Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1991-08-21
1999-01-12
Stucker, Jeffrey
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435 71, 435 72, 435974, 435975, 530350, 530826, C12Q 170
Patent
active
058586466
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a polypeptide having immunological activity for use as a diagnostic reagent and/or a vaccine component.
BACKGROUND ART
Diagnostic kits for use in screening individuals for infection with human immunodeficiency virus (HIV) infection frequently include reagents comprising HIV antigens which are used to detect antibodies using known immunological techniques including ELISA, Western Blot, latex agglutination and immuno-luminescent and immuno-fluorescent techniques.
The effectiveness of such techniques however depends upon selection of suitable immunological reagents and one particular difficulty which arises is that particular reagents are often specific to individual strains or groups of strains of HIV. Thus, for example, known diagnostic reagents based upon HIV-1 may fail to detect antibodies resulting from an infection of a patient with HIV-2.
Similarly, in the production of vaccines designed to protect individuals against HIV infection, the use of antigens derived from one particular strain of HIV may fail to provide adeguate protection against infection with other strains.
Synthesis and cleavage of the HIV-I pol precursor polyprotein is disclosed in "Processing Protease and Reverse Transcriptase from Human Immunodeficiency Virus Type I Polyprotein in Escherichia coli" by Jan Mous et. al., Journal of Virology, Apr. 1988, p. 1433-1436. The process disclosed in this reference results in the formation of a 92 kDa polypeptide consisting of protease (18 kDa), reverse transcriptase (64 kDa) and an amino-terminal portion of endonuclease (integrase) (10 kDa). The polyprotein is thus lacking the intact endonuclease sequence (25 kDa), and thus lacks substantial antigenic epitopes representing the endonuclease (integrase). Thus, the protein is unlikely to be suited for the preparation of diagnostic tests and vaccines for HIV-I.
It is an object of the present invention to overcome such problems.
DISCLOSURE OF INVENTION
It has now been found that the product of expressing a substantial part of the HIV-pol gene in a suitable host has antigenic properties which allows the above-mentioned problems to be overcome.
Thus according to one aspect of the present invention there is provided the use as an antigenic reagent in the diagnostic test or as a vaccine component of a polypeptide comprising a substantial portion of each of more than one of the constituent proteins coded for by the HIV-pol gene.
Diagnostic kits and vaccines comprising said polypeptide form further aspects of the present invention.
The HIV-pol gene codes for four enzymes, namely a protease, a reverse transcriptase, a ribonuclease referred to as RNAse H and an enzyme referred to as Integrase.
It is believed that during infection of a T cell by HIV a full length precursor is expressed which is then cut up into the discrete proteins listed above. These have the following activities and (it is thought) act in the order indicated:
______________________________________ Protease Precursor cleavage
Reverse Transcriptase
Preparation of viral DNA from viral
RNA
RNase H Destruction of viral RNA leaving
newly synthesised DNA
Integrase Insertion of said DNA into host cell
genome
______________________________________
According to a preferred aspect of the present invention, said constituent proteins are enzymes coded for by the HIV-pol gene and the polypeptide thus comprises a substantial portion of each of a plurality of enzymes selected from HIV-pol protease, HIV-pol reverse transcriptase, HIV-pol RNAse H and HIV-pol Integrase. Most preferably, the polypeptide comprises substantial portions of all four of said enzymes.
In vivo, the initial product of expressing the HIV-pol gene is cleaved into its individual elements by the protease. The active site for proteolytic activity occurs adjacent the NH.sub.2 -terminus of the expression product, corresponding to the 5'-end of the protease gene.
According to a preferred aspect of the present invention, the polypeptide omits at least that part of the amino acid seque
REFERENCES:
patent: 4745051 (1988-05-01), Smith
patent: 4751180 (1988-06-01), Cousens et al.
patent: 5194376 (1993-03-01), Kang
Farmerie--`Science`, vol. 236, Apr. 17, 1987.
Mous et al--`Journal of Virology`, Apr. 1988, pp. 1433-1436.
Matthews & Bolognesi--`Scientific American`, Oct. 1988--AIDS Vaccines.
Reagan--`The Lancet`--Jan. 27, 1990 p. 236.
Navia et al--`Nature`, vol. 337, Feb. 16, 1989--Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1.
Kang--`Advances in Virus Research`, vol. 35--Baculovirus Vectors For Expression of Foreign Genes.
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Hu et al--`Proc. Natl. Acad. Sci.,` Feb. 8, 1991--"Enzyme Activities in Four Different Forms of HIV-1 pol Gene Products".
Human Retroviruses and AIDS--1988--Dr. Gerald Myers, Chief Editor, Los Alamos National Laboratory, Los Alamos, NM 87545 U.S.A.
Farmerie et al, "Expression and Processing of the AIDS Virus Reverse Transcriptase in Escherichia coli", Science, vol. 236(17 Apr. 1987), pp. 305-308.
Mous et al, "Processing Protease and Reverse Transcriptase from Human Immunodeficiency Virus Type I Polyprotein in Escherichia coli", Journal of Virology, vol. 62, No. 4(Apr. 1988), pp. 1433-1436.
Stucker Jeffrey
University of Ottawa
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