Early and prolonged antiretroviral therapy is associated with an HIV-1-specific T-cell profile comparable to that of long-term non-progressors

Background

Intervention with antiretroviral treatment (ART) and control of viral replication at the time of HIV-1 seroconversion may curtail cumulative immunological damage. We have therefore hypothesized that ART maintenance over a very prolonged period in HIV-1 seroconverters could induce an immuno-virological status similar to that of HIV-1 long-term non-progressors (LTNPs).

Methodology/Principal Findings

We have investigated a cohort of 20 HIV-1 seroconverters on long-term ART (LTTS) and compared it to one of 15 LTNPs. Residual viral replication and reservoirs in peripheral blood, as measured by cell-associated HIV-1 RNA and DNA, respectively, were demonstrated to be similarly low in both cohorts. These two virologically matched cohorts were then comprehensively analysed by polychromatic flow cytometry for HIV-1-specific CD4⁺ and CD8⁺ T-cell functional profile in terms of cytokine production and cytotoxic capacity using IFN-γ, IL-2, TNF-α production and perforin expression, respectively. Comparable levels of highly polyfunctional HIV-1-specific CD4⁺ and CD8⁺ T-cells were found in LTTS and LTNPs, with low perforin expression on HIV-1-specific CD8⁺ T-cells, consistent with a polyfunctional/non-cytotoxic profile in a context of low viral burden.

Conclusions

Our results indicate that prolonged ART initiated at the time of HIV-1 seroconversion is associated with immuno-virological features which resemble those of LTNPs, strengthening the recent emphasis on the positive impact of early treatment initiation and paving the way for further interventions to promote virological control after treatment interruption.     

Viral replication capacity as a correlate of HLA B57/B5801-associated nonprogressive HIV-1 infection

HLA B57 and the closely related HLA B5801 are over-represented among HIV-1 infected long-term nonprogressors (LTNPs). It has been suggested that this association between HLA B57/5801 and asymptomatic survival is a consequence of strong CTL responses against epitopes in the viral Gag protein. Moreover, CTL escape mutations in Gag would coincide with viral attenuation, resulting in low viral load despite evasion from immune control. In this study we compared HLA B57/5801 HIV-1 infected progressors and LTNPs for sequence variation in four dominant epitopes in Gag and their ability to generate CTL responses against these epitopes and the autologous escape variants. Prevalence and appearance of escape mutations in Gag epitopes and potential compensatory mutations were similar in HLA B57/5801 LTNPs and progressors. Both groups were also indistinguishable in the magnitude of CD8+ IFN-gamma responses directed against the wild-type or autologous escape mutant Gag epitopes in IFN-gamma ELISPOT analysis. Interestingly, HIV-1 variants from HLA B57/5801 LTNPs had much lower replication capacity than the viruses from HLA B57/5801 progressors, which did not correlate with specific mutations in Gag. In conclusion, the different clinical course of HLA B57/5801 LTNPs and progressors was not associated with differences in CTL escape mutations or CTL activity against epitopes in Gag but rather with differences in HIV-1 replication capacity.     

Low TRBP levels support an innate human immunodeficiency virus type 1 resistance in astrocytes by enhancing the PKR antiviral response

Acute human immunodeficiency virus type 1 (HIV-1) replication in astrocytes produces minimal new virus particles due, in part, to inefficient translation of viral structural proteins despite high levels of cytoplasmic viral mRNA. We found that a highly reactive double-stranded (ds) RNA-binding protein kinase (PKR) response in astrocytes underlies this inefficient translation of HIV-1 mRNA. The dsRNA elements made during acute replication of HIV-1 in astrocytes triggers PKR activation and the specific inhibition of HIV-1 protein translation. The heightened PKR response results from relatively low levels of the cellular antagonist of PKR, the TAR RNA binding protein (TRBP). Efficient HIV-1 production was restored in astrocytes by inhibiting the innate PKR response to HIV-1 dsRNA with dominant negative PKR mutants, or PKR knockdown by siRNA gene silencing. Increasing the expression of TRBP in astrocytes restored acute virus production to levels comparable to those observed in permissive cells. Therefore, the robust innate PKR antiviral response in astrocytes results from relatively low levels of TRBP expression and contributes to their restricted infection. Our findings highlight TRBP as a novel cellular target for therapeutic interventions to block productive HIV-1 replication in cells that are fully permissive for HIV-1 infection.     

Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy

Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of 'covert' virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.     

Inefficient enhancement of viral infectivity and CD4 downregulation by human immunodeficiency virus type 1 Nef from Japanese long-term nonprogressors

It has been reported that patients infected with nef-defective human immunodeficiency virus type 1 (HIV-1) do not progress to AIDS; however, mutations that abrogate Nef expression are not common in long-term nonprogressors (LTNPs). We postulated that Nef function might be impaired in LTNPs, irrespective of the presence or absence of detectable amino acid sequence anomalies. To challenge this hypothesis we compared in vitro function of nef alleles that were derived from three groups of Japanese patients: LTNPs, progressors, and asymptomatic carriers (ACs). The patient-derived nef alleles were subcloned into a nef-defective infectious HIV-1 molecular clone and an expression vector. We first examined Nef-dependent enhancement of infection in a single-round infectivity assay by the use of MAGNEF cells, in which Nef is required more strictly for the infection than in the parent MAGI cells. All nef alleles from LTNPs showed reduced enhancement in the infectivity of nef-defective HIV-1 mutants compared to the nef alleles of progressors or ACs. Second, we found that nef alleles from LTNPs were less efficient in CD4 downregulation than those of progressors or ACs. Third, all nef alleles from LTNPs, progressors, and ACs reduced the cell surface expression of major histocompatibility complex class I to a similar level. Last, there was no correlation between Hck-binding activity of Nef and clinical grouping. In conclusion, we detected inefficient enhancement of HIV-1 infectivity and CD4 downregulation by HIV-1 nef alleles of LTNPs. It awaits further study to conclude that these characteristics of nef alleles are the cause or the consequence of the long-term nonprogression after HIV-1 infection.     

Identification of a Cluster of HIV-1 Controllers Infected with Low Replicating Viruses

Long term non-progressor patients (LTNPs) are characterized by the natural control of HIV-1 infection. This control is related to host genetic, immunological and virological factors. In this work, phylogenetic analysis of the proviral nucleotide sequences in env gene from a Spanish HIV-1 LTNPs cohort identified a cluster of 6 HIV-1 controllers infected with closely-related viruses. The patients of the cluster showed common clinical and epidemiological features: drug user practices, infection in the same city (Madrid, Spain) and at the same time (late 70’s-early 80’s). All cluster patients displayed distinct host alleles associated with HIV control. Analysis of the virus envelope nucleotide sequences showed ancestral characteristic, lack of evolution and presence of rare amino-acids. Biological characterization of recombinant viruses with the envelope proteins from the cluster viruses showed very low replicative capacity in TZMbl and U87-CD4/CCR5 cells. The lack of clinical progression in the viral cluster patients with distinct combinations of protective host genotypes, but infected by low replicating viruses, indicate the important role of the virus in the non-progressor phenotype in these patients.     

Direct evidence of lower viral replication rates in vivo in human immunodeficiency virus type 2 (HIV-2) infection than in HIV-1 infection

Studies have shown that human immunodeficiency virus type 2 (HIV-2) is less pathogenic than HIV-1, with a lower rate of disease progression. Similarly, plasma viral loads are lower in HIV-2 infection, suggesting that HIV-2 replication is restricted in vivo in comparison to that of HIV-1. However, to date, in vivo studies characterizing replication intermediates in the viral life cycle of HIV-2 have been limited. In order to test the hypothesis that HIV-2 has a lower replication rate in vivo than HIV-1 does, we quantified total viral DNA, integrated proviral DNA, cell-associated viral mRNA, and plasma viral loads in peripheral blood samples from groups of therapy-na?ve HIV-1-infected (n = 21) and HIV-2-infected (n = 18) individuals from Dakar, Senegal, with CD4(+) T-cell counts of >200/microl. Consistent with our previous findings, total viral DNA loads were similar between HIV-1 and HIV-2 and plasma viral loads were higher among HIV-1-infected individuals. Proportions of DNA in the integrated form were also similar between these viruses. In contrast, levels of viral mRNA were lower in HIV-2 infection. Our study indicates that HIV-2 is able to establish a stable, integrated proviral infection in vivo, but that accumulation of viral mRNA is attenuated in HIV-2 infection relative to that in HIV-1 infection. The differences in viral mRNA are consistent with the differences in plasma viral loads between HIV-1 and HIV-2 and suggest that lower plasma viral loads, and possibly the attenuated pathogenesis of HIV-2, can be explained by lower rates of viral replication in vivo.     

High-throughput screening uncovers a compound that activates latent HIV-1 and acts cooperatively with a histone deacetylase (HDAC) inhibitor

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cell-based system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4(+) T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.     

The extent of early viral replication is a critical determinant of the natural history of simian immunodeficiency virus infection.

Different patterns of viral replication correlate with the natural history of disease progression in humans and macaques infected with human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), respectively. However, the viral and host factors influencing these patterns of viral replication in vivo are poorly understood. We intensively studied viral replication in macaques receiving identical inocula of SIV. Marked differences in viral replication patterns were apparent within the first week following inoculation, a time prior to the development of measurable specific immune effector responses to viral antigens. Plasma viral RNA levels measured on day 7 postinoculation correlated with levels measured in the postacute phase of infection. Differences in the susceptibility of host cells from different animals to in vitro SIV infection correlated with the permissiveness of the animals for early in vivo viral replication and hence with the postacute set point level of plasma viremia. These results suggest that host factors that exert their effects prior to full development of specific immune responses are critical in establishing the in vivo viral replication pattern and associated clinical course in subjects infected with SIV and, by extension, with HIV-1.     

The effects of cytomegalovirus on human immunodeficiency virus replication in brain-derived cells correlate with permissiveness of the cells for each virus.

Human cytomegalovirus (HCMV) is commonly found in the brains of patients with AIDS and in some cases can be detected in the same cells as can human immunodeficiency virus type 1 (HIV-1). In this study, we analyzed the patterns of replication of HIV-1 and HCMV in singly infected cells and the effects of dual infection in human brain-derived cell lines of three different origins: neuroblastoma cell lines SK-N-MC and SY5Y; astrocytoma/glioblastoma cell lines U373-MG and Hs 683; and undifferentiated glioblastoma cell lines A172 and T98G. To bypass the restriction at the adsorption/penetration step in these CD4-negative cells, we used HIV-1 (amphotropic retrovirus) pseudotypes. These HIV-1 pseudotypes infected the majority of the cells in the cultures and expressed high levels of HIV-1 gene products in all except the SY5Y cells. The cell lines differed in the ability to support HCMV infection, but coinfection with HIV-1 had no effect on HCMV replication. The A172 cells were completely nonpermissive for HCMV gene expression, while HCMV replication in the singly infected T98G and SK-N-MC cell lines was restricted at the level of some early gene products. This resulted in complete and partial inhibition, respectively, of viral DNA synthesis. Dual infection of the A172, T98G, and SK-N-MC cells had no effect on HIV-1 replication. The other three cell lines, U373-MG, Hs 683, and SY5Y, were fully permissive for HCMV replication. In the U373-MG and Hs 683 cells, HCMV markedly inhibited the synthesis of HIV-1 gene products. In contrast, a transient stimulation of HIV-1 production followed by a repression was observed in the dually infected SY5Y cells. We conclude from these results that under conditions in which both HIV-1 and HCMV can undergo fully permissive infection, HCMV can repress HIV-1 gene expression. In cells in which HCMV replication is limited but HIV-1 replicates well, there is no effect on HIV-1 gene expression. However, activation of HIV-1, at least transiently, may occur in cells in which HIV-1 gene expression is limited. These studies suggest that a threshold level of some HIV-1 gene product(s) may obscure activation or promote repression of HIV replication by HCMV.     

Components of apoptotic pathways modulate HIV-1 latency in Jurkat cells

The ability of the human immunodeficiency virus type 1 (HIV-1) to establish latent infections serves as a major barrier for its cure. This process could occur when its host cells undergo apoptosis, but it is uncertain whether the components of the apoptotic pathways affect viral latency. Using the susceptible Jurkat cell line, we investigated the relationship of apoptosis-associated components with HIV-1 DNA levels using the sensitive real-time PCR assay. Here, we found that the expression of proapoptotic proteins, including Fas ligand (FasL), FADD, and p53, significantly decreased HIV-1 viral DNA in cells. In contrast, the expression of antiapoptotic molecules, such as FLIP, Bcl2, and XIAP, increased the levels of viral DNA. Furthermore, promoting cellular antiapoptotic state via the knockdown of Bax with siRNA and FADD with antisense mRNA or the treatment with the Caspase-3 inhibitor, Z-DEVD, also raised viral DNA. We also simultaneously measured viral RNA from supernatants of these cell cultures and found that HIV-1 latency is inversely proportional to viral replication. Furthermore, we demonstrated that HIV-1-infected cells that underwent the transient expression of FLIP- or XIAP-induced viral latency would then produce an increased level of viral RNA upon the reversal of these antiapoptotic effects via PMA treatment compared to LacZ control cells. Taken together, these data suggest that HIV-1 infection could be adapted to employ or even manipulate the cellular apoptotic pathway to its advantage: when the host cell remains in a pro-apoptotic state, HIV-1 favors active replication, while when the host cell prefers an anti-apoptotic state, the virus establishes viral latency and promotes latent reservoir seeding in a way which would enhance viral replication and cytopathogenesis when the cellular conditions shift to encourage the productive infection phase.