Evidence of CD8+ T-cell-mediated selective pressure on human immunodeficiency virus type 1 nef in HLA-B*57+ elite suppressors

Elite suppressors (ES) are human immunodeficiency virus type 1 (HIV-1)-infected patients who maintain viral loads of <50 copies/ml without treatment. The observation that the HLA-B*57 allele is overrepresented in these patients implies that HIV-1-specific CD8⁺ T cells play a key role in suppressing viral replication. We have previously shown that while CD8⁺ T-cell escape mutations are rarely seen in proviral Gag sequences in resting CD4⁺ T cells from peripheral blood, they are present in every clone amplified from the low levels of free virus in the plasma of HLA-B*57⁺ ES. In this study, we compared the pattern of mutations in Nef sequences amplified from peripheral blood CD4⁺ T cells and from plasma virus. We show that Nef mutations are present in plasma virus but are rare in the cellular sequences and provide evidence that these plasma Nef variants represent novel escape mutants. The results provide further evidence of CD8⁺ T-cell-mediated selective pressure on plasma virus in ES and suggest that there must be ongoing HIV-1 replication in spite of the very low viral loads seen for these patients.     

Efficacious Early Antiviral Activity of HIV Gag- and Pol-Specific HLA-B*2705-Restricted CD8+ T Cells

The association between HLA-B*2705 and the immune control of human immunodeficiency virus type 1 (HIV-1) has previously been linked to the targeting of the HLA-B*2705-restricted Gag epitope KRWIILGLNK (KK10) by CD8⁺ T cells. In order to better define the mechanisms of the HLA-B*2705 immune control of HIV, we first characterized the CD8⁺ T-cell responses of nine highly active antiretroviral therapy (HAART)-naïve B*2705-positive subjects. Unexpectedly, we observed a strong response to an HLA-B*2705-restricted Pol epitope, KRKGGIGGY (KY9), in 8/9 subjects. The magnitude of the KY9 response was only marginally lower than that of the KK10-specific response (median, 695 versus 867 spot-forming cells [SFC]/million peripheral blood mononuclear cells [PBMCs]; not significant [NS]), and viral escape mutants were observed in both KY9 and KK10, resulting from selection pressure driven by the respective CD8⁺ T-cell response. By comparing inhibitions of viral replication by CD8⁺ T cells specific for the Gag KK10, Pol KY9, and Vpr VL9 HLA-B*2705-restricted epitopes, we observed a consistent hierarchy of antiviral efficacy (Gag KK10 > Pol KY9 > Vpr VL9). This hierarchy was associated with early recognition of HIV-1-infected cells, within 6 h of infection, by KK10- and KY9-specific CD8⁺ T cells but not until 18 h postinfection by VL9-specific CD8⁺ T cells. There was no association between antiviral efficacy and proliferative capacity, cytotoxicity, polyfunctionality, or T-cell receptor (TCR) avidity. These data are consistent with previous studies indicating an important role for the B*2705-Gag KK10 response in the control of HIV but also suggest a previously unrecognized role played by the subdominant Pol-specific KY9 response in HLA-B*2705-mediated control of HIV and that the recognition of HIV-infected cells by CD8⁺ T cells early in the viral life cycle may be important for viral containment in HIV-infected individuals.Current human immunodeficiency virus (HIV) vaccine strategies are focused on emulating the protective effect observed for HIV-infected individuals carrying alleles such as B*2705 by inducing the virus-specific CD8⁺ T-cell responses that are thought to be responsible for delaying or preventing disease progression. Understanding why such alleles confer protection facilitates a rational approach to vaccine design. It has been hypothesized that the slow progression to AIDS exhibited by HLA-B*2705-positive (HLA-B*2705⁺) HIV-infected individuals is due to the immunodominant B*27-restricted CD8⁺ T-cell response toward the p24 Gag epitope KRWIILGLNK (KK10) (Gag residues 263 to 272). Escape from this epitope typically occurs late in infection and is associated with rapid progression to AIDS (14, 16). The commonly selected mutation R264K abrogates CD8⁺ T-cell recognition but also confers a substantial fitness cost to the virus, and the selection of compensatory mutations is required to restore viral replicative capacity (19, 29, 30). This has prompted the hypothesis that CD8⁺ T-cell responses that can drive escape mutations that reduce viral fitness are a contributing factor in the immune control of HIV, either by promoting the outgrowth of a viral quasispecies with a lower replicative capacity or by delaying the selection of escape mutations, both of which may slow the onset of AIDS (11, 21, 25).To better understand how CD8⁺ T cells can be most effective against HIV, recent studies have directly assessed the antiviral activity of CD8⁺ T cells via the viral suppression of HIV-infected CD4⁺ T cells during coculture. Such studies indicated that Gag-specific CD8⁺ T cells have a higher potency for viral suppression than Env-specific CD8⁺ T cells (10), supporting previous data indicating that broad CD8⁺ T-cell targeting of Gag epitopes was associated a with lower viral set point and, hence, slower progression to AIDS (20). A recent study of simian immunodeficiency virus (SIV) suggested that the protective effect of Gag-specific CD8⁺ T cells is mediated by the early presentation of Gag epitopes, processed from the viral Gag protein from incoming virions during infection, which can sensitize target cells for lysis by Gag-specific CD8⁺ T cells within 6 h of infection (26, 27). In addition, it was proposed previously that the ability of CD8⁺ T cells to secrete multiple cytokines may also be an important correlate of immune protection (6), and a further recent study demonstrated a more polyfunctional cytokine profile of Gag-specific B*2705-KK10 CD8⁺ T-cell responses than those of other HIV-specific CD8⁺ T-cell responses (1). The ability of CD8⁺ T cells to proliferate in response to the cognate epitope peptide has also been associated with immune control (1, 12). Other studies demonstrated the importance of lytic granule loading of CD8⁺ T cells for the effective elimination of HIV-infected cells (6, 22). However, the induction of a Gag KK10-specific CD8⁺ T-cell vaccine response in a B*2705-positive vaccinee did not protect against rapid progression following subsequent HIV-1 infection (5). This anecdotal case suggests the possibility that HLA-B*2705-associated immune control of HIV-1 may not be dependent on the Gag KK10-specific CD8⁺ T-cell response alone.Since current vaccine strategies hope to induce a protective effect, such as that observed for HLA-B*2705⁺ HIV-infected individuals, the study of the functional and phenotypic characteristics of B*2705-specific CD8⁺ T cells provides an opportunity to redefine the proposed correlates of immune protection essential for rational vaccine design. In this study we analyze three different specificities of HLA-B*2705-restricted CD8⁺ T cells from chronically HIV-infected individuals in order to directly compare antiviral activity with potential correlates of immune protection, including the kinetics of viral inhibition, cytokine profile, granzyme production, proliferative capacity, and cytotoxicity.     

Effective Downregulation of HLA-A*2 and HLA-B*57 by Primary Human Immunodeficiency Virus Type 1 Isolates Cultured from Elite Suppressors

Elite controllers or suppressors (ES) are human immunodeficiency virus type 1 (HIV-1)-infected patients who control viral replication to <50 copies/ml without antiretroviral therapy. Downregulation of HLA class I molecules is an important mechanism used by HIV-1 to evade the immune system. In this study, we showed that primary isolates from ES are as effective as isolates obtained from patients with progressive HIV-1 disease at downregulating HLA-A*2 and HLA-B*57 molecules on primary CD4⁺ T cells. Thus, a diminished ability of viral isolates from ES to evade HIV-specific immune responses probably does not contribute to the control of viral replication in these patients.Long-term nonprogressors (LTNP) are human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain normal CD4⁺ T-cell counts and remain asymptomatic for longer than 10 years without therapy (7). Although many LTNP have detectable levels of HIV-1 RNA in their plasma, patients known as elite suppressors (ES) have viral loads of <50 RNA copies/ml. Understanding the factors involved in the maintenance of LTNP and ES statuses may be critical for the development of effective vaccines and immunotherapeutic treatments. One such factor under investigation is the role of cytotoxic-T-lymphocyte (CTL) responses. Several studies have shown that the HLA-B*27 and -B*57 alleles are overrepresented in cohorts of ES (13, 16, 19, 28, 29, 34). These findings suggest important roles for major histocompatibility complex class I (MHC-I) restriction and CD8⁺ T-cell responses in the control of viremia. Indeed, multiple studies have documented qualitatively superior CD8⁺ T cell function in ES compared to that in chronic HIV progressors (CP) (2, 5, 12, 27, 28, 37, 47).Other studies suggest that some ES and LTNP are infected with attenuated viruses. One illustrative example comes from studies done on the Sydney Blood Bank Cohort, in which an LTNP donor transmitted an HIV-1 isolate with a large deletion in nef and the U3 region of the long terminal repeat to multiple recipients, all of whom became LTNP (11, 21). As in the Sydney Blood Bank Cohort studies, several other investigators have detected viruses with defective nef genes in LTNP and ES (1, 8, 18, 23, 25, 35, 36, 38, 43). In contrast, other studies showed that CD4⁺ T cells from ES could produce Gag when they were stimulated in vitro (20, 26), and full-length sequence analyses of plasma and proviral genomes revealed no evidence of significant deletions (30). Recent studies have suggested that plasma isolates (31) and replication-competent viruses (32) from HLA-B*57/B58*01 ES and LTNP, respectively, are less fit than isolates from B*57/B*5801 CP, but the difference in fitness observed is unlikely to fully explain the control of viral replication in these patients. Furthermore, we recently performed detailed genotypic and phenotypic analyses of replication-competent viruses isolated from ES and showed that these viruses were fully replication competent (6) Although nef is not required for viral replication in vitro, it has been strongly associated with pathogenesis in vivo (reviewed in reference 14). It is thus possible that some ES isolates are replication competent but have mutations in nef that result in diminished pathogenesis.nef has been shown to be involved in the downregulation of both CD4 (15) and MHC-I (41). Several studies have shown that nef-induced MHC-I downregulation has a major impact on CTL function. In a seminal study, a dramatic reduction in HLA-A*2 expression by CD4⁺ T cells infected with wild-type virus but not by those infected with a virus carrying a defective nef gene was demonstrated. This downregulation resulted in diminished killing of HIV-1-infected cells by CTL clones specific for an HLA-A*2-restricted HIV-1 Gag epitope (10). Similarly, nef-mediated MHC-I downregulation was shown to impair the ability of HIV-1-specific CTL clones to suppress viral replication (42, 44). While these findings strongly suggest that HIV-1 partially evades the immune response by inducing MHC-I downregulation, other studies have demonstrated that primary CD8⁺ T cells from some ES and CP could effectively respond to autologous viral replication in autologous CD4⁺ T cells (26).We tested the hypothesis that ES are infected with HIV-1 isolates that are less capable of downregulating MHC-I molecules. This could potentially cause the isolates to be more susceptible to CD8⁺ T-cell suppression of replication and may explain the superior CD8⁺ T-cell responses reported in prior ES studies (2, 5, 12, 27, 28, 37, 47). To date, fully characterized replication-competent isolates have been reported from just six ES subjects (1, 3, 6). We compared the MHC-I downregulation capacity of isolates from five of these ES to that of isolates obtained from resting CD4⁺ T cells of eight patients with progressive disease (viral load, >10,000 copies/ml). In order to develop a physiological model for HIV-1-induced MHC-I downregulation, we enriched primary CD4⁺ T cells from peripheral blood mononuclear cells (PBMC) from donors who were HLA-A*2 and/or HLA-B*57 positive by CD8⁺ T cell depletion with magnetic beads (Dynal), followed by activation in vitro with phytohemagglutinin for 3 days. For evaluation of HLA-A*2 downregulation, CD4⁺ T cells were obtained from HIV-seronegative donors. CD4⁺ T cells from ES were used for the evaluation of HLA-B*57 downregulation. This allele was as effectively downregulated in these ES as it was in multiple HLA-B*57 CP (data not shown). Following activation, the cells were infected with primary HIV-1 isolates from ES or CP by spinoculation (33). The primary isolates were obtained as previously described from latently infected CD4⁺ T cells (9). The median peak viral load and CD4⁺ T-cell nadir of the CP from whom viral isolates were obtained was 81,000 copies/ml and 279 cells/μl, respectively, and thus these isolates should be effective at HLA downregulation (22).At different time points, the cells were harvested and stained with either fluorescein isothiocyanate (FITC)-conjugated anti-HLA-A*2 (Becton Dickinson) and tricolor-conjugated anti-CD4 antibodies (Caltag) or biotinylated anti-HLA-B*57 antibody (One Lambda) followed by FITC-conjugated streptavidin, peridinin chlorophyll protein-Cy5.5-conjugated anti-CD4 antibody (Becton Dickenson), and allophycocyanin-conjugated anti-CD3 antibody. The cells were fixed and permeabilized with Cytofix/Cytoperm solution (Becton Dickenson). Intracellular staining was then performed with the phycoerythrin-conjugated Gag-specific monoclonal antibody Kc57 or an immunoglobulin G1 mouse isotype control (Beckman Coulter). A total of 100,000 to 500,000 events were analyzed for each sample. HLA typing of ES was performed as previously described (4). The HLA-specific antibodies were tested on cells from a panel of ES with known HLA types to confirm specificity.MHC-I downregulation was measured by comparing the mean fluorescence intensities (MFI) of HLA-A*2 and HLA-B*57 on HIV-1-infected versus noninfected CD4⁺ T cells. Infected cells were defined as cells that stained positive for intracellular Gag and had downregulated CD4 (Fig. ​(Fig.1).1). Uninfected CD4⁺ T cells were defined as cells that expressed high levels of CD4 and were negative for intracellular Gag protein. In order to standardize values, we determined relative MFI by dividing the MFI of the infected population by that of the CD4-positive, uninfected population. The Wilcoxon Mann-Whitney test was used to analyze the data.Open in a separate windowFIG. 1.Analysis of HLA-B*57 downregulation on HIV-1-infected cells. (A) CD8⁺ T-cell-depleted PBMC were stained with anti-HLA-B*57 and anti-CD4 monoclonal antibodies 3 days after infection with primary isolates from an ES (ES8) or a CP (CP2). Cells in quadrant 1 are uninfected CD4⁺ T cells, and cells in quadrant 4 (Gag-positive, low levels of CD4) are infected cells that have downregulated CD4. (PE, phycoerythrin; IgG, immunoglobulin G.) (B) The MFI of HLA-B*57 were compared for uninfected (quadrant 1) and infected (quadrant 4) cells from each sample.To determine if there was a difference in the ability of HIV-1 isolates cultured from ES versus CP to downregulate HLA-A*2, we measured the MFI of this molecule on infected CD4⁺ T cells that had downregulated CD4. On average, primary CD4⁺ T cells infected by ES viruses had levels of MHC-I downregulation of about two- to threefold, with relative MFI of 0.51, 0.37, and 0.30 on days 2, 3, and 4, respectively. Similarly, cells infected by isolates cultured from CP had relative MFI of 0.46, 0.36, and 0.33 on days 2, 3, and 4, respectively (Fig. ​(Fig.2B).2B). These differences were not significantly different at any time point.Open in a separate windowFIG. 2.(A) Relative MFIs of HLA-A*02 on cells infected with isolates from five ES (triangles) and eight CP (squares) on days 2 to 4 postinfection. The relative MFI is defined as the MFI of the infected cells divided by the MFI of the uninfected CD4⁺ T cells in each sample. The horizontal bars represent the median for each group. (B) Average relative MFI of HLA-A*02 for cells infected with isolates from ES and CP on each day. (C) Average relative MFI of HLA-A*02 for cells infected with the wild-type NL4-3-green fluorescent protein virus (diamonds) or the Nef⁻ Vpr⁻ mutant virus (circles).In order to rule out nonspecific downregulation of MHC-I on infected cells, we determined the MFI of HLA-DR and CD45 RO on cells infected with isolates from two subjects. The average relative MFI of the two proteins were 1.28 and 1.48, respectively, indicating that the MHC-I was in fact specifically downregulated. Since mutations in Nef have been shown to abrograte HLA downregulation, we also compared HLA-A2 downregulation by the HIV-1-based reporter construct NL4-3-green fluorescent protein and a Nef⁻ Vpr⁻ mutant vector (45, 46). As shown in Fig. ​Fig.2C,2C, no downregulation of HLA-A2 was seen at any point after infection with the Nef⁻ Vpr⁻ mutant virus, whereas infection with wild-type virus caused a degree of downregulation that was similar to that seen with primary isolates from ES and CP. Finally, we also looked at CD3 downregulation, as this molecule has been shown to be downregulated by Nef from HIV-2 and many simian immunodeficiency virus (SIV) isolates but not from HIV-1 (39). Furthermore, since SIVsmm nef isolated from sooty mangabeys with preserved CD4⁺ T-cell counts causes significantly more downregulation than SIVsmm nef from sooty mangabeys with CD4⁺ T-cell depletion (40), we determined whether isolates from ES also selectively downregulated this molecule. As shown in Fig. ​Fig.3A,3A, there was no significant downmodulation of CD3 after infection of cells with isolates from ES or CP.Open in a separate windowFIG. 3.(A) Relative MFI of CD3 on cells infected with isolates from five ES (triangles) and five CP (squares) on day 3 postinfection. The horizontal bars represent the median for each group. (B) Relative MFI of HLA-B*57 on cells infected with isolates from ES and CP.Epidemiologic studies have suggested that HLA-B alleles play a larger role than HLA-A alleles in determining the outcome of infection (17). Furthermore, while HLA-B*57 is the most overrepresented allele seen in ES, there have not been any studies looking at downregulation of this MHC-I protein. Activated CD4⁺ T cells from an HLA-B*5703-positive ES were infected with isolates from five ES and five CP, and the degree of HLA-B*57 downregulation was measured on day 3. As shown in Fig. ​Fig.3B,3B, the average relative MFI of cells infected with isolates from five ES was 0.53, which was not significantly different from the average relative MFI of 0.64 that was seen in cells infected with isolates from five progressors.While it appeared that there was generally more downregulation of HLA-A*2 than HLA-B*57, the studies were performed in cells from different donors, and this precluded a direct comparison of the MFI of the two MHC-I alleles. Two ES in our cohort were positive for both HLA alleles, and the degrees of downregulation of these proteins could thus be compared. CD4⁺ T cells from ES8 were infected with autologous virus (6), and cells from ES9 were infected with a primary isolate from the CP who transmitted virus to her (3). For patient ES8, HLA-A2 showed a greater degree of downregulation than HLA-B57 at day 3 (a relative MFI of 0.36 versus 0.62) (Fig. ​(Fig.4).4). In contrast, in ES9 the degrees of downregulation of the two proteins were nearly identical (a relative MFI of 0.35 for HLA-A2 versus 0.31 for HLA-B*57).Open in a separate windowFIG. 4.Comparison of the relative MFI of HLA-A*02 and HLA-B*57 on CD8⁺ T-cell-depleted PBMC from ES8 and ES9 that were infected with autologous virus (ES8) or with the primary isolate from the CP who transmitted the virus to ES9. The MFI of HLA-A*2 or HLA-B*57 on uninfected CD4⁺ T cells (top panels) and infected cells that had downregulated CD4 (bottom panels) are shown.This is the first study to look at downregulation of MHC-I proteins on CD4⁺ T cells infected with HIV-1 isolates cultured from ES CD4⁺ T cells. We used a physiological model where primary CD4⁺ T cells were infected with primary HIV-1 isolates. One advantage of this approach is that it accounts for HLA downregulation mediated by viral proteins such as Tat (24), as well as Nef. Similar amounts of MHC-I downregulation were seen for cells infected with replication-competent isolates cultured from ES and progressors. These results demonstrate that most ES are not infected by HIV-1 virions that are deficient in downregulating MHC-I compared to those of CP. Thus, it is likely that other factors enable ES to control viremia. The identification of these factors will have implications for the design of HIV-1 vaccines.     

Role of Natural Killer Cells in a Cohort of Elite Suppressors: Low Frequency of the Protective KIR3DS1 Allele and Limited Inhibition of Human Immunodeficiency Virus Type 1 Replication In Vitro

Natural killer (NK) cells are associated with the innate immune response and are important in many viral infections. Recent studies indicate that NK cells can control human immunodeficiency virus type 1 (HIV-1) replication. We studied the effect of NK cells on HIV-1 replication in a subpopulation of HIV-1-infected individuals termed elite suppressors (ES) or elite controllers. These patients maintain a clinically undetectable viral load without treatment and thus provide a fascinating cohort in which to study the immunological response to HIV-1. Using an autologous system, we analyzed the effects of NK cells and CD8⁺ T cells on viral replication in CD4⁺ T lymphoblasts. Although we had postulated that NK cells of ES would be highly effective at controlling viral replication, we found that NK cells from some, but not all, ES were capable of inhibiting replication in the presence of interleukin-2, and the inhibition was less robust than that mediated by CD8⁺ T cells. Additionally, we examined whether particular alleles of the KIR receptors, specifically KIR3DS1 and KIR3DL1, or allele-ligand combinations correlated with the control of HIV-1 replication by NK cells and whether any specific KIR alleles were overrepresented in ES. Our ES cohort did not differ from the general population with respect to the frequency of individual KIR. However, of the eight ES studied, the four exhibiting the most NK cell-mediated control of viral replication also had the fewest activating KIR and were haplotype A. Thus, the strong NK cell-mediated inhibition of viral replication is not necessary for the immunological control of HIV-1 in all ES.A small subset of untreated, human immunodeficiency virus type 1 (HIV-1)-infected individuals referred to as elite suppressors (ES) control viremia to levels that are undetectable by ultrasensitive commercial assays while maintaining high CD4⁺ T-cell counts (13, 37). While defective virus has been shown to account for the control of virus in some patients, examining multiple host factors in ES with replication-competent virus (9) already has provided critical information on the immune response to HIV-1 and may yield important insights into future therapies and vaccine development.Research on ES suggests that CD8⁺ T cells play a crucial role in an effective response to HIV-1. CD8⁺ T cells from ES are capable of controlling viral replication in autologous CD4⁺ T cells significantly better than CD8⁺ T cells from progressors (36), and only the former proliferate (29) and secrete multiple cytokines (8) in response to HIV-1 antigens. Furthermore, certain class I HLA alleles, such as HLA-B*27 and HLA-B*57, which appear to be important in the cytotoxic T-lymphocyte (CTL) response, are overrepresented in ES (15, 19, 21, 30, 32). A second, less well studied cytotoxic cell also may play a role in the control of HIV-1. Natural killer (NK) cells are part of the innate immune system and are an important component of the host response to many viral infections. They act on target cells via cytokine release and cytolysis in response to the integration of signals from inhibitory and activating receptors.The striking propensity of HIV-1 to evolve rapidly in response to immunologic or pharmacologic pressure suggests that the virus has the capability to evade the NK cell response, and indeed selection for evasive measures seems to have occurred. The virus-induced downregulation of HLA-A and -B molecules on infected cells provides some protection against the CTL response; at the same time, however, HLA-C molecules are not downregulated upon infection (12). NK cell interaction with HLA-C can inhibit NK cytotoxic effects, and thus the retention of HLA-C on infected cells can provide some protection against the NK cell response. Additionally, a variety of alterations in NK cell function have been observed during HIV-1 infection. NK cells of patients with chronic HIV-1 have altered phenotypes and effector capabilities: NK cells from viremic patients have an increased expression of inhibitory receptors, and there is an expansion of the defective CD56⁻ NK cells compared to the levels in patients on highly active antiretroviral therapy or in ES (7, 27). These changes may be due to alterations in the cytokine environment during infection, which can affect the activation of the NK cells (39); they also may be due to direct interactions between HIV-1 gene products and the NK cells (20). Although the precise cause is unknown, the result is the development of defective NK cells that express an altered receptor and NK cell marker phenotype.Studies specifically examining a role for NK cells in the response to HIV-1 have yielded conflicting results. During acute HIV-1 infection, the NK cell population is activated and expands, particularly the cytotoxic CD56^dim population (2, 3). This activation declines in the chronic phase, and at least one study suggests that the drop in the viral load (VL) of patients during acute infection occurs before the CD8⁺ T-cell response is fully activated; this could be attributed to the effect of NK cells (2). At the same time, the study of exposed, uninfected individuals shows a correlation between resistance to acquiring HIV-1 infection and NK cell activation levels, cytokine release, and cytotoxicity to NK cell-sensitive cell lines (33, 38). Additionally, a recent whole-genome association study identified three single-nucleotide polymorphisms that appear to be important for the host control of HIV-1 (16). Two of these may have an impact on NK cell function, one that is associated with HLA-B*57 and a second that correlates with higher HLA-C mRNA expression. Taken together, such data suggest that NK cells are important for preventing HIV-1 infection and/or reducing the magnitude of viral replication in acute infection, thereby contributing to the ability of ES to control viremia.In this study, we provide the first characterization of NK cells in patients who naturally control HIV-1 infection. Considering that the effectiveness of CD8⁺ T cells against viral replication is well documented, we directly compared the effect of NK cells to that of CD8⁺ T cells from ES on viral replication to put the effect of NK cells in perspective. We studied the NK cell response by measuring the change in p24 production when autologous effector cell populations were coincubated with infected CD4⁺ lymphoblasts with and without the addition of interleukin-2 (IL-2). Additionally, we examined the killer immunoglobulin-like receptors (KIR) and KIR ligand genotype of ES patients to determine whether any KIR are overrepresented in ES and whether KIR-ligand combinations correlated with the HIV-1 inhibitory activity of the NK cells from specific patients. Previous studies have identified correlations between the expression of certain KIR and progression to AIDS in chronic progressors (25, 26); however, a connection between KIR, KIR ligands, and the control of HIV-1 has yet to be identified in ES. The results of these studies significantly advance the understanding of the nature of NK cells and of their potential role in reducing HIV-1 replication.     

Functional consequences of human immunodeficiency virus escape from an HLA-B*13-restricted CD8+ T-cell epitope in p1 Gag protein

The observed association between HLA-B*13 and control of human immunodeficiency virus type 1 (HIV-1) infection has been linked to the number of Gag-specific HLA-B*13-restricted cytotoxic T-cell (CTL) responses identified. To date, the Gag escape mutations described that result in an in vitro fitness cost to the virus have been located within structural protein p24 only. Here we investigated the hypothesis that CTL escape mutations within other regions of HIV Gag may also reduce viral fitness and contribute to immune control. We analyzed an HLA-B*13-restricted CTL response toward an epitope in p1 Gag, RQANFLGKI_429-437 (RI9), where amino acid variation at Gag residues 436 and 437 is associated with HLA-B*13 expression. In this work, we assessed the impact of amino acid substitutions at these positions on CTL recognition and on HIV-1 fitness. We demonstrated that substitutions I437L and I437M largely abrogate CTL recognition and reduce viral fitness while variants K436R and I437V have only a marginal effect on recognition and fitness. Examination of the patterns of protein synthesis indicated that the loss of fitness in the I437L and I437M mutants is associated with the accumulation of unprocessed Gag precursors. A significant reduction in ribosomal frameshifting efficiency was observed with I437M, suggesting that this mechanism contributes to the observed reduced fitness of this virus. These studies illustrate the apparent trade-off available to the virus between evasion of CTL recognition in p1 Gag and the functional consequences for viral fitness.     

Naïve and Memory CD4 T Cells Differ in Their Susceptibilities to Human Immunodeficiency Virus Type 1 Infection following CD28 Costimulation: Implications for Transmission and Pathogenesis

In vitro evidence suggests that memory CD4⁺ cells are preferentially infected by human immunodeficiency virus type 1 (HIV-1), yet studies of HIV-1-infected individuals have failed to detect preferential memory cell depletion. To explore this paradox, we stimulated CD45RA⁺ CD4⁺ (naïve) and CD45RO⁺ CD4⁺ (memory) cells with antibodies to CD3 and CD28 and infected them with either CCR5-dependent (R5) or CXCR4-dependent (X4) HIV-1 isolates. Naïve CD4⁺ cells supported less X4 HIV replication than their memory counterparts. However, naïve cells were susceptible to R5 viral infection, while memory cells remained resistant to infection and viral replication. As with the unseparated cells, mixing the naïve and memory cells prior to infection resulted in cells resistant to R5 infection and highly susceptible to X4 infection. While both naïve and memory CD4⁺ subsets downregulated CCR5 expression in response to CD28 costimulation, only the memory cells produced high levels of the β-chemokines RANTES, MIP-1α, and MIP-1β upon stimulation. Neutralization of these β-chemokines rendered memory CD4⁺ cells highly sensitive to infection with R5 HIV-1 isolates, indicating that downregulation of CCR5 is not sufficient to mediate complete protection from CCR5 strains of HIV-1. These results indicate that susceptibility to R5 HIV-1 isolates is determined not only by the level of CCR5 expression but also by the balance of CCR5 expression and β-chemokine production. Furthermore, our results suggest a model of HIV-1 transmission and pathogenesis in which naïve rather than memory CD4⁺ T cells serve as the targets for early rounds of HIV-1 replication.     

Novel cytotoxic T-lymphocyte escape mutation by a three-amino-acid insertion in the human immunodeficiency virus type 1 p6Pol and p6Gag late domain associated with drug resistance

Cytolytic T lymphocytes (CTL) play a major role in controlling human immunodeficiency virus type 1 (HIV-1) infection. To evade immune pressure, HIV-1 is selected at targeted CTL epitopes, which may consequentially alter viral replication fitness. In our longitudinal investigations of the interplay between T-cell immunity and viral evolution following acute HIV-1 infection, we observed in a treatment-naïve patient the emergence of highly avid, gamma interferon-secreting, CD8⁺ CTL recognizing an HLA-Cw*0102-restricted epitope, NSPTRREL (NL8). This epitope lies in the p6^Pol protein, located in the transframe region of the Gag-Pol polyprotein. Over the course of infection, an unusual viral escape mutation arose within the p6^Pol epitope through insertion of a 3-amino-acid repeat, NSPT(SPT)RREL, with a concomitant insertion in the p6^Gag late domain, PTAPP(APP). Interestingly, this p6^Pol insertion mutation is often selected in viruses with the emergence of antiretroviral drug resistance, while the p6^Gag late-domain PTAPP motif binds Tsg101 to permit viral budding. These results are the first to demonstrate viral evasion of immune pressure by amino acid insertions. Moreover, this escape mutation represents a novel mechanism whereby HIV-1 can alter its sequence within both the Gag and Pol proteins with potential functional consequences for viral replication and budding.     

Higher homologous and lower cross-reactive Gag-specific T-cell responses in human immunodeficiency virus type 2 (HIV-2) than in HIV-1 infection

Human immunodeficiency virus type 2 (HIV-2) infection results in slower CD4⁺ T-cell decline, lower plasma viral load levels, and hence slower progression of the disease than does HIV-1 infection. Although the reasons for this are not clear, it is possible that HIV-2 replication is more effectively controlled by host responses. We used aligned pools of overlapping HIV-1 and HIV-2 Gag peptides in an enhanced gamma interferon enzyme-linked immunospot assay to compare the levels of homologous and cross-reactive Gag-specific T-cell responses between HIV-1- and HIV-2-infected patients. HIV-2-infected patients showed broader and stronger homologous Gag-specific T-cell responses than HIV-1-infected patients. In contrast, the cross-reactive T-cell responses in HIV-2-infected patients were both narrower and weaker than those in HIV-1-infected patients, in line with overall weaker correlations between homologous and heterologous T-cell responses among HIV-2-infected patients than among HIV-1-infected patients. Cross-reactive responses in HIV-2-infected patients tended to correlate directly with HIV-1/HIV-2 Gag sequence similarities; this was not found in HIV-1-infected patients. The CD4⁺ T-cell counts of HIV-2-infected patients correlated directly with homologous responses and inversely with cross-reactive responses; this was not found in HIV-1-infected patients. Our data support a model whereby high-level HIV-2-specific T-cell responses control the replication of HIV-2, thus limiting viral diversification and priming of HIV-1 cross-reactive T-cell responses over time. However, we cannot exclude the possibility that HIV-2 replication is controlled by other host factors and that HIV-2-specific T-cell responses are better maintained in the context of slow viral divergence and a less damaged immune system. Understanding the nature of immune control of HIV-2 infection could be crucial for HIV vaccine design.     

A Bipartite Membrane-Binding Signal in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Required for the Proteolytic Processing of Gag Precursors in a Cell Type-Dependent Manner

It is unclear whether proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to block virus assembly and release from the plasma membrane of COS cells but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as virus production. We have now addressed this issue with CD4⁺ T cells, which are natural target cells of HIV-1. In these cells, myristylation of Gag proteins was required for proteolytic processing of Gag proteins and production of extracellular viral particles. This result was not due to a lack of expression of the viral protease in the form of a Gag-Pol precursor or a lack of interaction between unmyristylated Gag and Gag-Pol precursors. The processing defect of unmyristylated Gag was partially rescued ex vivo by coexpression with wild-type myristylated Gag proteins in HeLa cells. The cell type-dependent processing of HIV-1 Gag precursors was also observed when another part of the plasma membrane binding signal, a polybasic region in the matrix protein, was mutated. The processing of unmyristylated Gag precursors was inhibited in COS cells by HIV-1 protease inhibitors. Altogether, our findings demonstrate that the processing of HIV-1 Gag precursors in CD4⁺ T cells occurs normally at the plasma membrane during viral morphogenesis. The intracellular environment of COS cells presumably allows activation of the viral protease and proteolytic processing of HIV-1 Gag proteins in the absence of plasma membrane binding.     

Dendritic cells reveal a broad range of MHC class I epitopes for HIV-1 in persons with suppressed viral load on antiretroviral therapy

Background

HIV-1 remains sequestered during antiretroviral therapy (ART) and can resume high-level replication upon cessation of ART or development of drug resistance. Reactivity of memory CD8⁺ T lymphocytes to HIV-1 could potentially inhibit this residual viral replication, but is largely muted by ART in relation to suppression of viral antigen burden. Dendritic cells (DC) are important for MHC class I processing and presentation of peptide epitopes to memory CD8⁺ T cells, and could potentially be targeted to activate memory CD8⁺ T cells to a broad array of HIV-1 epitopes during ART.

Principal Findings

We show for the first time that HIV-1 peptide-loaded, CD40L-matured DC from HIV-1 infected persons on ART induce IFN gamma production by CD8⁺ T cells specific for a much broader range and magnitude of Gag and Nef epitopes than do peptides without DC. The DC also reveal novel, MHC class I restricted, Gag and Nef epitopes that are able to induce polyfunctional T cells producing various combinations of IFN gamma, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1 beta and the cytotoxic de-granulation molecule CD107a.

Significance

There is an underlying, broad antigenic spectrum of anti-HIV-1, memory CD8⁺ T cell reactivity in persons on ART that is revealed by DC. This supports the use of DC-based immunotherapy for HIV-1 infection.     

Ligand-independent exhaustion of killer immunoglobulin-like receptor-positive CD8+ T cells in human immunodeficiency virus type 1 infection

Virus-specific CD8⁺ T cells play a central role in the control of viral infections, including human immunodeficiency virus type 1 (HIV-1) infection. However, despite the presence of strong and broad HIV-specific CD8⁺ T-cell responses in chronic HIV-1 infection, these cells progressively lose critical effector functions and fail to clear the infection. Mounting evidence suggests that the upregulation of several inhibitory regulatory receptors on the surface of CD8⁺ T cells during HIV-1 infection may contribute directly to the impairment of T-cell function. Here, we investigated the role of killer immunoglobulin receptors (KIR), which are expressed on NK cells and on CD8⁺ T cells, in regulating CD8⁺ T-cell function in HIV-1 infection. KIR expression was progressively upregulated on CD8⁺ T cells during HIV-1 infection and correlated with the level of viral replication. Expression of KIR was associated with a profound inhibition of cytokine secretion, degranulation, proliferation, and activation by CD8⁺ T cells following stimulation with T-cell receptor (TCR)-dependent stimuli. In contrast, KIR⁺ CD8⁺ T cells responded potently to TCR-independent stimulation, demonstrating that these cells are functionally competent. KIR-associated suppression of CD8⁺ T-cell function was independent of ligand engagement, suggesting that these regulatory receptors may constitutively repress TCR activation. This ligand-independent repression of TCR activation of KIR⁺ CD8⁺ T cells may represent a significant barrier to therapeutic interventions aimed at improving the quality of the HIV-specific CD8⁺ T-cell response in infected individuals.     

Dendritic cells are less susceptible to human immunodeficiency virus type 2 (HIV-2) infection than to HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) has been documented in vivo and may be an important contributor to HIV-1 transmission and pathogenesis. HIV-1-specific CD4⁺ T cells respond to HIV antigens presented by HIV-1-infected DCs and in this process become infected, thereby providing a mechanism through which HIV-1-specific CD4⁺ T cells could become preferentially infected in vivo. HIV-2 disease is attenuated with respect to HIV-1 disease, and host immune responses are thought to be contributory. Here we investigated the susceptibility of primary myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to infection by HIV-2. We found that neither CCR5-tropic primary HIV-2 isolates nor a lab-adapted CXCR4-tropic HIV-2 strain could efficiently infect mDCs or pDCs, though these viruses could infect primary CD4⁺ T cells in vitro. HIV-2-exposed mDCs were also incapable of transferring virus to autologous CD4⁺ T cells. Despite this, we found that HIV-2-specific CD4⁺ T cells contained more viral DNA than memory CD4⁺ T cells of other specificities in vivo. These data suggest that either infection of DCs is not an important contributor to infection of HIV-2-specific CD4⁺ T cells in vivo or that infection of DCs by HIV-2 occurs at a level that is undetectable in vitro. The frequent carriage of HIV-2 DNA within HIV-2-specific CD4⁺ T cells, however, does not appear to be incompatible with preserved numbers and functionality of HIV-2-specific CD4⁺ T cells in vivo, suggesting that additional mechanisms contribute to maintenance of HIV-2-specific CD4⁺ T-cell help in vivo.     

Role of human immunodeficiency virus (HIV)-specific T-cell immunity in control of dual HIV-1 and HIV-2 infection

Progressive immune dysfunction and AIDS develop in most cases of human immunodeficiency virus type 1 (HIV-1) infection but in only 25 to 30% of persons with HIV-2 infection. However, the natural history and immunologic responses of individuals with dual HIV-1 and HIV-2 infection are largely undefined. Based on our previous findings, we hypothesized that among patients with dual infection the control of HIV-1 is associated with the ability to respond to HIV-2 Gag epitopes and to maintain HIV-specific CD4⁺ T-cell responses. To test this, we compared the HIV-specific ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay responses of 19 dually infected individuals to those of persons infected with HIV-1 or HIV-2 only. Further, we assessed the functional profile of HIV Gag-specific CD4⁺ and CD8⁺ T cells from nine HIV dually infected patients by using a multicolor intracellular cytokine staining assay. As determined by ELISPOT assay, the magnitude and frequency of IFN-γ-secreting T-cell responses to gene products of HIV-1 were higher than those to gene products of HIV-2 (2.64 versus 1.53 log₁₀ IFN-γ spot-forming cells/10⁶ cells [90% versus 63%, respectively].) Further, HIV-1 Env-, Gag-, and Nef- and HIV-2 Gag-specific responses were common; HIV-2 Nef-specific responses were rare. HIV-specific CD4⁺ T helper responses were detected in nine of nine dually infected subjects, with the majority of these T cells producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and, to a lesser extent, interleukin-2. The HIV-1 plasma viral load was inversely correlated with HIV-2 Gag-specific IFN-γ-/TNF-α-secreting CD4⁺ and HIV-2 Gag-specific IFN-γ-secreting CD8⁺ T cells. In conclusion, the T-cell memory responses associated with containment of single HIV-1 and HIV-2 infection play a similar significant role in the immune control of dual HIV-1 and HIV-2 infection.     

Preservation of FoxP3+ regulatory T cells in the peripheral blood of human immunodeficiency virus type 1-infected elite suppressors correlates with low CD4+ T-cell activation

Elite suppressors (ES) are untreated human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain normal CD4⁺ T-cell counts and control viremia to levels that are below the limit of detection of current assays. The mechanisms involved in long-term control of viremia have not been fully elucidated. CD4⁺ CD25⁺ regulatory T cells (Tregs) downmodulate chronic inflammation by suppressing the activation and proliferation of effector lymphocytes. We found that while Tregs were functional in ES and patients on highly active antiretroviral therapy (HAART), ES maintained high levels of Tregs in peripheral blood mononuclear cells whereas patients on HAART had evidence of Treg depletion. We also demonstrated that Tregs can serve as reservoirs for HIV-1 in vivo. These data suggest that both direct infection by HIV-1 and tissue redistribution are possible explanations for declining FoxP3⁺ Tregs in progressive HIV-1 infection. Furthermore, the maintenance of Tregs may be one mechanism associated with the nonprogressive nature of HIV-1 infection in ES.     

Regulatory T Cell Suppression of Gag-Specific CD8+ T Cell Polyfunctional Response After Therapeutic Vaccination of HIV-1-Infected Patients on ART

We tested the hypothesis that therapeutic vaccination against HIV-1 can increase the frequency and suppressive function of regulatory, CD4⁺ T cells (Treg), thereby masking enhancement of HIV-1-specific CD8⁺ T cell response. HIV-1-infected subjects on antiretroviral therapy (N = 17) enrolled in a phase I therapeutic vaccine trial received 2 doses of autologous dendritic cells (DC) loaded with HIV-1 peptides. The frequency of CD4⁺CD25^hiFOXP3⁺ Treg in blood was determined prior to and after vaccination in subjects and normal controls. Polyfunctional CD8⁺ T cell responses were determined pre- and post-vaccine (N = 7) for 5 immune mediators after in vitro stimulation with Gag peptide, staphylococcal enterotoxin B (SEB), or medium alone. Total vaccine response (post-vaccine–pre-vaccine) was compared in the Treg(+) and Treg-depleted (Treg-) sets. After vaccination, 12/17 subjects showed a trend of increased Treg frequency (P = 0.06) from 0.74% to 1.2%. The increased frequency did not correlate with CD8⁺ T cell vaccine response by enzyme linked immunosorbent assay for interferon γ production. Although there was no significant change in CD8⁺ T cell polyfunctional response after vaccination, Treg depletion increased the polyfunctionality of the total vaccine response (P = 0.029), with a >2-fold increase in the percentage of CD8⁺ T cells producing multiple immune mediators. In contrast, depletion of Treg did not enhance polyfunctional T cell response to SEB, implying specificity of suppression to HIV-1 Gag. Therapeutic immunization with a DC-based vaccine against HIV-1 caused a modest increase in Treg frequency and a significant increase in HIV-1-specific, Treg suppressive function. The Treg suppressive effect masked an increase in the vaccine-induced anti-HIV-1-specific polyfunctional response. The role of Treg should be considered in immunotherapeutic trials of HIV-1 infection.     

Gag-Protease-Mediated Replication Capacity in HIV-1 Subtype C Chronic Infection: Associations with HLA Type and Clinical Parameters

The mechanisms underlying HIV-1 control by protective HLA class I alleles are not fully understood and could involve selection of escape mutations in functionally important Gag epitopes resulting in fitness costs. This study was undertaken to investigate, at the population level, the impact of HLA-mediated immune pressure in Gag on viral fitness and its influence on HIV-1 pathogenesis. Replication capacities of 406 recombinant viruses encoding plasma-derived Gag-protease from patients chronically infected with HIV-1 subtype C were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. Viral replication capacities varied significantly with respect to the specific HLA-B alleles expressed by the patient, and protective HLA-B alleles, most notably HLA-B*81, were associated with lower replication capacities. HLA-associated mutations at low-entropy sites, especially the HLA-B*81-associated 186S mutation in the TL9 epitope, were associated with lower replication capacities. Most mutations linked to alterations in replication capacity in the conserved p24 region decreased replication capacity, while most in the highly variable p17 region increased replication capacity. Replication capacity also correlated positively with baseline viral load and negatively with baseline CD4 count but did not correlate with the subsequent rate of CD4 decline. In conclusion, there is evidence that protective HLA alleles, in particular HLA-B*81, significantly influence Gag-protease function by driving sequence changes in Gag and that conserved regions of Gag should be included in a vaccine aiming to drive HIV-1 toward a less fit state. However, the long-term clinical benefit of immune-driven fitness costs is uncertain given the lack of correlation with longitudinal markers of disease progression.There is broad heterogeneity in the ability of HIV-infected individuals to control virus replication, ranging from elite controllers, who maintain undetectable viral loads without treatment, to rapid progressors, who progress to AIDS within 2 years of infection (9, 22, 32). Many interrelated factors, including host and viral genetic factors involved in antiviral immunity and the viral life cycle, may partially account for the differences in the course of disease progression (10, 11, 30, 41). The complex interplay between host genetic factors and viral factors is exemplified by human leukocyte antigen (HLA) class I-restricted cytotoxic T-lymphocyte (CTL) responses, which exert considerable immune pressure on the virus, resulting in escape mutations that affect the interaction of viral and host proteins, thereby influencing infection outcome.The exact mechanisms by which some HLA class I alleles, such as HLA-B*57 and HLA-B*27, are associated with slower progression to AIDS, while others, such as B*5802 and B*18, are associated with accelerated disease progression (6, 20, 42), are unclear. The magnitude and/or breadth of HLA-restricted CTL responses to the conserved Gag protein has been correlated inversely with disease progression or markers of disease progression in several studies (12, 21, 28, 31, 35, 43, 46), although there are some exceptions (4, 16, 37), while preferential targeting of the highly variable envelope protein (as occurs in HLA-B*5802-positive individuals) correlates with higher viral loads (21, 29). Protective HLA alleles restrict CTL responses that impose a strong selection pressure on a few specific Gag p24 epitopes, resulting in escape mutations (14) for which fitness costs have been demonstrated either through site-directed mutations introduced into a reference strain background (2, 8, 25, 38) or through in vivo reversion of these mutations after transmission to an HLA-mismatched individual (8, 24). Recent evidence suggests that Gag escape mutations with a fitness cost, particularly those in p24, are a significant determinant of disease progression: the transmitted number of HLA-B-associated polymorphisms in Gag was found to significantly impact the viral set point in recipients (although an associated fitness cost was not shown) (7, 15), and in a small number of infants, decreased fitness of the transmitted virus with HLA-B*5703/5801-selected mutations in Gag p24 epitopes resulted in slower disease progression (33, 39). Also, the number of reverting Gag mutations (thought to revert as a consequence of fitness costs) associated with individual HLA-B alleles was strongly correlated with the HLA-linked viral set point in chronically infected patients (26). A recent in vitro study showed that HLA-associated variation in Gag-protease, with resulting reduced replication capacity, may contribute to viral control in HIV-1 subtype B-infected elite controllers (27). Taken together, these studies suggest that CTL responses restricted by favorable HLA alleles select for escape mutations in conserved epitopes, particularly those in Gag, resulting in a fitness cost to HIV and therefore at least partly explaining the slower disease progression in individuals carrying these alleles.To date, many of the studies investigating the fitness cost of Gag escape mutations and their clinical relevance have concentrated on escape mutations associated with protective HLA alleles, have not assessed fitness consequences in the natural sequence background (in the presence of other escape and compensatory mutations), and/or have focused on a limited number of patients. Most importantly, the majority of studies have focused on HIV-1 subtype B. The present study is the first to use a large population-based approach and clinically derived Gag-protease sequences to investigate comprehensively the relationships between immune-driven sequence variation in Gag, viral replication capacity, and markers of disease progression in chronic infection with HIV-1 subtype C, the most predominant subtype in the epidemic. We assayed the replication capacity of recombinant viruses encoding patient Gag-protease in an HIV-1-inducible green fluorescent protein (GFP) reporter cell line and found associations between lower replication capacities, protective HLA alleles, protective HLA-associated mutations, lower baseline viral loads, and higher baseline CD4 counts. However, Gag-protease replication capacity did not correlate with the subsequent rate of CD4 decline.     

Preferential infection of dendritic cells during human immunodeficiency virus type 1 infection of blood leukocytes

Human immunodeficiency virus type 1 (HIV-1) transmission by the parenteral route is similar to mucosal transmission in the predominance of virus using the CCR5 coreceptor (R5 virus), but it is unclear whether blood dendritic cells (DCs), monocytes, or T cells are the cells initially infected. We used ex vivo HIV-1 infection of sorted blood mononuclear cells to model the in vivo infection of blood leukocytes. Using quantitative real-time PCR to detect full-length HIV-1 DNA, both sorted CD11c⁺ myeloid and CD11c⁻ plasmacytoid DCs were more frequently infected than other blood mononuclear cells, including CD16⁺ or CD14⁺ monocytes or resting CD4⁺ T cells. There was a strong correlation between CCR5 coreceptor use and preferential DC infection across a range of HIV-1 isolates. After infection of unsorted blood mononuclear cells, HIV-1 was initially detected in the CD11c⁺ DCs and later in other leukocytes, including clustering DCs and activated T cells. DC infection with R5 virus was productive, as shown by efficient transmission to CD4⁺ T cells in coculture. Blood DCs infected with HIV-1 in vitro and cultured alone expressed only low levels of multiply spliced HIV-1 RNA unless cocultured with CD4⁺ T cells. Early selective infection of immature blood DCs by R5 virus and upregulation of viral expression during DC-T-cell interaction and transmission provide a potential pathway for R5 selection following parenteral transmission.     

Preferential CTL targeting of Gag is associated with relative viral control in long-term surviving HIV-1 infected former plasma donors from China

It is generally believed that CD8⁺ cytotoxic T lymphocytes (CTLs) play a critical role in limiting the replication of human immunodeficiency virus type 1 (HIV-1) and in determining the outcome of the infection, and this effect may partly depend on which HIV product is preferentially targeted. To address the correlation between HIV-1-specific CTL responses and virus replication in a cohort of former plasma donors (FPDs), 143 antiretroviral therapy naive FPDs infected with HIV-1 clade B'' strains were assessed for HIV-1-specific CTL responses with an IFN-γ Elispot assay at single peptide level by using overlapping peptides (OLPs) covering the whole consensus clade B proteome. By using a Spearman''s rank correlation analysis, we found that the proportion of Gag-specific CTL responses among the total virus-specific CTL activity was inversely correlated with viral loads while being positively correlated to CD4 counts, as opposed to Pol- and Env-specific responses that were associated with increased viral loads and decreased CD4 counts. In addition, Vpr-specifc CTL responses showed a similar protective effect with Gag responses, but with a much lower frequency of recognition. Significantly, we also observed an association between HLA-A^*30/B^*13/Cw^*06 haplotype and lower viral loads that was probably due to restricted Gag-specific CTL responses. Thus, our data demonstrate the prominent role of Gag-specific CTL responses in disease control. The advantage of HLA-A^*30/B^*13/Cw^*06 haplotype in viral control may be associated with the contribution of Gag-specific CTL responses in the studied individuals.