HIV-1 integration sites in CD4+ T cells during primary, chronic, and late presentation of HIV-1 infection.

HIV-1 is capable of integrating its genome into that of its host cell. We examined the influence of the activation state of CD4+ T cells, the effect of antiretroviral therapy (ART), and the clinical stage of HIV-1 infection on HIV-1 integration site features and selection. HIV-1 integration sites were sequenced from longitudinally sampled resting and activated CD4+ T cells from 12 HIV-1-infected individuals. In total, 589 unique HIV-1 integration sites were analyzed: 147, 391, and 51 during primary, chronic, and late presentation of HIV-1 infection, respectively. As early as during primary HIV-1 infection and independent of the activation state of CD4+ T cells collected on and off ART, HIV-1 integration sites were preferentially detected in recurrent integration genes, genes associated with clonal expansion of latently HIV-1-infected CD4+ T cells, cancer-related genes, and highly expressed genes. The preference for cancer-related genes was more pronounced at late stages of HIV-1 infection. Host genomic features of HIV-1 integration site selection remained stable during HIV-1 infection in both resting and activated CD4+ T cells. In summary, characteristic HIV-1 integration site features are preestablished as early as during primary HIV-1 infection and are found in both resting and activated CD4+ T cells.

Long-term experimental evolution of HIV-1 reveals effects of environment and mutational history.

An often-returning question for not only HIV-1, but also other organisms, is how predictable evolutionary paths are. The environment, mutational history, and random processes can all impact the exact evolutionary paths, but to which extent these factors contribute to the evolutionary dynamics of a particular system is an open question. Especially in a virus like HIV-1, with a large mutation rate and large population sizes, evolution is expected to be highly predictable if the impact of environment and history is low, and evolution is not neutral. We investigated the effect of environment and mutational history by analyzing sequences from a long-term evolution experiment, in which HIV-1 was passaged on 2 different cell types in 8 independent evolutionary lines and 8 derived lines, 4 of which involved a switch of the environment. The experiments lasted for 240-300 passages, corresponding to approximately 400-600 generations or almost 3 years. The sequences show signs of extensive parallel evolution-the majority of mutations that are shared between independent lines appear in both cell types, but we also find that both environment and mutational history significantly impact the evolutionary paths. We conclude that HIV-1 evolution is robust to small changes in the environment, similar to a transmission event in the absence of an immune response or drug pressure. We also find that the fitness landscape of HIV-1 is largely smooth, although we find some evidence for both positive and negative epistatic interactions between mutations.

In vivo and in vitro escape from neutralizing antibodies 2G12, 2F5, and 4E10.

Recently, passive immunization of human immunodeficiency virus (HIV)-infected individuals with monoclonal antibodies (MAbs) 2G12, 2F5, and 4E10 provided evidence of the in vivo activity of 2G12 but raised concerns about the function of the two membrane-proximal external region (MPER)-specific MAbs (A. Trkola, H. Kuster, P. Rusert, B. Joos, M. Fischer, C. Leemann, A. Manrique, M. Huber, M. Rehr, A. Oxenius, R. Weber, G. Stiegler, B. Vcelar, H. Katinger, L. Aceto, and H. F. Gunthard, Nat. Med. 11:615-622, 2005). In the light of MPER-targeting vaccines under development, we performed an in-depth analysis of the emergence of mutations conferring resistance to these three MAbs to further elucidate their activity. Clonal analysis of the MPER of plasma virus samples derived during antibody treatment confirmed that no changes in this region had occurred in vivo. Sequence analysis of the 2G12 epitope relevant N-glycosylation sites of viruses derived from 13 patients during the trial supported the phenotypic evaluation, demonstrating that mutations in these sites are associated with resistance. In vitro selection experiments with isolates of four of these individuals corroborated the in vivo finding that virus strains rapidly escape 2G12 pressure. Notably, in vitro resistance mutations differed, in most cases, from those found in vivo. Importantly, in vitro selection with 2F5 and 4E10 demonstrated that resistance to these MAbs can be difficult to achieve and can lead to selection of variants with impaired infectivity. This remarkable vulnerability of the virus to interference within the MPER calls for a further evaluation of the safety and efficacy of MPER-targeting therapeutic and vaccination strategies.

Low human immunodeficiency virus envelope diversity correlates with low in vitro replication capacity and predicts spontaneous control of plasma viremia after treatment interruptions.

Genetic diversity of viral isolates in human immunodeficiency virus (HIV)-infected individuals varies substantially. However, it remains unclear whether HIV-related disease progresses more rapidly in patients harboring virus swarms with low or high diversity and, in the same context, whether high or low diversity is required to induce potent humoral and cellular immune responses. To explore whether viral diversity predicts virologic control, we studied HIV-infected patients who received antiretroviral therapy (ART) for years before undergoing structured treatment interruptions (STI). Viral diversity before initiation of ART and the ability of the patients to contain viremia after STI and final cessation of treatment was evaluated. Seven out of 21 patients contained plasma viremia at low levels after the final treatment cessation. Clonal sequences encompassing the envelope C2V3C3 domain derived from plasma prior to treatment, exhibited significantly lower diversity in these patients compared to those derived from patients with poor control of viremia. Viral diversity pre-ART correlated with the viral replication capacity of rebounding virus isolates during STI. Neutralizing antibody activity against autologous virus was significantly higher in patients who controlled viremia and was associated with lower pretreatment diversity. No such association was found with binding antibodies directed to gp120. In summary, lower pretreatment viral diversity was associated with spontaneous control of viremia, reduced viral replication capacity and higher neutralizing antibody titers, suggesting a link between viral diversity, replication capacity, and neutralizing antibody activity.

Humoral immunity to HIV-1: kinetics of antibody responses in chronic infection reflects capacity of immune system to improve viral set point.

We analyzed the humoral immune response in 46 patients following structured treatment interruption (STI) to investigate the general potential of therapeutic vaccination in chronic HIV-1 infection. Evoked antibody titer increases to glycoprotein 120 (gp120) and p24 were low during 4 short-term STIs and only reached significance during a fifth long-term interruption. Although induction of binding antibodies to viral antigens was not associated with potent suppression of viremia, we observed that individuals with a rapid and high response to p24, and to a lesser extent also to gp120, lowered their viral set points significantly. Of note, the increase of the anti-p24 response correlated with specific CD4 T helper frequency to this antigen. Despite induction of binding antibody responses, which correlated with improved viral control, the increase in neutralizing activity was marginal and did not lead to this enhanced viral suppression. However, a subgroup of patients who potently suppressed viremia independently of STI had significantly higher pre-existing neutralization titers, suggesting a role of humoral immunity in conferring potent protection. In summary, measuring the kinetics of antibody responses provided a marker to validate the responsiveness and capacities of the immune system of HIV-1-infected individuals and reflected the patients' ability to decrease viral set points.