Distinct viral reservoirs in individuals with spontaneous control of HIV-1.

Sustained, drug-free control of HIV-1 replication is naturally achieved in less than 0.5% of infected individuals (here termed 'elite controllers'), despite the presence of a replication-competent viral reservoir1. Inducing such an ability to spontaneously maintain undetectable plasma viraemia is a major objective of HIV-1 cure research, but the characteristics of proviral reservoirs in elite controllers remain to be determined. Here, using next-generation sequencing of near-full-length single HIV-1 genomes and corresponding chromosomal integration sites, we show that the proviral reservoirs of elite controllers frequently consist of oligoclonal to near-monoclonal clusters of intact proviral sequences. In contrast to individuals treated with long-term antiretroviral therapy, intact proviral sequences from elite controllers were integrated at highly distinct sites in the human genome and were preferentially located in centromeric satellite DNA or in Krüppel-associated box domain-containing zinc finger genes on chromosome 19, both of which are associated with heterochromatin features. Moreover, the integration sites of intact proviral sequences from elite controllers showed an increased distance to transcriptional start sites and accessible chromatin of the host genome and were enriched in repressive chromatin marks. These data suggest that a distinct configuration of the proviral reservoir represents a structural correlate of natural viral control, and that the quality, rather than the quantity, of viral reservoirs can be an important distinguishing feature for a functional cure of HIV-1 infection. Moreover, in one elite controller, we were unable to detect intact proviral sequences despite analysing more than 1.5 billion peripheral blood mononuclear cells, which raises the possibility that a sterilizing cure of HIV-1 infection, which has previously been observed only following allogeneic haematopoietic stem cell transplantation2,3, may be feasible in rare instances.

Assessing the impact of autologous neutralizing antibodies on rebound dynamics in postnatally SHIV-infected ART-treated infant rhesus macaques.

The presence of antibodies against HIV in infected children is associated with a greater capacity to control viremia in the absence of therapy. While the benefits of early antiretroviral treatment (ART) in infants are well documented, early ART may interfere with the development of antibody responses. In contrast to adults, early treated children lack detectable HIV-specific antibodies, suggesting a fundamental difference in HIV pathogenesis. Despite this potential adverse effect, early ART may decrease the size of the latent reservoir established early in infection in infants, which can be beneficial in viral control. Understanding the virologic and immunologic aspects of pediatric HIV is crucial to inform innovative targeted strategies for treating children living with HIV. In this study, we investigate how ART initiation time sets the stage for trade-offs in the latent reservoir establishment and the development of humoral immunity and how these, in turn, affect posttreatment dynamics. We also elucidate the biological function of antibodies in pediatric HIV. We employ mathematical modeling coupled with experimental data from an infant nonhuman primate Simian/Human Immunodeficiency Virus (SHIV) infection model. Infant Rhesus macaques (RMs) were orally challenged with SHIV.C.CH505 375H dCT four weeks after birth and started treatment at different times after infection. In addition to viral load measurements, antibody responses and latent reservoir sizes were measured. We estimate model parameters by fitting viral load measurements to the standard HIV viral dynamics model within a nonlinear fixed effects framework. This approach allows us to capture differences between rhesus macaques (RMs) that develop antibody responses or exhibit high latent reservoir sizes compared to those that do not. We find that neutralizing antibody responses are associated with increased viral clearance and decreased viral infectivity but decreased death rate of infected cells. In addition, the presence of detectable latent reservoir is associated with less robust immune responses. These results demonstrate that both immune response and latent reservoir dynamics are needed to understand post-rebound dynamics and point to the necessity of a comprehensive approach in tailoring personalized medical interventions.

Assessing the impact of autologous virus neutralizing antibodies on viral rebound time in postnatally SHIV-infected ART-treated infant rhesus macaques.

While the benefits of early antiretroviral therapy (ART) initiation in perinatally infected infants are well documented, early initiation is not always possible in postnatal pediatric HIV infections. The timing of ART initiation is likely to affect the size of the latent viral reservoir established, as well as the development of adaptive immune responses, such as the generation of neutralizing antibody responses against the virus. How these parameters impact the ability of infants to control viremia and the time to viral rebound after ART interruption is unclear and has never been modeled in infants. To investigate this question we used an infant nonhuman primate Simian/Human Immunodeficiency Virus (SHIV) infection model. Infant Rhesus macaques (RMs) were orally challenged with SHIV.C.CH505 375H dCT and either given ART at 4-7 days post-infection (early ART condition), at 2 weeks post-infection (intermediate ART condition), or at 8 weeks post-infection (late ART condition). These infants were then monitored for up to 60 months post-infection with serial viral load and immune measurements. To gain insight into early after analytic treatment interruption (ATI), we constructed mathematical models to investigate the effect of time of ART initiation in delaying viral rebound when treatment is interrupted, focusing on the relative contributions of latent reservoir size and autologous virus neutralizing antibody responses. We developed a stochastic mathematical model to investigate the joint effect of latent reservoir size, the autologous neutralizing antibody potency, and CD4+ T cell levels on the time to viral rebound for RMs rebounding up to 60 days post-ATI. We find that the latent reservoir size is an important determinant in explaining time to viral rebound in infant macaques by affecting the growth rate of the virus. The presence of neutralizing antibodies can also delay rebound, but we find this effect for high potency antibody responses only. Finally, we discuss the therapeutic implications of our findings.

Comparative analysis of within-host dynamics of acute infection and viral rebound dynamics in postnatally SHIV-infected ART-treated infant rhesus macaques.

Viral dynamics of acute HIV infection and HIV rebound following suspension of antiretroviral therapy may be qualitatively similar but must differ given, for one, development of adaptive immune responses. Understanding the differences of acute HIV infection and viral rebound dynamics in pediatric populations may provide insights into the mechanisms of viral control with potential implications for vaccine design and the development of effective targeted therapeutics for infants and children. Mathematical models have been a crucial tool to elucidate the complex processes driving viral infections within the host. Traditionally, acute HIV infection has been modeled with a standard model of viral dynamics initially developed to explore viral decay during treatment, while viral rebound has necessitated extensions of that standard model to incorporate explicit immune responses. Previous efforts to fit these models to viral load data have underscored differences between the two infection stages, such as increased viral clearance rate and increased death rate of infected cells during rebound. However, these findings have been predicated on viral load measurements from disparate adult individuals. In this study, we aim to bridge this gap, in infants, by comparing the dynamics of acute infection and viral rebound within the same individuals by leveraging an infant nonhuman primate Simian/Human Immunodeficiency Virus (SHIV) infection model. Ten infant Rhesus macaques (RMs) orally challenged with SHIV.C.CH505 375H dCT and given ART at 8 weeks post-infection. These infants were then monitored for up to 60 months post-infection with serial viral load and immune measurements. We use the HIV standard viral dynamics model fitted to viral load measurements in a nonlinear mixed effects framework. We find that the primary difference between acute infection and rebound is the increased death rate of infected cells during rebound. We use these findings to generate hypotheses on the effects of adaptive immune responses. We leverage these findings to formulate hypotheses to elucidate the observed results and provide arguments to support the notion that delayed viral rebound is characterized by a stronger CD8+ T cell response.

Assessing the impact of autologous neutralizing antibodies on viral rebound in postnatally SHIV-infected ART-treated infant rhesus macaques.

While the benefits of early antiretroviral therapy (ART) initiation in perinatally infected infants are well documented, early ART initiation is not always possible in postnatal pediatric HIV infections, which account for the majority of pediatric HIV cases worldwide. The timing of onset of ART initiation is likely to affect the size of the latent viral reservoir established, as well as the development of adaptive immune responses, such as the generation of neutralizing antibody responses against the virus. How these parameters impact the ability of infants to control viremia and the time to viral rebound after ART interruption is unclear. To gain insight into the dynamics, we utilized mathematical models to investigate the effect of time of ART initiation via latent reservoir size and autologous virus neutralizing antibody responses in delaying viral rebound when treatment is interrupted. We used an infant nonhuman primate Simian/Human Immunodeficiency Virus (SHIV) infection model that mimics breast milk HIV transmission in human infants. Infant Rhesus macaques (RMs) were orally challenged with SHIV.C.CH505 375H dCT and either given ART at 4-7 days post-infection (early ART condition), at 2 weeks post-infection (intermediate ART condition), or at 8 weeks post-infection (late ART condition). These infants were then monitored for up to 60 months post-infection with serial viral load and immune measurements. We develop a stochastic mathematical model to investigate the joint effect of latent reservoir size, the autologous neutralizing antibody potency, and CD4+ T cell levels on the time to viral rebound and control of post-rebound viral loads. We find that the latent reservoir size is an important determinant in explaining time to viral rebound by affecting the growth rate of the virus. The presence of neutralizing antibodies also can delay rebound, but we find this effect for high potency antibody responses only.

The latent reservoir of inducible, infectious HIV-1 does not decrease despite decades of antiretroviral therapy.

HIV-1 persists in a latent reservoir in resting CD4+ T cells despite antiretroviral therapy (ART). The reservoir decays slowly over the first 7 years of ART (t1/2 = 44 months). However, whether decay continues with long-term ART is unclear. Recent integration site studies indicate gradual selection against inducible, intact proviruses, raising speculation that decades of ART might allow treatment interruption without viral rebound. Therefore, we measured the reservoir in 42 people on long-term ART (mean 22 years) using a quantitative viral outgrowth assay. After 7 years of ART, there was no long-term decrease in the frequency of inducible, replication-competent proviruses but rather an increase with an estimated doubling time of 23 years. Another reservoir assay, the intact proviral DNA assay, confirmed that reservoir decay with t1/2 of 44 months did not continue with long-term ART. The lack of decay reflected proliferation of infected cells. Most inducible, replication-competent viruses (79.8%) had env sequences identical to those of other isolates from the same sample. Thus, although integration site analysis indicates changes in reservoir composition, the proliferation of CD4+ T cells counteracts decay, maintaining the frequency of inducible, replication-competent proviruses at roughly constant levels over the long term. These results reinforce the need for lifelong ART.

Heightened resistance to host type 1 interferons characterizes HIV-1 at transmission and after antiretroviral therapy interruption.

Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their antiviral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally derived HIV-1 isolates from the plasma and CD4+ T cells of 26 individuals sampled longitudinally after transmission or after antiretroviral therapy (ART) and analytical treatment interruption. We determined the concentration of IFNα2 and IFNß that reduced viral replication in vitro by 50% (IC50) and found consistent changes in the sensitivity of HIV-1 to IFN-I inhibition both across individuals and over time. Resistance of HIV-1 isolates to IFN-I was uniformly high during acute infection, decreased in all individuals in the first year after infection, was reacquired concomitant with CD4+ T cell loss, and remained elevated in individuals with accelerated disease. HIV-1 isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just before ART initiation. However, viruses that rebounded after treatment interruption displayed the highest degree of IFNα2 and IFNß resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control affected by both ART and analytical treatment interruption. Although elevated at transmission, host innate pressures are the highest during viral rebound, limiting the viruses that successfully become reactivated from latency to those that are IFN-I resistant.

HIV-1 latent reservoir size and diversity are stable following brief treatment interruption.

BACKGROUND: The effect of a brief analytical treatment interruption (ATI) on the HIV-1 latent reservoir of individuals who initiate antiretroviral therapy (ART) during chronic infection is unknown. METHODS: We evaluated the impact of transient viremia on the latent reservoir in participants who underwent an ATI and at least 6 months of subsequent viral suppression in a clinical trial testing the effect of passive infusion of the broadly neutralizing Ab VRC01 during ATI. RESULTS: Measures of total HIV-1 DNA, cell-associated RNA, and infectious units per million cells (IUPM) (measured by quantitative viral outgrowth assay [QVOA]) were not statistically different before or after ATI. Phylogenetic analyses of HIV-1 env sequences from QVOA and proviral DNA demonstrated little change in the composition of the virus populations comprising the pre- and post-ATI reservoir. Expanded clones were common in both QVOA and proviral DNA sequences. The frequency of clonal populations differed significantly between QVOA viruses, proviral DNA sequences, and the viruses that reactivated in vivo. CONCLUSIONS: The results indicate that transient viremia from ATI does not substantially alter measures of the latent reservoir, that clonal expansion is prevalent within the latent reservoir, and that characterization of latent viruses that can reactivate in vivo remains challenging. TRIAL REGISTRATION: ClinicalTrials.gov NCT02463227FUNDING. Funding was provided by the NIH.

Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells.

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection.

Designing and Interpreting Limiting Dilution Assays: General Principles and Applications to the Latent Reservoir for Human Immunodeficiency Virus-1.

Limiting dilution assays are widely used in infectious disease research. These assays are crucial for current human immunodeficiency virus (HIV)-1 cure research in particular. In this study, we offer new tools to help investigators design and analyze dilution assays based on their specific research needs. Limiting dilution assays are commonly used to measure the extent of infection, and in the context of HIV they represent an essential tool for studying latency and potential curative strategies. Yet standard assay designs may not discern whether an intervention reduces an already miniscule latent infection. This review addresses challenges arising in this setting and in the general use of dilution assays. We illustrate the major statistical method for estimating frequency of infectious units from assay results, and we offer an online tool for computing this estimate. We recommend a procedure for customizing assay design to achieve desired sensitivity and precision goals, subject to experimental constraints. We consider experiments in which no viral outgrowth is observed and explain how using alternatives to viral outgrowth may make measurement of HIV latency more efficient. Finally, we discuss how biological complications, such as probabilistic growth of small infections, alter interpretations of experimental results.