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.

Widespread B cell perturbations in HIV-1 infection afflict naive and marginal zone B cells.

Perturbations in B cells are a hallmark of HIV-1 infection. This is signified by increased numbers of exhausted CD21neg memory B cells, driven by continuous antigen-specific and bystander activation. Using high-dimensional flow cytometry, we demonstrate that this exhausted phenotype is also prevalent among peripheral antigen-inexperienced naive and marginal zone (MZ) B cells in acute and chronic HIV-1 infection. A substantial fraction of naive and MZ B cells exhibit down-regulated CD21 levels and diminished response to B cell receptor (BCR)-dependent stimulation. Compared with CD21pos subsets, the CD21neg naive and MZ B cells differ in the expression of chemokine receptors and activation markers. Effective antiretroviral treatment normalizes peripheral naive and MZ B cell populations. Our results emphasize a more widely spread impairment of B cells in HIV-1 infection than previously appreciated, including antigen-inexperienced cells. This highlights the importance of monitoring functional capacities of naive B cells in HIV-1 infection, as exhausted CD21neg naive B cells may severely impair induction of novel B cell responses.

Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4(+) T cells.

The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4(+) T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4(+) T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4(+) T cells were over-represented in gene hotspots identified in CD4(+) T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4(+) T cells.

HIV rebounds from latently infected cells, rather than from continuing low-level replication.

Rapid rebound of plasma viremia in patients after interruption of long-term combination antiretroviral therapy (cART) suggests persistence of low-level replicating cells or rapid reactivation of latently infected cells. To further characterize rebounding virus, we performed extensive longitudinal clonal evolutionary studies of HIV env C2-V3-C3 regions and exploited the temporal relationships of rebounding plasma viruses with regard to pretreatment sequences in 20 chronically HIV-1-infected patients having undergone multiple 2-week structured treatment interruptions (STI). Rebounding virus during the short STI was homogeneous, suggesting mono- or oligoclonal origin during reactivation. No evidence for a temporal structure of rebounding virus in regard to pretreatment sequences was found. Furthermore, expansion of distinct lineages at different STI cycles emerged. Together, these findings imply stochastic reactivation of different clones from long-lived latently infected cells rather than expansion of viral populations replicating at low levels. After treatment was stopped, diversity increased steadily, but pretreatment diversity was, on average, achieved only >2.5 years after the start of STI when marked divergence from preexisting quasispecies also emerged. In summary, our results argue against persistence of ongoing low-level replication in patients on suppressive cART. Furthermore, a prolonged delay in restoration of pretreatment viral diversity after treatment interruption demonstrates a surprisingly sustained evolutionary bottleneck induced by punctuated antiretroviral therapy.

Potent human immunodeficiency virus-neutralizing and complement lysis activities of antibodies are not obligatorily linked.

To evaluate the contribution of complement-mediated lysis to the in vivo activities of neutralizing antibodies, we analyzed the influence of complement activation on treatment success in a recent passive immunization trial with the neutralizing monoclonal antibodies 2G12, 2F5, and 4E10. Administration of monoclonal antibodies led to an immediate, high activation of the complement system even in the absence of viremia in the 14 participating human immunodeficiency virus-infected individuals. Lysis activity measured in patient plasma increased during passive immunization; however, the increases were modest and only partially attributable to the administration of antibodies. We found that unlike neutralization activity, lysis activity was not associated with treatment success in this trial. Compared to complement lysis mounted by the polyclonal antibody response in vivo, monoclonal antibodies were weak inducers of this activity, suggesting that polyclonal responses are more effective in reaching the required threshold of complement activation. Importantly, strong neutralization activity of the monoclonal antibodies did not predict complement lysis activity against patient and reference viruses, suggesting that these activities are not linked. In summary, our data support the notion that the in vivo activities of 2G12, 2F5, and 4E10 are likely due to direct neutralization or Fc receptor-mediated mechanisms such as phagocytosis and antibody-dependent cellular cytotoxicity.

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.

Complement lysis activity in autologous plasma is associated with lower viral loads during the acute phase of HIV-1 infection.

BACKGROUND: To explore the possibility that antibody-mediated complement lysis contributes to viremia control in HIV-1 infection, we measured the activity of patient plasma in mediating complement lysis of autologous primary virus. METHODS AND FINDINGS: Sera from two groups of patients-25 with acute HIV-1 infection and 31 with chronic infection-were used in this study. We developed a novel real-time PCR-based assay strategy that allows reliable and sensitive quantification of virus lysis by complement. Plasma derived at the time of virus isolation induced complement lysis of the autologous virus isolate in the majority of patients. Overall lysis activity against the autologous virus and the heterologous primary virus strain JR-FL was higher at chronic disease stages than during the acute phase. Most strikingly, we found that plasma virus load levels during the acute but not the chronic infection phase correlated inversely with the autologous complement lysis activity. Antibody reactivity to the envelope (Env) proteins gp120 and gp41 were positively correlated with the lysis activity against JR-FL, indicating that anti-Env responses mediated complement lysis. Neutralization and complement lysis activity against autologous viruses were not associated, suggesting that complement lysis is predominantly caused by non-neutralizing antibodies. CONCLUSIONS: Collectively our data provide evidence that antibody-mediated complement virion lysis develops rapidly and is effective early in the course of infection; thus it should be considered a parameter that, in concert with other immune functions, steers viremia control in vivo.

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.