HAART Resistance Mutations and ENV/GAG Characterization in Venezuelan  Individuals with HIV Persistence

E. Castro1, M. Bouchard1, M. Moreno1, L. Deibis1, G. Echeverría1 and the Genotyping Clinical Study Group2. 1Immunology Institute, Central University of Venezuela, Caracas; 2Department of Infectology, University Hospital, Caracas & 2The Immunology Center from the Venezuelan Social Security Institute (Venezuela).

Background: HAART regimens for HIV were introduced in Venezuela since 1999. Our previous study showed resistance mutations (RM) among treated patients to reverse transcriptase inhibitors (RTIs) in 26% and to protease inhibitors (PIs) in 9%. Also, 2 different B/F recombinant strains were reported. The interest of this survey is to access information about RM and simultaneous characterization of env/gag genes within a group of individuals receiving HAART with HIV persistence. Material and Methods: As part of an ongoing study for HIV-1 genotyping, outpatients with documented virological failure and persistent HIV evolution were selected. Proviral DNA was isolated and amplified by nested PCR for env, gag and pol regions, respectively. HIV  characterization of env and gag regions was performed with Heteroduplex Mobility Assay (HMA). Genotyping methods included both probe hybridization by LIPA (Innogenetics) and nucleotide sequencing with the Big Dye Terminator Kit (ABI PRISM 377). Results: A total of 10 individuals (f= 2; m= 8) were studied, all sexually infected, 60% diagnosed since 1998, 60% at CDC C category and 50% with CD4+ counts between 200-499 cel/mm3. HMA migration patterns corresponded to subtype B in both env and gag regions. Analysis of pol sequences also revealed subtype B in all cases. Resistance mutations (RM) to  RTIs  as: M41L; D67N; T69S; V108X/R; V118I/X; M184V; T215Y were found in 80% of the isolates. Additionally, mutations E44D; V75X and G190E were only verified in two isolates with highly polymorphic HMAgag patterns. The two remaining samples with no RM to RTIs did show a wide number of other mutations as well as highly polymorphic HMAgag patterns and complex quasispecies in the HMAenv gels. Concerning RM to PIs, in all studied isolates (7/7) different mutations as: L1OI/F; M36I; M46I/L; I54V; L63P; V82A; L90M were found. Also RM D30N was demonstrated in one individual with highly polymorphic HMAenv pattern. Conclusion: According to these preliminary results, infrequent RM in our setting, to both RTIs and PIs, where found in isolates that harbor important polymorphism in either env or gag genes. An interesting finding was the two isolates with complex quasispecies in env and high polymorphism in gag, in the absence of RM to RTIs. Beyond the fact that selective pressure of antiretroviral drugs have a fundamental roll in the emerging of RM, a monitoring of other genome sites as gag and env genes before and during HAART, might add important information to our understanding of HIV persistence.