-III), and LAV-2.
REFERENCES:
patent: 4629783 (1986-12-01), Cosand et al
Alizon Marc
Chamaret Solange
Cosand Wesley L.
Houghton Raymond L.
Montagnier Luc
Genetic Systems Corporation
Parkin Jeffrey S.
Scheiner Laurie
Townsend and Townsend / and Crew LLP
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