Elite Suppressors Harbor Low Levels of Integrated HIV DNA and High Levels of 2-LTR Circular HIV DNA Compared to HIV+ Patients On and Off HAART

Elite suppressors (ES) are a rare population of HIV-infected individuals that are capable of naturally controlling the infection without the use of highly active anti-retroviral therapy (HAART). Patients on HAART often achieve viral control to similar (undetectable) levels. Accurate and sensitive methods to measure viral burden are needed to elucidate important differences between these two patient populations in order to better understand their mechanisms of control. Viral burden quantification in ES patients has been limited to measurements of total DNA in PBMC, and estimates of Infectious Units per Million cells (IUPM). There appears to be no significant difference in the level of total HIV DNA between cells from ES patients and patients on HAART. However, recovering infectious virus from ES patient samples is much more difficult, suggesting their reservoir size should be much smaller than that in patients on HAART. Here we find that there is a significant difference in the level of integrated HIV DNA in ES patients compared to patients on HAART, providing an explanation for the previous results. When comparing the level of total to integrated HIV DNA in these samples we find ES patients have large excesses of unintegrated HIV DNA. To determine the composition of unintegrated HIV DNA in these samples, we measured circular 2-LTR HIV DNA forms and found ES patients frequently have high levels of 2-LTR circles in PBMC. We further show that these high levels of 2-LTR circles are not the result of inefficient integration in ES cells, since HIV integrates with similar efficiency in ES and normal donor cells. Our findings suggest that measuring integration provides a better surrogate of viral burden than total HIV DNA in ES patients. Moreover, they add significantly to our understanding of the mechanisms that allow viral control and reservoir maintenance in this unique patient population

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

Case report: mechanisms of HIV elite control in two African women

Background The majority of people living with HIV require antiretroviral therapy (ART) for controlling viral replication, however there are rare HIV controllers who spontaneously and durably control HIV in the absence of treatment. Understanding what mediates viral control in these individuals has provided us with insights into the immune mechanisms that may be important to induce for a vaccine or functional cure for HIV. To date, few African elite controllers from high incidence settings have been described. We identified virological controllers from the CAPRISA 002 cohort of HIV-1 subtype C infected women in KwaZulu Natal, South Africa, two (1%) of whom were elite controllers. We examined the genetic, clinical, immunological and virological characteristics of these two elite HIV controllers in detail, to determine whether they exhibit features of putative viral control similar to those described for elite controllers reported in the literature. Case presentation In this case report, we present clinical features, CD4+ T cell and viral load trajectories for two African women over 7 years of HIV infection. Viral load became undetectable 10 months after HIV infection in Elite Controller 1 (EC1), and after 6 weeks in Elite Controller 2 (EC2), and remained undetectable for the duration of follow-up, in the absence of ART. Both elite controllers expressed multiple HLA Class I and II haplotypes previously associated with slower disease progression (HLA-A*74:01, HLA-B*44:03, HLA-B*81:01, HLA-B*57:03, HLA-DRB1*13). Fitness assays revealed that both women were infected with replication competent viruses, and both expressed higher mRNA levels of p21, a host restriction factor associated with viral control. HIV-specific T cell responses were examined using flow cytometry. EC1 mounted high frequency HIV-specific CD8+ T cell responses, including a B*81:01-restricted Gag TL9 response. Unusually, EC2 had evidence of pre-infection HIV-specific CD4+ T cell responses. Conclusion We identified some features typical of elite controllers, including high magnitude HIV-specific responses and beneficial HLA. In addition, we made the atypical finding of pre-infection HIV-specific immunity in one elite controller, that may have contributed to very early viral control. This report highlights the importance of studying HIV controllers in high incidence settings

Transient Nature of Long-Term Nonprogression and Broad Virus-Specific Proliferative T-Cell Responses with Sustained Thymic Output in HIV-1 Controllers

HIV-1(+) individuals who, without therapy, conserve cellular anti-HIV-1 responses, present with high, stable CD4(+) T-cell numbers, and control viral replication, facilitate analysis of atypical viro-immunopathology. In the absence of universal definition, immune function in such HIV controllers remains an indication of non-progression.CD4 T-cell responses to a number of HIV-1 proteins and peptide pools were assessed by IFN-gamma ELISpot and lymphoproliferative assays in HIV controllers and chronic progressors. Thymic output was assessed by sjTRECs levels. Follow-up of 41 HIV-1(+) individuals originally identified as "Long-term non-progressors" in 1996 according to clinical criteria, and longitudinal analysis of two HIV controllers over 22 years, was also performed. HIV controllers exhibited substantial IFN-gamma producing and proliferative HIV-1-specific CD4 T-cell responses to both recombinant proteins and peptide pools of Tat, Rev, Nef, Gag and Env, demonstrating functional processing and presentation. Conversely, HIV-specific T-cell responses were limited to IFN-gamma production in chronic progressors. Additionally, thymic output was approximately 19 fold higher in HIV controllers than in age-matched chronic progressors. Follow-up of 41 HIV-1(+) patients identified as LTNP in 1996 revealed the transitory characteristics of this status. IFN-gamma production and proliferative T-cell function also declines in 2 HIV controllers over 22 years.Although increased thymic output and anti-HIV-1 T-cell responses are observed in HIV controllers compared to chronic progressors, the nature of nonprogressor/controller status appears to be transitory

A Stochastic Model of Latently Infected Cell Reactivation and Viral Blip Generation in Treated HIV Patients

Motivated by viral persistence in HIV+ patients on long-term anti-retroviral treatment (ART), we present a stochastic model of HIV viral dynamics in the blood stream. We consider the hypothesis that the residual viremia in patients on ART can be explained principally by the activation of cells latently infected by HIV before the initiation of ART and that viral blips (clinically-observed short periods of detectable viral load) represent large deviations from the mean. We model the system as a continuous-time, multi-type branching process. Deriving equations for the probability generating function we use a novel numerical approach to extract the probability distributions for latent reservoir sizes and viral loads. We find that latent reservoir extinction-time distributions underscore the importance of considering reservoir dynamics beyond simply the half-life. We calculate blip amplitudes and frequencies by computing complete viral load probability distributions, and study the duration of viral blips via direct numerical simulation. We find that our model qualitatively reproduces short small-amplitude blips detected in clinical studies of treated HIV infection. Stochastic models of this type provide insight into treatment-outcome variability that cannot be found from deterministic models

Dissecting the Dynamics of HIV-1 Protein Sequence Diversity

10.1371/journal.pone.0059994PLoS ONE84

Elite Suppressor–Derived HIV-1 Envelope Glycoproteins Exhibit Reduced Entry Efficiency and Kinetics

Elite suppressors (ES) are a rare subset of HIV-1–infected individuals who are able to maintain HIV-1 viral loads below the limit of detection by ultra-sensitive clinical assays in the absence of antiretroviral therapy. Mechanism(s) responsible for this elite control are poorly understood but likely involve both host and viral factors. This study assesses ES plasma-derived envelope glycoprotein (env) fitness as a function of entry efficiency as a possible contributor to viral suppression. Fitness of virus entry was first evaluated using a novel inducible cell line with controlled surface expression levels of CD4 (receptor) and CCR5 (co-receptor). In the context of physiologic CCR5 and CD4 surface densities, ES envs exhibited significantly decreased entry efficiency relative to chronically infected viremic progressors. ES envs also demonstrated slow entry kinetics indicating the presence of virus with reduced entry fitness. Overall, ES env clones were less efficient at mediating entry than chronic progressor envs. Interestingly, acute infection envs exhibited an intermediate phenotypic pattern not distinctly different from ES or chronic progressor envs. These results imply that lower env fitness may be established early and may directly contribute to viral suppression in ES individuals

Effects of prostratin on Cyclin T1/P-TEFb function and the gene expression profile in primary resting CD4(+ )T cells

BACKGROUND: The latent reservoir of human immunodeficiency virus type 1 (HIV-1) in resting CD4(+ )T cells is a major obstacle to the clearance of infection by highly active antiretroviral therapy (HAART). Recent studies have focused on searches for adjuvant therapies to activate this reservoir under conditions of HAART. Prostratin, a non tumor-promoting phorbol ester, is a candidate for such a strategy. Prostratin has been shown to reactivate latent HIV-1 and Tat-mediated transactivation may play an important role in this process. We examined resting CD4(+ )T cells from healthy donors to determine if prostratin induces Cyclin T1/P-TEFb, a cellular kinase composed of Cyclin T1 and Cyclin-dependent kinase-9 (CDK9) that mediates Tat function. We also examined effects of prostratin on Cyclin T2a, an alternative regulatory subunit for CDK9, and 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and repress its kinase activity. RESULTS: Prostratin up-regulated Cyclin T1 protein expression, modestly induced CDK9 protein expression, and did not affect Cyclin T2a protein expression. Although the kinase activity of CDK9 in vitro was up-regulated by prostratin, we observed a large increase in the association of 7SK snRNA and the HEXIM1 protein with CDK9. Using HIV-1 reporter viruses with and without a functional Tat protein, we found that prostratin stimulation of HIV-1 gene expression appears to require a functional Tat protein. Microarray analyses were performed and several genes related to HIV biology, including APOBEC3B, DEFA1, and S100 calcium-binding protein genes, were found to be regulated by prostratin. CONCLUSION: Prostratin induces Cyclin T1 expression and P-TEFb function and this is likely to be involved in prostratin reactivation of latent HIV-1 proviruses. The large increase in association of 7SK and HEXIM1 with P-TEFb following prostratin treatment may reflect a requirement in CD4(+ )T cells for a precise balance between active and catalytically inactive P-TEFb. Additionally, genes regulated by prostratin were identified that have the potential to regulate HIV-1 replication both positively and negatively

Functional Cure of SIVagm Infection in Rhesus Macaques Results in Complete Recovery of CD4+ T Cells and Is Reverted by CD8+ Cell Depletion

Understanding the mechanism of infection control in elite controllers (EC) may shed light on the correlates of control of disease progression in HIV infection. However, limitations have prevented a clear understanding of the mechanisms of elite controlled infection, as these studies can only be performed at randomly selected late time points in infection, after control is achieved, and the access to tissues is limited. We report that SIVagm infection is elite-controlled in rhesus macaques (RMs) and therefore can be used as an animal model for EC HIV infection. A robust acute infection, with high levels of viral replication and dramatic mucosal CD4+ T cell depletion, similar to pathogenic HIV-1/SIV infections of humans and RMs, was followed by complete and durable control of SIVagm replication, defined as: undetectable VLs in blood and tissues beginning 72 to 90 days postinoculation (pi) and continuing at least 4 years; seroreversion; progressive recovery of mucosal CD4+ T cells, with complete recovery by 4 years pi; normal levels of T cell immune activation, proliferation, and apoptosis; and no disease progression. This “functional cure” of SIVagm infection in RMs could be reverted after 4 years of control of infection by depleting CD8 cells, which resulted in transient rebounds of VLs, thus suggesting that control may be at least in part immune mediated. Viral control was independent of MHC, partial APOBEC restriction was not involved in SIVagm control in RMs and Trim5 genotypes did not impact viral replication. This new animal model of EC lentiviral infection, in which complete control can be predicted in all cases, permits research on the early events of infection in blood and tissues, before the defining characteristics of EC are evident and when host factors are actively driving the infection towards the EC status

Control of Stochastic Gene Expression by Host Factors at the HIV Promoter

The HIV promoter within the viral long terminal repeat (LTR) orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat), this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright) and inactive (Off) expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-κB elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-κB elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-κB sites exhibit distinct properties, with κB site I serving a stronger activating role than κB site II. Moreover, Sp1 site III plays a particularly important role in the recruitment of both p300 and RelA to the promoter. Finally, analysis of 362 clonal cell populations infected with the viral variants revealed that mutations in any of the Sp1 sites yield a 6-fold higher frequency of clonal bifurcation compared to that of the wild-type promoter. Thus, each Sp1 and NF-κB site differentially contributes to the regulation of viral gene expression, and Sp1 sites functionally “dampen” transcriptional noise and thereby modulate the frequency and maintenance of this model of viral latency. These results may have biomedical implications for the treatment of HIV latency