EP1735471A2

EP1735471A2 - Method of examining the functional genic variability of hiv and kit for implementing same

Info

Publication number: EP1735471A2
Application number: EP05757257A
Authority: EP
Inventors: Sophie Lebel-Binay; Elisabeth Dam; Luc Boblet; Dominique Costantini
Original assignee: Eurofins Viralliance Inc
Current assignee: Eurofins Viralliance Inc
Priority date: 2004-04-16
Filing date: 2005-04-15
Publication date: 2006-12-27
Also published as: WO2005108606A2; US20060292553A1; WO2005108606A3; CA2563394A1; FR2869045A1; CN1969048A

Abstract

The invention relates to a method of analysing a sample that may contain HIV virus. The inventive method comprises the following steps: (a) extraction of the viral RNA; (b) inverse transcription of the RNA obtained in step (a) and amplification of same with a first primer pair, thereby enabling the production of an amplified reverse transcription product comprising all or part of at least two successive genes of the genome of a HIV virus; (c) sequencing of the amplified reverse transcription product; (d) amplification of the amplified reverse transcription product obtained in step (b) with a second primer pair; (e) homologous recombination of the amplification product prepared in step (d) with a vector; (f) functional analysis of viral proteins encoded by all or part of at least two genes; and (g) measurement of the replicative capacity of the recombinant viruses obtained in step (e).

Description

METHOD OF STUDYING THE GENE AND FUNCTIONAL VARIABILITY OF HIV AND KIT FOR ITS IMPLEMENTATION.

The present invention relates to the field of analysis of the Human Immunodeficiency Virus type 1 (HIV-1). More particularly, the invention relates to a method and its means of implementation for the investigation of the genetic and functional variability of HIV. The Human Immunodeficiency Virus type 1 (HIV-1) is an enveloped retrovirus whose genome codes in particular for three distinct enzymes: Reverse Transcriptase which transcribes viral RNA into double stranded DNA, the integrase which allows integration of viral DNA into the genome of the target cell and the protease which is necessary for the maturation of virions. The viral enzymes, Reverse Transcriptase (RT) and protease (PR) will be the main targets of anti-retroviral drugs. Currently about fifteen anti-retroviral molecules inhibiting Reverse Transcriptase and protease are used in clinical practice. The combined use of these inhibitors leads to large decreases in viral replication, but these combinations of drugs are often complicated by significant side effects, poor compliance and the development of anti-retroviral resistant strains. One of the major causes of failure of treatments for the Human Immunodeficiency Virus (HIV) is the emergence of mutant viruses resistant to antiviral treatments which appear when the suppression of viral replication is incomplete. Escape from drug pressure causes mutations in enzymes and viral proteins (RT and PR) which play an important role in replication and viral infectivity (Fitness). Resistant viruses can exhibit reduced infectivity compared to "wild" viruses. Clinically, it seems important to have at the same time information on the genetic and functional variability on the 2 main targets since these treatments are associated in clinical practice. Today, such a tool is not available. The tests present are not sufficient, from the same viral sample of patient, to explore at the same time the known and unknown mutations and the infectivity in the presence or in the absence of drugs this on at least 2 gene targets of interest. The various current diagnostic tests make it possible to judge either the genetic variability (genotype) or the functional variability (phenotype and replicative capacity) of the viruses of patients infected with HIV with antivirals (Figure

1) - Genotypic Resistance Tests - Phenotypic Resistance Tests - Viral Replication Tests - Associated Tests

Genotypic resistance tests. In genotypic resistance tests, the viral RNA, derived from the plasma, is extracted and the regions coding for reverse transcriptase and protease are analyzed. Today, their analysis method is based on the position of the mutated amino acid preceded and followed by a letter indicating the “wild” amino acid and the mutant respectively. For example, for the resistance mutation to lamivudine M184V, Valine replaces Methionine at position 184 of the RT. Current tests mainly use automatic sequencing techniques for target genes. These tests detect all the mutations present in the sequenced region but cannot interpret them all. In fact, only known mutations are interpreted according to algorithms which are updated regularly by committees of international experts. These algorithms have become more and more complex with the growing therapeutic combinations and more than 15 drugs available on the market. Other tests, such as the "Line Probe Assay" (LiPA) or the "Gene Chips" offered by the Affymetrix Company, are based on hybridization techniques and use specific probes limited to the identification of certain mutations. The interpretation of genotypic test results is complex due to the difficulty of estimating the cumulative effects of multiple mutations, some of which may have additive effects while others will restore sensitivity.

Phenotypic Resistance tests. The principle of phenotypic resistance tests is based on the in vitro measurement of the growth of a patient's virus in the presence of drugs. In the phenotypic tests, the viral RNA, derived from the plasma is extracted then the regions coding for the reverse transcriptase and / or the protease are amplified by PCR. The amplicons are recombined in vitro in a defective vector to form a viral particle. This virus particle is cultured in the presence of increasing concentrations of drugs. The results are expressed as a “fold change” ratio of the IC 50 (or IC90) vis-à-vis a control virus, which corresponds to the concentration of the drug which inhibits 50% (or 90%) of replication. viral compared to the reference wild-type virus. The resistance level is defined according to sensitivity thresholds (eut off). Three main phenotypic resistance tests are currently available on the market: PhenoSense ™ (Virology, USA), Antivirogram ™ (Virco, Belgium) and Phenoscript ™ (VIRalliance, France). These tests give information on the susceptibility of the drugs towards their target but do not prejudge the impact of sentinel mutations on the evolution of the resistance of the virus. These two pieces of information, genotype and phenotype, could be combined to validate the technique, but in this case the methodology includes a step comprising the construction of a recombination vector by ligation (Parkin et al 2004, Antimicrob. Agents Chemother. 48: 437 ) or requires multiple cycles of infection for phenotyping (PCT patent application 00233638). These two technical approaches can introduce a bias in the representativeness of the patient's virus. On the other hand, the current phenotyping tests are not designed to be compatible with the genotyping tests in use except for internal use. Viral replication tests. Resistance mutations induced by protease and reverse transcriptase inhibitors are known to modify the replicative capacity of HIV viruses. Tests for determining the replicative capacity in vitro are based on the use of a recombinant plasmid, transfected and then amplified in cell culture. After normalization of the amount of virus, the viral supernatant is used to infect new cells. The replicative capacity is then evaluated over a given period, corresponding to a single cycle or several replication cycles depending on the methodology used. The replicative capacity of a mutated variant is generally expressed compared to that of a wild variant. The qualification of a highly infectious virus is today disconnected from its genetic and functional variability from the same patient sample.

Associated tests. PCT patent application 00233638 describes the possibility of producing a phenotype and a genotype from the same amplification product. However, the phenotyping technique used does not describe a single cycle of viral replication. Firstly, it requires viral production in permissive cells, secondly allowing the reinfection of indicator cells to measure IC50 (PCT patent application WO9727480). These stages are not very representative of the patient's initial viral populations due to multiple cycles infection without the pressure of drug selection with the risk of evolution of the initial virus. PCT patent application WO2004003513 proposes a method of genotyping, phenotyping and moreover of replicative capacity, centered on the construction by ligation of a recombination vector containing a reporter gene and the sequence studied. This method is also less representative of the reality of the behavior of the virus during the patient's infection. Cloning by ligation is interesting for the recombination yield, but can introduce biases in the selection of the patient's initial viral populations. The tests described in PCT patent application W00238792 make it possible to measure the infectivity (replicative capacity) and the phenotype from the same recombination vector. In particular, the large fragment (> 2800 bp) coding for part of the gag gene and regions of the reading frame of the pol gene coding for the protease and reverse transcriptase, can be used to determine the replicative capacity of the virus. However, this large fragment, in current practice, does not make it possible to obtain an amplification success rate and a replication rate sufficient to allow the measurement of infectivity. The following steps are therefore not feasible, which limits the use of this large fragment.

The methods of studying the HIV virus described in the prior art are limited by the difficulty in achieving both a good representativeness of the behavior of the virus (homologous recombination), a sufficient level of amplification and replication including for viruses very mutated. The method of 1 invention now allows to inform and better interpret the virus data and the patient treatment data from the same sample. There is indeed today an important need for a strategy allowing, starting from the same biological sample of a patient reached by the

HIV, to obtain a measure of the genotypic, phenotypic resistance and the replicative capacity of the virus. The purpose of the present invention is precisely to offer a new strategy making it possible, from the same biological sample of a patient infected with HIV, to obtain a measurement of the genotypic, phenotypic resistance and the replicative capacity of the virus. , so as to have a better understanding of the patient's situation and therefore to achieve better therapeutic orientation. This object is achieved according to the invention thanks to a method of analysis of a sample capable of containing an HIV virus, comprising the following steps: a) the extraction of viral RNA from a biological sample capable of containing a virus

HIV; b) reverse transcription of the RNA obtained in step (a) and amplification with a first pair of primers making it possible to obtain an amplified reverse transcription product comprising all or part of at least two genes successive genomes of an HIV virus; said method further comprising: - step (c), and / or - the following steps (d), (e), (f) and (g), below: c) sequencing the amplified product reverse transcription obtained in step (b) so as to establish a genotype of the HIV virus present in said sample and identify the mutations possibly present in said amplified reverse transcription product; d) amplification of the reverse transcription product obtained in step (b) with a second pair of primers, complementary to the first pair used in step b, and capable of generating an amplification product capable of to be inserted by homologous recombination into a defective retroviral vector in the region corresponding to the amplified reverse transcription product prepared in step (b); e) homologous recombination of said amplification product prepared in step (d) with said defective vector; f) functional analysis of the viral proteins encoded by all or part of said at least two successive genes of the reverse transcription product recombined in a vector in step (d); g) measuring the replicative capacity of the recombinant viruses obtained in step (e) in the presence or absence of one or more drugs.

The method of the invention is remarkable in that it offers the possibility of measuring the impact of anti-retroviral treatments, judged both on: gene variation recording known mutations, associated mutations and unknown mutations; - the functional variation recording the infectivity or replicative capacity of the virus in the presence or absence of anti-retrovirals; It makes it possible to study both on the same biological sample from a patient the impact of an antiviral agent on the genetic and functional variability on its initial target, for example the viral enzyme for which the anti-retroviral was designed, but also the tool will provide information in parallel on the same data, gene and functional variability, on one or more targets of interest. The method of the invention also makes it possible to be very representative of the behavior of the patient's virus, it is also representative for evaluating the genetic and functional variability of one or more targets of HIV-1 virus belonging to subtypes B and not -B. Each parameter, genotype, phenotype, replicative capacity, taken individually, provides information on resistance, and according to the invention, the virus tested is the closest to its natural behavior. The invention makes it possible, from the same biological sample, to measure the 3 key parameters of resistance: genotype, phenotype and replication capacity. It ensures efficiency in the isolation of viral populations, allows the normalization of reconstituted viruses and a quantitative analysis of the results. The 3 components allow better clinical interpretation and mutual understanding of these items to make them a combined tool of clinical utility. The method of the invention relates more particularly to a method of analyzing samples capable of containing HIV viruses belonging to subtypes B and not B. Thus, the RNA is that of an HIV virus belonging to subtypes B and not B.

For carrying out step (a), the method of the invention is more particularly interested in samples derived from a sample from a patient. It can be a blood or serum sample, but it can also come from a biological fluid or a biopsy or any other tissue preparation. The biological sample can also come from a viral culture. In general, a biological sample corresponds to all types of samples containing one or more variants of HIV, in particular HIV-1. By HIV-1 virus is meant, as indicated above, any viral strain belonging to subtype B and not B.

For carrying out step (b), the method of the invention is more particularly interested in a first pair of primers allowing the production of an amplified reverse transcription product, also referred to below as amplicon, comprising all or part of at least two genes useful in the study of resistance to anti-retrovirals. Thus, step (b) implements a pair of primers which makes it possible to prepare an amplicon characterized by: - The presence at each of its ends of areas preserved to allow the amplification of the viral populations, the potential presence of mutations of interest.

A preferred embodiment of the invention comprises, in step (b), the implementation of a first pair of primers allowing the obtaining of an amplicon, comprising all or part of the gag gene and of pol gene encoding the protease and reverse transcriptase involved in the replicative capacity of the virus, and capable of conferring on the virus a therapeutic resistance. In step (b), this involves obtaining an amplicon having, in addition to the above characteristics: - part of the nucleic acid sequence coding for gag and including the cleavage sites, - all of the sequence coding for the protease, - the sequence coding for the reverse transcriptase going at least up to codon 340. The amplicon as defined above has a size less than 2800bp, preferably between 2200 and 2700bp and most preferably between 2300 and 2600 bp. Thus, advantageously, the amplification of step (b) of the method of the invention, implements a pair of primers framing a nucleic sequence, complementary in 5 ′ to the phylogenetically conserved region of the gag gene including the sites of cleavage, containing the entire nucleic acid sequence coding for the protease, complementary to 3 'of the phylogenetically conserved region of the gene coding for reverse transcriptase. More particularly, the pair of primers used in step (b) frames a nucleic acid sequence: complementary in 5 ′ to the phylogenetically conserved region of the gag gene comprised between the codon 102 of the protein pl7 (position 1093 on the genome) and codon 76 of the p24 protein (position

1415 on the genome), and complementary in 3 'to the phylogenetically conserved region of the gene coding for reverse transcriptase, between codon 325

(position 3520) and codon 421 (position 3811 on the genome). Most preferably, the pair of primers used in step (b) frames a nucleic acid sequence: complementary in 5 'to the phylogenetically conserved region of the gag gene comprised between codon 126 (position 1165 on the genome) of the protein pl7 and codon 21 of the protein p24

(position 1250 on the genome), and complementary 3 'to the phylogenetically conserved region of the gene coding for reverse transcriptase, lying between codon 335 (position 3550 on the genome) and codon 395 (position

3751 on the genome). The amplification of step (b) of the method of the invention is carried out with a pair of primers having a size between 10 and 50 nucleotides, preferably between 20 and 30 nucleotides. Advantageously, the amplification of step (b) is carried out with a pair of primers chosen from the group comprising: - as sense primer, that represented by one of the sequences SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 5 (Table 1) - as antisense primer, that represented by one of the sequences SEQ ID NO. 2, SEQ ID NO. 4 and

SEQ ID NO. 6 (Table 1) fragments or analogs of these sequences. By analogues is meant either sequences carrying one or more mutations without affecting its hybridization capacities under stringency conditions generally encountered during PCR, or sequences which are situated from 1 to 10, 1 to 5 or 1 to 3 nucleotides upstream or downstream of said primer sequences. Very specifically, the invention relates to the implementation in step (b) of a pair of primer chosen from the group comprising: - the pair of primers Ri of sequences SEQ ID NO.l and SEQ ID NO. 2 of table 1 - the pair of primers R2 of sequences SEQ ID NO.3 and SEQ ID NO.4 of table 1 - the pair of primers R3 of sequences SEQ ID NO.5 and SEQ ID NO.6 of table 1 .

The sequencing step (c) of the method of the invention makes it possible to identify the known, unknown and associated mutations from the data available in the literature.

In step (d), the amplification of the amplified reverse transcription product obtained in step (b) uses a pair of primers which makes it possible to prepare an amplicon characterized by: - the presence at each of its ends of areas conserved to allow recombination with the retroviral vector, the potential presence of mutations of interest. In step (d), the amplification of the amplified reverse transcription product obtained in step (b) comprising all or part of the gag gene and of the pol gene coding for the protease and the reverse transcriptase is advantageously carried out with a second pair of primers, complementary to the first pair used in step (b), making it possible to obtain an amplicon further comprising: part of the nucleic acid sequence coding for gag and including the cleavage sites, - the entire sequence coding for the protease, - the sequence coding for the reverse transcriptase going at least up to codon 340. The amplicon as defined above has a size less than 2800bp, preferably between 2200 and 2700 bp and most preferably between 2300 and 2600 bp. Thus, advantageously, the amplification of step (d) of the method of the invention, implements a pair of primers framing a sequence of nucleic acids, complementary in 5 ′ to the phylogenetically conserved region of the gag gene including cleavage sites, containing the entire nucleic acid sequence encoding the protease, 3 'complementary to the phylogenetically conserved region of the gene encoding reverse transcriptase. More particularly, the pair of primers implemented in step (d) frames a nucleic acid sequence: complementary in 5 ′ to the phylogenetically conserved region of the gag gene comprised between the codon 102 of the protein pl7 (position 1093 on the genome) and codon 76 of the p24 protein (position

(position 3520) and codon 421 (position 3811 on the genome). Most preferably, the pair of primers used in step (d) frames a nucleic acid sequence: complementary in 5 ′ to the phylogenetically conserved region of the gag gene comprised between codon 126 (position 1165 on the genome) of the protein pl7 and codon 21 of the protein p24

3751 on the genome). The amplification of step (d) of the method of the invention is carried out with a pair of primers having a size of between 10 and 50 nucleotides, preferably between 20 and 30 nucleotides. Advantageously, the amplification of step (d) is carried out with a pair of primers chosen from the group comprising: - as sense primer, that represented by one of the sequences SEQ ID NO. 7 and SEQ ID NO. 9 (Table 4) - as antisense primer, that represented by one of the sequences SEQ ID NO. 8 and SEQ ID NO. 10

(Table 4) fragments or analogs of these sequences. By analogues is meant either sequences carrying one or more mutations without affecting its hybridization capacities under stringency conditions generally encountered during PCR, or sequences which are situated from 1 to 10, 1 to 5 or 1 to 3 nucleotides upstream or downstream of said primer sequences. Very specifically, the invention relates to the implementation in step (d) of a pair of primers chosen from the group comprising: - the pair of primers Ni of sequences SEQ ID NO.7 and SEQ ID NO. 8 of table 4 - the pair of primers N2 of sequences SEQ ID NO.9 and SEQ ID NO.10 of table 4.

The functional analysis of step (f) of the method of the invention consists more particularly in infecting HIV target cells with the recombinant viruses produced in step (e) in the presence or absence of one or more drugs. By way of example, there may be mentioned the infection of HIV target cells, in the presence or absence of one or more drugs, containing an indicator gene, independent of the retroviral vector, the expression of which is linked to the infection viral. The measurement of the replicative capacity in step (g) of the method of the invention consists more particularly in measuring the expression of an indicator gene in response to infection by the recombinant virus produced in step (e ) compared to a reference virus. By way of example, mention may be made of the methods integrating the optical density values resulting from an enzymatic reaction linked to a revealing gene independent of the vector and present in the infected cells.

According to a particular embodiment, the method of the invention comprises, in the case where steps (c) and (f) and (g) have been carried out: h) the processing of data relating to: - the possible presence of mutations determined in step (c), and - in the functional analysis of the viral proteins in step (f), and - in the replicative capacity of step (g), to obtain the characteristics of the virus and / or of treatment. As an example of such processing of these data, we can cite the algorithms for interpretation of the mutations identified in (c), the values of the concentrations of drugs inhibiting 50% (IC50) or 90% (IC90) of viral replication. obtained in (f), the comparison of the replicative capacity of the recombinant virus in (g) with a reference virus in the absence of drugs. These allow a reciprocal interpretation of the characteristics of the virus and treatment, so as to have a new individual and collective epidemiological monitoring tool.

The invention also relates to primers and combinations thereof for the amplification of HIV nucleic acid sequences as defined above.

The method of the invention makes it possible to have a new test capable, from the same biological sample of an HIV patient, of obtaining a measurement of the genotypic, phenotypic resistance and the replicative capacity of the virus, that is ie the gene variation recording the known mutations, the associated mutations and the unknown mutations and the functional variation recording the infectivity or replicative capacity of the virus in the presence or absence of anti-retrovirals (FIG. 2). The invention therefore also relates to a kit for implementing a method as described, comprising one or more primers defined above. Such a kit also includes a method for analyzing the 3 data obtained using the method of the invention: genotype, phenotype and replicative capacity, etc.

Other advantages and characteristics of the invention will appear in the examples which follow where reference will be made to the appended drawings in which: FIG. 1 represents the main stages of the analyzes of the genotype, of the phenotype and of the replicative capacity. FIG. 2 represents the compatibility of the analyzes of the genotype, of the phenotype and of the replicative capacity according to the method of the invention. FIG. 3 shows a better efficiency of amplification of the viral populations: The RNA originating from 4 patients (A, B, C, D) were retrotrancrits and amplified under the conditions described in the PCT patent application WO 0238792 (test 1) or under the conditions defined by the present invention in its example 1 (test 2). FIG. 4 represents the DO / P24 curve obtained in a replicative capacity test for a patient virus (virus 1) and a reference virus. FIG. 5 represents the position of the primer pairs of the invention on the HIV genome. In addition, the figure shows the organization of the gag and pol genes of HIV-1: - Gag gene:. Matrix: pl7. Capside (core): p24. Nucleocapsid (CA): p2, p7, pi, p6 - Pol gene:. Protease (PR): plO Cleavage and maturation gag and pol. Reverse transcriptase (RT): p51:

Reverse transcription. RNAse H: pl5: RNAse H activity. Integrase (Int): p31: Proviral DNA integration Figure 6 shows the regions amplified by the various commercial genotyping and phenotyping tests described in Example 3. Example l_j Gene and functional analysis of the protease and reverse transcriptase. The method according to the present invention is characterized in the first place by the generation of a specific nucleic acid compatible with both the genotyping techniques and the phenotyping techniques. To generate this first specific amplicon, we proceed as follows:

1) The viral RNA contained in a biological sample is extracted. The biological sample can be derived from a patient sample. It can be a blood or serum sample, but it can also come from a biological fluid or a biopsy or any other tissue preparation. The biological sample can also come from a viral culture. In general, a biological sample corresponds to all types of samples containing one or more HIV-1 variants. By HIV-1 virus is meant any viral strain belonging to subtypes B and not B. 2) We retrotranscribe and amplify the RNA obtained in (1) with one of the pairs of specific primers from Table 1 below to obtain an amplicon characterized by: - The presence, in 5 'and 3', of areas preserved to allow the amplification of viral populations; the presence of all the mutations of interest already described; - the presence of part of the nucleic acid sequence coding for gag and including the cleavage sites; - the entire sequence coding for the protease; - the sequence coding for reverse transcriptase going at least up to codon 340.

Table 1

FIG. 3 shows that, on 4 patients, the patent method gives a better amplification of the viral populations than under the conditions of the previous patent. On the other hand, on a series of 26 patients, the patent method allows the amplification of all the samples while the method described in the previous patent only allows the effective amplification of 16 samples out of the 26 tested (4 are negative and 6 are too weakly amplified). Tables 2 and 3 below show two examples of plasma samples from patients 1 and 2 respectively, for which the amplicons generated by the method of the invention above have enabled the genotype to be produced according to the Trugene and Viroseq techniques and of the Phenotype using the Phenoscript technique.

Table 2

Table 3

Example Analysis of the replicative capacity of HIV viruses originating from patients with protease and reverse transcriptase mutations. In this example, the method of the invention comprises the following steps: 1) The viral RNA contained in a biological sample is extracted.

2) The RNA obtained in (1) is retrotranscribed and amplified with a pair of specific primers making it possible to regularly amplify a specific amplicon, comprising at least 2 genes of interest in the study of resistance to retrovirals, as described in Example 1.

3) The preparation of a new amplicon from the amplicon obtained in step (2) above using a new pair of primers described in Table 4 below. This new amplicon is characterized by: - The presence, in 5 'and 3', of conserved zones to allow recombination with the retroviral vector the presence of all the mutations of interest already described; - the presence of part of the nucleic acid sequence coding for gag and including the cleavage sites; - the entire sequence coding for the protease; - the sequence coding for reverse transcriptase going at least up to codon 340. Table 4

The primers of the patent method described in Table 4 allow better recombination in patients with numerous mutations with a new retroviral vector deleted from part of the gag gene, from the region of the pol reading frame coding for the protease and part of the HIV-1 reverse transcriptase as the recombination described under the conditions of the prior patent. In Tables 5 and 6 below, the mutations identified in the protease and reverse transcriptase gene are listed for a series of 6 patients. Table 7 gives the mean values of replicative capacity obtained for these 6 patients in two independent tests with the method of the present invention. Under the conditions described in PCT patent application WO 0238792), in this series of patients with numerous mutations, the recombination with the retroviral vector was not effective enough to produce a sufficient level of recombinant viruses and to allow analysis of the replicative capacity. Table 5: list of the main mutations identified for protease inhibitors (PI) in the six patients studied.

Table 6; list of the main mutations identified for reverse transcriptase inhibitors (RTI) in the six patients studied.

Table 7

The primers of the patent method described in Table 4 allow, on another series of 4 patients, in addition the production of a quantity of recombinant viruses greater than the recombination described under the conditions of the prior patent as well as the normalization of infection of indicator cells using, for example, p24 antigen assay. Table 8 below describes that the amount of p24 produced by the recombinant viruses of 4 patients according to the patent method (vector GRF) is greater than that of the previous patent (vector GPR).

Table 8

The primers in Table 4 also make it possible to measure the replicative capacity on a range of recombinant viruses in comparison to a reference according to quantitative methods integrating the optical density values resulting from an enzymatic reaction linked to a revealing gene independent of the vector and present in the infected cells (Figure

4). The primers of Table 4 finally make it possible to obtain good reproducibility of the measurement of the replicative capacity on two control samples, one of which exhibits mutations known to decrease the replicative capacity. Table 9 below reports the reproducibility of the replicative capacity value on 2 control samples with known mutations in the protease. 3 independent tests. Table 9

Example 3: Compatibility of the amplicon of examples 1 and 2 with the various commercial tests.

1) Compatibility with the main commercial genotyping tests. The 4 main commercial tests are described in an article by W. Cavert & HH Balfour (Detection of antiretroviral resistance in HIV-1 Clin Lab Med 2003 23: 915). 1.1) Trugene ™ kit (Bayer visible Genetics Inc,) (Patent application WO 02/070731; Grant et. Al. Accuracy of the Trugene HIV-1 Genotyping kit J Clin Microbiol 2003 41: 1586).

The Trugene HIV-1 Genotyping Kit is used to determine the genotype of virus types B and not B. The RNA is extracted from the plasmas of patients according to conventional techniques, the viral RNA is transcribed and amplified by PCR with specific primers of the pol gene allowing the amplification of a nucleic acid sequence of 1300 bp comprising for the protease codons 1 to 99 and comprising for the reverse transcriptase the codons 1 to 247. The RT-PCR product thus obtained is used in each of the 16 sequencing reactions using the principle of the CLIP ™ reaction, a sequencing technique using labeled primers (dye primers). Four different primer pairs are used for this sequencing method, two pairs for protease sequencing and two other pairs for reverse transcriptase sequencing. Each sequence reaction is initiated by a specific labeled primer (CLIP ™) and then interrupted by the corresponding labeled nucleotide. All the synthesized fragments are then separated on an electrophoresis gel and analyzed by an automatic sequencer, specifically indicating the fragments ending in each of the four labeled nucleotides. The sequences, once reconstituted, are compared to the sequence of a reference virus, using alignment software. 1.2) ViroSeq ™ (Applied Biosystems) (Mracna M et. Al. J Clin Microbiol. 2001. 39: 4323). The RNA is extracted from the plasmas of patients according to a conventional extraction technique based on the affinity of the RNA vis-à-vis Sicily columns, the viral RNA is retrotranscribed and amplified by PCR with primers specific for the pol gene allowing the amplification of a 1800 bp nucleic acid sequence comprising for the protease codons 1 to 99 and comprising for the reverse transcriptase codons 1 to 335. The DNA thus obtained is then sequenced with 7 different primers using Big Dye ™ Terminator technology, a sequencing technique using labeled nucleotides (dye terminators). The sequence reaction is initiated by each specific unlabeled primer and then interrupted by each of the labeled nucleotides. All the synthesized fragments are then separated on an electrophoresis gel. The color of the fluorochrome will then be recorded by an automatic sequencer (ABI Prism 377 DNA sequencer), specifically indicating the fragments ending in each of the four labeled nucleotides. The sequences, once reconstituted, are compared to the sequence of a reference virus, using alignment software.

1.3) GeneSeq ™ (ViroLogic) (Parkin NT et. Al., Antimicrob.Agents Chemother. 2004. 48: 437). This technology uses resistance vectors built for the PhenoSense phenotypic study test. The nucleic acid sequence of the vector comprising, for the protease codons 1 to 99 and comprising, for the reverse transcriptase the codons 1 to 305, is analyzed by sequencing using different combinations of fluorescent probes, the nucleic acids are then deposited on an electrophoresis gel and analyzed by an automatic sequencer. The sequences obtained are compared with those obtained for reference viruses.

1.4) GenoSure ™ (Virco) (PCT patent application WO 01/81624). The RNA is extracted from the plasmas of patients according to conventional extraction techniques, the viral RNA is retrotranscribed and amplified by PCR with primers specific for the pol gene allowing the amplification of a nucleic acid sequence of 1800 bp comprising for the protease the codons 1 to 99 and comprising for the reverse transcriptase the codons 1 to 415. The DNA thus obtained is then sequenced according to the Big Dye ™ Terminator technology, previously described.

2) Compatibility with the main commercial phenotyping tests. A recent summary by M. Youle "Clinical Issues in HIV" published in December 2003 on the site www.hivandhepatitis.com describes the main resistance tests. Three tests are currently available:

Phenoscript ™ (Viralliance); Antivirogram ™ (Virco); PhenoSense ™ (ViroLogic).

2.1) The test developed by Virco, Antivirogram ™, is not compatible with the method of the invention since the 2200 bp nucleic acid fragment amplified from the patient's RNA comprises the protease from codon 10 to codon 99 and all of the reverse transcriptase. (Hertogs et. Al. 1998. Antimicrob.Agents Chemother).

2.2) PhenoSense ™ (ViroLogic) (Parkin NT et. Al., Antimicrob.Agents Chemother. 2004. 48: 437; Petropoulos et. Al., Antimicrob.Agents Chemother. 2000. 44: 920). The PhenoSense test is carried out using viral RNA extracted from the plasma of an HIV patient. The regions of the pol gene coding for the protease and the reverse transcriptase are amplified by RT-PCR to obtain a nucleic acid sequence of 1500 bp comprising the cleavage sites of the gag protein (p7-pl-p6) the entire region coding for the protease and the reverse transcriptase region from codon 1 to codon 313. This sequence is then ligated into an HIV retroviral vector containing a reporter gene (Luciferase) and deleted in the HIV envelope protein. The retroviral vector is then co-transfected into 293T cells with a vector encoding the envelope protein MLV (Murine Leukemia Virus). The viruses produced are used to infect new cells in the presence or absence of antiretrovirals. Luciferase activity in infected cells in the presence of drugs is compared to luciferase activity in the absence of drugs, which allows the calculation of concentrations inhibiting 50% of viral production (IC50). Table 10 below summarizes the characteristics of the nucleic acid sequences amplified in the main tests described above. Table 10

FIG. 6 shows the regions amplified by the various commercial genotyping and phenotyping tests described in example 3. The amplicon according to the present invention, designated “new amplicon” in FIG. 6 is compatible with all of these tests.

Example 4: Determination of a score as a tool to aid therapeutic decision.

From a biological sample of an HIV patient, the method of the invention makes it possible to take into account in a score system the measures of gene and functional variability of an HIV virus belonging to subtypes B and not B. The measurement of gene variability is carried out from the specific nucleic acid sequence amplified according to the method of the invention, then analyzed according to the usual sequencing techniques (Trugene, ViroSeq). The mutations in the protease and reverse transcriptase genes identified by these tests are interpreted according to algorithms regularly updated by expert committees. A first score of 0 to 2 is assigned to each of the 3 resistance levels determined by the interpretation. Table 11 below summarizes the interpretations given by the Trugene and ViroSeq tests according to the mutations identified and the antiretrovirals (ARVs) used and assigns a genotypic score corresponding to the importance of the resistance. Table 11

The measurement of the functional variability of an HIV virus consists in analyzing the replicative capacity of the virus in the presence (phenotype) or in the absence of antiretrovirals (Fitness). The principle of phenotypic resistance tests is based on the in vitro measurement of the growth of a patient's virus in the presence of drugs, compared to a reference virus (resistance index). The resistance level is defined according to sensitivity thresholds (eut off). A second score of 0 to 2 is assigned to each of the 3 resistance levels determined by the interpretation. Table 12 summarizes the interpretations given by the main Phenotypic tests PhenoSense and Phenoscript according to their sensitivity thresholds and assigns a phenotypic score corresponding to the importance of the resistance. Table 12 below reports the assignment of a phenotypic score according to the interpretation of the phenotypic thresholds. Table 12

The replicative capacity of a virus, or fitness, in the absence of antiretroviral is measured in comparison with a reference virus. It provides information on the ability of the virus to replicate and is expressed as a% of the reference virus. By way of examples, there may be mentioned a virus having a fitness of 100% which will be considered to have a high replicative activity and a virus having a fitness of 10% which will be considered to have a low replicative activity. The combination of the two scores, genotype, phenotype and the percentage of replicative capacity from the same biological sample should allow a better interpretation of clinical data and provide a tool to aid therapeutic decision. In practice, the addition of genotypic and phenotypic scores for a given antiretroviral, combined with the measurement of replicative capacity, can help guide the therapeutic choice. The genotypic + phenotypic score will be between 0 and 4 for each ARV and will be accompanied by a% replicative capacity. Thus, for an ARV in progress at the time of sampling if the genotypic + phenotypic score is less than 1 and the replicative capacity is high (100% or more), the molecule must be stopped because the virus is resistant and retains a strong capacity to replicate in the presence of this ARV. For an ARV in progress at the time of sampling if the genotypic + phenotypic score is less than 1, and the replicative capacity low (10%), the continuation of the molecule may be preferred to the cessation of treatment by the clinician by absence of therapeutic alternative.

Claims

1) Method for analyzing a sample capable of containing an HIV virus, comprising the following steps: a) extracting the viral RNA from a biological sample capable of containing an HIV virus; b) reverse transcription of the RNA obtained in step (a) and amplification with a first pair of primers making it possible to obtain an amplified reverse transcription product comprising all or part of at least two genes successive genomes of an HIV virus; said method further comprising: - step (c), and / or - the following steps (d), (e), (f) and (g), below: c) sequencing the amplified product reverse transcription obtained in step (b) so as to establish a genotype of the HIV virus present in said sample and identify the mutations possibly present in said reverse transcript; d) amplification of the reverse transcription product obtained in step (b) with a second pair of primers, complementary to the first pair used in step b, and capable of generating an amplification product capable of to be inserted by homologous recombination into a defective retroviral vector in the region corresponding to the amplified reverse transcription product prepared in step (b); e) homologous recombination of said amplification product prepared in step (d) with said defective vector; f) functional analysis of the viral proteins encoded by all or part of said at least two successive genes of the reverse transcription product recombined in a vector in step (d); g) measuring the replicative capacity of the recombinant viruses obtained in step (e) in the presence or absence of one or more drugs.

2) A method according to claim 1, characterized in that in step (b), the first pair of primers makes it possible to obtain an amplified product of reverse transcription, comprising all or part of at least two genes useful in the study of resistance to anti-retrovirals.

3) A method according to one of claims 1 or 2, characterized in that in step

(b), the first pair of primers makes it possible to prepare an amplified product of reverse transcription exhibiting: - at each of its ends, conserved zones to allow the amplification of the viral populations, - potentially mutations of interest.

4) A method according to one of claims 1 to 3, characterized in that in step (b), the first pair of primers makes it possible to prepare an amplified reverse transcription product comprising all or part of the gag gene and of the pol gene encoding protease and reverse transcriptase involved in the replicative capacity of the virus, and capable of imparting therapeutic resistance to the virus.

5) A method according to claim 4, characterized in that in step (b) an amplified reverse transcription product is obtained having: part of the nucleic acid sequence coding for gag and including the cleavage sites, - the entire sequence coding for the protease, - the sequence coding for the reverse transcriptase going at least up to codon 340. 6) A method according to any one of the preceding claims, characterized in that in step (b) an amplified reverse transcription product is obtained having a size less than 2800 bp, preferably between 2200 and 2700 bp and most preferably between 2300 and 2600 bp.

7) A method according to any one of claims 4 to 6, characterized in that in step (b) the first pair of primers frames a nucleic sequence, complementary in 5 ′ to the phylogenetically conserved region of the gag gene including cleavage sites, containing the entire nucleic acid sequence encoding the protease, and complementary 3 'to the phylogenetically conserved region of the gene encoding reverse transcriptase.

8) A method according to any one of claims 4 to 7, characterized in that in step (b) the first pair of primers frames a nucleic acid sequence: complementary in 5 ′ to the phylogenetically conserved region of the gag gene comprised between the codon 102 of the protein pl7 (position 1093 on the genome) and the codon 76 of the p24 protein (position

(position 3520) and codon 421 (position 3811 on the genome).

9) A method according to any one of claims 4 to 8, characterized in that in step

(b) the first pair of primers frames a nucleic acid sequence: complementary in 5 ′ to the phylogenetically conserved region of the gag gene comprised between codon 126 (position 1165 on the genome) of the protein pl7 and codon 21 of the p24 protein

(position 1250 on the genome), and complementary 3 'to the phylogenetically conserved region of the gene coding for reverse transcriptase, between codon 335

(position 3550 on the genome) and codon 395 (position

3751 on the genome).

10) A method according to any one of claims 4 to 9, characterized in that in step

(b) the amplification is carried out with a pair of primers chosen from the group comprising: - as sense primer, that represented by one of the sequences SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 5 - as antisense primer, that represented by one of the sequences SEQ ID NO. 2, SEQ ID NO. 4 and

SEQ ID NO. 6 fragments or analogs of these sequences. 11) A method according to any one of claims 4 to 10, characterized in that in step (b) the amplification is carried out with a pair of primers chosen from the group comprising: - the pair of primers sequences SEQ ID NO.l and SEQ ID NO.2 - the pair of primers of sequences SEQ ID NO.3 and SEQ ID NO.4 - the pair of primers of sequences SEQ ID NO.5 and SEQ ID NO.6 .

12) A method according to any one of the preceding claims, characterized in that the step (c) of sequencing makes it possible to identify the known, unknown and associated mutations from the data available in the literature.

13) A method according to any one of the preceding claims, characterized in that in step (d), the amplification of the amplified reverse transcription product obtained in step (b) implements a pair of primers which allows you to prepare an amplicon with: - at each of its ends, conserved zones to allow recombination with the retroviral vector - potentially mutations of interest.

14) A method according to claim 13, characterized in that in step (d), the amplification of the amplified reverse transcription product obtained in step (b) comprises all or part of the gag gene and of the pol gene coding for the protease and the reverse transcriptase, is carried out with a second pair of primers, complementary to the first pair implemented in step (b), and making it possible to obtain an amplicon comprising: - part of the sequence of nucleic acids coding for gag and including the cleavage sites, - the entire sequence coding for the protease, - the sequence coding for the reverse transcriptase going at least up to codon 340.

15) A method according to one of claims 13 or 14, characterized in that the amplicon has a size less than 2800bp, preferably between 2200 and 2700 bp and most preferably between 2300 and 2600 bp.

16) A method according to one of claims 13 to 15, characterized in that the amplification of step (d) implements a pair of primers framing a sequence of nucleic acids, complementary in 5 'to the phylogenetically conserved region of the gag gene including the cleavage sites, containing the entire nucleic acid sequence coding for the protease, complementary to 3 'of the phylogenetically conserved region of the gene coding for reverse transcriptase.

17) A method according to one of claims 13 to 16, characterized in that the pair of primers used in step (d) frames a sequence of nucleic acids: - complementary in 5 'to the phylogenetically region conserved of the gag gene included between codon 102 of the protein pl7 (position 1093 on the genome) and codon 76 of the protein p24 (position 1415 on the genome), and - complementary in 3 'to the phylogenetically conserved region of the coding gene for reverse transcriptase, between codon 325 (position 3520) and codon 421 (position 3811 on the genome).

18) A method according to one of claims 13 to 17, characterized in that the pair of primers used in step (d) frames a sequence of nucleic acids: - complementary in 5 'to the phylogenetically region conserved of the gag gene included between codon 126 (position 1165 on the genome) of the protein pl7 and codon 21 of the protein p24 (position 1250 on the genome), and - complementary in 3 'to the phylogenetically conserved region of the coding gene for reverse transcriptase, between codon 335 (position 3550 on the genome) and codon 395 (position 3751 on the genome). 19) A method according to one of claims 13 to 18, characterized in that the amplification of step (d) is carried out with a pair of primers chosen from the group comprising: - as sense primer, that represented by one of the sequences SEQ ID NO. 7 and SEQ ID NO. 9 - as antisense primer, that represented by one of the sequences SEQ ID NO. 8 and SEQ ID NO. 10 fragments or analogs of these sequences.

20) A method according to one of claims 13 to 19, characterized in that the amplification of step (d) is carried out with a pair of primers chosen from the group comprising: - the pair of primers of sequences SEQ ID NO.7 and SEQ ID NO.8 - the pair of sequence primers SEQ ID NO.9 and SEQ ID NO.10.

21) A method according to any one of the preceding claims, characterized in that the functional analysis of step (f) consists in infecting HIV target cells with the recombinant viruses produced in step (e) in the presence or in the absence of one or more drugs.

22) A method according to any one of the preceding claims, characterized in that the measurement of the replicative capacity in step (g) consists in measuring the expression of an indicator gene in response to infection by the virus recombinant produced in step (e) in comparison to a reference virus. 23) A method according to any one of the preceding claims, characterized in that in the case where steps (c) and (f) and (g) have been carried out, said method comprises: h) the processing of the relative data: - the possible presence of mutations determined in step (c), and - the functional analysis of the viral proteins in step (f), and - the replicative capacity of step (g), to obtain characteristics of the virus and / or treatment.

24) A set of primers capable of being implemented in a method according to any one of claims 1 to 23, characterized in that it comprises or consists of a pair of primers framing a nucleic sequence, 5 'complementary to the phylogenetically conserved region of the gag gene including the cleavage sites, containing the entire nucleic acid sequence coding for the protease, and 3' complementary to the phylogenetically conserved region of the gene coding for the reverse transcriptase .

25) A set of primers according to claim 24, characterized in that said pair of primers frames a nucleic acid sequence: complementary in 5 ′ to the phylogenetically conserved region of the gag gene comprised between codon 102 of the protein pl7 (position 1093 on the genome) and codon 76 of the p24 protein (position

1415 on the genome), and 3 'complementary to the phylogenetically conserved region of the gene coding for reverse transcriptase, between codon 325

(position 3520) and codon 421 (position 3811 on the genome).

26) A set of primers according to one of claims 24 or 25, characterized in that said pair of primers frames a nucleic acid sequence: complementary in 5 ′ to the phylogenetically conserved region of the gag gene comprised between the codon 126 (position 1165 on the genome) of the protein pl7 and codon 21 of the protein p24 (position 1250 on the genome), and complementary in 3 ′ to the phylogenetically conserved region of the gene coding for reverse transcriptase, between the codon 335 (position 3550 on the genome) and codon 395 (position 3751 on the genome).

27) A set of primers according to one of claims 24 to 26, characterized in that said pair is chosen from the group comprising: - as sense primer, that represented by one of the sequences SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 5 - as antisense primer, that represented by one of the sequences SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6 fragments or analogs of these sequences. 28) A set of primers according to one of claims 24 to 27, characterized in that said pair is chosen from the group comprising: - the pair of primers sequences SEQ ID NO.l and SEQ ID NO.2 - the pair of primers of sequences SEQ ID NO.3 and SEQ ID NO.4 - the pair of primers of sequences SEQ ID NO.5 and SEQ ID NO.6.

29) A set of primers capable of being implemented in a method according to any one of claims 1 to 23, characterized in that it comprises or consists of a pair of primers chosen from the group comprising : - as sense primer, that represented by one of the sequences SEQ ID NO. 7 and SEQ ID NO. 9 - as antisense primer, that represented by one of the sequences SEQ ID NO. 8 and SEQ ID NO. - fragments or analogs of these sequences.

30) A set of primers according to claim 29, characterized in that said pair of primers is chosen from the group comprising: - the pair of primers with sequences SEQ ID NO.7 and SEQ ID NO.8 - the pair of primers of sequences SEQ ID NO.9 and SEQ ID NO.10.

31) A primer set according to any one of claims 24 to 30, characterized in that said pair of primers makes it possible to amplify a sequence having a size less than 2800bp, of preferably between 2200 and 2700 bp and most preferably between 2300 and 2600 bp.

32) A kit for implementing a method according to any one of claims 1 to 23, characterized in that it comprises at least one set of primers according to any one of claims 24 to 31.