Title:  Methodologies for the detection of human immunodeficiency virus nucleic acids employing oligonucleotide primer derived from the HIV-1 MVP5180/91 genome

United States Patent:  6,335,158

Assignee:  Dade Behring Marburg GmbH (Marburg, DE)

Filed:  August 10, 1998

Foreign Application Priority Data:  Feb 23, 1994[DE] (44 05 810)

The present invention is directed toward nucleic-acid based methodologies for the detection of human immunodeficiency virus (HIV) nucleic acids in a sample. A novel HIV-1 isolate, designated MVP5180/91, was isolated from a West African Cameroonian patient with immunodeficiency. Nucleic acid and amino acid sequence comparisons of this isolate, with other HIV-1 strains of subtypes A-E and HIV-2 isolates, demonstrated that this virus shares only limited homology with other known HIV-1 and -2 isolates. However, this virus does display some genetic relatedness to another Cameroonian isolate designated ANT-70. These viruses form the basis for a new HIV-1 group which has been designated subtype O. An immunologically important epitope, corresponding to amino acids 601-623 of the MVP5180/91 transmembrane envelope glycoprotein, was identified. Labeled nucleic acids can be prepared from the nucleotide sequence encoding this region and employed in standard hybridization assays to detect HIV-1 nucleic acids. Alternatively, oligonucleotide primers can also be prepared from this region and employed in polymerase chain reaction (PCR) assays to detect viral-specific nucleic acids.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide an immunologically active peptide comprising at least 15 consecutive amino acids selected from the amino acids in the following sequence (SEQ ID NO:1):

VWGIRQLRARLQALETLIQNQQRLNLWGXKGKLIXYTSVKWNTSWSGR,

wherein X is C or S. This peptide detects antibodies against retroviruses of the HIV type.

The invention further relates to a kit for detecting antibodies against viruses which cause immune deficiency comprising the above described peptide.

The invention further relates to a diagnostic agent for detecting an antibody against a retrovirus that causes immune deficiency, the diagnostic agent comprising the above described peptide and a detectable label that is capable of detecting the binding of the peptide with the antibody.

In another embodiment, the invention relates to a method of detecting the presence of anti-retrovirus antibodies in a sample, the method comprising contacting the sample with the above described diagnostic agent and detecting the presence of antibody bound to the diagnostic agent as a result of the contacting.

Another embodiment of the invention relates to an immunogen comprising (a) an amount of the above described peptide and (b) a physiologically-acceptable excipient therefor, wherein said amount is sufficient to elicit an immune response that is protective of a susceptible mammal against retrovirus infection.

In another embodiment, the invention relates to a method of immunizing a mammal against retrovirus infection, comprising administering to the mammal an effective amount of the above described immunogen.

Another embodiment of the present invention relates to an isolated DNA molecule which encodes the above described peptide.

Another embodiment relates to a method of detecting in a sample nucleic acids encoding a retrovirus that causes immune deficiency, comprising the steps of: (a) hybridizing a labeled DNA molecule to nucleic acids encoding a retrovirus in said sample, wherein said labeled DNA molecule is prepared by labeling the above described DNA molecule with a detectable label, and (b) detecting the hybridizing by means of said detectable label.

In another embodiment, the invention relates to a method of detecting in a sample nucleic acids encoding a retrovirus that causes immune deficiency, comprising subjecting said nucleic acids to a Polymer Chain Reaction (PCR), wherein the PCR employs at least two oligonucleotide primers that anneal to a nucleic acid encoding a retrovirus that causes immune deficiency, wherein one of the primers is complementary to a first nucleotide sequence comprising the sequence of the above described DNA molecule, or its complementary sequence, wherein the other primer is complementary to a second nucleotide sequence comprising a nucleic acid molecule encoding a retrovirus that causes immune deficiency, whereby a geometrically amplified product is obtained only when the first and second nucleotide sequences occur within the same nucleic acid molecule encoding a retrovirus that causes immune deficiency.

Claim 1 of 2 Claims

What is claimed is:

1. A method of detecting the presence of nucleic acid of a human immunodeficiency virus in a sample, comprising the steps of:

(a) providing a sample suspected of containing one or more nucleic acids encoding a protein of an immunodeficiency virus;

(b) contacting the sample of step a with suitable PCR reagents that comprise at least a first and a second oligonucleotide primer that can anneal to the immunodeficiency virus nucleic acid, wherein the first primer sequence is complementary to a nucleic acid sequence from the region within amino acids 601 to 623 of the transmembrane protein of the MVP5180/91 strain of HIV-1 and the second primer is complementary to a known nucleotide sequence of a protein from HIV-1 or HIV-2; and

(c) detecting the presence of a geometrically amplified product after incubation under conditions suitable for amplification using both primers.

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