# US 7,767,800 B2 · Nucleic acids obtained from LAVMAL, a variant African AIDS virus

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- Number: US 7,767,800 B2
- Document ID: US7767800B2
- Status: Granted
- Kind: B2
- Application number: 10/417,217
- Filed: 2003-04-17
- Published: 2003-12-04
- Granted: 2010-08-03
- Earliest claimed: 1986-06-23
- Assignees: Institut Pasteur
- Inventors: Marc Alizon; Pierre Sonigo; Simon Wain-Hobson; Luc Montagnier
- Web page: https://patentgrep.com/patent/US7767800B2

## CPC

- C12Q 1/702: {for retroviruses}
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12Q 1/703: {Viruses associated with AIDS}
- G01N 33/56988: {HIV or HTLV}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C12N 2740/10021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2740/10022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2740/16021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2740/16051: Methods of production or purification of viral material
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

A variant of a LAV virus, designated LAVMAL and capable of causing AIDS. The cDNA and antigens of the LAVMAL virus can be used for the diagnosis of AIDS and pre-AIDS.

## Claims

### Claim 1

A purified RNA or DNA of HIV-1 encoding an HIV-1 polypeptide or containing an HIV-1 regulatory element and comprising an RNA or DNA that hybridizes under stringent conditions to at least one of the following cDNA fragments of LAVMAL:
A. a sequence having nucleotides 1 to 96, which is the long terminal repeat R region of LAVMAL;
B. a sequence having nucleotides 97 to 179, which is the 5′ long terminal repeat U5 region of LAVMAL;
C. a sequence having nucleotides 8676 to 9133, which is the 3′ long terminal repeat U3 region of LAVMAL;
D. a sequence having nucleotides 9134 to 9229, which is the 3′ long terminal repeat U3 region of LAVMAL;
E. a sequence having nucleotides 5405 to 5620, which is the tat coding region of LAVMAL;
F. a sequence having nucleotides 5134 to 5421, which is the vpr coding region of LAVMAL;
G. a sequence having nucleotides 8380 to 9006, which is the nef coding region of LAVMAL;
H. a sequence having nucleotides 350 to 1864, which is the gag coding region of LAVMAL;
I. a sequence having nucleotides 1663 to 4668, which is the pol coding region of LAVMAL;
J. a sequence having nucleotides 5799 to 8375, which is the env coding region of LAVMAL;
K. a sequence having nucleotides 764 to 1501, which is the gag p25 coding region of LAVMAL;
L. a sequence having nucleotides 1502 to 1864, which is the gag p13 coding region of LAVMAL;
M. a sequence having nucleotides 5799 to 5885, which corresponds to amino acids 1-33 of the env coding region of LAVMAL;
N. a sequence having nucleotides 5886 to 7337, which corresponds to amino acids 34 to 530 of the env coding region of LAVMAL;
O. a sequence having nucleotides 5895 to 6176, which corresponds to amino acids 37 to 130 of the env coding region of LAVMAL;
P. a sequence having nucleotides 6399 to 6635, which corresponds to amino acids 211 to 289 of the env coding region of LAVMAL;
Q. a sequence having nucleotides 7212 to 7337, which corresponds to amino acids 488 to 530 of the env coding region of LAVMAL;
R. a sequence having nucleotides 7215 to 7604, which corresponds to amino acids 490 to 620 of the env coding region of LAVMAL; and
S. a sequence having nucleotides 7782 to 7844, which corresponds to amino acids 680 to 700 of the env coding region of LAVMAL,
wherein the stringent hybridization conditions comprise hybridization in 50% formamide, 5×SSC at 42° C., and washing in 0.1×SSC, 0.1% SDS at 65° C. two times for 30 minutes, and wherein the RNA or DNA is not ARV2, and wherein the purified RNA or DNA of HIV-1 is not an RNA or DNA of LAVBRU.

### Claim 2

A cDNA fragment of LAVMAL produced by the process comprising treating a cDNA sequence that comprises a nucleotide sequence of FIGS. 7A-7I with at least one restriction enzyme, wherein the restriction enzyme is selected from the group consisting of AvaI, BamHI, BqlII, EcoRI, HindIII, HincII, KpnI, NdeI, PstI, SacI, and XbaI.

### Claim 3 (depends on 2)

An RNA fragment corresponding to the cDNA fragment of claim 2.

### Claim 4

A purified RNA or DNA of HIV-1, wherein the HIV-1 is LAVMAL or a variant of LAVMAL, comprising an RNA or DNA that hybridizes under stringent conditions to at least one of the following cDNA fragments of LAVMAL:
A. a sequence having nucleotides 1 to 96, which is the long terminal repeat R region of LAVMAL;
B. a sequence having nucleotides 97 to 179, which is the 5′ long terminal repeat U5 region of LAVMAL;
C. a sequence having nucleotides 8676 to 9133, which is the 3′ long terminal repeat U3 region of LAVMAL;
D. a sequence having nucleotides 9134 to 9229, which is the 3′ long terminal repeat U3 region of LAVMAL;
E. a sequence having nucleotides 5405 to 5620, which is the tat coding region of LAVMAL;
F. a sequence having nucleotides 5134 to 5421, which is the vpr coding region of LAVMAL;
G. a sequence having nucleotides 8380 to 9006, which is the nef coding region of LAVMAL;
H. a sequence having nucleotides 350 to 1864, which is the gag coding region of LAVMAL;
I. a sequence having nucleotides 1663 to 4668, which is the pol coding region of LAVMAL;
J. a sequence having nucleotides 5799 to 8375, which is the env coding region of LAVMAL;
K. a sequence having nucleotides 764 to 1501, which is the gag p25 coding region of LAVMAL;
L. a sequence having nucleotides 1502 to 1864, which is the gag p13 coding region of LAVMAL;
M. a sequence having nucleotides 5799 to 5885, which corresponds to amino acids 1-33 of the env coding region of LAVMAL;
N. a sequence having nucleotides 5886 to 7337, which corresponds to amino acids 34 to 530 of the env coding region of LAVMAL;
O. a sequence having nucleotides 5895 to 6176, which corresponds to amino acids 37 to 130 of the env coding region of LAVMAL;
P. a sequence having nucleotides 6399 to 6635, which corresponds to amino acids 211 to 289 of the env coding region of LAVMAL;
Q. a sequence having nucleotides 7212 to 7337, which corresponds to amino acids 488 to 530 of the env coding region of LAVMAL;
R. a sequence having nucleotides 7215 to 7604, which corresponds to amino acids 490 to 620 of the env coding region of LAVMAL; and
S. a sequence having nucleotides 7782 to 7844, which corresponds to amino acids 680 to 700 of the env coding region of LAVMAL,
wherein the stringent hybridization conditions comprise hybridization in 50% formamide, 5×SSC at 42° C., and washing in 0.1×SSC, 0.1% SDS at 65° C. two times for 30 minutes, and wherein the RNA or DNA is not ARV2, corresponds to either an ORF encoding polypeptides with the same immunogenic or immunological properties as the polypeptides of LAVMAL or to an HIV-1 regulatory element, and wherein the purified RNA or DNA of HIV-1 is not an RNA or DNA of LAVBRU.

### Claim 5 (depends on 1)

The purified RNA or DNA of HIV-1 of claim 1, wherein the purified RNA or DNA of HIV-1 is a cDNA.

### Claim 6 (depends on 1)

The purified RNA or DNA of claim 1, wherein the purified RNA or DNA comprises the sequence of at least one of the LAVMAL cDNA fragments A though S of claim 1.

### Claim 7 (depends on 5)

A method for the detection of proviral DNA or viral RNA of LAV in host tissues comprising:
(i) extracting DNA or RNA from host tissues suspected of containing proviral DNA or viral RNA of LAV,
(ii) immobilizing the DNA or RNA from (i) to a support;
(iii) hybridizing the DNA or RNA from (i) with at least one cDNA fragment, wherein the cDNA fragment is the cDNA of claim 5, under stringent conditions that allow the cDNA of claim 5 and one of proviral LAV DNA and LAV RNA to form a stable hybridized complex,
(iv) washing the support under stringent conditions; and
(v) detecting the presence of a stable hybridized complex.

### Claim 8 (depends on 7)

A method for the detection of proviral DNA or viral RNA of LAV as claimed in claim 7, further comprising, after step (i) but before step (ii), the steps:
(1) treating the DNA extracted from host tissues with a restriction enzyme to produce restriction fragments, and
(2) separating the restriction fragments by size.

### Claim 9 (depends on 5)

A method for detecting LAVMAL virion RNA comprising
i) immobilizing a sample on a substrate;
ii) i) hybridizing the sample with a probe under stringent conditions, wherein the probe is a LAVMAL DNA fragment that is the cDNA of claim 5;
iii) washing the sample under stringent conditions; and
iv) detecting if the probe bound LAVMAL virion RNA,
wherein the probe is labeled with at least one of a radionucleotide, an enzyme, or a fluorescent reagent.

### Claim 10 (depends on 2)

A method for the detection of proviral DNA or viral RNA of LAV in host tissues comprising:
(i) extracting DNA or RNA from host tissues suspected of containing proviral DNA or viral RNA of LAV,
(ii) immobilizing the DNA or RNA from (i) on a support;
(iii) hybridizing the DNA or RNA from (i) with at least one cDNA fragment of claim 2,
under stringent conditions that allow cDNA of LAVMAL and one of proviral LAV DNA and LAV RNA to form a stable hybridized complex,
(iv) washing the DNA or RNA from (i) under stringent conditions; and
(v) detecting the presence of a stable hybridized complex.

### Claim 11 (depends on 2)

A method for detecting LAVMAL virion RNA comprising:
i) immobilizing a sample on a substrate;
ii) hybridizing the sample with a probe under stringent conditions, wherein the probe is a cDNA of fragment of claim 2;
iii) washing the sample under stringent conditions;
iv) detecting if the probe bound LAVMAL virion RNA,
wherein the probe is labeled with at least one of a radionucleotide, an enzyme, or a fluorescent reagent.