Ultra-deep characterization of viral quasispecies in HIV infection

Abstract: HIV-1 has the ability to rapidly diversify and adapt to changes in its environment, such as evading the host immune response, altering cell tropism, and developing resistance to antiretroviral drugs. Minority HIV-1 variants have been shown to be of clinical significance, especially those with non-nucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutations or determinants of CXCR4 phenotype (X4-virus). In this thesis a next generation sequencing technology, ultra-deep pyrosequencing (UDPS), has been used to dissect HIV-1 quasispecies in infected patients to study the evolution of drug resistance and cell tropism. The depth of UDPS depends on the number of viral templates that can be successfully extracted and amplified from a plasma sample, the error rate of PCR and UDPS, and the efficiency of cleaning the UDPS data from such errors. For this reason, we developed an experimental design that allows high recovery of HIV-1 templates and an efficient data cleaning strategy. Our data cleaning strategy reduced the UDPS error rate approximately 10-fold. We carefully evaluated the performance of our UDPS protocol and found that the repeatability of detection of major as well as minor variants in patient plasma samples was good. This indicated that the experimental noise introduced during RNA extraction, cDNA synthesis, PCR and UDPS was low. However, for rare variants in vitro PCR recombination and effects of sequence direction need to be considered. Finally, the design of primers for PCR amplification is of special importance during UDPS, since we observed that primer-related selective amplification can skew the frequency estimates of genetic variants. We investigated the levels of pre-existing drug resistance mutations in plasma samples from five treatment-naive patients. In four of five patients we found low levels of pre-existing drug resistance mutations at two positions (M184I, T215A/I), whereas other mutations (M184V, Y181C, Y188C and T215Y/F) were not detected. During treatment failure and treatment interruption, we found almost complete replacement of wild-type and drug-resistant variants, respectively. This implies that the proportion of minority variants with drug resistance in patients with previous treatment failure or transmitted drug resistance can be too low to be detectible even with highly sensitive UDPS. In another study, the HIV-1 populations from three patients with HIV-1 populations that switched coreceptor use were investigated longitudinally. UDPS analysis showed that the X4-virus that emerged after coreceptor switch was not detected during primary HIV-1 infection (PHI) and that the X4 population most probably evolved from the CCR5-using population during the course of infection rather than was transmitted as minor variants. Moreover, one to three major variants were found during PHI, supporting that infection usually is established with one or just a few viral particles. The frequency and type of errors that occurred during UDPS were investigated. The errors that remained after data cleaning were significantly more often transitions than transversions, which indicates that a substantial proportion of errors were introduced during PCR rather than UDPS itself. This affects the limits of detection of minority mutations since UDPS analyses of HIV-1 are preceded by a PCR step. To further reduce the UDPS error rate we developed a new, improved methodology, based on re-sequencing of molecularly tagged template molecules. Preliminary results showed that this method has the potential to increase the sensitivity of UDPS analyses 1000-fold and thus is close to error-free. Taken together, this thesis adds knowledge on the use of UDPS to gain new insights in HIV evolution and resistance and is relevant for the possible future clinical use of this technology.