Association between specific HIV-1 Env traits and virologic control in vivo

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Abstract

HIV RNA levels are influenced by genetic characteristics of both the host and the virus. Here we applied machine learning techniques to determine if plasma-derived HIV-1 amino acid sequences can be used to predict spontaneous virologic control. We studied the relationship between HIV-1 env genotype and viral load in 20 chronically infected patients undergoing treatment interruptions (SSITT, Swiss-Spanish Intermittent Treatment Trial) and in 104 primary HIV infected (PHI) patients before antiretroviral therapy (cART) and where applicable also after treatment stop. Extensive longitudinal sampling during the interruptions was performed in nine SSITT patients. Sequences obtained from these nine patients during the first virus rebound were used as a training data set and revealed a strong genetic signature (accuracy 98.6% in cross-validation) associated with control of viremia at levels below 5000 copies/mL of viral RNA maintained for at least 2 months after the final cART stop. The simple sequence pattern at gp120 positions 268E/358T was confirmed to be predictive of control in the clonal sequences originating from these patients during all subsequent rebounds. Sequences from the remaining 11 SSITT patients with less frequent sampling and from the PHI patients were used for external validation. High sensitivities (71–100%) and negative predictive values (80–100%) but low positive predictive values (12–40%) were achieved in the patient-wise analysis which was based on presence of the genetic pattern in all clones. These results suggest that presence of virus lacking the amino acid pattern 268E/358T is associated with VL >5000 at baseline of PHI and with low probability of spontaneous virologic control after treatment stop. Conversely, however, presence of 268E/358T does not predict control of viremia. These residues in HIV gp120 might affect in vivo HIV-1 fitness either at the level of Env function or influence susceptibility to adaptive or innate immune response.

Introduction

Empirical approaches to development of HIV vaccines have so far, despite huge efforts, not been successful. Among the major obstacles are the enormous genetic variability and the high degree of glycosylation of the outer HIV envelope proteins enabling the virus to escape from immune recognition. Moreover, there is a narrow window of opportunity to clear the initial infection because latency is established within the first weeks, and destruction of CD4+ T cells begins early after infection (Johnston and Fauci, 2008). HIV induces a natural immune response involving neutralizing antibodies and cytotoxic T lymphocytes. However, these responses are typically too weak to eliminate the virus and the targeted epitope sequences are continuously mutated to evade the immune system. The envelope glycoproteins gp120 and gp41 comprise the key determinants presenting their surface to the host's humoral immune system. They inevitably undergo continuous evolution driven by positive selection, and are therefore the most variable gene products of HIV. Eventually mutations arise resulting in escape from host immune recognition (Yamamoto and Matano, 2008). These mutations often confer considerable fitness cost to the virus which may subsequently be alleviated by selection of compensatory mutations (Goulder and Watkins, 2004).

To explore intrapatient HIV evolution in the region of gp120 we conducted longitudinal clonal sequencing of the envelope C2-V3-C3 domain in HIV-1 infected patients with long-term suppression of plasma viremia who interrupted combination antiretroviral therapy (cART) within a well-controlled structured treatment interruption trial (Swiss Spanish Intermittent Treatment Trial). After successful cART for years, these patients underwent four cycles consisting of 2 weeks treatment interruption and 8 weeks retreatment before cART was completely discontinued for a prolonged time period. During the strictly defined short interruptions HIV RNA usually rebounded within 2 weeks (Fischer et al., 2003a). Patients who maintained plasma viremia at levels below 5000 copies/mL of viral RNA for at least 2 months after the final treatment stop were classified as “controllers” (Fagard et al., 2003). Analysis of the sequences derived from plasma before the initiation of antiretroviral therapy (cART) revealed that lower viral diversity was associated with spontaneous control of viremia after treatment stop as well as with reduced in vitro replication capacity and higher plasma titers of neutralizing antibodies against autologous virus (Joos et al., 2005). Thus, viral properties appeared to influence disease progression being at least in part responsible for spontaneous control of viremia after cessation of cART. We also performed extensive longitudinal clonal studies of plasma HIV RNA to characterize the rebounding virus during the short interruptions and after the prolonged treatment break in a subset of patients (Joos et al., 2008). Phylogenetic analysis exploiting the temporal relation of rebounding virus to pre-treatment sequences was in accord with mono- or oligoclonal reactivation of distinct virus populations from long-lived latently infected cells in contrast to expansion of virus populations replicating at a low level. Furthermore, viral diversity was restored only very slowly after the final treatment stop. This implies that a sustained evolutionary bottleneck was induced by suppressive cART. In the long term, evolution of the envelope sequences spanning the V3 loop and its flanking regions appeared to be driven by positive selection (Joos et al., 2007). Interestingly the adaptive mutations occurred almost exclusively at solvent exposed amino acid positions on the virion surface which confer a high external accessibility according to the 3D structure of the envelope gp120 (Huang et al., 2005) suggesting that these sites evolved in response to selection pressure induced by neutralizing antibodies or adaptations in the receptor interaction necessitated by shifting cellular tropism. However, in this analysis we detected widely different patterns of positively selected sites within the C2-C3 region of gp120 in individual patients by codon-based likelihood methods (Kosakovsky Pond and Frost, 2005).

Here we aimed at identifying common genetic characteristics of rebounding viruses across 20 chronically HIV-1 infected patients having undergone multiple planned treatment interruptions. We employed machine learning techniques to determine if a shared genetic signature could be found in plasma virus rebounding during the first interruption after years of continuous suppressive therapy. Next, we searched for specific signatures associated with high or low distance from the most recent common ancestor (MRCA) in the phylogeny which would reflect virus populations replicating at different speed. In addition, we sought to determine if plasma-derived HIV amino acid sequences could be used to predict spontaneous control of viremia. We found evidence of a link between HIV Env sequence and viral load by decision tree learning and aimed to validate this genetic signature by analyzing amino acid sequences originating from different patient groups.

Section snippets

Study subjects

Blood samples were obtained from chronically infected patients enrolled into the Swiss-Spanish Intermittent Treatment Trial (SSITT) at the University Hospital Zürich. Two patients were excluded because they did not complete the study. We selected those patients from whom a pre-treatment plasma sample was available provided that PCR amplification was successful. All patients had long-term combination antiretroviral therapy (cART) and undetectable viral loads (<50 copies/mL) for at least 6 months

Results

To study the association between HIV-1 env genotype and viral load in chronically infected patients we took advantage of the Swiss-Spanish Intermittent Treatment Trial (SSITT) and followed 20 patients longitudinally. The first samples were obtained during the chronic phase of infection before any treatment was started. Subsequently patients received combination antiretroviral therapy for a median of 2.7 (range 1.5–3.4) years and then underwent 4 treatment interruption cycles (2 weeks

Discussion

It is well accepted that the replicative capacity of HIV in vivo is influenced by both genetic characteristics of the host and those of the virus. Interactions between virus and the host's immune system are complex and better understanding of the underlying mechanisms is important for planning optimized treatment strategies and for vaccine development. In a comprehensive longitudinal study we examined the virus characteristics of HIV envelope in a well-defined group of chronically HIV infected

Acknowledgements

The study was supported by the Swiss National Science Foundation (SNF) (grant no. 3345-65168 and 3100AO-103748/1 to HFG and grant no 320000-116035 to HFG), by the Swiss HIV Cohort Study (SNF grant no. 33CSC0-10878), SHCS project no. 290 and a grant from the EMDO Foundation (both to HFG), and a research grant from the Kanton of Zürich. The authors are grateful for the training received at the 14th International Bioinformatics Workshop on Virus Evolution and Molecular Epidemiology, September

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