Background:  Deep sampling of the HIV-1 quasispecies in infected patients is essential for low frequency detection of wild type versus drug-resistant clones and of CCR5- versus CXCR4-tropic HIV-1 clones for correct administration of the R5 antagonist, Maraviroc. We compared 3 cloning methods to produce patient-derived env polymerase chain reaction (PCR) product:  classical restriction site and ligation cloning in bacteria, recombination in eukaryotic cells, and recombination in yeast.

Methods:  An env fragment containing gp120 and most of gp41 (~2.5 kb) was real-time polymerase chain reaction (RT-PCR) amplified from plasma of 10 ART-experienced individuals. The same PCR product was introduced into env expression vector for pseudotyped virus production using bacterial restriction enzyme/ligation cloning or in chimeric viruses via recombination in mammalian cells or yeast. We used both Sanger and pyro454 sequencing to measure genetic diversity of HIV-1 clones directly from plasma as well as cloned via bacteria, mammalian, and yeast cloning systems. Finally, co-receptor usage was determined by infecting U87.CD4.CCR5 or U87.CD4.CXCR4 cells.

Results:  Phylogenetic analyses on about 1000 clones indicate that yeast-based recombination best approximates the diversity and structure of the HIV-1 quasispecies in the plasma samples. In contrast, a stringent bottleneck and frequent re-sampling resulted from bacterial cloning and even more, from recombination in mammalian cells. Using the phenotypic assays, R5 virus clones predominated in the quasispecies from all 10 plasma samples. Higher frequencies of X4 variants (>20%) were identified when sampled by all methods but only the yeast-based approach could sufficiently sample the quasispecies for detection of low frequency X4 variants (1 to 20%). Due to resampling error, several X4 lineages found in plasma and sampled by the yeast method, were absent in the phylogenetic trees derived from bacteria cloning and mammalian recombination methods.

Conclusions:  The success of CCR5 antagonists in HIV-1 therapy is dependent on detection of X4 HIV-1 variants in the patient’s quasispecies. However, a significant limitation of current detection methods is related to a cloning bottleneck and resampling error as opposed to the threshold of phenotypic assay. Chimeric env viruses derived from yeast-based recombination/cloning best approximated the HIV-1 genetic diversity in plasma and were at least 4- to 200-fold more diverse than viruses derived from more classical cloning approaches.