Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1990-06-20
2002-11-26
Woodward, Michael P. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C530S324000, C530S325000, C530S326000, C514S012200, C514S013800
Reexamination Certificate
active
06485900
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
With the discovery that the diseases called lymphadenopathy syndrome and acquired immune deficiency disease (AIDS) are caused by an infectious retrovirus designated lymphadenopathy virus (LAV), human T-cell lymphotropic virus-III (HTLV-III), AIDS-related virus (ARV), or immune deficiency-associated virus (IDAV), there has become an immediate need to be able to detect potential vectors of the disease, such as blood from diseased individuals, which may be employed for transfusions or from which specific blood factors may be isolated.
To detect potential vectors of the disease, it is necessary to have viral proteins and/or antibodies to such proteins. Because of the hazards associated with growing the LAV/HTLV-III retrovirus, there is significant interest in establishing means for obtaining the viral proteins or their immunologic equivalents, which means do not necessitate handling large volumes of live, potentially infectious virus. In choosing alternatives, one must be concerned with the fact that the viruses have been reported to be highly polymorphic, frequently changing as the retrovirus is passaged.
2. Brief Description of the Relevant Literature
The various antigens of the retrovirus are described by Saxinger et al.,
Science
(1985) 227:1036-1038. See also Gallo et al.,
ibid
. (1984) 224:500; Sarangadharn et al.,
ibid
. 224:506; Barre-Sinoussi et al.,
ibid
. (1983) 220:868; Montagnier et al., in Human T-Cell Leukemia/Lymphoma Virus, Gallo, Essex, Gross, eds. (Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y.), 1984, p. 363. These may include, but are not limited to, p13, p18, p25, p36, gp43, p55, gp65, gp110, etc., where the numbers may differ depending upon the reporter.
Hopp and Woods,
Proc. Natl. 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 been shown not to have a high predictive value. 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.
The complete nucleotide sequence of LAV is reported by Wain-Hobson et al.,
Cell
(1985) 40:9. The complete sequence for HTLV-III is reported by Muesing et al.,
Nature
(1985) 313:450, while the complete sequence for ARV is reported by Sanchez-Pescador et al.,
Science
(1985) 227:484. All three viruses exhibit substantial nucleotide homology and are similar with respect to morphology, cytopathology, requirements for optimum reverse transcriptase activity, and at least some antigenic properties (Levy et al.,
Science
(1984) 225:840; Shupbach et al.,
Science
(1984) 224:503), and hence should be considered isolates of the same virus. See also, Chang et al.,
Science
(1985) 228:93.
SUMMARY OF THE INVENTION
Peptide sequences capable of immunologically mimicking proteins encoded in the gag and/or env regions of the LAV/HTLV-III retrovirus are provided as reagents for use in the screening of blood and blood products for prior exposure to the retrovirus. The peptides are of at least 5 amino acids and can be used in various specific binding assays for the detection of antibodies to LAV/HTLV-III virus, for the detection of LAV/HTLV-III antigens, or as immunogens.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
For the purpose of this disclosure, a virus is considered to be the same as or equivalent to LAV/HTLV-III if it substantially fulfills the following criteria:
(a) The virus is tropic for T-lymphocytes, especially T-helper cells (CD4
+
, according to the international nomenclature defined in Bernard et al., eds.
Leucocyte Typing
, New York: Springer Verlag, 1984);
(b) The virus is cytopathic for infected CD4
+
cells (rather than transforming, as are HTLV-I and -II);
(c) The virus encodes an RNA-dependent DNA polymerase (reverse transcriptase) which is Mg
2+
-dependent (optimum concentration 5 mM), has a pH optimum of 7.8, is not inhibitable by actinomycin D, and can employ oligo(dT)
12-18
as a primer for reverse transcription from its 3′ LTR;
(d) The virus bands in a sucrose gradient at a density of approximately 1.16;
(e) The virus can be labeled with [
3
H]-uridine;
(f) The virus is substantially cross-reactive immunologically with the proteins encoded by the gag and env regions of LAV/HTLV-III; and
(g) The virus shares substantial nucleotide homology (approximately 75-100%) and amino acid sequence homology (approximately 75-100%) with LAV or HTLV-III.
Novel peptides are provided which immunologically mimic proteins encoded by the LAV/HTLV-III retrovirus, particularly proteins encoded by the gag and/or env regions of the viral genome. To accommodate strain-to-strain variations among different isolates, adjustments for conservative substitutions and selection among the alternatives where non-conservative substitutions are involved, may be made. These peptides can be used individually or together for detection of the virus or of antibodies to the virus in a physiological sample. Depending upon the nature of the test protocol, the peptides may be labeled or unlabeled, bound to a solid surface, conjugated to a carrier or other compounds, or the like.
The peptides of interest will be derived from the peptides encoded by the gag region or the env region. These peptides will be primarily derived from p55 or fragments thereof, e.g., p25 and p18, or gp150 and fragments thereof, e.g., gp41. These peptides will be given Roman numerals, but will also be given numerical designations which are arbitrarily associated with the manner in which they were produced.
For the gag region, of particular interest are the coding regions extending from about base pair (bp) 450 to bp 731, particularly from about bp 450 to bp 545 (97) and bp 696 to bp 731 (71); from about bp 900 to bp 1421, particularly from about bp 921 to bp 1016, including bp 921 to bp 1010; bp 972 to bp 1016 (92); and bp 936 to bp 995 (17); or from about bp 1158 to about bp 1400, particularly bp 1164 to bp 1250 (90); bp 1278 to bp 1385 (88); and bp 1320 to bp 1385 (15), of the LAV/HTLV-III retrovirus. (Numbering according to Wain-Hobson-et al., supra.)
For the env region, the regions of particular interest will be those polypeptides encoded within the bp 7210 to bp 7815 regions, particularly within bp 7231 to bp 7794, more particularly within about bp 7246 through bp 7317 (36), bp 7516 through bp 7593 (39), particularly bp 7543 through bp 7593 (79) and bp 7561 through 7593 (78), bp 7708 through bp 7779 (23), bp 7630 through bp 7689 (40), bp 7498 through bp 7554 (56).
The peptides of interest will include at least five, sometimes six, sometimes eight, sometimes 12, usually fewer than about 50, more usually fewer than about 35, and preferably fewer than about 25 amino acids included within a sequence coded for by the LAV/HTLV-III retrovirus. in each instance, desirably the oligopeptide will be as small as possible, while still maintaining substantially all of the sensitivity of the larger peptide. In some instances it may be desirable to join two or more oligopeptides which are non-overlapping in the same peptide structure or as individual peptides, which separately or together provide equivalent sensitivity to the parent.
The peptides may be modified by introducing conservative or non-conservative substitutions in the peptides, usually fewer than 20 number percent, more usually fewer than 10 number percent of
Bio-Rad Laboratories
Townsend and Townsend / and Crew LLP
Woodward Michael P.
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