Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
  • C12Q1/702—Specific hybridization probes for retroviruses
  • C12Q1/703—Viruses associated with AIDS
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

• the present invention relates to a method for analyzing the phenotypic characteristics which exhibit certain strains of virus, in particular of HIV virus.
• Genotypic tests which are rapid and widely available for detecting the presence of mutations in the viral genes have been developed for example to detect mutations present in the genes coding for the protease or the reverse transcriptase of HIV. Although some mutations have been associated with a particular inhibitor of viral activity, many others are associated with treatment with multiple molecules. In addition, with the development of new inhibitor molecules, the genotyping of variants of viruses that escape treatment becomes more and more complex. This makes it difficult to assess and know against which inhibitors the viruses become resistant or still retain a slight susceptibility.
• phenotypic tests which measure directly in the culture medium, the modification of the phenotypic characteristic of the virus, such as the inhibition of viral activity, for example in the presence of compounds inhibiting said viral activity, provide a quantitative index of resistance .
• phenotypic characteristics whose analysis proves to be interesting, in particular from a medical point of view, are those which confer on a virus its resistance towards inhibitory agents capable of blocking at least one mechanism involved in the viral activity, those which confer on a virus its replicative capacity, those which confer on a virus its tropism towards particular targets or those which confer on a virus its aptitude to be neutralized by molecules, such as antibodies, chemokines or inhibitors.
• the present invention makes it possible to test several distinct but complementary phenotypic characteristics of HIV, using a rapid method based on the reconstruction of a recombinant virus from samples taken from infected patients and involving a single cycle of replication. viral.
• This invention is part of a decision support and optimization process for the therapeutic strategy in patients infected with HIV who are in a situation of virological failure of antiretroviral treatment, or in patients naive in antiretroviral treatment. Only a complete knowledge of the different phenotypic characteristics of the virus can effectively help the clinician to make the therapeutic decisions best suited to the particular situation of his patient.
• phenotypic characteristics of the HIV virus whose analysis is of particular interest from a medical point of view, are those linked to the expression of genes, capable of undergoing at least one mutation, located in the GAG, ENV, or POL regions. of the viral genome, such as those listed below:
• HIV viruses can be linked to the function of all regions of the viral genome, whether they are the parts coding for proteins or regions involved in the various mechanisms or stages of the viral replicative cycle. In particular, it is important to evaluate the effect produced by the mutations in the genes coding for the protease, the reverse transcriptase, the integrase or the envelope on the replication of the viruses and very particularly in the viruses having developed resistance to antiviral agents. Its analysis makes it possible to measure the replicative capacity of a virus, also called infectivity or "fitness".
• NRTI non-nucleoside reverse transcriptase inhibitors
• HIV is linked to the expression of part of the POL region coding for integrase. Its analysis provides indications for the adjustment of antiretroviral treatments by integrase inhibitors.
• HIV is linked to the expression of the HIV envelope glycoprotein and in particular to the expression of the transmembrane subunit of said glycoprotein encoded by a part of the ENV gene.
• This phenotypic characteristic is also linked to the expression of at least part of the ENV region of HIV viruses which codes for polypeptides which participate in the binding with coreceptors of the target cell.
• the envelope / co-receptor interaction allows the entry of the HIV virus into said target cell.
• Inhibitory agents interfere with the co-receptor by inhibiting its interaction with the HIV envelope.
• This phenotypic characteristic is also linked to the expression of at least part of the ENV region coding for polypeptides of the envelope of HIV viruses, which participate in the binding with one or more receptors of the target cell, in particular with CXCR4 or CCR5 coreceptors
• This phenotypic characteristic is also linked to the expression of all or part of the ENV region which codes for the envelope polypeptides of HIV viruses.
• This phenotypic characteristic is also linked to the expression of the envelope proteins of HIV viruses. Its analysis makes it possible to assess the susceptibility of viruses to the inhibitory action of antibodies or substances naturally present in the body and present in serum or in other fluids.
• the first tests to detect, for example, the phenotypic characteristic of resistance of HIV viruses to antiviral treatments were carried out using primary isolates and peripheral blood lymphocytes (PBL) stimulated by phytoagglutinin (PHA) according to a laborious and difficult to reproduce procedure. .
• PBL peripheral blood lymphocytes
• PHA phytoagglutinin
• An innovative alternative to these tests, a recombinant virus test, hereinafter called RVA was proposed by Kellam and Larder in 1994.
• This RVA analysis method measured the resistance of a recombinant virus carrying reverse transcriptase isolated from the plasma of a patient carrying the virus by co-transfection of the sequences thereof duly amplified by a polymerase chain reaction (PCR). ), with a clone of virus obtained in the laboratory, which is deleted from its reverse transcriptase and is competent for replication in a variety of well-established cell lines.
• PCR polymerase chain reaction
• Another drawback for detecting, for example, a phenotypic characteristic of resistance which is present in the methods of analysis of the prior art stems from the fact that the simultaneous presence of several mutations capable of conferring resistance with respect to the various retroviral inhibitors reduces the replicative capacity of the virus.
• the inventors have developed a new method for analyzing a phenotypic characteristic of HIV viruses which requires only a single replication cycle. This method requiring only a single cycle of viral replication is implemented by the choice of culture conditions for the recombinant viruses obtained.
• These culture conditions relate to either the control of the culture time, chosen to prevent viral replication beyond the first cycle, this culture time is between 12 hours to 72 hours, preferably 24 hours to 48 hours.
• the culture conditions also relate to the choice of cells of the first cellular system, they are chosen so that they are not permissive to viral infection, for example such cells do not have the CD4 receptor necessary for entry of the HIV virus into the cell.
• examples of such cells are HeLa or 293T cells.
• these culture conditions also relate to the recombinant viruses constructed according to the method of the invention, which have a deficit in envelope protein. These viruses, once produced in the first cellular system, are in fact incapable of re-infecting the cells of this first cellular system.
• This analysis method is based on the construction of a recombinant virus (RAV) obtained by co-transfection and homologous recombination with: a) the DNA sequences obtained from a
• a biological medium such as plasma, serum, saliva, sperm or other secretions
• the method developed by the inventors is rapid, it requires approximately seven days to be carried out and can therefore be used for routine determinations such as measuring the susceptibility of patients infected with HIV to inhibitors of viral activity.
• the inventors have already described in US Pat. No. 6,103,462 a first application of this analysis method, based on the formation of a particular recombinant virus, to determine the susceptibility of an HIV virus to protease inhibitors .
• the new methods of analysis implemented within the framework of the present invention HIV are based on the determination of phenotypic characteristics of the HIV viruses associated with mutations likely to be present at least in a gene chosen from the group comprising the genes gag, pol, protease, reverse transcriptase, RNAse H, integrase, vif, vpr, tat, rev, vpu, env, nef, cis-active sequences, LTR, dimerization sequences, splicing regulatory sequences, RRE using ad-hoc recombinant viruses.
• the invention therefore relates to a method for analyzing a phenotypic characteristic of HIV viruses present in a biological sample of a patient, said phenotypic characteristic resulting from one or more mutations in the viral genome capable of influencing viral infection, characterized in that it comprises: a) the extraction of the nucleic acids contained in a biological sample, b) at least one amplification by PCR of a segment of the nucleic acids of step (a), each with a pair d primers framing a nucleic acid sequence of the viral genome capable of carrying at least one mutation, c) the preparation of a vector comprising the parts of a genome of an HIV virus necessary for viral replication with the exception of segment amplified in step (b) and optionally with the exception of the gene coding for the envelope protein, d) the transfection of a first cellular host with: the nucleic acids obtained with et ape (b),
• a second vector comprising a gene coding for an envelope protein if the envelope gene is deleted from the vector prepared in step (c), to obtain, by homologous recombination, a chimeric virus, e) culturing said first cellular host under conditions allowing viral particles to be produced during a single replication cycle, f) infection with the viral particles obtained in step (e) of at least one second cell host capable of being infected with an HIV virus or an HIV pseudotyped virus and possibly comprising a marker gene which can be activated only following viral infection, and g) detection and / or quantification of the marker expressed in step (f) in order to highlight at least one phenotypic characteristic of the HIV viruses present in the biological sample.
• PCR of step (b) is carried out with a pair of primers framing a nucleic acid sequence comprising all or part of a region of the viral genome chosen from: gag, pol, protease, reverse transcriptase, RNAse H, integrase , vif, vpr, tat, rev, vpu, env, nave, cis-active sequences, LTR, dimerization sequences, splicing regulatory sequences or the Rev response element (RRE).
• a pair of primers framing a nucleic acid sequence comprising all or part of a region of the viral genome chosen from: gag, pol, protease, reverse transcriptase, RNAse H, integrase , vif, vpr, tat, rev, vpu, env, nave, cis-active sequences, LTR, dimerization sequences, splicing regulatory sequences or the Rev response element (RRE).
• the PCR amplification of step (b) is carried out with a pair of primers framing a nucleic acid sequence coding for a part of the gag protein of the human immunodeficiency virus and a nucleic acid sequence coding for the protease, capable of carrying at least one mutation in the gene coding for the protease and, in that the vector of step (c ) is built from a genome of an HIV virus where all or part of the gene encoding the protease is deleted.
• step (b) the amplification of step (b) according to the analysis method of the invention, of a nucleic acid sequence capable of carrying at least one mutation in the gene coding for the protease is carried out with a pair of primers having a size of between 10 and 50 oligonucleotides, comprising the sequences Fit A -: (5 'TCA CCT AGA ACT TTA AAT GC 3') (SEQ ID No: 1) and Pro A-: (5 'GGC AAA TAC TGG AGT ATT GTA TG3 '3') (SEQ ID No: 2), or consisting of fragments thereof, or sequences analogous to these carrying mutations of one or more nucleotides which do not modify essentially their capacity to hybridize the region of the protease gene carrying the mutation (s), followed by a second amplification with a pair of primers having a size of between 10 and 50 oligonucleotides, comprising the sequences: Fit B: ( 5 'AGA ACT T
• step (b) the amplification of step (b) according to the analysis method of the invention, of a nucleic acid sequence capable of carrying at least one mutation in the gene coding for the protease is carried out with a pair of primers: Fit A -: (5 'TCA CCT AGA ACT TTA AAT GC 3') (SEQ ID No: 1) and
• Fit B (5 'AGA ACT TTA AAT GCA TGG GT 3') (SEQ ID No: 3) and
• Pro B- (5 'GGA GTA TTG TAT GGA TTT TCA GG 3') (SEQ ID No: 4), to obtain a DNA segment of 1488 base pairs, extending between residues 1237 and 2725 inclusive, and the vector of step (c) is a retroviral vector deleted from the region of the pol reading frame coding for the HIV-1 protease extending from residues 1505 to 2565 inclusive, deleted from the envelope region and comprising a unique MluI restriction site.
• the PCR amplification of step (b) of the analysis method according to the invention is carried out with a pair of primers framing a nucleic acid sequence capable of carrying at least one mutation in the gene coding for reverse transcriptase
• the transfection of step (c) is carried out with a first vector constructed from a genome of a virus HIV where all or part of the gene coding for reverse transcriptase is deleted.
• (b) according to the method of analysis of the invention, with a pair of primers framing a nucleic acid sequence capable of carrying at least one mutation in the gene coding for reverse transcriptase is carried out with a pair of primers having a size of between 10 and 50 oligonucleotides, comprising the sequences MJ3 (5 'AGT AGG ACC TAC ACC TGT CA 3') (SEQ ID No: 5) and RT-EXT (5 'TTC CCA ATG CAT ATT GTG AG 3') (SEQ ID No: 6), or consisting of fragments thereof, or similar sequences thereof ci carrying mutations of one or more nucleotides which do not essentially modify their capacity to hybridize the region of the transcriptase gene carrying at least one mutation, followed by a second amplification step with a pair of primers comprising the sequences: A35 (5 'TTG GTT GCA TAA ATT TTC CCA TTA GTC CTA TT 3') (SEQ
• step (b) the amplification of step (b) according to the analysis method of the invention, with a pair of primers framing a nucleic acid sequence capable of carrying at least one mutation in the gene coding for the reverse transcriptase is performed with a pair of primers:
• RT-EXT (5 'TTC CCA ATG CAT ATT GTG AG 3') (SEQ ID No: 6), followed by a second amplification step with a pair of primers:
• step (c) is a retroviral vector deleted from the region of the pol reading frame coding for HIV-1 reverse transcriptase, extending from residues 2618 to 2872 inclusive, and comprising a single restriction site MluI.
• the invention also relates to an analysis method according to which the amplification of step (b) is carried out with a pair of primers framing a nucleic acid sequence coding for part of the gag protein, for the protease and for part of the reverse transcriptase of the human immunodeficiency virus capable of carrying at least one mutation in the nucleic acid sequence coding for the gag protein, or for the protease or for reverse transcriptase.
• step (b) the amplification of step (b) with a pair of primers framing a nucleic acid sequence coding for a part of the gag protein, for the protease and for a part of the reverse transcriptase of the virus.
• human immunodeficiency likely to carry at least one mutation in the nucleic acid sequence coding for the gag protein, or for the protease or for the reverse transcriptase is carried out with the pair of primers: gag + 1 (5 'AGGGGCAAATGGTACATCA 3') (SEQ ID No: 31) and
• step ( c) Fit B + (SEQ ID No: 1) and RT-IN (SEQ ID No: 8) to obtain a DNA segment of 2825 base pairs, extending between residues 1237 and 4062 and the transfection of step ( c) is carried out with a retroviral vector deleted from part of the gag gene and regions of the pol reading frame coding for the protease and part of the HIV-1 reverse transcriptase extending from residues 1507 to 3870 inclusive, deleted in the envelope region and having a unique Nrul restriction site.
• the virus resulting from the above-mentioned transfection can be used to determine the infectious or replicative capacity of the virus exhibiting mutations in the reverse transcriptase and / or the protease.
• the quantification of the viral particles according to this last method of analysis is accomplished by the measurement of the p24 antigen. It should be noted that a similar analysis method can in no case be carried out using a vector deleted simply from the reverse transcriptase sequence since these non-viable recombinant viruses can still produce the p24 antigen.
• a similar analysis method can in no case be carried out using a vector deleted simply from the reverse transcriptase sequence since these non-viable recombinant viruses can still produce the p24 antigen.
• the invention also relates to an analytical method for determining the susceptibility of an HIV virus to a reverse transcriptase inhibitor compound, comprising whether or not to add said reverse transcriptase inhibitor compound, optionally at different concentrations, to the second host. cell, prior to infection thereof with the viral particles obtained in step (e), and comprising in step (g) comparing the expression of the marker gene with and without a transcriptase inhibitor compound reverse
• the PCR amplification of step (b) is carried out with a pair of primers framing a nucleic acid sequence capable of carrying at least one mutation in the gene coding for integrase
• the vector of step (c) is a retroviral vector deleted from all or part of the gene coding for integrase.
• the amplification of step (b) according to the analysis method of the invention with a pair of primers framing a nucleic acid sequence capable of carrying at least one mutation in the gene coding for integrase is carried out with the pair of primers having a size between 10 and 50 oligonucleotides, comprising the sequences: INT B + 5'GTTACTAATAGAGGAAGACAAA3 '(SEQ ID No: 9) and INT B- 5'TTTTGGTGTTATTAATGCT3' (SEQ ID No: 10), or sequences similar to these carrying mutations of one or more nucleotides which do not modify essentially their capacity to hybridize the region of the integrase gene carrying at least one mutation, followed by a second amplification step, with the pair of primers: ' INT V + 5' CACCCTAACTGACACAACAA3 '(SEQ ID No: 11 ) and INT V- 5 ⁇ AGGCCTTTCTTATAGCAGA3 '(SEQ ID
• step (b) the amplification of step (b) according to the analysis method of the invention with a pair of primers framing a nucleic acid sequence capable of carrying at least one mutation in the gene coding for the integrase is carried out with the pair of primers:
• step (c) is a retroviral vector deleted from the entire region of the pol reading frame coding for the HIV-1 integrase extending from residues 4228 to 5093 inclusive and from the region coding for the viral envelope between positions 6343 and 7611 inclusive.
• the invention also relates to an analytical method for determining the susceptibility of an HIV virus to an integrase inhibitor compound, comprising whether or not to add said integrase inhibitor compound, optionally at different concentrations, during the course of the invention. step (e), before step (f) and comprising in step (g) comparing the expression of the marker gene with and without an integrase inhibitor compound.
• the PCR amplification of step (b) is carried out with a pair of primers framing a nucleic acid sequence capable of bringing about minus a mutation in the gene coding for the envelope protein
• the vector of step (c) is a retroviral vector constructed from a genome of an HIV virus where all or part of the gene coding for the protein envelope is deleted.
• the vector of step (c) is a retroviral vector deleted from the entire region coding for the extracellular portion of the gp41 subunit of the HIV-1 envelope, extending from residues 7745 to 8263 inclusive, from the region of the HIV-1 genome that constitutes the Rev response element (RRE).
• RRE Rev response element
• the amplification of step (b) with a pair of primers framing a nucleic acid sequence capable of carrying at least one mutation in the gene coding for the envelope protein is carried out with a pair of primers having a size between 10 and 50 oligonucleotides, comprising either the sequences: FIN-A: 5 'TCAAATATTACAGGGCTGCT3' (SEQ ID No: 13) and FIN-B: 5 'TAGCTGAAGAGGCACAGG3' (SEQ ID No: 14), or the sequences FuA: 5 'AAGCAATGTATGCCCCTCCCAT3' (SEQ ID No: 23) and FuB: 5 'GGTGGTAGCTGAAGAGGCACAGG3' (SEQ ID No: 24) or constituted by fragments thereof or by sequences analogous thereto carrying mutations d '' one or more nucleotides which do not essentially modify their capacity to hybridize the region of the envelope gene carrying at least one mutation followed by a second amplification step
• step (c) 5'TCCACCTTCTTCTTCGATT3 '(SEQ ID No: 16), to obtain a DNA segment of 965 base pairs extending from residues 7553 to 8517 inclusive and the vector of step (c) is a vector retroviral deleted from the entire region encoding the extracellular portion of the gp41 subunit of the HIV-1 envelope, extending from residues 7745 to 8263 inclusive, and has a unique Mull restriction site.
• the method of analysis according to the invention allows the amplification of sequences of the envelope region of HIV virus whatever their subtype and in particular of viruses of subtypes A, B, C, D and E), using for the amplification of step (b) with a pair of primers framing a nucleic acid sequence capable of carrying at least one mutation in the gene coding for the envelope protein is carried out with a pair of primers:
• FuA 5 ⁇ AGCAATGTATGCCCCTCCCAT3 '(SEQ ID No: 23) and FuB: 5 'GGTGGTAGCTGAAGAGGCACAGG3' (SEQ ID No: 24) followed by a second amplification step, carried out with the primer: FuC: 5 ⁇ TATGAGGGACAATTGGAGAAGTGA3 '(SEQ ID No:
• step (c) said mixing being preferably carried out in a ratio between (10%: 90%) and (90%: 10%) and most preferably between (60%: 40%) and (40%: 60%), to obtain a DNA segment of 805 base pairs extending from residues 7635 to 8440 inclusive and the vector of step (c) is a retroviral vector deleted from the entire region coding for the extracellular portion of the subunit HIV-1 envelope gp41, spanning residues 7745 to 8263 inclusive, and has a unique Mull restriction site.
• the amplification of step (b) with a pair of primers framing a nucleic acid sequence capable of carrying at least one mutation in the gene coding for the envelope protein is carried out with a pair of primers having a size between 10 and 50 oligonucleotides, comprising the sequences: NEU-A: 5 'TAGAAAGAGCAGAAGACAGTGGCAATG3' (SEQ ID No: 17) and FIN-B: 5 'TAGCTGAAGAGGCACAGG3' (SEQ ID No: 14) or FuB: 5 ' GGTGGTAGCTGAAGAGGCACAGG3 '(SEQ ID No: 24) or consisting of fragments thereof, or alternatively by sequences analogous to these carrying mutations of one or more nucleotides which do not essentially modify their capacity to hybridize the region of the envelope gene carrying at least one mutation, followed by a second stage of amplification, with the pair of primers having a size between 10 and 50 oligonucleotides,
• step (b) the amplification of step (b) with a pair of primers framing a nucleic acid sequence capable of carrying at least one mutation in the gene coding for the envelope protein is carried out with a pair of primers: NEU-A: 5 'TAGAAAGAGCAGAAGACAGTGGCAATG3' (SEQ ID NO: 1 .
• step (c) 5'TCCACCTTCTTCTTCGATT3 '(SEQ ID No: 16), to obtain a DNA fragment between 2106 and 2320 base pairs extending from residues 6197- 6222 to residues 6197-6222 inclusive and the vector of step (c) is a retroviral vector deleted from the whole region encoding the majority of the gpl20 subunit and the extracellular portion of the gp41 of the HIV-1 envelope, extending from residues 6480 to 8263 inclusive, and has a unique Mull restriction site.
• step (b) the amplification of step (b) with a pair of primers framing a nucleic acid sequence capable of carrying at least one mutation in the gene coding for the envelope protein is carried out with a pair of primers: NEU-A: 5 'TAGAAAGAGCAGAAGACAGTGGCAATG3' (SEQ ID NO: 1 .
• FuB 5 'GGTGGTAGCTGAAGAGGCACAGG3' (SEQ ID No: 24), followed by a second amplification step, with the primers: NEU-C: 5 'GTGGGTCACAGTCTATTATGGGG3' (SEQ ID No: 24), followed by a second amplification step, with the primers: NEU-C: 5 'GTGGGTCACAGTCTATTATGGGG3' (SEQ ID No: 24), followed by a second amplification step, with the primers: NEU-C: 5 'GTGGGTCACAGTCTATTATGGGG3' (SEQ ID No: 24), followed by a second amplification step, with the primers: NEU-C: 5 'GTGGGTCACAGTCTATTATGGGG3' (SEQ ID No: 24), followed by a second amplification step, with the primers: NEU-C: 5 'GTGGGTCACAGTCTATTATGGGG3' (SEQ ID No: 24), followed by a second amplification
• FuD2 5'TCTGTCTTGCTCTCCACCTTCTTCTT3 '(SEQ ID No: 27), said mixing being preferably carried out in a ratio between (10%: 90%) and (90%: 10%) and most preferably between (60%: 40%) and (40%: 60%), to obtain a DNA fragment of 2118 base pairs extending from residues 6322 to 8440 inclusive and the vector of step (c) is a retroviral vector deleted from the entire coding region for the majority of the sub- gpl20 unit and the extracellular portion of gp41 of the HIV-1 envelope, spanning residues 6480 to 8263 inclusive, and has a unique Mull restriction site.
• (b) with a pair of primers framing a nucleic acid sequence capable of carrying at least one mutation in the gene coding for the envelope protein is carried out with a pair of primers having a size of between 10 and 50 oligonucleotides , comprising the sequences: E00: 5 'TAGAAAGAGCAGAAGACAGTGGCAATGA3' (SEQ ID No: 19) and ES8B: 5 'CACTTCTCCAATTGTCCCTCA3' (SEQ ID No: 20), or consisting of fragments thereof, or also sequences analogous to these carrying mutations of one or more nucleotides which do not essentially modify their capacity to hybridize the region of the envelope gene carrying at least one mutation, followed by a second amplification step, with a pair primers having a size between 10 and 50 oligonucleotides comprising the sequences: E20:
• step (b) the amplification of step (b) with a pair of primers framing a nucleic acid sequence capable of carrying at least a mutation in the gene coding for the envelope protein is carried out with a pair of primers:
• E00 5'TAGAAAGAGCAGAAGACAGTGGCAATGA3 '(SEQ ID No: 19) and ES8B: 5' CACTTCTCCAATTGTCCCTCA3 '(SEQ ID No:
• E20 5'GGGCCACACATGCCTGTGTACCCACAG3 '(SEQ ID No: 21) and
• step (c) is a deleted retroviral vector of the region, coding for domains ranging from loop VI to loop V3 of the HIV-1 envelope extending from 6617 to 7250 inclusive and has a unique Nhel restriction site.
• the invention also relates to an analytical method for determining the susceptibility of an HIV virus to a fusion inhibitor compound targeting the HIV-1 gp41 protein, comprising carrying out the amplification of step (b) either with the pair of primers SEQ ID No: 13, SEQ ID No: 14 followed by a second amplification with the pair of primers SEQ ID No: 15 SEQ ID No: 16, or with the pair of primers SEQ ID No: 17 SEQ ID No: 18 followed by a second amplification with the pair of primers SEQ ID No: 18, SEQ ID No: 16, to add or not to add said fusion inhibitor compound, optionally at different concentrations, during the culture of the cell host obtained in step (e), before step (f) and comprising in step (g) comparing the expression of the marker gene with and without a fusion inhibitor compound targeting the HIV-1 gp41.
• the invention also relates to an analytical method for determining the susceptibility of an HIV virus to a compound which inhibits entry of said HIV virus into a target cell, comprising carrying out the amplification of step (b) with the pair. of primers SEQ ID No: 17 and SEQ ID No: 18 followed by a second amplification with the pair of primers SEQ ID No: 18 and SEQ ID No: 16, whether or not to add said entry inhibitor compound, optionally at different concentrations, to the cell host obtained in step (e) before the infection in step (f) and comprising in step (g) the comparison of the expression of the marker gene with and without entry inhibitor compound.
• the invention also relates to an analysis method for determining the susceptibility of an HIV virus to the inhibitory action of antibodies, comprising carrying out the amplification of step (b) with the pair of primers SEQ ID No: 17 SEQ ID No: 18 followed by a second amplification with the pair of primers SEQ ID No: 18, SEQ ID No: 16, to add or not to add said antibodies during the culture step (e), optionally at concentrations different and comprising in step (g) the comparison of the expression of the marker gene with and without antibody.
• the invention also relates to an analytical method for determining the tropism of an HIV virus for a cellular receptor, comprising performing amplification of step (b) with the pair of primers SEQ ID No: 17 SEQ ID No: 18 followed by a second amplification with the pair of primers SEQ ID No: 18, SEQ ID No: 16, to carry out the infection of step (f) with the viral particles obtained in step (e) on two distinct cellular hosts and comprising in step (g) the comparison of the expression of the marker gene by each of the two separate cell hosts.
• the cellular hosts used for the infection of step (f) according to the analysis method of the invention are chosen from cellular hosts expressing the CCR5 receptor or the CXCR4 receptor.
• the invention also relates to an analytical method for determining the susceptibility of an HIV virus to an inhibitor compound targeting the HIV-1 co-receptors, comprising carrying out the amplification of step (b) with the pair of primers.
• SEQ ID No: 17 SEQ ID No: 18 followed by a second amplification with the pair of primers SEQ ID No: 18, SEQ ID No: 16, to add or not to said inhibitor compound targeting the HIV-1 co-receptors, possibly at different concentrations, during stage (e) of culture, the infection of stage (f) being carried out on two distinct cellular hosts and comprising in stage (g) the comparison of the expression of the marker gene by each of the two separate cellular hosts.
• the invention also relates to an analytical method for determining the tropism of an HIV virus for a cellular receptor, comprising performing amplification of step (b) with the pair of primers SEQ ID No: 19 and SEQ ID No: 20, followed by a second amplification with the pair of primers SEQ ID No: 21 and SEQ ID No: 22, to infect in step (f) two distinct cellular hosts with the viral particles obtained in step (e) and comprising in step (g) the comparison of the expression of the marker gene by each of the two hosts separate cells.
• the invention also relates to an analytical method for determining the susceptibility of an HIV virus to an inhibitor compound targeting the HIV-1 co-receptors, comprising carrying out the amplification of step (b) with the pair of primers.
• the invention also relates to an analytical method for determining the infectivity or the replicative capacity of an HIV virus, which consists in comparing in step (g) the expression of the marker gene by the second infected cellular host with the viral particles. obtained by applying steps (a) to (f) to a biological sample from a patient, and expression of the marker gene by the same second cellular host infected with reference viral particles obtained in applying steps (a) to (f) to a sample containing a reference virus.
• the reference viral particles originating from a reference virus are viral particles obtained by applying steps (a) to (f) to a biological sample from the same patient at a stage prior to or before the therapeutic treatment. .
• the invention also relates to an analysis method for determining the virulence of an HIV virus comprising carrying out the amplification of step (b) either with the pair of primers SEQ ID No: 13, SEQ ID No: 14 followed by a second amplification with the pair of primers SEQ ID No: 15 SEQ ID No: 16, or with the pair of primers SEQ ID No: 17 SEQ ID No: 18 followed by a second amplification with the pair d primers SEQ ID No: 18, SEQ ID No: 16, and to measure, during the infection of step (f), the cytopathogenic effect produced on the second cellular host.
• the cytopathogenic effect produced during the infection of step (f) on the second cellular host is measured by means of cytotoxicity techniques such as measuring the induction of syncytia, the induction of apoptosis. or by flow cytometry.
• the invention also relates to an analytical method for determining the susceptibility of an HIV virus to hydroxyurea, comprising whether or not to add hydroxyurea, optionally at concentrations different, either during the culture step (e) or at the second cell host and to perform in step (g) the comparison of the expression of the marker gene with and without hydroxyurea.
• the duration of the culture step (e) according to the method of the invention is between 12 and 72 hours, very preferably it is between 24 and 48 hours.
• the subject of the invention is also a kit for implementing a method for analyzing a phenotypic characteristic of HIV viruses present in a biological sample of a patient, characterized in that it comprises: i) a pair primers framing a nucleic acid sequence of the viral genome capable of carrying at least one mutation, ii) a vector comprising the parts of a genome of an HIV virus necessary for viral replication with the exception of the segment amplified with the primers defined in (i) and of the gene coding for the envelope protein, iii) a second vector comprising a gene coding for an envelope protein, iv) a first cellular host capable of being infected with an HIV virus, v) a second cell host capable of being infected with an HIV virus and comprising a marker gene which can be activated only following viral infection, vi) the products and reagents necessary for carrying out the amplification by PCR, vii) products and reagents for the detection of the expressed marker.
• the kit according to the invention comprises: i) the pairs of primers of sequences:
• a deleted retroviral vector from the region of the pol reading frame coding for the HIV-1 protease extending from residues 1505 to 2565 inclusive, deleted from the region of envelope and comprising a single MluI restriction site, iii) a virus pseudotyped with a gene coding for an envelope protein.
• a first cellular host capable of being infected with an HIV virus v) a second cellular host capable of being infected with an HIV virus and comprising a marker gene which can be activated only following viral infection, vi) the products and reagents necessary to carry out the amplification by PCR, vii) products and reagents allowing the detection of the expressed marker.
• the kit according to the invention comprises: i) the pairs of primers of sequences: SEQ ID No: 5 and SEQ ID No: 7
• a deleted retroviral vector from the region of the pol reading frame coding for the transcriptase reverse of HIV-1, extending from residues 2618 to 2872 inclusive, and comprising a single MluI restriction site, iii) a virus pseudotyped by a gene coding for an envelope protein, iv) a first cellular host capable of be infected with an HIV virus, v) a second cellular host capable of being infected with an HIV virus and comprising a marker gene which can be activated only following viral infection, vi) the products and reagents necessary for carrying out the amplification by PCR, vii) the products and reagents allowing the detection of the expressed marker.
• the kit according to the invention comprises: i) the pairs of primers of sequences:
• HIV-1 extending from residues 4228 to 5093 inclusive and of the region coding for the viral envelope between positions 6343 and 7611 inclusive, iii) a virus pseudotyped by a gene coding for an envelope protein, iv) a first cell host capable of being infected with an HIV virus, v) a second cell host capable of being infected with an HIV virus and comprising a marker gene which can be activated only following viral infection, vi) the products and reagents necessary to carry out the amplification by PCR, vii) the products and reagents allowing the detection of the expressed marker.
• the kit according to the invention comprises: i) the pairs of primers of sequences
• the kit according to the invention comprises: i) the pairs of sequence primers - SEQ ID No: 17 and SEQ ID No: 14
• the kit according to the invention comprises: i) the pairs of primers of sequences
• the kit according to the invention comprises i) the pairs of primers of sequences - SEQ ID No: 23 and SEQ ID No: 24
• the kit according to the invention comprises: i) the pairs of primers of sequences
• Figure 1 shows schematically the plasmid pSRT.
• the region coding for the reverse transcriptase of pNL4-3xcenv is deleted by means of digestion with BalI-SnaBI. Linearization of the resulting pSRT is accomplished by the use of Nru I.
• Figure 2 illustrates the curves of the dose response effects obtained for two patients versus AZT and 3TC, before and after treatment with reverse transcriptase inhibitors.
• the curves show the inhibition of infection by a recombinant virus of P4 cells treated with either zidovuidine (AZT, panels A and C) or lamivudine (3TC, panels B and D), according to the technique described later in the hardware and methods section.
• ZT zidovuidine
• lamivudine 3TC, panels B and D
• FIG. 3 is a diagram of the first steps (a and b) of a particular implementation of the method of the invention.
• the diagram illustrates the steps of extraction (a) and amplification (b) of the reverse transcriptase sequences extracted from the plasma of a patient by RT PCR of the method of the invention as well as the construction diagrams of the plasmid pRVA / RT used later in step (d).
• FIG. 4 is a diagram of steps (c) to (g) of a particular implementation of the method of the invention. This diagram illustrates step (d) of cotransfection into HeLa / 293T cells of the amplified nucleic acids of step (b), of a first plasmid p43xcsn ⁇ env RT constructed from a genome of an HIV virus.
• step (e) making it possible to produce viral particles, the step (f) of transfection of the viral particles obtained in step (e) into P4 cells, previously incubated in the presence or not of serial dilutions of different reverse transcriptase inhibitors, said P4 indicator cells having a expression system of the gene coding for the beta-galactosidase enzyme activatable exclusively by the tat activation sequences expressed by the recombinant virus, and step (g) of detection and / or quantification of beta-galactosidase by means of the CPRG substrate.
• FIG. 5 illustrates the results of the phenotypic analysis obtained in the presence of the T20 fusion inhibitor for viruses present in patient plasmas (P1, P2 and P3) and reference viral prototypes (NL 43 and the resistant clone SUN) .
• FIG. 6 illustrates the results of measurement of the replicative capacity of viruses L1, L2 and L3 extracted from different samples.
• RNA is isolated from patient plasma using a Roche Amplicor ® kit (Roche).
• Amplification of the region coding for reverse transcriptase is carried out using the external primers MJ3 (5 'AGT AGG ACC TAC ACC TGT CA 3') (Sequence SEQ ID No: 5, in appendix) and RT-EXT (5 'TTC CCA ATG CAT ATT GTG AG 3') (Sequence SEQ ID No: 6, in appendix) and internal primers A35 (5 'TTG GTT GCA TAA ATT TTC CCA TTA GTC CTA TT 3') (Sequence SEQ ID No: 7, in appendix) and RT-IN (5 'TTC CCA ATG CAT ATT GTG AG 3') (SEQ ID No: 8, in appendix) with an initial cycle at 50 ° C (30 minutes) and 94 ° C (2 minutes), followed by 40 cycles at 94 ° C (30 seconds), 55 ° C (30 seconds) and 68 ° C (90 seconds) and a final extension step at 98 ° C for 10
• the nucleotide sequences of the regions of reverse transcriptase coding are determined by automatic sequencing of the termination of the dideoxinucleotide chain of the crude PCR products.
• the molecular clones of HIV-1 used in the analysis method are derived from pNL4-3.
• the plasmid deleted in reverse transcriptase is constructed by a modification of pNL4-3xc ⁇ env mut e to carry unique SnaBI restriction sites in position 3872 and of Nru I in position 3892.
• the enzymes Ba I and SnaB I are used to remove the region coding for reverse transcriptase (between positions 2618 and 2872) and linearization of the resulting plasmid pSRT is carried out by means of the enzyme Nru I.
• the expression of the envelope glycoprotein VSV-G in the transfected cells is ensured by the plasmid pVSV which contains the coding sequence vsv-g under the control of a CMV promoter.
• HeLa, 293 T and P4 cells are cultured in DMEM medium supplemented with 10% fetal calf serum (FCS), 50 IU / ml of penicillin and 50 ⁇ g / ml of streptomycin.
• P4 cells are Hela-CD4, LTR-LacZ cells in which the expression of beta-galactosidase is strictly inducible by the HIV transactivating Tat protein, therefore allowing precise quantification of the infectivity or the replicative capacity.
• HIV-I viruses based on a single cycle of replication (Chameau, P., Mirambeau, G., Roux, P., Paulous, S., Bue, H. and Clavel, F. (1994) "HIV-1 reverse transcription. A termination step at the center of the genome ". J Mol Biol 241 (5), 651-62.).
• P4 cells are cultured in the presence of 500 ⁇ g / ml of geneticin.
• the determination of the susceptibility of an HIV virus to a reverse transcriptase inhibitor is carried out as follows: 293 T cells are transfected with 7.5 ⁇ g of plasmid pSRT linearized with NruI, 0.1 ⁇ g of the plasmid pVSV-G and 0.5 and 1 ⁇ g of the product resulting from the PCR reaction of the reverse transcription of HIV. The transfection precipitate is removed from the cells after 18 hours of incubation and from the growth medium. fee is added.
• the supernatant is clarified by centrifugation (500 g, 15 minutes) and transferred to P4 indicator cells which have been pre-incubated with serial dilutions of a reverse transcriptase inhibitor, in triplicate wells, for four hours.
• the range of inhibitor concentrations used varies among the compounds.
• the signal produced by the activation of the marker gene was developed with CPRG for 48 hours, as for the analysis of the susceptibility to a reverse transcriptase inhibitor and the IC50 index was calculated using the equation of effect. median.
• the plasmid pSRT carrying a deletion in the region coding for pol ranging from codon 24 of reverse transcriptase (base 2618) to codon 432 of reverse transcriptase (base 3872) includes all the mutations associated with a resistance phenomenon known to date.
• the homologous sequences of the reverse transcriptase product derived from PCR extend 88 base pairs upstream and 186 bases downstream of the deletion in pSRT.
• the transfections for determining the susceptibility to reverse transcriptase inhibitors are carried out with a 293T cell line having a high capacity to be transfected rather than with HeLa cells. This is not a problem because the cells are removed from the virus-containing supernatant by centrifugation before the transfer of P4 cells.
• the transfer conditions are optimized by means of a chessboard test.
• the variation in the ratio of the plasmid / product resulting from PCR does not significantly modify the quantity of p24 or reverse transcriptase produced or the speed of reaction with CPRG. Since the circular plasmid, pVSV-G appears to be extremely toxic to 293T cells, the amount thereof has been reduced from 3 ⁇ g to 0.1 ⁇ g in the transfection mixture, leading to high yields of p24
• the inhibitor concentration ranges used are chosen according to the cellular toxicity of each compound and the IC50 / IC90 ratio for the susceptibility of resistant isolates (Table 1). For example, as the IC50 index for abacavir for P4 cells is approximately 250 ⁇ M while the IC50 index for this compound for the native strain of the virus is approximately 3 ⁇ M, the detection of resistance is limited by toxicity. A range of four serial dilutions, starting at 200 ⁇ M, has been used for abacavir, allowing the detection of up to 60 times more resistance.
• IC50 is used rather than the IC90 index because detection of the IC90 index for resistant viruses could require toxic compound levels for most reverse transcriptase inhibitors.
• the method of analysis to determine the susceptibility of the HIV virus to reverse transcriptase inhibitors gives a Standard Deviation of the geometric mean for 20 tests between 1.78 (abacavir) and 2.7 (D4T and AZT). Median standard deviation for reverse transcriptase inhibitors (RTIs) tested
• IR Resistance Index
• NL43 is a reference virus suitable for comparison with clinical isolates
• a panel of samples taken from patients with ordinary treatment was tested on 3 RVA replicates for their susceptibility to reverse transcriptase inhibitors.
• the median ICso found for 22 viruses tested tends to be slightly higher than that found for the NL43 virus with a median IR around 0.92 (for stavudine) and 1.22 (lamivudine).
• the IR is below the defined limit of 5 on the total of inhibitors for most of the samples tested, the range of IR seems wide, especially for non-nucleoside inhibitors.
• the inter-test variation for the determination of the susceptibility of HIV viruses to reverse transcriptase inhibitors indicates that in some cases there is a difference greater than 5 between the maximum IR and the minimum IR found for repeated determinations.
• the Resistance Index is the ratio of the IC 50 in the sample compared to that of NL43 determined in parallel.
• Reverse transcriptase inhibitors show agreement with the genotypic profiles of the samples.
• Sample R2 which shows a high degree of resistance compared to all the compounds tested, has multiple mutations including those of the multi-compound resistance complex (62V, 751, 77L, 116Y and 151M) which confers resistance to RTI nucleotides, 3TC resistance associated with the 184V mutation and mutations known to induce reduced susceptibility to NNRTI (181C, 190A).
• the multi-compound resistance complex 62V, 751, 77L, 116Y and 151M
• Sample R3 shows a high resistance level to 3TC, again modulated by the mutation 184V, with a considerable variation of the IR for AZT which probably reflects the inconsistent nature of the suppression of resistance induced by 215F by 184V.
• the R5 sample remains sensitive to AZT despite a 41L mutation and a 215Y mutation because of the effects of the 1001 mutation which, in combination with 103N, is responsible for the high resistance levels observed with efavirenz and nevirapine.
• step (b) The recombinations obtained using for the amplification of step (b) the pair of primers FuA: 5 ⁇ AGCAATGTATGCCCCTCCCAT3 '(SEQ ID No: 23) and FuB: 5' GGTGGTAGCTGAAGAGGCACAGG3 '(SEQ ID No: 24) followed by a second amplification, carried out with the primer: FuC: 5 ⁇ TATGAGGGACAATTGGAGAAGTGA3 '(SEQ ID No: 25) and a mixture of the following primers:
• FuDl 5'TCTGTCTCTCTCTCCACCTTCTTCTT3 '(SEQ ID No: 26)
• FuD2: 5'TCTGTCTTGCTCTCCACCTTCTTCTT3' (SEQ ID No: 27) are very effective.
• a viral production corresponding to 50-400 ng / ml of p24 is obtained after transfection.
• For the infection of the target cells between 0.5 and 5 ng p24 / well (96-well plates) are used. The increase in optical density obtained after infection of target cells is linear if the infection is carried out under these conditions
• fusion inhibitors In order to validate the analysis method concerning the susceptibility of HIV viruses to fusion inhibitors, two fusion inhibitors were used: a peptide derived from the distal helix sequence in HIV gp41 (called DP178 or T20) and a derivative of betulinic acid (RPR103611). For each inhibitor, the reduction in sensitivity of one or more resistant viruses (previously identified by other authors) compared to two reference viruses: a primary plasma virus T5A1 and a virus suitable for in vitro culture (LAI) a been performed. All viruses were produced by recombination.
• the highly resistant DIM virus shows an increase in the IC50 value of 80 times compared to T5A1 and more than 100 times compared to LAI.
• the partially resistant NL4.3 virus is characterized by an increase in IC50 of 10 times compared to T5A1 and of 12.5 times compared to LAI.
• V.1.1 Onlytprnni nat i on de la susr.ppt i hi 1 it é a VTH pi asmat i qip à l f inhihifpnr dp fusi on T? 0
• Viral RNA is extracted from the plasmas of HIV positive patients.
• a first PCR amplification is carried out with the pair of primers ED3_E01-.
• a second amplification is carried out by PCR with the pair of primers E10_FuB to obtain a DNA fragment of 2200 base pairs s' extending between residues 6322 and 8522.
• the transfection of a first cellular host is carried out with: - the nucleic acids obtained after the second amplification.
• the retroviral vector deleted from the entire region coding for the majority of the gpl20 subunit and of the extacellular portion of the gp41 of the HIV envelope, extending from residues 6480 to 8263 inclusive and comprising a unique restriction site MluI.
• the first cell host is cultured under conditions allowing the production of viral particles.
• Infection is carried out with the viral particles produced in the preceding step, of a second cellular host (comprising a marker gene which can be activated following viral infection) in the presence of an increasing dose of T20 fusion inhibitor.
• the expressed marker is quantified in order to highlight the level of susceptibility of HIV viruses present in biological samples to the T20 fusion inhibitor.
• the increase in the IC50 relative to LAI and to T5A1 is greater than 100 times.
• step (b) The recombinations obtained using for the amplification of step (b) a pair of primers NEU- A: 5'TAGAAAGAGCAGAAGACAGTGGCAATG3 '(SEQ ID No: 17) and
• FuB 5 'GGTGGTAGCTGAAGAGGCACAGG3' (SEQ ID No: 24), followed by a second amplification, with the primers:
• NEU-C 5'GTGGGTCACAGTCTATTATGGGG3 '(SEQ ID No: 18) and a mixture of primers FuDl: 5' TCTGTCTCTCTCTCCACCTTCTTCTT3 '
• a viral production corresponding to 50-400 ng / ml of p24 after transfection is obtained.
• For the infection of the target cells between 0.5 and 5 ng p24 / well (96-well plates) are used.
• the increase in optical density obtained after infection of target cells is linear if the infection is carried out under these conditions.
• a first PCR amplification is carried out with the pair of primers FITA and PROA-
• a second PCR amplification is carried out with the pair of primers FITB and PROB- to obtain a DNA fragment of 1488 base pairs extending between residues 1237 and 2725.
• the transfection of a first cellular host is carried out with: the nucleic acids obtained after the second amplification. - the retroviral vector deleted from a region coding for part of Gag and all of the protease and comprising a unique MluI restriction site.
• the culture of the first cell host is carried out under conditions allowing the production of viral particles.
• the quantity of recombinant viral particles in the supernatant of this culture is measured by an Elisa test assaying the quantity of p24 antigen.
• Infection is carried out with the viral particles produced in the preceding step, of a second cellular host (comprising a marker gene which can be activated following viral infection), by increasing dilutions of the viral supernatant
• Quantification of the expressed marker is carried out in order to demonstrate the level of infectivity of the recombinant virus.
• the replicative capacity of the recombinant virus is calculated by establishing a regression line from the measured values and retaining the slope of this line.
• the viral replicative capacity is expressed relative to that of a laboratory reference virus or relative to the recombinant virus carrying the viral sequences originating from the same patient before establishment of the treatment and before development of the resistance. The results obtained are illustrated in FIG. 6 in the appendix.
• the L1, L2 and L3 viruses correspond to recombinant viruses carrying protease sequences obtained at three different times in the same patient.
• the L1 virus carries protease sequences obtained before treatment. Each point corresponds to the optical density measured after infection of P4 indicator cells by a dilution of each of the 3 viruses.
• the replicative capacity of L2 is expressed by the ratio of the slope of L2 to that of Ll, and the replicative capacity of L3 by the ratio of the slope of L3 to that of Ll.
• the reference virus is the pre-therapeutic virus L1. When the pre-therapeutic virus is lacking, the replicative capacity of a patient virus can be expressed relative to a laboratory reference virus such as pNL4-3.

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