Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1995-04-19
2002-01-08
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C424S188100, C424S208100
Reexamination Certificate
active
06337179
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a virus capable of inducing lymphadenopathies (hereinafter “LAS”) and acquired immuno-depressive syndromes (hereinafter “AIDS”), to antigens of this virus, particularly in a purified form, and to a process for producing these antigens, particularly antigens of the envelope of this virus. The invention also relates to polypeptides, whether glycosylated or not, produced by the virus and to DNA sequences which code for such polypeptides. The invention further relates to cloned DNA sequences hybridizable to genomic RNA and DNA of the lymphadenopathy associated virus (hereinafter “LAV”) of this invention and to processes for their preparation and their use. The invention still further relates to a stable probe including a DNA sequence which can be used for the detection of the LAV virus of this invention or related viruses or DNA proviruses in any medium, particularly biological, and in samples containing any of them.
An important genetic polymorphism has been recognized for the human retrovirus which is the cause of AIDS and other diseases like LAS, AIDS-related complex (hereinafter “ARC”) and probably some encephalopathies (for review, see Weiss, 1984). Indeed all of the isolates, analyzed until now, have had distinct restriction maps, even those recovered at the same place and time [Benn et al., 1985]. Identical restriction maps have only been observed for the first two isolates which were designated LAV which was deposited at the Collection Nationale des Cultures de Micro-organismes (C.N.C.M), 28 Rue du Dr. Roux, Paris, France, on Jul. 15, 1983, under the Accession Number I-232. [Alizon et al., 1984] and human T-cell lymphotropic virus type 3 (hereinafter “HTLV-3”) [Hahn et al., 1984] and which appear to be exceptions. The genetic polymorphism of the AIDS virus was better assessed after the determination of the complete nucleotide sequence of LAV [Wain-Hobson et al., 1985], HTLV-3 [Ratner et al., 1985; Muesing et al., 1985] and a third isolate designated AIDS-associated retrovirus (hereinafter “ARV 2”) [Sanchez-Pescador et al., 1985]. In particular, it appeared that, besides the nucleic acid variations responsible for the restriction map polymorphism, isolates could differ significantly at the protein level, especially in the envelope (up to 13% of difference between ARV and LAV), by both amino acids substitutions and reciprocal insertions-deletions [Rabson and Martin, 1985].
Nevertheless, such differences did not go so far as to destroy the immunological similarity of such isolates as evidenced by the capabilities of their similar proteins, (e.g., core proteins of similar nature, such as the p25 proteins, or similar envelope glycoproteins, such as the 110-120 kD glycoproteins) to immunologically cross-react. Accordingly, the proteins of any of said LAV viruses can be used for the in vitro detection of antibodies induced in vivo and present in biological fluids obtained from individuals infected with the other LAV variants. Therefore, these viruses are grouped together as a class of LAV viruses (hereinafter “LAV-1 viruses”).
SUMMARY OF THE INVENTION
In accordance with this invention, a new virus has been discovered that is responsible for diseases clinically related to AIDS and that can be classified as a LAV-1 virus but that differs genetically from known LAV-1 viruses to a much larger extent than the known LAV-1 viruses differ from each other. The new virus is basically characterized by the cDNA sequence which is shown in
FIGS. 7A
to
7
I, and this new virus is hereinafter generally referred to as “LAV
ELI
”.
Also in accordance with this invention, variants of the new virus are provided. The RNAs of these variants and the related cDNAs derived from said RNAs are hybridizable to corresponding parts of the cDNA of LAV
ELI
. The DNA of the new virus also is provided, as well as DNA fragments derived therefrom hybridizable with the genomic RNA of LAV
ELI
such DNA and DNA fragments particularly consisting of the cDNA or cDNA fragments of LAV
ELI
or of recombinant DNAs containing such cDNA or cDNA fragments.
DNA recombinants containing the DNA or DNA fragments of LAV
ELI
or its variants are also provided. It is of course understood that fragments which would include some deletions or mutations which would not substantially alter their capability of also hybridizing with the retroviral genome of LAV
ELI
are to be considered as forming obvious equivalents of the DNA or DNA fragments referred to hereinabove.
Cloned probes are further provided which can be made starting from any DNA fragment according to the invention, as are recombinant DNAs containing such fragments, particularly any plasmids amplifiable in procaryotic or eucaryotic cells and carrying said fragments. Using cloned DNA containing a DNA fragment of LAV
ELI
as a molecular hybridization probe—either by marking with radionucleotides or with fluorescent reagents—LAV virion RNA may be detected directly, for example, in blood, body fluids and blood products (e.g., in antihemophylic factors such as Factor VIII concentrates). A suitable method for achieving such detection comprises immobilizing LAV
ELI
on a support (e.g., a nitrocellulose filter), disrupting the virion and hybridizing with a labelled (radiolabelled or “cold” fluorescent- or enzyme-labelled) probe. Such an approach has already been developed for Hepatitis B virus in peripheral blood [according to Scotto J. et al.
Hepatology
(1983), 3, 379-384).
Probes according to the invention can also be used for rapid screening of genomic DNA derived from the tissue of patients with LAV related symptoms to see if the proviral DNA or RNA present in their tissues is related to LAV
ELI
. A method which can be used for such screening comprises the following steps: extraction of DNA from tissue, restriction enzyme cleavage of said DNA, electrophoresis of the fragments and Southern blotting of genomic DNA from tissues and subsequent hybridization with labelled cloned LAV proviral DNA. Hybridization a situ can also be used. Lymphatic fluids and tissues and other non-lymphatic tissues of humans, primates and other mammalian species can also be screened to see if other evolutionary related retroviruses exist. The methods referred to hereinabove can be used, although hybridization and washings would be done under non-stringent conditions.
The DNA according to the invention can be used also for achieving the expression of LAV viral antigens for diagnostic purposes, as well as for the production of a vaccine against LAV. Fragments of particular advantage in that respect will be discussed later. The methods which can be used are multifold:
a) DNA can be transfected into mammalian cells with appropriate selection markers by a variety of techniques, such as calcium phosphate precipitation, polyethylene glycol, protoplast-fusion, etc.
b) DNA fragments corresponding to genes can be cloned into expression vectors for
E. coli,
yeast or mammalian cells and the resultant proteins purified.
c) The proviral DNA can be “shot-gunned” (fragmented) into procaryotic expression vectors to generate fusion polypeptides.
Recombinants, producing antigenically competent fusion proteins, can be identified by simply screening the recombinants with antibodies against LAV
ELI
antigens. Particular reference in this respect is made to those portions of the genome of LAV
ELI
which, in the figures, are shown to belong to open reading frames and which encode the products having the polypeptidic backbones shown.
Different polypeptides which appear in
FIGS. 7A
to
7
I are still further provided. Methods disclosed in European application O 178 978 and in PCT application PCT/EP 85/00548, filed Oct. 18, 1985, are applicable for the production of such peptides from LAV
ELI
. In this regard, polypeptides are provided containing sequences in common with polypeptides comprising antigenic determinants included in the proteins encoded and expressed by the LAV
ELI
genome. Means
Alizon Marc
Montagnier Luc
Sonigo Pierre
Wain-Hobson Simon
Institut Pasteur
Parkin Jeffrey S.
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