WO2001057245A2 - Hiv-1 resistance assay - Google Patents

Abstract

Present invention involves a recombination assay for the HIV envelope genes, gp120, gp41, and gp160. It further involves env-deleted proviral clones, the optimization of the PCR amplification of the corresponding env-genes and the subsequent sequencing of these genes. These techniques have been applied on several HIV-1(NL4.3) strains selected in vitro in the presence of increasing concentrations of inhibitors of HIV entry and evaluated for the phenotypic resistance of these recombined viruses. This phenotypic resistance has been correlated with genotypic resistance. Present invention also involves a recombination assay for the integrase gene.

Description

HIV-1 RESISTANCE ASSAY

The current treatment of human immunodeficiency virus (HIV) infection focuses primarily on inhibition of the viral enzymes reverse transcriptase (RT) and protease (PRO). Still, combination therapies based on these compounds are not able to completely suppress virus replication. Residual replication in the presence of the selective pressure of antiviral drugs allows the emergence of drug-resistant strains, resulting in therapeutic failure (Larder et al, 1998, Vandamme et ah, 1999a). To assess the implications of HIV drug resistance on patient management, assays to evaluate drug-resistance of clinical HIV isolates against the current RT and PRO inhibitors are becoming more widely used (Kuritz es, 1999). Whereas genotypic drug resistance assays are fast and relatively cheap to monitor the presence of known resistance-related mutations, only phenotypic assays can measure the overall viral response to the drug and can determine the possible synergistic or antagonistic effects of a combined set of mutations. The fastest and most reproducible replication-based phenotypic assay is the 'Recombinant Virus Assay', devised by Kellam and Larder (Kellam and Larder, 1994). The Recombinant Virus Assay (RVA) is based on the generation of viable virus by homologous recombination of a polymerase chain reaction (PCR)-pool of patient-derived viral RT gene into a RT-deleted proviral clone of a cell- culture adapted HIN-1 strain. This technique was designed for the susceptibility testing of clinical isolates towards RT inhibitors. Maschera et al. (1995) extended this method to the protease gene. A combined RT and PRO RVA has also been developed (Hertogs et al, 1998). The resulting recombined virus retains the sensitivity towards RT and/or protease inhibitors of the clinical isolate, while the inhibition of the HIV -induced cytopathicity can now be measured by fast and cheap tests, designed for laboratory strains in a semi- automated manner.

Since virus drug resistance is the main cause of therapeutic failure, the development of new drugs, preferentially acting on new targets in the HIV replication cycle is of high priority in anti-HIV research. A potentially powerful target, in addition to RT and PRO, is HIV entry, the first event in the virus replicative cycle. Several compounds that inhibit viral entry have been described. These molecules act at different stages of HIV entry. Polyanionic structures in general inhibit the binding of gpl20 to CD4 on the surface of the host cell (Baba et al., 1988). HIV co-receptor antagonists have also been described as HIV inhibitors. The low molecular mass bicyclams are highly potent and selective CXCR4 antagonists (De Clercq et al, 1992, 1994; De Vreese et al, 1996a, b; Schols et al, 1997). The bicyclam AMD3100 is currently subject of phase II clinical trials (Hendrix et al., 1999, Vartanian, 2000). AMD3100 not only inhibits the replication of X4 strains, but may also prevent the switch from the less pathogenic R5 strains to the more pathogenic X4 HIV strains (Este et al., 1999). Other inhibitors of viral entry interact with the fusion process itself. T-20, a synthetic peptide segment of the ectodomain of gp41, has now proceeded to phase II clinical trials (Kilby et al, 1998, Drucker, 2000).

Another interesting target in the replicative cycle of HIV is the viral enzyme integrase, since integration is an indispensable step during HIV replication and no human counterpart of the enzyme is known. Several anti-HIV compounds were initially proffered as integrase inhibitors. However, only one authentic series of integrase inhibitors, the diketo acids (e.g. L-731,988) have been identified until now (Hazuda et al., 2000).

Given that the entry inhibitors AMD3100 and T-20 are already introduced in clinical studies, methods to evaluate the susceptibility of clinical virus isolates towards inhibitors of HIV entry are required. Therefore, we have developed recombination assays for the HIV envelope genes, gp!20, gp41 and gpl60. Here, we describe the construction of the different env-deleted pro viral clones, the optimization of the PCR amplification of the corresponding env-genes and the subsequent sequencing of these genes. Furthermore, we applied these techniques on several HIV-1(NL4.3) strains selected in vitro in the presence of increasing concentrations of inhibitors of HIV entry and evaluated the phenotypic resistance of these recombined viruses, and correlated this phenotypic resistance with genotypic resistance.

Since HIV-1 integration is likely to be a future target for novel anti-HIV agents, we have designed as well a recombination assay for the integrase gene. We applied the technique using a wild- type HIV-I(III_B) strain to obtain a proof-of-principle regarding the recombination of the integrase gene. ILLUSTRATIVE DESCRIPTION OF THE EMBODIMENTS

Selection of HIV-KNL4.3) strains resistant to virus cell entry inhibitors The HIV-1(NL4.3) strains resistant to L-CA (L-CA^res), BRI2923 (BRI2923^res) and AMD3100 (AMD3100^res) were selected after serial passage (23 to 63 passages) of HIV-1(NL4.3) in the presence of increasing concentrations of the respective compounds (Table 1). The experiment was initiated at a low multiplicity of infection (moi: 0.01) and a drug concentration equal to 5-fold its IC5₀. Every 3 to 4 days the culture was monitored for the appearance of CPE. When CPE was observed, the cell-free culture supernatant was used to reinfect fresh, uninfected cells in the presence of equal or higher concentrations of the compound. When no virus breakthrough was observed, the cell culture was subcultivated in the presence of the same concentration of the compound. Gradually, after subsequent passaging, the concentration of the compound required for the prevention of virus break-trough increased significantly to 30 μg/ml for L-CA, 20 μg/ml for BRI2923 and 500 μg/ml for AMD3100 (Table 1). These final concentrations were 4- and 67 fold higher than the concentration required to inhibit the replication of wild-type HIV-1(NL4.3) by 50% (IC50) for L-CA and BRI2923, respectively. For AMD3100, the final concentration was 125,000 times higher than the IC₅₀ value for wild-type HIV-1(NL4.3) (Table 1).

Genotypic analysis of the gp!20 genes of HIV-KNL4.3) strains resistant to virus cell entry inhibitors

The DNA sequencing analysis of the gpl20 encoding region of the drug-resistant

HIV-1(NL4.3) strains revealed several mutations that were not present in the wild-type strain (Table 5). Noteworthy are the amino acid changes from serine (S) (at position 160) and threonine (T) (at position 372 and 378) in the L-CA-resistant strain towards asparagine (N). The amino acid residues S and T are possible O-glycosylation sites, while N is a potential site for N-glycosylation. These carbohydrate moieties are important for the three-dimensional structure of gpl20 and are known to play an important role in the immune escape of HIV (Cheng-Mayer et al., 1999).

It has been reported before that polyanionic compounds, like dextran sulfate, can select for the mutation from N to aspartic acid (D) (negatively charged) at position 295 of gpl20 (N295D) (Este et al, 1997). This mutation is also found in the L-CA^res strain. In contrast, the polycationic molecule AMD3100 selects for histidine (H) (positively charged) at this position. The five amino acid deletion (FNSTW) in the V4 loop is associated with in vitro resistance towards various binding and fusion inhibitors: it is found in the BRI2923- and AMD3100-resistant strains. AMD3100 resistance selection elicits extensive mutations in the V3 loop, known to be important for interaction with the CXCR4 co-receptor (Verrier et al., 1999), a logical strategy for the virus to escape inhibitors of the virus-CXCR4 interaction.

Genotypic analysis of the gp41 genes of HIV-KNL4.3) strains resistant to virus cell entry inhibitors

The genotypic analysis of gp41 revealed mutations only for the BRI2923^res strain, and not for the L-CA- and AMD3100^res strains (Table 6). The substitutions of alanine (A) by valine (V) at position 22 and of leucine (L) by serine (S) at position 33 of the gp41-gene were found in the BRI2923^res strain, as well as the mixtures proline/leucine (P/L) and alanine/threonine (A/T) at the positions 216 and 308 of the gp41 gene, respectively.

Evaluation of phenotypic resistance and cross-resistance of the selected HIV-1 strains resistant to virus cell entry inhibitors

We evaluated the antiviral activity of the virus cell entry inhibitors L-CA, BRI2923, AMD3100 and T-20 against resistant HIV-l(NL4.3)-derived strains that were selected in vitro in the presence of L-CA, BRI2923 and AMD3100 (L-CA^res, BRI2923^res and AMD3100^res strains) (Table 7). Resistance of the selected strain against the drug used for selection (shown in bold on the diagonal line in Table 7) was very high for the AMD3100^res strain, but much lower for BRI2923^res and L-CA^res strains. This can be explained by lower final concentrations of the latter products during resistance selection (Table 1).

The L-CA^res strain showed little cross-resistance towards the polyanionic compound BRI2923, while the BRI2923^res strain was highly cross-resistant to the fusion inhibitor T-20, but still sensitive to L-CA and AMD3100. The AMD3100^res strain displayed cross- resistance towards BRI2923, but not towards L-CA and T-20.

L-CA only lost inhibitory activity against the L-CA^res strain. BRI2923 lost activity towards all resistant strains evaluated, although this was less pronounced in case of the L-CA^res strain. The CXCR4 antagonist AMD3100 only lost antiviral activity (158-fold) against the AMD3100^res strain, whereas T-20 was less active against the BRI2923^res strain only.

Evaluation of resistance and cross-resistance of the different env-recombined viruses

Evaluation of resistance and cross-resistance of the gpl20-recombined viruses The sensitivity of the L-CA^res g/?720-recombined strain, as well as the AMD3100^res g/../20-recombined strain matched the sensitivity of the in vitro selected L-CA^res and AMD3100^res strains for all compounds evaluated (L-CA, BRI2923, AMD3100 and T-20) (Table 7 and 8). However, gpi20-recombination of the BRI2923^res strain in the wild-type background of NL4.3 did not reproduce the resistance phenotype of the in vitro selected strain against BRI2923 and T-20. The BRI2923^res gpi 20-recombined strain displayed the same sensitivity as the NL4.3 wild-type gpi20-recombined strain to these virus cell entry inhibitors (Table 8). The wild-type gpi20-recombined strain, as well as the L-CA^res gp!20- recombined and AMD3100^res g/?i20-recombined viruses, showed a 5- to 10-fold lower sensitivity to the inhibitory effect of T-20 in comparison to the NL4.3 wild- type, the L- CA^res and the AMD3100^res in vitro selected strains.

Evaluation of resistance and cross-resistance of the gp41 -recombined viruses The L-CA^res-, BRI2923^res- and AMD3100^res gp41 -recombined viruses displayed the same sensitivity to L-CA, BRI2923 and AMD3100 as the wild-type HIV-1(NL4.3) gp41 -recombined strain. However, T-20 lost its antiviral activity against the BRI2923^res g/? 7-recombined strain, but not against the other gp41 -recombined strains; i.e. NL4.3^WT, L-CA^res and AMD3100^res (Table 9). Evaluation of resistance and cross-resistance of the gpl60-recombined viruses

The recombination of the gpl60-gene of the AMD3100^res strain fully rescued the resistance phenotype of the original in vitro selected strain. This finding is totally in line with the expectations, since gp720-recombination was already sufficient to rescue the resistance profile of this in vitro selected virus towards the four compounds evaluated. The BRI2923^res g/?7<50-recombined strain, however, displayed wild-type sensitivity to BRI2923, but not to T-20. L-CA and AMD3100 did not lose activity against the in vitro selected BRI2923^res strain, nor against the BRI2923^res gpl 60-recombined strain. gpl (50-recombination has not been carried out for the L-CA^res strain (Table 10). Genotypic analysis of the gag genes of HIV-KNL4.3') strains resistant to virus cell entry inhibitors

The genotypic analysis of gag revealed mutations only for the BRI2923^res strain, and not for the L-CA^res and AMD3100^res strains (Table 11). The substitutions of glutamic acid (E) by lysine (K) at position 12 and of lysine (K) by arginine (R) at position 27, as well as the mutations from valine (V) to isoleucine (I) at position 35 and from glycine (G) to arginine (R) at position 62 were found in the BRI2923 ^res strain. In addition, the mixture of alanine/glutamic acid (A/E) was found at position 125 of the gag gene in the BRI2923^res strain. The latter mutation is located in the p24 coding sequences of the gag gene.

Recombination of the integrase gene

To obtain a proof-of principle that this chimeric virus technology could also be applied to the integrase gene, the integrase gene of the HIV-1(III_B) strain was placed in the genetic background of the HIV-1(NL4.3) integrase-deleted clone. The homologous recombination resulted in viable virus and recombination was demonstrated by sequencing analysis. The sensitivity of the resulting virus was evaluated for a nucleoside RT inhibitor (zidovudine), a non-nucleoside RT inhibitor (nevirapine), a protease inhibitor (ritonavir) and an integrase inhibitor [ L-731,988 (diketo acid)]. As expected, the antiviral activities of these compounds against the HIV-l(IIl_B)-integrase-recombined NL4.3 strain, the HIV-I(III_B) and the HIV-1(NL4.3) strains were identical (Table 12).

Examples

Example 1. Compounds, Cells and Virus strains

L-Chicoric Acid (L-CA) was synthesized according to the method described elsewhere (Zhao and Burke, 1998). The origin of the dendrimer BRI2923 is described elsewhere (Witvrouw et al, 2000). The bicyclam AMD3100 was kindly provided by Geoffrey Henson, AnorMED (Langley, BC, Canada) and was synthesized as described (Bridger et al, 1995). T-20 was a gift from S. Brown (AIDS Research Alliance, West Hollywood, CA). MT-4 cells (Miyoshi et al., 1982) were grown and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2mM L-glutamine, 0.1% sodium bicarbonate and 20 μg gentamycin per ml.

The HIV-1 NL4.3 strain is a molecular clone (Adachi et al., 1986) obtained from the National Institutes of Health (Bethesda, MD). The HIV-1(NL4.3) strains resistant to the entry inhibitors L-CA (L-CA^res), BRI2923 (BRI2923^res) and AMD3100 (AMD3100^res) were selected after serial passage of HIV-1(NL4.3) in the presence of increasing concentrations of the respective compounds (Table 1). The AMD3100^res strain was previously selected in our laboratory (De Vreese et al, 1996a,b). The origin of HIV-1(III_B) has been described (Popovic et al, 1984).

Example 2: Drug susceptibility assay

The inhibitory effect of the various antiviral drugs on the HIV-induced cytopathicity of the different strains in human lymphocyte MT-4 cell culture was determined by the MTT assay (Pauwels et al, 1988). This assay is based on the reduction of the yellow colored 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehydrogenase of metabolically active cells to a blue formazan derivative, which can be measured spectrophotometrically. The 50% cell culture infective dose (CCID₅₀) of the different HIV virus strains was determined by titration of the virus stock using MT-4 cells. For the drug-susceptibility assays MT-4 cells were infected with 100-300 CCID₅0 of the HIV strains in the presence of five-fold dilutions of the antiviral drugs. The concentration of the compound achieving 50% protection against the cytopathic effect (CPE) of the various HIV strains, which is defined as the 50% inhibitory concentration (IC₅o), was determined.

Example 3.: Optimizing the PCR amplification and the sequencing of the different genes

PCR Amplification of gpl20 encoding sequences

MT-4 cells were infected with HIV laboratory strains resistant to virus cell entry inhibitors. DNA extraction of proviral DNA was performed using the QIAamp blood Kit (Qiagen, Westburg, Leusden, The Netherlands). A 2105-nucϊeotide base pair fragment (codons 1 to 445) of gpl20 was amplified in a nested PCR using Expand ™ High Fidelity PCR system (Boehringer Mannheim, Roche, Germany), which is composed of an enzyme mix containing thermostable Taq DNA and Pwo DNA polymerase with 3 '-5' exonuclease proofreading capacity. The outer PCR reaction was performed on a Perkin Elmer Gene Amp PCR system 9600 and the inner PCR on a Biometra Trioblock (Westburg) using the primers AV310 (5'-AGC AGG ACA TAA T/CAA GGT AGG-3' corresponding to position 5447-5467 of NL4.3) and AV311 (5' GGA GAA GTG AAT TAT ATA AG/AT ATA AAG TAG-3' corresponding to position 7630-7659 of NL4.3), followed by the primers AV312 (5'-AGA A/GGA C/TAG ATG GAA CAA GCC CCA G-3' corresponding to position 5549-5573 of NL4.3) and AV313 (5'-GAC CTG GAG GAG GAA/G ATA TGA G/AGG A-3' corresponding to position 7605-7629 of NL4.3). The outer cycling conditions were as follows: a first denaturation step of 2 min at 95 °C followed by 40 cycles of 30 sec 95°C, 30 sec 50°C, 2 min 68°C. A final extension was performed at 72°C for 10 min. For the inner cycling, the following conditions were used: after 2 min at 95°C, 30 cycles of 30 sec 95°C, 30 sec 58°C, 2 min 68°C and 10 min 72°C extension (Table 2).

Sequencing of the gpl20-coding region

PCR products were purified using the PCR purification kit (Qiagen, Westburg, Leusden,

The Netherlands). To carry out the sequencing reaction, the ABI PRISM™ Dye terminator cycle sequencing core kit (Perkin Elmer, Brussels, Belgium) was used. The primers used to sequence the gpl20 gene were: AV304 (5'-ACA TGT GGA AAA ATG ACA TGG T-3' corresponding to position 6504-6525 of NL4.3), AV305 (5'-GAG TGG GGT TAA TTT TAG ACA TGG-3' corresponding to position 6552-6575 of NL4.3), AV306 (5'-TGT CAG CAC AGT ACA ATG TAC ACA-3' corresponding to position 6946-6969 of NL4.3), AV307 (5'-TCT TCT TCT GCT AGA CTG CCA T-3' corresponding to position 6987-7008 of NL4.3), AV308 (5'-TCC TCA GGA GGG GAC CCA GAA ATT-3' corresponding to position 7313-7336 of NL4.3), AV309 (5'-CAG TAG AAA AAT TCC CCT CCA CA-3' corresponding to position 7333-7355 of NL4.3) and AV313 (5'-GAC CTG GAG GAG GAA/G ATA TGA G/AGG A-3' corresponding to position 7605-7629 of NL4.3) (Table 2). The samples were loaded on the ABI PRISM 310 Genetic Analyser (Perkin Elmer, Brussels, Belgium). The sequences were analyzed using the software program Geneworks 2.5.1 (Intelligenetics Inc., Oxford, UK). PCR Amplification of gp41 encoding sequences

A 1560-nucleotide base pair fragment (corresponding to position 7353-8913 of NL4.3) was amplified by PCR using Expand ™ High Fidelity PCR system. The PCR reaction was performed on a Biometra Trioblock using the primers AV320 (5'-ATT GTA/G GAG GA/GG AAT TTT TCT ACT G-3' corresponding to position 7353-7378 of NL4.3) and AV321 (5' TTG CTA G CTT GTG ATT GCT/C CCA TG-3' corresponding to position 8890-8913 of NL4.3). The cycling conditions were as follows: a first denaturation step of 2 min at 95°C followed by 40 cycles consisting of 15 sec 95°C, 30 sec 55°C, 2 min 68°C. A final extension was performed at 72°C for 10 min (Table 2).

Sequencing of the gp41 -coding region

The primers used to sequence the gp41 gene were: AV322 (5' -AAG CAA TGT ATG CCC CTC C-3' corresponding to position 7518-7537 of NL4.3), AV323 (5'-CTG CTC CC/TA AGA ACC CAA-3' corresponding to position 7764-7782 of NL4.3), AV324 (5'-GGC AAA GAG AAG AGT GGT-3' corresponding to position 7723-7741 of NL4.3), AV325 (5'-GTA TCT TTC CAC AGC TAG-3' corresponding to position 7946-7964 of NL4.3), AV326 (5'-TTG GGG T/CTG CTC TGG AAA AC-3' corresponding to position 8008- 8028 of NL4.3), AV327 (5'-TTT TAT ATA CCA CAG CCA-3' corresponding to position 8237-8255 of NL4.3), AV328 (5'-ATA ATG ATA GTA GGA GG-3' corresponding to position 8279-8296 of NL4.3), AV329 (5'-GTC CCA GAA GTT CCA CA-3' corresponding to position 8549-8566 of NL4.3), AV330 (5'-GGA G/ACC TGT GCC TCT TCA-3' corresponding to position 8505-8523 of NL4.3), AV331 (5'-TCT CAT TCT TTC CCT TA-3' corresponding to position 8825-8842 of NL4.3) (Table 2).

PCR Amplification of the gplόO encoding sequences

A 3577 nucleotide base pair fragment (corresponding to position 5447-9024 of NL4.3) was amplified by PCR using Expand ™ High Fidelity PCR system. The PCR reaction was performed on a Biometra Trioblock using the primers AV310 (5'-AGC AGG ACA TAA T/CAA GGT AGG-3' corresponding to position 5447-5467 of NL4.3) and AV319 (5' - GCT G/C CC TTA/G TAA GTC ATT GGT CT-3' corresponding to position 9001-9024 of NL4.3). The cycling conditions were as follows: a first denaturation step of 2 min at 95°C followed by 40 cycles consisting of 15 sec 95°C, 30 sec 55°C, 3 min 40 sec 68°C. A final extension was performed at 72°C for 10 min (Table 2). PCR Amplification of gag encoding sequences

A 1424-nucleotide base pair fragment (corresponding to position 59-1483 of NL4.3) was amplified by PCR using AmpliTaq (Perkin Elmer, Brussels, Belgium). The PCR reaction was performed on a Biometra Trioblock using the primers AV14 (5'-CTC TCT CGA CGC AGG ACT CGG CTT GCT GAA-3') corresponding to position 59-88 of NL4.3 and BVR2 (5'-GCC AG/AA TC/TT TCC CTA AAA AAT TAG CC-3') corresponding to position 1457-1483 of NL4.3. The cycling conditions were as follows: a first denaturation step of 2 min at 95°C followed by 40 cycles consisting of 15 sec 95°C, 30 sec 55°C, 90 sec 72°C. A final extension was performed at 72°C for 10 min (Table 3). Sequencing of the gag-coding region

The primers used to sequence the pi 7 and p24 encoding regions of the gag gene were: AV13 (5'-CTG CGA ATC GTT CTA GCT CCC TGC TTG CCC -3') corresponding to position 245-274 of NL4.3, AV26 (5'-GCT ATG TCA CTT CCC CTT GGT TCT C -3') corresponding to position 829-853 of NL4.3, AV54 (5'-GAG ACC ATC AAT GAG GAA GCT GC-3') corresponding to position 774-796 of NL4.3, AV58, (5'-GGC TAA TTT TTT AGG-3') corresponding to position 1457-1471 of NL4.3, AV159 (5'-GGG GTT AAA TAA AAT AGT AAG-3') corresponding to position 971-991 of NL4.3, AV103 (5'- GCC ATA TCA CCT AGA ACT TT-3') corresponding to position 603-622 of NL4.3, GAG-1 (5'-AGA AAC ATC AGA AGG CTG TAG-3') corresponding to position 320-340 of NL4.3, BVR3 (5'-TTT CCA ACA GCC CTT TTT CCT AG-3') corresponding to position 1368-1390 of NL4.3, BVR4 (5'-AGA CAC CAA G/AGA AGC-3') corresponding to position 352-366 of NL4.3, BVR5 (5'-TCC T/CTC TGA TAA TGC TG-3') corresponding to position 669-685 of NL4.3 and BVR6 (5'-TAG AAG AAA TGA TGA CAG C-3') corresponding to position 1195-1213 of NL4.3 (Table 3)

PCR Amplification of integrase encoding sequences

A 2578-nucleotide base pair fragment (corresponding to position 3003-5581 of NL4.3) was amplified by PCR using Expand ™ High Fidelity PCR system. The PCR reaction was performed on a Biometra Trioblock using the primers MW3 (5' -TAT GTA GGA/G TCT GAC/T TTA GAA ATA GGG-3' corresponding to position 3117-3144 of NL4.3) and MW4 (5'-TAA CAC TAG GCA A/GAG GTG GCT T-3' corresponding to position 5524-5546 of NL4.3). The cycling conditions were as follows: a first denaturation step of 2 min at 95°C followed by 40 cycles consisting of 15 sec 95°C, 30 sec 60°C, 3 min 68°C. A final extension was performed at 72°C for 10 min (Table 4).

Sequencing of the integrase-coding region The primers used to sequence the integrase gene were: IN-PCRA (5'- GGAGGAAATGAACAAGTAGAT-3' corresponding to position 4181-4202 of NL4.3), HP4392C (5'-TCTACTTGTCCATGCATGGCTTC-3' corresponding to position 4377- 4399 of NL4.3), IN-SEQ1 (5'-TTAAGATGTTCAGCCTGATCT-3' corresponding to position 4733-4753 of NL4.3), IN-SEQ2 (5'-CATTGTCTGTATGTACTGTTT-3' corresponding to position 4567-4587 of NL4.3), IN-SEQ3 (5'- GGATATATAGAAGCAGAAGTAA-3' corresponding to position 4479-4500 of NL4.3), IN-SEQ4 (5'-GAACATCTTAAGACAGCAGTA-3' corresponding to position 4743-4753 of NL4.3), IN-SEQ5 (5'-AAGCTCCTCTGGAAAGGTGAA-3' corresponding to position 4953-4973 of NL4.3), and IN-PCRB (5'-CCTTGAAATATACATATGGTG-3' corresponding to position 5125-5145 of NL4.3) (Table 4).

Example 4. : Construction of the different deleted clones.

Construction of the gpl20-deleted clone. The proviral molecular clone pNL4.3 (Adachi et al, 1986) was used as starting material. This clone consists of the plasmid pUC18, wherein the complete HIV-1 genome flanked with chromosomal DNA is inserted. To generate the gp 20-deleted clone, pNL4.3 was digested with the restriction enzymes Sal I and Mam I. The vector was purified by gel extraction (using β-agarase I) and subsequent phenol/chloroform extraction. A 23/19 base pair linker containing the Sal I and Mam I restriction sites was ligated into the vector to recirculize the plasmid (Fig. la). Escherichia coli bacteria (DH5α) were transformed with this gp720-deleted clone. For use in recombination experiments large-scale plasmid DNA preparations were linearized by Sal I digestion and recombined with PCR amplified gp!20 genes from the selected strains resistant to virus cell entry inhibitors (Fig. lb).

Construction of the gp41 -deleted clone.

To generate the g/? 7-deleted clone, pNL4.3 was digested with the restriction enzymes

Mam I and Cel II. A 22/25 base pair linker containing the Xba I restriction site was ligated into the vector to recirculize the plasmid (Fig. 2a). For use in recombination experiments large-scale plasmid DNA preparations were linearized by Xba I digestion and recombined with PCR amplified gp41 -genes derived from the HIV strains selected in the presence of virus cell entry inhibitors (Fig. 2b).

Construction of the gplβO-deleted clone. pNL4.3 was digested with the restriction enzymes Sal I and Cel II to generate the gpl 60- deleted clone. A 28/28 base pair linker containing the Sal I and Cel II restriction sites was ligated into the vector to recirculize the plasmid (Fig. 3a). For use in recombination experiments large-scale plasmid DNA preparations were linearized by Sal I digestion and recombined with PCR amplified gpl60-ge s derived from the HIV strains selected in the presence of virus cell entry inhibitors (Fig. 3b).

Construction of the integrase-deleted clone. To generate the tegrase-deleted clone, pNL4.3 was digested with the restriction enzymes Pin Al and Van 91 1. A 21/14 base pair linker containing the Xba I restriction site was ligated into the vector to recirculize the plasmid (Fig. 4a). For use in recombination experiments large-scale plasmid DNA preparations were linearized by Xba I digestion and recombined with PCR amplified integrase genes from certain virus strains (Fig. 4b).

Construction of the gag -deleted clone. pNL4.3 has been digested with the restriction enzymes BssH II and Spe I to generate the ?77-deleted clone. A linker sequence, which contains besides the BssH II and Spe I restriction sites, the rest of the packaging signal and the novel Mlu I restriction site, has been created by means of PCR amplification (Figures 6 and 7). PCR amplification of the pNL4.3 sequence with the primers AV14 and PC-VAB, followed by digestion of the resulting PCR product with the endonucleases BssH II and Spe I resulted in a unique linker sequence, which has been ligated into the vector to recirculize the plasmid. For use in recombination experiments large-scale plasmid DNA preparations were linearized by Mlu I digestion and can be recombined with PCR amplified gαg-genes derived from the HIV strains selected in the presence of virus cell entry inhibitors. Construction of the gag/gpl60-deleted clone.

The described gp76^~0-deleted clone has been digested with the restriction enzymes BssH II and Spe I to generate the pi7/gp7<50-deleted clone (Figure 5a). The linker sequence as described above in the construction of the gαg-deleted clone, was ligated into the vector to recirculize the p77/gp76O-deleted plasmid. For use in recombination experiments large-scale plasmid DNA preparations of the p77/gp76O-deleted clone were linearized in parallel. One batch was digested with Mlu I at the gαg-deletion, whereas another batch was digested with Sal I at the env-deletion. These linearized plasmids can be co-transfected in the appropriate cell line with the PCR-derived gag and env sequences derived from the HIV strains selected in the presence of virus cell entry inhibitors (Figure 5b).

Due to the low percentage of occurrence of homologous recombination after electroporation, tranfection of cells by means of lipofection may be a better way of introducing DNA in the target cells. Since, 293 cells are known to have a high tranfection efficiency, these cells can be used for lipofection, when MT-4 or other target cells for HIV are added after this tranfection.

Generation of the linker sequence for the pi 7- and pl7/gp!60-deleted clone A 126 nucleotide base pair fragment of pNL4.3 was amplified by PCR using Expand ™ High Fidelity PCR system. The PCR reaction was performed on a Biometra Trioblock using the primers AV14 (5'-CTC TCT CGA CGC AGG ACT CGG CTT GCT GAA-3') and PC-VAB (5' ACT AGT AGT TCC TGC TAT GTC ACG CGT CTC TCT CCT TCT AGC C 3'). PC-VAB was specifically designed to insert the Mlu I and Spe I restriction sites in the linker sequence. Therefore, a tail with the required sequence was added to the obligatory sequence to amplify the fragment in front of the start codon of pi 7, i.e. the packaging signal (Figure 6). The cycling conditions were as follows: 40 cycles consisting of 15 sec 95°C, 30 sec 55°C and 30 sec 68°C. A final extension was performed at 72°C for 10 min. The resulting PCR fragment was digested with the restriction endonucleases BssH II and Spe I and subsequently purified by phenol/chloroform extraction. Example 5.: Co- transfection of the different sequences derived from the wild-type and resistant strains with their corresponding delected clone.

MT-4 cells were subcultured at a density of 500.000 cells/ml on the day before transfection. Cells were pelleted and resuspended in phosphate-buffered saline at a concentration of 3.125 xlO⁶ cells/ml. For each transfection 2.5 xlO⁶ cells (0.8ml) were used. Transfections were performed by electroporation using an EASYJECT (Eurogentec, Seraing, Belgium) and electroporation cuvettes (Eurogentec, Seraing, Belgium). For gpl20 recombination experiments, MT-4 cells were cotransfected with 10 μg of the linearized gp720-deleted NL4.3 clone and 2 μg (3 tubes of 50 μl) purified and concentrated AV312- AV313 inner PCR product (PCR Purification Kit, Qiagen, Westburg, Leusden, The Netherlands). For gp41 recombination experiments, cells were cotransfected with 10 μg of the linearized gp41 -deleted NL4.3 clone and 2 μg purified and concentrated AV320- AV321 PCR product, whereas the AV310-AV319 PCR product was co-transfected with the g/?7<50-deleted clone in gplόO recombination experiments. In the integrase recombination assay, MW3-MW4 PCR products were co-transfected with the integrase- deleted clone. The electroporation conditions were 300 μF and 300 V for all transfections. After 30 minutes incubation at room temperature, the transfected cell suspension in 5 ml of culture medium was incubated at 37°C in a humidified atmosphere with 5% CO₂. The recombinant virus was harvested by centrifugation, when full CPE was observed in the culture (about 6 days post-transfection) and stored in 1 ml aliquots at -80°C for subsequent infectivity and drug susceptibility determinations (IC₅₀) in the MT-4/MTT assay.

DISCUSSION

In view of the strong correlation between the appearance of drug-resistant strains and therapeutic failure, the availibility of new drugs effective at new targets within the HIV replication cycle (other than RT and PRO) and thus being effective against the current drug-resistant viruses should allow more potent combination strategies (Larder et al, 1998, Vandamme et al, 1999a). An attractive and promising new target is virus-cell binding and virus-cell fusion. Several compounds inhibiting viral entry into cells have been described. Another interesting target for new inhibitors is the viral enzyme integrase, since integration is an essential step for HIV replication and since no human homologue is known to play a role during normal cell metabolism. Still, only one authentic integrase inhibitor in cell culture has so far been reported (Hazuda et al, 2000). Since inhibitors of viral entry, i.e. AMD3100 and T-20 are already subject of clinical phase II studies, the development of phenotypic drug resistance assays, that can measure the sensitivity of patient isolates towards these new compounds, is mandatory. Knowledge on development of HIV-1 resistance against entry inhibitors in cell culture will help the interpretation of clinical data. Here, we describe the construction of gpl20-, gp41- and gplόO recombination assays and the application on several drug-resistant HIV-1 strains selected in vitro in the presence of entry inhibitors. In addition, we describe the development of an integrase recombination assay.

The study of resistance development towards compounds inhibiting viral entry can contribute to the elucidation of their molecular mode of action. Drug-resistant HIV-1(NL4.3) strains were selected in vitro in the presence of increasing concentrations of the entry inhibitors L-CA (L-CA^res), BRI2923 (BRI2923^res) and AMD3100 (AMD3100^res) (Table 1). Mutations in the gpl20 genes of these in vitro selected strains were detected by sequencing analysis (Table 5). Moreover, the strain BRI2923^res contained mutations in the gp41-g&ne (Table 6). Antiviral drug susceptibility assays, such as the MT-4/MTT assay, allow the investigation of resistance and cross-resistance patterns of these mutant strains to various anti-HIV drugs by determining their 50% inhibitory concentrations (IC₅₀) (Pauwels et al, 1988, Vandamme et al, 1999b) (Table 7). The env-recombination techniques described, wherein gpl20-, gp41- and gpl60- coding sequences can be recombined into the genetic background of a proviral clone deleted for the corresponding gene, are helpful to further delineate to what extent some regions are responsible for the observed resistance of the selected resistant strains. These different env- recombination assays were applied on HIV-1(NL4.3) virus strains selected in the presence of L-CA, BRI2923 and AMD3100. Comparison of the degree of resistance of the in vitro selected strain with the corresponding recombinant virus strain may be considered as indicative for the role of the recombined region in the observed phenotypic resistance (Tables 8-10). Recombination of gp!20 (and gplόO) fully rescued the phenotypic resistance of the virus strain selected in the presence of L-CA. L-CA was originally proffered as an integrase inhibitor, because of its inhibitory activity in an oligonucleotide-based integrase assay (McDougall et al, 1998). However, in cell culture this compound was found to inhibit HIV replication by interfering with viral entry (Pluymers et al, 2000). This finding was corroborated by the appearance of mutations in gpl 20, and not in integrase, in the viral strain selected in the presence of this compound. gp720-recombination in a wild-type background resulted in a virus displaying the same resistance profile as the in vitro selected strain. Since the BRI2923^res gpl20- and gplόO- recombined strains displayed wild-type sensitivity towards the compound BRI2923, it appears that resistance against BRI2923 is attributed to mutations in other genes distinct from the genes encoding the envelope glycoproteins. The close proximity of the matrix protein MA (pl7) to the lipid bilayer supports the idea that a direct interaction takes place between the endodomain of the glycoprotein gp41 and the matrix domain of gag (Freed et al, 1998). Therefore, we have looked for mutations in the gag encoding sequences of the BRI2923^res strain. Mutations were found in the basic domain, that is known to play a role in membrane binding (Zhou et al., 1994), in the pl7 encoding part of the gag gene of BRI2923^res and not in the NL4.3^WT, L-CA^res and AMD3100^res strains (Table 11).

To further asses the impact of these mutations on the observed resistance of the BRI2923^res strain, recombination assays have been developed, wherein the pl7 and a small part of the p24 encoding part of the gag gene by itself and in combination with the ercv-region can be placed in a proviral clone. To construct the double deleted clone, the pl7/p24 encoding part of the gag gene has been excised out the gp760-deleted clone. The resulting clone has then been linearized at the position of the gαg-deletion and in parallel at the position of the env-deletion. These linearized plasmids were co-transfected with the PCR-derived gag and env sequences. The gag sequences recombine in the plasmid linearized at the gαg-deletion, whereas the env sequences recombine in the plasmid linearized at the env-deletion. Viable virus particles will only be generated by an additional recombination step during virus replication, when one RNA strand of the HIV virion contains the gαg-recombined plasmid and the other RNA strand contains the env-recombined plasmid. Moreover, one has to take care that the packaging signal situated upstream from the gag sequences is not disrupted by endonuclease digestion, required to cut out the pl7/p24 encoding region. Since the start codon of the gag polyprotein is located directly behind the end of the packaging signal, we used a unique restriction site located in the packaging signal. The double deleted vector has been completed via the reintroduction of the packaging signal by means of designing a linker sequence that contains the rest of the packaging signal. By generating a novel unique restriction site in this linker sequence at the start codon of the gag polyprotein, the resulting deleted clone can be linearized without disturbing the packaging signal. The same primers and linker sequences have been used to develop the recombination assay for the pl7/p24 encoding region solely. In this case however, the disruption of the packaging signal does not affect recombination, since no additional recombination step during viral replication is required.

Since the CXCR4 antagonist AMD3100 and the fusion inhibitor T-20 have proceeded to clinical Phase II trials, we assessed the use of these recombination assays for phenotypic evaluation of clinical isolates that may have developed resistance at the level of the envelope glycoproteins. Since complete resistance of the AMD3100 resistant virus strain could be fully rescued by gp720-recombination, we conclude that env-recombination is a powerful diagnostic tool to assess the susceptibility/resistance profile of clinical isolates to AMD3100. When applied on clinical isolates, gp7<50-recombination will be more complete than that of gpl 20. Because resistance towards the fusion inhibitor T-20 is reflected by mutations in the gp41 gene (Rimsky et al, 1998), the gp41- or gp76O-recombination assay can be used for susceptibility testing of clinical isolates of patients under treatment with this compound. The mutations in the gp41 gene of the BRI2923^res strain were responsible for the observed cross-resistance of the BRI2923^res strain against T-20, since gp 7-recombination of the BRI2923^res strain resulted in a virus with the same sensitivity towards T-20 as the in vitro selected and the BRI2923^res g_Jp7(50-recombined strain.

The wild-type gp720-recombined strain, as well as the L-CA^res gp720-recombined and AMD3100^res gp720-recombined viruses, showed a 5- to 10-fold lower sensitivity to the inhibitory effect of T-20 in comparison to the NL4.3 wild-type and the L-CA^res and AMD3100^res in vitro selected strains. Since this is the case for all gp720-recombined viruses, (with the exception of the BRI2923^res g/?720-recombined strain), the reduced sensitivity can be explained by the genetic background of the gp720-deleted clone, probable the gp41 gene of the plasmid pNL4.3. Indeed, the strain obtained directly after transfection of MT-4 cells with the plasmid pNL4.3 showed a 10-times lower susceptibility to T-20 in comparison with the NL4.3 wild-type strain that is adapted to cell culture and was used for the selection (data not shown).

Besides e/zv-recombinations, we introduced the integrase gene of a HIV-1(III_B) strain in the integrase-deleted proviral pNL4.3 clone. Viable virus was obtained and evaluauted The resulting virus showed the same sensitivity for several compounds [ i.e. zidovudine, nevirapine, ritonavir and diketo acid (L-708,988) ] compared to the HIV-I(III_B) and HIV-1(NL4.3) strain (Table 12). Homologeous recombination was demonstrated by sequencing analysis, providing a proof-of -principle regarding the integrase-recombination assay. The env- and integrase recombination assays are research tools for the study of the mode of action of new compounds inhibiting HIV entry or integration. These recombination assays delineate the region(s) responsible for the observed phenotypic resistance of a particular drug-resistant HIV strain. In addition, the recombination technique described here could also be used to investigate the unknown mechanism of action of new drugs. A battery of gene-recombinations, recombining each gene separately should greatly facilitate those mechanism of action studies. We have demonstrated for the first time the design and the application of the gpl20-, gp41-, gplόO- and z^'/itegrαse-recombination assays. This technique can additionally be extended to other genes of the HIV genome encoding proteins that will act as targets of antiviral therapy e.g. the matrix protein (MA, pl7), capsid antigen (CA, p24), nucleocapsid (NC, p7), regulatory proteins, etc. Moreover, the recombination methods, mentioned above, can as well be designed for viruses other than HIV. In conclusion, we used chimeric virus technology for env as a research tool to prove that L- CA exclusively targets gpl 20 or the interaction of gpl 20 with a cellular receptor. We also showed that resistance against BRI2923 involves another gene, namely MA (pi 7). Furthermore, since the resistance of the AMD3100 resistant virus strain can be completely reproduced by gp720-recombination, gpl20 may be considered as the sole viral (glyco)protein involved in the anti-HIV action of this compound. The ercv-recombination technique may be a powerful diagnostic tool to assess the susceptibility of clinical isolates to AMD3100.

Table 1. In vitro selection of HTV-1(NL4.3'. strains resistant to virus cell entry inhibitors

L-CA^res BRI2923^res AMD3100^π

Final concentration 30 20 500 of the compound (4)^a (67) (125.000) used for selection

(μg/ml)

Number of passages 23 30 63

fold increase in IC₅0 against wild-type HIV-1(NL4.3)

Table 2. Optimizing the different env PCR's

Table 3. Optimizing the sas PCR

Λ 0 Λ

Λ

J

s)

Table 4. Optimizing the integrase PCR

Table 5. Genotypic analysis of the gp!20 genes of drug-resistant HIV-1(NL4.3) strains

Region V2 V3

Amino acid 147 154 160 270 272 274 276 280 290 292 295 299

NL4.3^WT F V S N N R S Q I K N A Λ 0 Λ L-CA^res , . N , _Λ . . . D .

BRI2923^res L E . • . . . . Q .

AMD3100^res L + Y S T R H V H T Λ

t J

Region C3 V4 C4 V5 C5 s)

Amino acid 323 366 367 368 369 370 372 378 379 387 389 393 412 435 459

NL4.3 ^WT F F N S T W T T E P R F Q S V

L-CA^res N N Q •

BRI2923^res Y _ _ _ _ . . E V . . .

AMD3100^res • - - - - — • L • E P I

Table 6. Genotypic analysis of the ep41 genes of drug-resistant HIV-KNL4.3) strains

position of NL4.3 2 22 33 49 126 210 213 216 308

NL4.3 ^WT V A L E N L P P A

BRI2923^res • V S • • • P/L A/T

One letter code for the amino acids Clarification of the symbols

Alanine A . no change in amino acid

Arginine R + insertion of an amino acid

Asparagine N - deletion of an amino acid

Aspartic acid D

Cysteine C

Glutamine Q

Glutamic acid E

Glycine G

Histidine H

Isoleucine I

Leucine L

Lysine K

Methionine M

Phenylalanine F

Proline P

Serine S

Threonine T

Tryptophane w

Tyrosine Y

Valine V Table 7. Drug-susceptibility of HTV-l(NL4.3)-derived strains that were in vitro selected in the presence of virus cell entry inhibitors in comparison tot the wild-type HIV-1(NL4.3) strain

IC₅₀ ^a (fold resistance)

Virus strains L-CA BRI2923 AMD3100 T-20 Λ 0 Λ

NL4.3^W 5.6±3.5 0.29+0.22 0.012+0.005 0.029+0.012

Λ NL4.3 L-CA^resc 31.7+2.6 1.00+0.11 0.014+0.003 0.042+0.014

(5.7) (3.4) (1.2) (1.4) to σs ⁾

NL4.3/BRI2923^{res d} 14.8 3.4+2.0 0.032+0.030 2.17+0.82 (2.6) (11.7) (2.7) (74.8) s)

NL4.3/AMD3100^{res e} 6.1 5.1+3.3 1.9+1.4 0.080+0.034 (1.1) (17.6) (158.3) (2.8)

^a 50% inhibitory concentration (IC₅₀) (in μg/ml), or concentration of the compound required to inhibit the cytopathic effect of the different HIV strains by 50% in MT-4 cells. ^b ratio of the IC₅₀ of the compound against the in vitro selected HIV-l(NL4.3)-derived strain to the IC₅₀ of the compound against the HIV-1(NL4. wild-type strain. ^c HIN-l(NL4.3)-derived in vitro selected resistant (^res) strain in the presence of L-CA. ^d HIV-l(NL4.3)-derived in vitro selected resistant (^res) strain in the presence of BRI2923. ^e HIV-l(NL4.3)-derived in vitro selected resistant (^res) strain in the presence of AMD3100.

Table 8. Drug-sensitivity of the gp720-recombined viruses derived from the HIV-l(NL4.3)-derived strains that were selected in vitro in the presence of virus cell entry inhibitors

IC_S0 ^a (fold resistance) ^b

Virus strains L-CA BRI2923 AMD3100 T-20 Λ R120/NL4.3^wr 6.8+1.7 0.21+0.11 0.0093+0.0066 0.15+0.01 0 Λ

R120 L-CA^{res c} 30.1+20.3 0.86+0.30 0.016+0.006 0.20+0.04 Λ (4.4) (4.1) (1.7) (1.3) t

-4

R120/BRI2923^{res d} 13.4 0.43+0.27 0.0062+0.0052 0.24+0.14 J (2.0) (2.0) (0.7) (1.6)

s) R120/AMD3100^{res e} 5.0+1.6 4.2+2.3 0.65+0.43 0.43+0.28 (0.7) (20) (69.9) (2.9)

^a 50% inhibitory concentration (IC50) (in μg/ml), or concentration of the compound required to inhibit the cytopathic effect of differe HIV strains by 50% in MT-4 cells. ^b ratio of the IC₅₀ of the compound against the gp720-recombined in vitro selected strain to the IC₅o of the compound against the HIV-1(NL4. g/?720-recombined wild-type strain. ^c HIV-1(NL4.3) wild-type strain recombined with the gpl20 gene of the in vitro selected resistant (^res) strain in the presence of L-CA. ^d HTV-1(NL4.3) wild-type strain recombined with the gp!20 gene of the in vitro selected resistant (^res) strain in the presence of BRI2923. ^e HIN-1(NL4.3) wild-type strain recombined with the gp!20 gene of the in vitro selected resistant (^res) strain in the presence of AMD3100.

Table 9. Drug-sensitivity of the gp41 -recombined viruses derived from the HIV-l(NL4.3)-derived strains that were selected in vitro in the presence of entry inhibitors

IC_S0 ^a (fold resistance) ^b

Virus strains L-CA BRI2923 AMD3100 T-20 Λ R41/NL4.3^WT 1.53 0.20+0.09 0.0032+0.0002 0.025+0.004 0 Λ

R41/L-CA^resc 1.83 0.19+0.05 0.0032+0.0006 0.025+0.005 Λ (1.2) (1.0) (1.0) (1.0) to

R41/BRI2923^{res d} 0.73 0.39 0.0042 0.61+0.06 J (0.5) (2.0) (1.3) (24.4)

s) R41/AMD3100^{res e} 2.43 0.34+0.24 0.0033+0.0019 0.017+0.016 (1.6) (1.7) (1.0) (0.7)

^a 50% inhibitory concentration (ICso) (in μg/ml), or concentration of the compound required to inhibit the cytopathic effect of differe HIV strains by 50% in MT-4 cells. ^b ratio of the IC₅o of the compound against the gp41 -recombined in vitro selected strain to the IC₅0 of the compound against the HIV-1(NL4.3) gp4 recombined wild-type strain. ^c HIV-1(NL4.3) wild-type strain recombined with the gp41 gene of the in vitro selected resistant (^res) strain in the presence of L-CA. ^d HIV-1(NL4.3) wild-type strain recombined with the gp41 gene of the in vitro selected resistant (^res) strain in the presence of BRI2923. ^e HIV-1(NL4.3) wild-type sttain recombined with the gp41 gene of the in vitro selected resistant (^res) strain in the presence of AMD3100.

Table 10. Drug-sensitivity of the gp7(5Q-recombined viruses derived from the HIV-l(NL4.3)-derived strains that were selected in vitro in the presence of entry inhibitors

IC_S0 ^a (fold resistance) ^b

Virus strains L-CA BRI2923 AMD3100 T-20 Λ 0 Λ R160/NL4.3^WT 2.4 0.23+0.13 0.0054+0.0022 0.029+0.0039

Λ R160/BRI2923^{res c} 6.7 0.6+0.01 0.0083+0.0067 0.70+0.17 (2.8) (2.6) (1.5) (24.1) to

R160/AMD3100^{res d} 3.6 3.13+0.73 0.74+0.24 0.03+0.0001 (1.5) (13.6) (137) (1.0) s)

^a 50% inhibitory concentration (ICso) (in μg/ml) or concentration of the compound required to inhibit the cytopathic effect of different H strains by 50% in MT-4 cells. ^b ratio of the IC₅₀ of the compound against the gp7<50-recombined in vitro selected sttain to the IC50 of the compound against the HIV-1 (NL4 gpl 60-recombined wild-type sttain. ^c HIV-1(NL4.3) wild-type sttain recombined with the gplόO gene of the in vitro selected resistant (^res) strain in the presence of BRI2923. ^d HIV-1(NL4.3) wild-type strain recombined with the gpl 60 gene of the in vitro selected resistant (^res) sttain in the presence of AMD3100.

Table 11. Genotypic analysis of the gag genes of drug-resistant HIV-1 (NL4.3 sttains

Table 12. Susceptibility of the HIV-KIIIR integrase-recombined HIV-HNIAS-) strain in comparison to the HIV-KHIR and HIV-KNL4.3) sttains

compound class of R IN/III_B ^a HIV-I(IIIB) HIV-1(NL4.3) compound

zidovudine (AZT) NRTI^b 0.0012+0.001 0.0011+0.001 0.0010+0.002 nevirapine (BOE/BIRG587) NNRTf 0.029+0.007 0.019+0.005 0.020+0.006 ritonavir (ABT/A84538) PROI^d 0.049+0.004 0.089+0.006 0.060+0.005 diketo acid (L-708,988) INI^e 5.5+0.5 5.0+0.4 10.0+0.8

^a HIV-1(III_B) integrase-recombined HIV-1(NL4.3) strain ^b Nucleoside reverse transcriptase inhibitor ^c Non-nucleoside reverse transcriptase inhibitor ^d Protease inhibitor ^e Integrase inhibitor CITED LITERATURE

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Claims

What is claimed is:

1. A method for determining susceptibility of an HIV isolate from HIV infected cell to anti-HIV activity of compounds, comprising:

- Excising a gpl 20, gp41, gpl 60, integrase or gag encoding polynucleotide sequence from a proviral molecular HIV clone;

- Recirculizing the gpl20, gp41, gplόO, integrase or gag deleted proviral molecular HIV clone with a base pair linker containing selected restriction sites; - DNA extraction of proviral DNA or RNA extraction of HIV virus;

- PCR amplifying a gpl 20, a gp41, a gpl 60, an integrase or a gag encoding sequence from HIV infected cell;

- Linearising the gpl 20, gp41, gpl 60, integrase or gag deleted clone by selected restriction enzymes; - Co-transfecting of an eukaryotic cell with the amplified DNA isolates encoding gpl 20, gp41, gpl 60, integrase or gag and with said linearised proviral molecular HIV clone (vector) which is deleted in the corresponding encoding polynucleotide sequence and culturing the co-transfected cell whenever homologous recombination is obtained; - Harvesting the chimeric HIV stock containing the HIV polynucleotide sequence encoding gpl20, gp41, gpl60, integrase or gag;

- Evaluating phenotypic resistance of said chimeric HIV stock to the compounds with said anti-HIV activity or to combinations of the compounds with said anti-HIV activity.

2. A method as claimed in claim 1, wherein in recirculisation of the gpl20, gp41, gpl60, integrase or gag deleted proviral molecular HIV clone is carried out by a base pair linker containing selected restriction sites and said base pair having a sequence which can homologously recombine with the gpl 20, gp41, gpl 60, integrase or gag or mutant or fragment thereof.

3. The method of claim 1, wherein the gpl 20 deleted proviral molecular HIV clone is recirculised by ligation into the vector of a base pair linker which contains a Sal I and Mam I restriction site which can homologously recombine with the gpl 20 gene or a mutant or fragment thereof.

4. The method of claim 1, wherein the gp41 deleted proviral molecular HIV clone is recirculised by ligation into the vector of a base pair linker which contains a Xba I restriction site and which can homologously recombine with the gp41 gene or a mutant or fragment thereof.

5. The method of claim 1, wherein the gpl 60 deleted proviral molecular HIV clone is recirculised by ligation into the vector of a base pair linker which contains a Sal I and Cell II restriction site and which can homologously recombine with the gpl 60 gene or a mutant or fragment thereof.

6. The method of claim 1, wherein integrase deleted proviral molecular HIV clone is recirculised by ligation into the vector of a base pair linker which contains a Xba I restriction site and which can homologously recombine with the integrase gene or a mutant or fragment thereof.

7. The method of claim 1, wherein or the gag deleted proviral molecular HIV clone is recirculised by ligation into the vector of a base pair linker which contains a Mlu I restriction site and which can homologously recombine with the gag gene or a mutant or fragment thereof.

8. The method of claim 3, wherein the gpl 20 deleted proviral molecular HIV clone is recirculised by ligation into the vector of a 23/19 base pair linker.

9. The method of claim 4, wherein the gp41 deleted proviral molecular HIV clone is recirculised by ligation into the vector of a 22/25 base pair linker.

10. The method of claim 5, wherein the gpl60 deleted proviral molecular HIV clone is recirculised by ligation into the vector of a 29/28 base pair linker.

11. The method of claim 6, wherein integrase deleted proviral molecular HIV clone is recirculised by ligation into the vector of a 21/14 base pair linker.

12. The method of claim 7, wherein or the gag deleted proviral molecular HIV clone is recirculised by ligation into the vector of a 100/100 base pair linker.

13. A method as claimed in claim 2 or 3, wherein the gpl 20 deleted proviral molecular HIV clone is recirculised by ligation into the vector of a base pair linker having the sequences 3' GTCACCTGTTTAATCTACA 5" and 5'

TCGACAGTGGACAAATTAGATGT 3" or a fragment thereof.

14. A method as claimed in claim 2 or 4, wherein the gp41 deleted proviral molecular HIV clone is recirculised by ligation into the vector of a base pair linker having the sequence 3' AGTAGATCTCTTACTCTGCTCGACT 5' and 5' TCATCTAGAGAATGAGACGAGC 3' or a fragment thereof.

15. A method as claimed in claim 2 or 5, wherein the gpl60 deleted proviral molecular HIV clone is recirculised by ligation into the vector of a base pair linker having the sequence 3' GACATTCCCTTTCTTACTCTGCTCGACT 5' and 5' TCGACTGTAAGGGAAAGAATGAGACGAGC 3 or a fragment thereof.

16. A method as claimed in claim 2 or 6, wherein integrase deleted proviral molecular HIV clone is recirculised by ligation into the vector of a base pair linker having the sequence 3' AGATCTCAGAGGTA 5' and 5' CCGGTCTAGAGTCTCCATAGA 3'.

17. A method as claimed in claim 2 or 7, wherein or the gag deleted proviral molecular HIV clone is recirculised by ligation into the vector of a base pair linker having the sequence 5' CGC GCA CGG CAA GAG GCG AGG GGC GGC GAC TGG TGA GTA CGC CAA AAA TTT TGA CTA GCG GAG GCT AGA AGG AGA GAG ACG CGT GAC ATA GCA GGA ACT A 3'and 3' GT GCC GTT CTC CGC TCC CCG CCG CTG ACC ACT CAT GCG GTT TTT AAA ACT GAT CGC CTC CGA TCT TCC TCT CTC TGC GCA CTG TAT CGT CCTTGA TGA TC 5' or a fragment thereof.

18. The method according to the claim 1 or 2, wherein the deleted proviral molecular HIV clones are produced by excising a gpl 20 encoding polynucleotide sequence from a proviral molecular HIV clone by digestion of said proviral molecular HIV clone by restriction enzymes Sal I and Mam I and recirculising by ligating a base pair containing Sal I and Mam I restriction sites into said proviral molecular clone.

19. The method according to the claim 1 or 2, wherein the deleted proviral molecular HIV clones are produced by excising a gp41 encoding polynucleotide sequence from said proviral molecular HIV clone by digestion of said proviral molecular HIV clone by restriction enzymes Mam I and Cell II and recirculising by ligating a base pair containing a Xba I restriction site into said proviral molecular clone.

20. The method according to the claims 1 or 2, wherein the deleted proviral molecular HIV clones are produced by excising a gpl 60 encoding polynucleotide sequence from said proviral molecular HIV clone by digestion of said proviral molecular HIV clone by restriction enzymes Sal I and Cell II and recirculising by ligating a base pair containing the Sal I and Cell II restriction sites into said proviral molecular clone.

21. The method according to the claim 1 to 2, wherein the deleted proviral molecular HIV clones are produced by excising an integrase encoding polynucleotide sequence from said proviral molecular HIV clone by digestion of said proviral molecular HIV clone by restriction enzymes Pin Al and Van 91 1 and recirculising by ligating a base pair containing the Sal I and Cell II restriction sites into said proviral molecular clone.

22. The method according to the claim 1 or 2, wherein the deleted proviral molecular HIV clones are produced by excising a gag encoding polynucleotide sequence from said proviral molecular HIV clone by digestion of said proviral molecular HIV clone by restriction enzymes BssH II and Spe I and recirculising by ligating a base pair containing the Mlu I restriction site into said proviral molecular clone.

23. The method according to the claim 1 or 2, wherein the deleted proviral molecular HIV clones are produced by excising a gag/gpl60 encoding polynucleotide sequence from said proviral molecular HIV clone by digestion of said gpl 60 deleted proviral molecular clone by restriction enzymes BssH II and Spe I and recirculising by ligating a base pair containing the Mlu I restriction site into said proviral molecular clone.

24. The method according any of the claims 1 to 23, wherein the gpl 20 deleted clone is linearised by Sal I digestion, the gp41 deleted clone is linearised by Xba I digestion, the gpl 60 deleted clone is linearised by Sal I digestion or the integrase deleted clone is linearised by Xba I digestion, the gag deleted clone by Mlu I digestion or the gag/gpl60 deleted clone by Mlu I digestion.

25. The method according to any of the claims 1, 2, 3, 8, 13 or 24, wherein the gpl20 encoding sequence from HIV infected cell are PCR amplified using at least one primer selected from the group the of gpl 20 primers AV310 (5'-AGC AGG ACA TAA T/CAA GGT AGG-3', AV311 (5'GGA GAA GTG AAT TAT ATA AG/AT ATA AAG TAG-3', AV312 (5' -AGA A/GGA C/TAG ATG GAA CAA GCC CCA G-3' or AV313 (5'-GAC CTG GAG GAG GAA/G ATA TGA G/AGG A-3' .

26. The method according to any of the claims 1, 2, 4, 9, 14, 19 or 24 wherein the gp41 encoding sequence from HIV infected cell are PCR amplified using at least one primer selected from the group the of gp41 primers AV320 (5' -ATT GTA/G GAG GA/GG AAT TTT TCT ACT G-3' and AV321 (5' -TTG CTA/G CTT GTG ATT

GCT/C CCA TG-30

27. The method according to any of the claims 1, 2, 5, 10, 15, 20 or 24, wherein the gpl 60 encoding sequence from HIV infected cell are PCR amplified using at least one primer selected from the group of gpl 60 primers AV310 (5'-AGC AGG ACA TAA T/CAA GGT AGG-3' and AV319 (5' -GCT G/C CC TTA/G TAA GTC ATT

GGT CT-30

28. The method according to any of the claims 1, 2, 6, 11, 16, 21, or 24, wherein the integrase encoding sequence from HIV infected cell are PCR amplified using at least one primer selected from the group of the integrase primers MW3 (5' -TAT GTA

GGA/G TCT GAC/T TTA GAA ATA GGG-3^* and MW4 (5'-TAA CAC TAG GCA A/GAG GTG GCT T-30

29. The method according to any of the claims 1, 2, 7, 12, 17, 22 or 24, wherein the gag- encoding sequence from HIV infected cell are PCR amplified using at least one primer selected from the group of the GAG primers AV14 (5'-CTC TCT CGA CGC AGG ACT CGG CTT GCT GAA-3') and BVR2 (5'-GCC AG/AA TC/TT TCC CTA AAA AAT TAG CC-3').

30. A method as claimed in any of the claims 1 to 29, wherein the proviral molecular HIV clone consists of the plasmid pUC18 with the complete HIV genome and flanked with chromosomal DNA.

31. A method as claimed in any of the claims 1 to 29, wherein the proviral molecular HIV clone is pNL4.3.

32. A method as claimed in any of the claims 1 to 31, wherein the co-transfected cell is an eukaroytic cell.

33. A method as claimed in any of the claims 1 to 31, wherein the co-transfected cell is a human cell.

34. A method as claimed in any of the claims 1 to 31, wherein the co-transfected cell is a MT-4 cell or a peripheral mononuclear cell.

35. The method of any of the claims 1 to 34, further comprising sequencing of the gpl20- encoding region, of the gp41 -encoding region, of the gpl60-encoding region, of the integrase-encoding region or of the gag-encoding region.

36. The method of claims 35, wherein the gpl20 gene were sequenced using al least one or several of the group of gpl20 sequencing primers consisting of AV304 (5'-ACA

TGT GGA AAA ATG ACA TGG T-3' , AV305 (5'-GAG TGG GGT TAA TTT TAC ACA TGG-3', AV306 (5'-TGT CAG CAC AGT ACA ATG TAC ACA-3', AV307 (5'-TCT TCT TCT GCT AGA CTG CCA T-3', AV308 (5'-TCC TCA GGA GGG GAC CCA GAA ATT-3' , AV309 (5'-CAG TAG AAA AAT TCC CCT CCA CA-3' andAV313 (5'-GAC CTG GAG GAG GAA/GATATGA G/AGGA-3'.

37. The method of claims 35, wherein the gp41 gene were sequenced using at least one or several primers of the group of the gp41 sequencing primers consisting of AV322 (5'-AAG CAA TGT ATG CCC CTC C-3', AV323 (5'-CTG CTC CC/TA AGA ACC CAA-3', AV324 (5'-GGC AAA GAG AAG AGT GGT-3' AV325 (5'-GTA TCT

TTC CAC AGC TAG-3', AV326 (5'-TTG GGG T/CTG CTC TGG AAA AC-3', AV327 (5'-TTT TAT ATA CCA CAG CCA-3', AV328 (5'-ATA ATG ATA GTA GGA GG-3', AV329 (5'-GTC CCA GAA GTT CCA CA-3', AV330 (5'-GGA G/ACC TGT GCC TCT TCA-3' and AV331 (5'-TCT CAT TCT TTC CCT TA-30 38. The method of claims 35, wherein the wherein the gpl60 gene were sequenced using al least one or several of the group of the gpl 20 sequencing primers and using al least one or several of the group of gp41 sequencing primers.

39. The method of claims 35, wherein the pl7 and p24 encoding regions of the gag gene were sequenced using al least one or several of the primers AVI 3 (5' -CTG CGA

ATC GTT CTA GCT CCC TGC TTG CCC -3'), AV26 (5'-GCT ATG TCA CTT CCC CTT GGT TCT C -3'), AV54 (5' -GAG ACC ATC AAT GAG GAA GCT GC- 3'), AV58, (5'-GGC TAA TTT TTT AGG-3'), AV159 (5'-GGG GTT AAA TAA AAT AGT AAG-3'), AV103 (5'-GCC ATA TCA CCT AGA ACT TT-3'), GAG-1 (5'-AGA AAC ATC AGA AGG CTG TAG-3'), BVR3 (5'-TTT CCA ACA GCC CTT TTT CCT AG-3'), BVR4 (5'-AGA CAC CAA G/AGA AGC-3'), BVR5 (5'-

TCC T/CTC TGA TAA TGC TG-3') and BVR6 (5'-TAG AAG AAA TGA TGA CAG C-3').

40. The method of claims 35, wherein the integrase gene were sequenced using at least one or several of the primers: IN-PCRA (5'-GGA GGA AAT GAA CAA GTA GAT-

. 3', HP4392C (5'-TCT ACT TGT CCA TGC ATG GCTTC-3', IN-SEQ1 ( 5'-TTA

AGA TGT TCA GCC TGA TCT-3' , IN-SEQ2 (5'- CAT TGT CTG TAT GTA CTG

TTT-3' , IN-SEQ3 (5'- GGA TAT ATA GAA GCA GAA GTA A-3' , IN-SEQ4 (5'-

GAA CAT CTT AAG ACA GCA GTA-3' , IN-SEQ5 (5'- AAG CTC CTC TGG AAA GGT GAA-3' and IN-PCRB (5'- CCT TGA AAT ATA CAT ATG GTG-3').

41. The method of claim 1 to 40, further comprising comparing data of the phenotypic resistance of said HIV stock containing the selected HIV polynucleotide sequence of said HIV isolate to anti-HIV activity of said compounds or of said combinations of said compounds with data of the phenotypic resistance of said HIV stock containing a wild type HIV polynucleotide sequence to anti-HIV activity of said compounds or of said combinations of said compounds.

42. The method of claim 41, further comprising repeating said resistance test for al least one more time and constructing at least two more data sets.

43. The method of claim 42, further comprising calculation of the fold decrease in susceptibility to said compound with anti-HIV activity or to combinations of said compounds of the chimeric viruses in comparison to the wild-type chimeric strains.

44. The method of any of the claim 1 to 43, wherein said DNA extraction of proviral DNA or RNA extraction of HIV virus is obtainable from an HIV patient.

45. The method of any of the claims 1 to 45, wherein said HIV isolate is selected from an in vitro culture of cells infected with a population of a HIV virus strain that was cultured in the presence of compounds or combinations of compounds with putative or with established anti-HIV activity.

46. The use of the method of claim 45 to study molecular target and resistance profile of action of said compounds with anti-HIV activity.

47. The use of method of claim 44, to adapt the chemotherapy administered to an HIV patient.

48. A genetic information data set on anti-HIV resistance, obtainable by the method claim 44 or 46.

49. The genetic information data set of claim 48, stored in the memory of computer controlling a gene-sequencing machine to screen for mutations in genes of HIV isolates.

50. The use of said genetic information data set of claim 47 or 49 that confer resistance to said anti-HIV compound to influence anti-HIV therapy.

51. The method according to claim 1 to claim 45, wherein the phenotypic sensitivity of said recombined viruses to inhibitors inducing mutations in HIV envelope proteins is assessed.

52. A method according to claim 1 to claim 45, wherein the phenotypic sensitivity of recombined viruses containing two resistant virus segments, i.e. (gpl60 and pl7/p24) to inhibitors inducing mutations in HIV envelope proteins (gpl 20, gp41, gpl 60) is assessed.

53. A method according to claim 1 or claim 45, wherein the phenotypic sensitivity of said recombined viruses to inhibitors of HIV integrase (IN, p32) is assessed.

54. A kit for determining susceptibility of an HIV isolate from HIV infected cell to anti- HIV activity of compounds, wherein a amplification primer cocktail comprises primers of the sequence: AV310 (5'-AGC AGG ACA TAA T/CAA GGT AGG-3' , AV311 (5'GGA GAA GTG AAT TAT ATA AG/AT ATA AAG TAG-3', AV312 (5'-AGA A/GGA C/TAG ATG GAA CAA GCC CCA G-3', AV313 (5'-GAC CTG GAG GAG GAA/G ATA TGA G/AGG A-3', AV320 (5'-ATT GTA/G GAG GA/GG

AAT TTT TCT ACT G-3', AV321 (5' -TTG CTA/G CTT GTG ATT GCT/C CCA TG-3', AV310 (5'-AGC AGG ACA TAA T/CAA GGT AGG-3', AV319 (5' -GCT G/C CC TTA/G TAA GTC ATT GGT CT-3', MW3 (5'-TAT GTA GGA/G TCT GAC/T TTA GAA ATA GGG-3', MW4 (5'-TAA CAC TAG GCA A/GAG GTG GCT T-3', AV14 (5'-CTC TCT CGA CGC AGG ACT CGG CTT GCT GAA-3') and

BVR2 (5' -GCC AG/AA TC/TT TCC CTA AAA AAT TAG CC-3').

55. The kit of claim 54, further comprising linkers of the group consisting of a base pair linker having the sequences 3' GTCACCTGTTTAATCTACA 5' and 5' TCGACAGTGGACAAATTAGATGT 3' or a fragment thereof, a base pair linker having the sequence 3" AGTAGATCTCTTACTCTGCTCGACT 5' and 5' TCATCTAGAGAATGAGACGAGC 3' or a fragment thereof, a base pair linker having the sequence 3' GACATTCCCTTTCTTACTCTGCTCGACT 5' and 5' TCGACTGTAAGGGAAAGAATGAGACGAGC 3 or a fragment thereof, a base pair linker having the sequence 3' AGATCTCAGAGGTA 5' and 5'

CCGGTCTAGAGTCTCCATAGA 3' or a fragment thereof and a base pair linker having the sequence 5' CGC GCA CGG CAA GAG GCG AGG GGC GGC GAC TGG TGA GTA CGC CAA AAA TTT TGA CTA GCG GAG GCT AGA AGG AGA GAG ACG CGT GAC ATA GCA GGA ACT A 3'and 3' GT GCC GTT CTC CGC TCC CCG CCG CTG ACC ACT CAT GCG GTT TTT AAA ACT GAT CGC

CTC CGA TCT TCC TCT CTC TGC GCA CTG TAT CGT CCΪTGA TGA TC 5' or a fragment thereof.

56. The kit of claim 55, further comprising a MT-4 or peripheral mononuclear cells line.

57. The kit of claim 56, further comprising sequencing primers selected from the group consisting of AV304 (5 '-AC A TGT GGA AAA ATG ACA TGG T-3' , AV305 (5'-

GAG TGG GGT TAA TTT TAC ACA TGG-3', AV306 (5' -TGT CAG CAC AGT ACA ATG TAC ACA-3', AV307 (5'-TCT TCT TCT GCT AGA CTG CCA T-3', AV308 (5' -TCC TCA GGA GGG GAC CCA GAA ATT-3' , AV309 (5' -CAG TAG AAA AAT TCC CCT CCA CA-3', AV313 (5'-GAC CTG GAG GAG GAA G ATA TGA G/AGG A-3', AV322 (5' -AAG CAA TGT ATG CCC CTC C-3', AV323 (5'-

CTG CTC CC/TA AGA ACC CAA-3', AV324 (5'-GGC AAA GAG AAG AGT GGT-3' AV325 (5'-GTA TCT TTC CAC AGC TAG-3', AV326 (5'-TTG GGG T/CTG CTC TGG AAA AC-3\ AV327 (5'-TTT TAT ATA CCA CAG CCA-3', AV328 (5'-ATA ATG ATA GTA GGA GG-3', AV329 (5'-GTC CCA GAA GTT CCA CA-3', AV330 (5'-GGA G/ACC TGT GCC TCT TCA-3', AV331 (5'-TCT

CAT TCT TTC CCT TA-3', AV13 (5'-CTG CGA ATC GTT CTA GCT CCC TGC TTG CCC -3'), AV26 (5'-GCT ATG TCA CTT CCC CTT GGT TCT C -3'), AV54 (5'-GAG ACC ATC AAT GAG GAA GCT GC-3'), AV58, (5'-GGC TAA TTT TTT AGG-3'), AV159 (5'-GGG GTT AAA TAA AAT AGT AAG-3'), AV103 (5'-GCC ATA TCA CCT AGA ACT TT-3'), GAG-1 (5'-AGA AAC ATC AGA AGG CTG

TAG-3'), BVR3 (5'-TTT CCA ACA GCC CTT TTT CCT AG-3'), BVR4 (5'-AGA CAC CAA G/AGA AGC-3'), BVR5 (5'-TCC T/CTC TGA TAA TGC TG-3'), BVR6 (5'-TAG AAG AAA TGA TGA CAG C-3'), IN-PCRA (5'-GGA GGA AAT GAA CAA GTA GAT-3', HP4392C (5'-TCT ACT TGT CCA TGC ATG GCTTC-3', IN-SEQl ( 5'-TTA AGA TGT TCA GCC TGA TCT-3' , IN-SEQ2 (5'- CAT TGT

CTG TAT GTA CTG TTT-3' , IN-SEQ3 (5'- GGA TAT ATA GAA GCA GAA GTA A-3' , IN-SEQ4 (5'- GAA CAT CTT AAG ACA GCA GTA-3' , IN-SEQ5 (5'- AAG CTC CTC TGG AAA GGT GAA-3' and IN-PCRB (5'- CCT TGA AAT ATA CAT ATG GTG-3').