Viral suppression of multiple escape mutants by de novo CD8+ T cell responses in a human immunodeficiency virus-1 Infected elite suppressor

Elite suppressors or controllers (ES) are HIV-1 infected patients who maintain undetectable viral loads without treatment. While HLA-B*57-positive ES are usually infected with virus that is unmutated at CTL epitopes, a single, dominant variant containing CTL escape mutations is typically seen in plasma during chronic infection. We describe an ES who developed seven distinct and rare escape variants at an HLA-B*57-restricted Gag epitope over a five year period. Interestingly, he developed proliferative, de novo CTL responses that suppressed replication of each of these variants. These responses, in combination with low viral fitness of each variant, may contribute to sustained elite control in this ES

Maintenance of viral suppression in HIV-1–infected HLA-B*57+ elite suppressors despite CTL escape mutations

Rare human immunodeficiency virus 1–infected individuals, termed elite suppressors (ES), maintain plasma virus levels of <50 copies/ml and normal CD4 counts without therapy. The major histocompatibility complex class I allele group human histocompatibility leukocyte antigen (HLA)-B*57 is overrepresented in this population. Mutations in HLA-B*57–restricted epitopes have been observed in ES, but their significance has remained unclear. Here we investigate the extent and impact of cytotoxic T lymphocyte (CTL) escape mutations in HLA-B*57+ ES. We provide the first direct evidence that most ES experience chronic low level viremia. Sequencing revealed a striking discordance between the genotypes of plasma virus and archived provirus in resting CD4+ T cells. Mutations in HLA-B*57–restricted Gag epitopes were present in all viruses from plasma but were rare in proviruses, suggesting powerful selective pressure acting at these epitopes. Surprisingly, strong CD8+ T cell interferon-γ responses were detected against some mutant epitopes found in plasma virus, suggesting the development of de novo responses to viral variants. In some individuals, relative CD8+ T cell interleukin-2 responses showed better correlation with the selection observed in vivo. Thus, analysis of low level viremia reveals an unexpectedly high level of CTL escape mutations reflecting selective pressure acting at HLA-B*57–restricted epitopes in ES. Continued viral suppression probably reflects CTL responses against unmutated epitopes and residual or de novo responses against epitopes with escape mutations

Evolution of the HIV-1 nef gene in HLA-B*57 Positive Elite Suppressors

Elite controllers or suppressors (ES) are HIV-1 infected patients who maintain viral loads of < 50 copies/ml without antiretroviral therapy. CD8+ T cells are thought to play a key role in the control of viral replication and exert selective pressure on gag and nef in HLA-B*57 positive ES. We previously showed evolution in the gag gene of ES which surprisingly was mostly due to synonymous mutations rather than non-synonymous mutation in targeted CTL epitopes. This finding could be the result of structural constraints on Gag, and we therefore examined the less conserved nef gene. We found slow evolution of nef in plasma virus in some ES. This evolution is mostly due to synonymous mutations and occurs at a rate similar to that seen in the gag gene in the same patients. The results provide further evidence of ongoing viral replication in ES and suggest that the nef and gag genes in these patients respond similarly to selective pressure from the host

A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection

Factors Associated With the Control of Viral Replication and Virologic Breakthrough in a Recently Infected HIV-1 Controller

HIV-1 controllers are patients who control HIV-1 viral replication without antiretroviral therapy. Control is achieved very early in the course of infection, but the mechanisms through which viral replication is restricted are not fully understood. We describe a patient who presented with acute HIV-1 infection and was found to have an HIV-1 RNA level of < 100 copies/mL. She did not have any known protective HLA alleles, but significant immune activation of CD8 + T cells and natural killer (NK) cells was present, and both cell types inhibited viral replication. Virus cultured from this patient replicated as well in vitro as virus isolated from her partner, a patient with AIDS who was the source of transmission. Virologic breakthrough occurred 9 months after her initial presentation and was associated with an increase in CD4 + T cell activation levels and a significant decrease in NK cell inhibitory capacity. Remarkably, CD8 + T cell inhibitory capacity was preserved and there were no new escape mutations in targeted Gag epitopes. These findings suggest that fully replication-competent virus can be controlled in acute HIV-1 infection in some patients without protective HLA alleles and that NK cell responses may contribute to this early control of viral replication

Relationships of PBMC microRNA expression, plasma viral load, and CD4+ T-cell count in HIV-1-infected elite suppressors and viremic patients

<p>Abstract</p> <p>Background</p> <p>HIV-1-infected elite controllers or suppressors (ES) maintain undetectable viral loads (< 50 copies/mL) without antiretroviral therapy. The mechanisms of suppression are incompletely understood. Modulation of HIV-1 replication by miRNAs has been reported, but the role of small RNAs in ES is unknown. Using samples from a well-characterized ES cohort, untreated viremic patients, and uninfected controls, we explored the PBMC miRNA profile and probed the relationships of miRNA expression, CD4+ T-cell counts, and viral load.</p> <p>Results</p> <p>miRNA profiles, obtained using multiple acquisition, data processing, and analysis methods, distinguished ES and uninfected controls from viremic HIV-1-infected patients. For several miRNAs, however, ES and viremic patients shared similar expression patterns. Differentially expressed miRNAs included those with reported roles in HIV-1 latency (miR-29 family members, miRs -125b and -150). Others, such as miR-31 and miR-31*, had no previously reported connection with HIV-1 infection but were found here to differ significantly with uncontrolled HIV-1 replication. Correlations of miRNA expression with CD4+ T-cell count and viral load were found, and we observed that ES with low CD4+ T-cell counts had miRNA profiles more closely related to viremic patients than controls. However, expression patterns indicate that miRNA variability cannot be explained solely by CD4+ T-cell variation.</p> <p>Conclusions</p> <p>The intimate involvement of miRNAs in disease processes is underscored by connections of miRNA expression with the HIV disease clinical parameters of CD4 count and plasma viral load. However, miRNA profile changes are not explained completely by these variables. Significant declines of miRs-125b and -150, among others, in both ES and viremic patients indicate the persistence of host miRNA responses or ongoing effects of infection despite viral suppression by ES. We found no negative correlations with viral load in viremic patients, not even those that have been reported to silence HIV-1 in vitro, suggesting that the effects of these miRNAs are exerted in a focused, cell-type-specific manner. Finally, the observation that some ES with low CD4 counts were consistently related to viremic patients suggests that miRNAs may serve as biomarkers for risk of disease progression even in the presence of viral suppression.</p

Improvements and Limitations of Humanized Mouse Models for HIV Research: NIH/NIAID “Meet the Experts” 2015 Workshop Summary

The number of humanized mouse models for the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and other infectious diseases has expanded rapidly over the past 8 years. Highly immunodeficient mouse strains, such as NOD/SCID/gamma chainnull (NSG, NOG), support better human hematopoietic cell engraftment. Another improvement is the derivation of highly immunodeficient mice, transgenic with human leukocyte antigens (HLAs) and cytokines that supported development of HLA-restricted human T cells and heightened human myeloid cell engraftment. Humanized mice are also used to study the HIV reservoir using new imaging techniques. Despite these advances, there are still limitations in HIV immune responses and deficits in lymphoid structures in these models in addition to xenogeneic graft-versus-host responses. To understand and disseminate the improvements and limitations of humanized mouse models to the scientific community, the NIH sponsored and convened a meeting on April 15, 2015 to discuss the state of knowledge concerning these questions and best practices for selecting a humanized mouse model for a particular scientific investigation. This report summarizes the findings of the NIH meeting

HIV-1 persistence following extremely early initiation of antiretroviral therapy (ART) during acute HIV-1 infection: An observational study

Background: It is unknown if extremely early initiation of antiretroviral therapy (ART) may lead to long-term ART-free HIV remission or cure. As a result, we studied 2 individuals recruited from a pre-exposure prophylaxis (PrEP) program who started prophylactic ART an estimated 10 days (Participant A; 54-year-old male) and 12 days (Participant B; 31-year-old male) after infection with peak plasma HIV RNA of 220 copies/mL and 3,343 copies/mL, respectively. Extensive testing of blood and tissue for HIV persistence was performed, and PrEP Participant A underwent analytical treatment interruption (ATI) following 32 weeks of continuous ART. Methods and findings Colorectal and lymph node tissues, bone marrow, cerebral spinal fluid (CSF), plasma, and very large numbers of peripheral blood mononuclear cells (PBMCs) were obtained longitudinally from both participants and were studied for HIV persistence in several laboratories using molecular and culture-based detection methods, including a murine viral outgrowth assay (mVOA). Both participants initiated PrEP with tenofovir/emtricitabine during very early Fiebig stage I (detectable plasma HIV-1 RNA, antibody negative) followed by 4-drug ART intensification. Following peak viral loads, both participants experienced full suppression of HIV-1 plasma viremia. Over the following 2 years, no further HIV could be detected in blood or tissue from PrEP Participant A despite extensive sampling from ileum, rectum, lymph nodes, bone marrow, CSF, circulating CD4+ T cell subsets, and plasma. No HIV was detected from tissues obtained from PrEP Participant B, but low-level HIV RNA or DNA was intermittently detected from various CD4+ T cell subsets. Over 500 million CD4+ T cells were assayed from both participants in a humanized mouse outgrowth assay. Three of 8 mice infused with CD4+ T cells from PrEP Participant B developed viremia (50 million input cells/surviving mouse), but only 1 of 10 mice infused with CD4+ T cells from PrEP Participant A (53 million input cells/mouse) experienced very low level viremia (201 copies/mL); sequence confirmation was unsuccessful. PrEP Participant A stopped ART and remained aviremic for 7.4 months, rebounding with HIV RNA of 36 copies/mL that rose to 59,805 copies/mL 6 days later. ART was restarted promptly. Rebound plasma HIV sequences were identical to those obtained during acute infection by single-genome sequencing. Mathematical modeling predicted that the latent reservoir size was approximately 200 cells prior to ATI and that only around 1% of individuals with a similar HIV burden may achieve lifelong ART-free remission. Furthermore, we observed that lymphocytes expressing the tumor marker CD30 increased in frequency weeks to months prior to detectable HIV-1 RNA in plasma. This study was limited by the small sample size, which was a result of the rarity of individuals presenting during hyperacute infection. Conclusions: We report HIV relapse despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete or complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission. However, the small numbers of latently infected cells in individuals treated during hyperacute infection may be associated with prolonged ART-free remission