HIV-1 entry into cells requires interactions with certain coreceptors in addition to CD4. Several studies have shown that the CCR5 acts as the major coreceptor for primary non-T-cell-line-adapted viruses[10, 11]. Virus variants use CCR5 co-receptor for entry was R5 phenotype, also referred to as non-syncytium-inducing (NSI) viruses. X4 phenotype referred to SI phenotype and T-cell-line-adapted HIV-1 strains instead use the CXCR4 as coreceptor[12, 13]. The coreceptor antagonist such as Maraviroc, is a new class of drugs approved to apply in combination with other antiretroviral drugs for the treatment of adults with CCR5-tropic HIV-1, who have been treated with other HIV medications and who have evidence of elevated levels of HIV in their blood (viral load). We determined the coreceptor usage of 13 drug resistant variants based the GHOST cell line. Interesting, there were 8(61.5%) drug resistant variants were use CCR5 and CXCR4 coreceptor for entry cells (R5X4). In our previous work, we found that 40%-50% patients with HIV-1 subtype B' infection native to HAART treatment, would switch from R5 phenotype to R5X4 phenotype with disease progression. Therefore, the R5X4 phenotype would more general in drug resistant variants from HAART treatment individuals, suggested that the inhibitor of CCR5 may be less useful in these individuals.
CD4 T cell counts of all R5X4 phenotype were less than 200 cells/ul (mean is 97.9 ± 50.2), while that of R5 phenotype range from 144 to 348 cells/ul (mean is 236.2 ± 91.6, P = 0.04). There was no difference between R5X4 phenotype viral load (4.91 ± 1.58) and R5 phenotype viral load (5.67 ± 0.33, P = 0.32). It have been proved that the emergence of SI viruses is associated with an accelerated decrease in CD4 T cell count, rapid disease progression and the establishment of AIDS[16, 17]. In HAART treatment individuals with HIV-1 subtype B' infected, the emergence of variants use CXCR4 coreceptor may be imply the severe disease progression.
It is believed that replication capacity is an important determinant of HIV-1 pathogenicity and transmissibility. We analysis the replication properties of 13 drug-resistant variants using PBMC assay. The data show all drug resistant variants were able to yield productive infections in PBMCs. Suggested that drug-resistant HIV-1 isolates can bear adaptive mutations that allow for wild-type-level replicative function, thereby overcoming the potential defects associated with the genetic changes necessary for drug resistance. Interestingly, the p24 antigen level of drug-resistant variants were lower than 10 ng at 7 day after infection, while the drug sensitivity variants usually reach 10 ng at 7 day after infection in our lab previous work (data unpublished). This result was coincides with several other reports [18–20], that the drug resistant HIV variants showed a decreased replication capacity than wild-type strains in the absence of antiretroviral therapy.
The drug resistance mutation in protease (PR) and reverse transcriptase (RT) genes responsible for escape drug inhibition and some may responsible for moderate viral fitness. We compared the drug resistance mutation between first variants and fourth variants in the absence of drugs. Only 7 out of 37 mutations were lost and emerged 2 mutations compare the first and fourth variants. Interestingly, the mainly primary mutations such as T215F, Y181C were stable in vitro environment in the absence of antiretroviral drugs. These data suggested that the drug resistant variants were stable in vitro culture in the absence of drugs. Simon also show that in vitro culture did not reduce the level of drug resistance. Therefore, when design the second-line HAART therapy for HAART failure individuals, it should substitute the drugs belong to new class such as fusion inhibitors or integrase inhibitors, instead of the same class.
Due to the decreased replication capacity of drug resistant variants, there is a hypothesis that when antiretroviral treatment is interrupted or terminated, drug-resistant variants in the quasispecies are rapidly replaced by the most fit wild-type virus. And it has been noted that after cessation of treatment following failure, resistant virus is often replaced by wild-type virus. These have clinical relevance in terms of selecting optimal therapies and reducing the rate of progression, as well as on the modeling of the epidemiology of transmission of resistant HIV-1strains. However, the drug resistant mutations in vitro in absence of drugs were persistent and only 7 mutations were lost after passage suggested that the drug resistant variants were predominant in culture. On the other hand, reversion to the wild type amino acid appears to occur infrequently. Our data also show that the drug resistant mutation to wild type can occur at 41, 62, 69, 101, 103, 108, 181, ect. Therefore, the cessation of treatment should be carefully and discussed more. Furthermore, even less fit, drug resistant variants will probe the sequence space and fitness landscape under drug pressure and eventually evolve as a more fit isolate, i.e. one that is stable in the quasispecies upon removal of drug pressure and that can be transmitted to new human recipients. Here, we use HIV-1 clinical variants instead of recombinant infectious clones to measure viral biological phenotype, and should be noted that our drug resistant variants were HIV-1 quasispecies, since the quaisispace variants responsible for the whole viral replicative capacity under the microenvironment.