Comparison of human immunodeficiency virus (HIV)-specific T-cell responses in HIV-1- and HIV-2-infected individuals in Senegal

Human immunodeficiency virus type 2 (HIV-2) infection is typically less virulent than HIV-1 infection, which may permit the host to mount more effective, sustained T-cell immunity. We investigated antiviral gamma interferon-secreting T-cell responses by an ex vivo Elispot assay in 68 HIV-1- and 55 HIV-2-infected Senegalese patients to determine if differences relate to more efficient HIV-2 control. Homologous HIV-specific T cells were detected in similar frequencies (79% versus 76%, P = 0.7) and magnitude (3.12 versus 3.08 log(10) spot-forming cells/10(6) peripheral blood mononuclear cells) in HIV-1 and HIV-2 infection, respectively. Gag-specific responses predominated in both groups (>/=64%), and significantly higher Nef-specific responses occurred in HIV-1-infected (54%) than HIV-2-infected patients (22%) (P < 0.001). Heterologous responses were more frequent in HIV-1 than in HIV-2 infection (46% versus 27%, P = 0.04), but the mean magnitude was similar. Total frequencies of HIV-specific responses in both groups did not correlate with plasma viral load and CD4(+) T-cell count in multivariate regression analyses. However, the magnitude of HIV-2 Gag-specific responses was significantly associated with lower plasma viremia in HIV-1-infected patients (P = 0.04). CD4(+) T-helper responses, primarily recognizing HIV-2 Gag, were detected in 48% of HIV-2-infected compared to only 8% of HIV-1-infected patients. These findings indicate that improved control of HIV-2 infection may relate to the contribution of T-helper cell responses. By contrast, the superior control of HIV-1 replication associated with HIV-2 Gag responses suggests that these may represent cross-reactive, higher-avidity T cells targeting epitopes within Gag regions of functional importance in HIV replication.     

Induction of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses in HIV vaccine trial participants who subsequently acquire HIV-1 infection

Candidate human immunodeficiency virus type 1 (HIV-1) vaccines designed to elicit T-cell immunity in HIV-1-uninfected persons are under investigation in phase I to III clinical trials. Little is known about how these vaccines impact the immunologic response postinfection in persons who break through despite vaccination. Here, we describe the first comprehensive characterization of HIV-specific T-cell immunity in vaccine study participants following breakthrough HIV-1 infection in comparison to 16 nonvaccinated subjects with primary HIV-1 infection. Whereas none of the 16 breakthrough infections possessed vaccine-induced HIV-1-specific T-cell responses preinfection, 85% of vaccinees and 86% of nonvaccinees with primary HIV-1 infection developed HIV-specific T-cell responses postinfection. Breakthrough subjects' T cells recognized 43 unique HIV-1 T-cell epitopes, of which 8 are newly described, and 25% were present in the vaccine. The frequencies of gamma interferon (IFN-gamma)-secreting cells recognizing epitopes within gene products that were and were not encoded by the vaccine were not different (P = 0.64), which suggests that responses were not anamnestic. Epitopes within Nef and Gag proteins were the most commonly recognized in both vaccinated and nonvaccinated infected subjects. One individual controlled viral replication without antiretroviral therapy and, notably, mounted a novel HIV-specific HLA-C14-restricted Gag LYNTVATL-specific T-cell response. Longitudinally, HIV-specific T cells in this individual were able to secrete IFN-gamma and tumor necrosis factor alpha, as well as proliferate and degranulate in response to their cognate antigenic peptides up to 5 years postinfection. In conclusion, a vaccinee's ability to mount an HIV-specific T-cell response postinfection is not compromised by previous immunization, since the CD8+ T-cell responses postinfection are similar to those seen in vaccine-na?ve individuals. Finding an individual who is controlling infection highlights the importance of comprehensive studies of breakthrough infections in vaccine trials to determine whether host genetics/immune responses and/or viral characteristics are responsible for controlling viral replication.     

Consistent patterns in the development and immunodominance of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T-cell responses following acute HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T-cell responses generated during acute infection play a critical role in the initial control of viremia. However, little is known about the viral T-cell epitopes targeted during acute infection or about their hierarchy in appearance and relative immunodominance over time. In this study, HIV-1-specific CD8+ T-cell responses in 18 acutely infected individuals expressing HLA-A3 and/or -B7 were characterized. Detailed analysis of CD8 responses in one such person who underwent treatment of acute infection followed by reexposure to HIV-1 through supervised treatment interruptions (STI) revealed recognition of only two cytotoxic T-lymphocyte (CTL) epitopes during symptomatic acute infection. HIV-1-specific CD8+ T-cell responses broadened significantly during subsequent exposure to the virus, ultimately targeting 27 distinct CTL epitopes, including 15 different CTL epitopes restricted by a single HLA class I allele (HLA-A3). The same few peptides were consistently targeted in an additional 17 persons expressing HLA-A3 and/or -B7 during acute infection. These studies demonstrate a consistent pattern in the development of epitope-specific responses restricted by a single HLA allele during acute HIV-1 infection, as well as persistence of the initial pattern of immunodominance during subsequent STI. In addition, they demonstrate that HIV-1-specific CD8+ T-cell responses can ultimately target a previously unexpected and unprecedented number of epitopes in a single infected individual, even though these are not detectable during the initial exposure to virus. These studies have important implications for vaccine design and evaluation.     

Impact of mucosal inflammation on cervical human immunodeficiency virus (HIV-1)-specific CD8 T-cell responses in the female genital tract during chronic HIV infection

The female genital tract is the major route of heterosexual human immunodeficiency virus (HIV) acquisition and transmission. Here, we investigated whether HIV-specific CD8 T-cell-mediated immune responses could be detected in the genital mucosa of chronically HIV-infected women and whether these were associated with either local mucosal HIV shedding or local immune factors. We found that CD8⁺ T-cell gamma interferon responses to Gag were detectable at the cervix of HIV-infected women but that the magnitude of genital responses did not correlate with those similarly detected in blood. This indicates that ex vivo HIV responses in one compartment may not be predictive of those in the other. We found that increased genital tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) levels correlated significantly with levels of Gag-specific CD8⁺ T cells at the cervix. Women who were detectably shedding virus in the genital tract had significantly increased cervical levels of TNF-α, IL-1β, IL-6, and IL-8 compared to women who were not detectably shedding virus. We were, however, unable to detect any association between the magnitude of cervical HIV-specific responses and mucosal HIV shedding. Our results support the hypothesis that proinflammatory cytokines in the female genital tract may promote HIV replication and shedding. In addition, we further show that inflammatory cytokines are associated with increased levels of HIV-specific CD8 effector cells at the genital mucosa but that these were not able to control genital HIV shedding.     

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.     

Early therapy of vertical human immunodeficiency virus type 1 (HIV-1) infection: control of viral replication and absence of persistent HIV-1-specific immune responses

Studies of potent antiretroviral combination regimens were undertaken in young infants to evaluate the potential for long-term suppression of viral replication and to evaluate the immune consequences of such therapies. Early combination antiretroviral therapy led to a loss of plasma viremia, cultivable virus, and labile extrachromosomal replication intermediates. Despite preservation of immune function, persistent human immunodeficiency type 1 (HIV-1)-specific immune responses were not detected in most infants. The absence of detectable, persisting immune responses in most HIV-1-infected infants treated early contrasts with what is typically seen in adults who are treated early. These results are consistent with the notion that early combination antiretroviral therapy of HIV-1-infected infants allows the long-term suppression of viral replication.     

Selective depletion of high-avidity human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells after early HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells in early infection are associated with the dramatic decline of peak viremia, whereas their antiviral activity in chronic infection is less apparent. The functional properties accounting for the antiviral activity of HIV-1-specific CD8+ T cells during early infection are unclear. Using cytokine secretion and tetramer decay assays, we demonstrated in intraindividual comparisons that the functional avidity of HIV-1-specific CD8+ T cells was consistently higher in early infection than in chronic infection in the presence of high-level viral replication. This change of HIV-1-specific CD8+ T-cell avidity between early and chronic infections was linked to a substantial switch in the clonotypic composition of epitope-specific CD8+ T cells, resulting from the preferential loss of high-avidity CD8+ T-cell clones. In contrast, the maintenance of the initially recruited clonotypic pattern of HIV-1-specific CD8+ T cells was associated with low-level set point HIV-1 viremia. These data suggest that high-avidity HIV-1-specific CD8+ T-cell clones are recruited during early infection but are subsequently lost in the presence of persistent high-level viral replication.     

Comprehensive analysis of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon-secreting CD8+ T cells in primary HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) T cells provide an important defense in controlling HIV-1 replication, particularly following acquisition of infection. To delineate the breadth and potency of these responses in patients upon initial presentation and before treatment, we determined the fine specificities and frequencies of gamma interferon (IFN-gamma)-secreting CD8(+) T cells recognizing all HIV-1 proteins in patients with primary infection. In these subjects, the earliest detected responses were directed predominantly against Nef, Tat, Vpr, and Env. Tat- and Vpr-specific CD8(+) T cells accounted for the greatest frequencies of mean IFN-gamma spot-forming cells (SFC). Nef-specific responses (10 of 21) were more commonly detected. A mean of 2.3 epitopes were recognized with various avidities per subject, and the number increased with the duration of infection (R = 0.47, P = 0.031). The mean frequency of CD8(+) T cells (985 SFC/10(6) peripheral blood mononuclear cells) correlated with the number of epitopes recognized (R = 0.84, P < 0.0001) and the number of HLA-restricting alleles (R = 0.79, P < 0.0001). Neither the total SFC frequencies nor the number of epitopes recognized correlated with the concurrent plasma viral load. Seventeen novel epitopes were identified, four of which were restricted to HLA alleles (A23 and B72) that are common among African descendents. Thus, primary HIV-1 infection induces strong CD8(+)-T-cell immunity whose specificities broaden over time, but their frequencies and breadth do not correlate with HIV-1 containment when examined concurrently. Many novel epitopes, particularly directed to Nef, Tat, and Env, and frequently with unique HLA restrictions, merit further consideration in vaccine design.     

Primary human immunodeficiency virus type 1 (HIV-1) infection during HIV-1 Gag vaccination

Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1 gag DNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences from gag, pol, and nef confirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while the gag DNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016.     

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.     

Transient mobilization of human immunodeficiency virus (HIV)-specific CD4 T-helper cells fails to control virus rebounds during intermittent antiretroviral therapy in chronic HIV type 1 infection

Immune control of human immunodeficiency virus (HIV) is not restored by highly active antiretroviral therapies (HAART) during chronic infection. We examined the capacity of repeated structured therapeutic interruptions (STI) to restore HIV-specific CD4 and CD8 T-cell responses that controlled virus production. Eleven STI (median duration, 7 days; ranges, 4 to 24 days) were performed in three chronically HIV-infected patients with CD4 counts above 400/mm(3) and less than 200 HIV RNA copies/ml after 18 to 21 months of HAART; treatment resumed after 1 week or when virus became detectable. HIV-specific T-cell responses were analyzed by proliferation, gamma interferon (IFN-gamma) production, and enzyme-linked immunospot assays. Seven virus rebounds were observed (median, 4,712 HIV-1 RNA copies/ml) with a median of 7 days during which CD4 and CD8 counts did not significantly change. After treatment resumed, the viral load returned below 200 copies/ml within 3 weeks. Significant CD4 T-cell proliferation and IFN-gamma production against HIV p24 appeared simultaneously with or even before the virus rebounds in all patients. These CD4 responses lasted for less than 3 weeks and disappeared before therapeutic control of the virus had occurred. Increases in the numbers of HIV-specific CD8 T cells were delayed compared to changes in HIV-specific CD4 T-cell responses. No delay or increase in virus doubling time was observed after repeated STI. Iterative reexposure to HIV during short STI in chronically infected patients only transiently mobilized HIV-specific CD4 T1-helper cells, which might be rapidly altered by virus replication. Such kinetics might explain the failure at delaying subsequent virus rebounds and raises concerns about strategies based on STI to restore durable HIV-specific T-cell responses in chronic HIV infection.     

Simian immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV-1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive plasma

To date, only a small number of anti-human immunodeficiency virus type 1 (HIV-1) monoclonal antibodies (MAbs) with relatively broad neutralizing activity have been isolated from infected individuals. Adequate techniques for defining how frequently antibodies of these specificities arise in HIV-infected people have been lacking, although it is generally assumed that such antibodies are rare. In order to create an epitope-specific neutralization assay, we introduced well-characterized HIV-1 epitopes into the heterologous context of simian immunodeficiency virus (SIV). Specifically, epitope recognition sequences for the 2F5, 4E10, and 447-52D anti-HIV-1 neutralizing monoclonal antibodies were introduced into the corresponding regions of SIVmac239 by site-directed mutagenesis. Variants with 2F5 or 4E10 recognition sequences in gp41 retained replication competence and were used for neutralization assays. The parental SIVmac239 and the neutralization-sensitive SIVmac316 were not neutralized by the 2F5 and 4E10 MAbs, nor were they neutralized significantly by any of the 96 HIV-1-positive human plasma samples that were tested. The SIV239-2F5 and SIV239-4E10 variants were specifically neutralized by the 2F5 and 4E10 MAbs, respectively, at concentrations within the range of what has been reported previously for HIV-1 primary isolates (J. M. Binley et al., J. Virol. 78:13232-13252, 2004). The SIV239-2F5 and SIV239-4E10 epitope-engrafted variants were used as biological screens for the presence of neutralizing activity of these specificities. None of the 92 HIV-1-positive human plasma samples that were tested exhibited significant neutralization of SIV239-2F5. One plasma sample exhibited >90% neutralization of SIV239-4E10, but this activity was not competed by a 4E10 target peptide and was not present in concentrated immunoglobulin G (IgG) or IgA fractions. We thus confirm by direct analysis that neutralizing activities of the 2F5 and 4E10 specificities are either rare among HIV-1-positive individuals or, if present, represent only a very small fraction of the total neutralizing activity in any given plasma sample. We further conclude that the structures of gp41 from SIVmac239 and HIV-1 are sufficiently similar such that epitopes engrafted into SIVmac239 can be readily recognized by the cognate anti-HIV-1 monoclonal antibodies.     

Analysis of total human immunodeficiency virus (HIV)-specific CD4(+) and CD8(+) T-cell responses: relationship to viral load in untreated HIV infection

Human immunodeficiency virus (HIV)-specific T-cell responses are thought to play a key role in viral load decline during primary infection and in determining the subsequent viral load set point. The requirements for this effect are unknown, partly because comprehensive analysis of total HIV-specific CD4(+) and CD8(+) T-cell responses to all HIV-encoded epitopes has not been accomplished. To assess these responses, we used cytokine flow cytometry and overlapping peptide pools encompassing all products of the HIV-1 genome to study total HIV-specific T-cell responses in 23 highly active antiretroviral therapy na?ve HIV-infected patients. HIV-specific CD8(+) T-cell responses were detectable in all patients, ranging between 1.6 and 18.4% of total CD8(+) T cells. HIV-specific CD4(+) T-cell responses were present in 21 of 23 patients, although the responses were lower (0.2 to 2.94%). Contrary to previous reports, a positive correlation was identified between the plasma viral load and the total HIV-, Env-, and Nef-specific CD8(+) T-cell frequency. No correlation was found either between viral load and total or Gag-specific CD4(+) T-cell response or between the frequency of HIV-specific CD4(+) and CD8(+) T cells. These results suggest that overall frequencies of HIV-specific T cells are not the sole determinant of immune-mediated protection in HIV-infection.     

Monocytotropic human immunodeficiency virus type 1 (HIV-1) variants detectable in all stages of HIV-1 infection lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture.

We previously demonstrated a correlation between the presence of syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variants showing tropism for cell line H9 and the occurrence of rapid CD4 cell decline and progression to AIDS. In contrast, in stable asymptomatic individuals, we detected only isolates with low replication rates that were non-syncytium-inducing (NSI) and nontropic for the H9 cell line. Here, we investigated the monocytotropism of established HIV-1 isolates with a panel of isolates and with biological HIV-1 clones with distinct phenotypes. Moreover, the prevalence and biological phenotypes of monocytotropic HIV-1 variants in the course of HIV-1 infection were analyzed in comparative primary isolation studies on peripheral blood lymphocytes (PBL) and monocyte-derived macrophages (MDM). In cell-free infection studies with MDM from eight blood donors, 13 of 17 NSI isolates but only 4 of 14 SI isolates were able to infect MDM. NSI isolates also infected significantly more different donors than SI variants (median, 3 of 8 versus 0 of 8). This enhanced monocytotropism of NSI isolates was confirmed in experiments with biological HIV-1 clones with distinct phenotypes recovered from the same donor. To investigate the prevalence and biological phenotypes of monocytotropic variants in different stages of HIV-1 infection, sequential isolates from peripheral blood mononuclear cell samples from nine asymptomatic individuals, five of whom progressed to AIDS and seven of whom had a known time of seroconversion, were recovered by cocultivation with both PBL and MDM. Monocytotropic variants were obtained from 37 of 42 time points. All monocytotropic variants were NSI in PBL culture and non-T-cell-line tropic, even when SI, T-cell-line-tropic HIV-1 variants could be recovered from the same patient sample by cocultivation with PBL. We conclude that monocytotropic HIV-1 variants mostly have an NSI phenotype in PBL and, in contrast to SI variants, are present at all stages of HIV-1 infection. These results suggest an important role for monocytotropic variants in the persistence of HIV-1 infection.     

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.     

Robust NK cell-mediated human immunodeficiency virus (HIV)-specific antibody-dependent responses in HIV-infected subjects

Antibody-dependent cellular cytotoxicity (ADCC) is a potentially effective adaptive immune response to human immunodeficiency virus (HIV) infection. The study of ADCC responses has been hampered by the lack of simple methods to quantify these responses and map effective epitopes. We serendipitously observed that standard intracellular cytokine assays on fresh whole blood from a cohort of 26 HIV-infected subjects identified non-T lymphocytes expressing gamma interferon (IFN-gamma) in response to overlapping linear peptides spanning HIV-1 proteins. The effector cells were CD3(-) CD4(-) CD8(-) CD14(-) CD2(+) CD56(+/-) NK lymphocytes and degranulated granzyme B and perforin in response to antigen stimulation. Serum transfer assays demonstrated that the specific response was mediated by immunoglobulin G. Fresh blood samples from half of the HIV-infected cohort demonstrated robust HIV peptide-specific IFN-gamma expression by NK cells, predominately to Env, Pol, and Vpu HIV-1 proteins. Responses were readily mapped to define minimal epitopes utilizing this assay. Antibody-dependent, HIV-specific NK cell recognition, involving components of both innate and adaptive immune systems, represents a potentially effective immune response to induce by vaccination.     

Efficient in vitro expansion of human immunodeficiency virus (HIV)-specific T-cell responses by gag mRNA-electroporated dendritic cells from treated and untreated HIV type 1-infected individuals

Developing an immunotherapy to keep human immunodeficiency virus type 1 (HIV-1) replication suppressed while discontinuing highly active antiretroviral therapy (HAART) is an important challenge. In the present work, we evaluated in vitro whether dendritic cells (DC) electroporated with gag mRNA can induce HIV-specific responses in T cells from chronically infected subjects. Monocyte-derived DC, from therapy-naïve and HAART-treated HIV-1-seropositive subjects, that were electroporated with consensus codon-optimized HxB2 gag mRNA efficiently expanded T cells, secreting gamma interferon (IFN-γ) and interleukin 2 (IL-2), as well as other cytokines and perforin, upon restimulation with a pool of overlapping Gag peptides. The functional expansion levels after 1 week of stimulation were comparable in T cells from HAART-treated and treatment-naïve patients and involved both CD4⁺ and CD8⁺ T cells, with evidence of bifunctionality in T cells. Epitope mapping of p24 showed that stimulated T cells had a broadened response toward previously nondescribed epitopes. DC, from HAART-treated subjects, that were electroporated with autologous proviral gag mRNA equally efficiently expanded HIV-specific T cells. Regulatory T cells did not prevent the induction of effector T cells in this system, whereas the blocking of PD-L1 slightly increased the induction of T-cell responses. This paper shows that DC, loaded with consensus or autologous gag mRNA, expand HIV-specific T-cell responses in vitro.Studies of immune responses generated in human immunodeficiency virus type 1 (HIV-1)-infected individuals suggest that CD8⁺ T cells play an important role in the defense against the virus. In acute HIV infection, the appearance of HIV-specific CD8⁺ T cells is associated with a decline in viremia (11, 32). More-direct evidence for the role of CD8⁺ T cells in viral control is deduced from studies of simian immune deficiency virus (SIV)-infected rhesus macaques in which the depletion of the CD8⁺ T cells results in an increase of the viral load and rapid disease progression (41, 55), although this is not always the case (35). Among HIV-infected humans, long-term nonprogressors (LTNP) with an undetectable viral load have higher levels of multifunctional HIV-specific CD8⁺ T cells in comparison to patients with rapidly progressive disease (53). Conversely, the HIV-specific CD8⁺ T cells from rapid progressors release low levels of interleukin 2 (IL-2) and high levels of gamma interferon (IFN-γ), they have a reduced proliferative capacity, and their perforin expression is impaired or exhausted (42, 69). Moreover, during primary and chronic infection, viral escape mutations are often observed as a consequence of immunological pressure mediated by SIV- and HIV-specific CD8⁺ T cells (3, 12, 20, 23, 50). During this process of viral adaptation, all the previous variants are stored as proviral DNA (46).Although current highly active antiretroviral therapy (HAART) may suppress viral replication and protect against disease progression, it is unable to eliminate the proviral latent reservoir. Moreover, as a consequence of low or absent HIV antigenic stimulation, HIV-1-specific cytotoxic T lymphocyte (CTL) responses tend to wane during HAART (16, 39). Therapy interruption invariably results in a viral rebound to pretreatment levels, indicating that no protective immunity has been built up during therapy (38). On the other hand, the partial immune reconstitution, induced by HAART, opens a window of opportunity to boost T-cell immunity by therapeutic vaccination. Clearly, it is not sufficient to enhance the response against the circulating virus. To minimize the risk of escape, it is equally important that immune responses against the entire latent reservoir are activated (49).Dendritic cells (DC) are the most powerful antigen-presenting cells (APC) that can stimulate effective immune responses both in vitro and in vivo (5, 9, 62). In the context of DC-based immunotherapy, many groups have used DC expressing HIV antigens (e.g., pulsed with peptides, transduced with different vectors, or loaded with apoptotic infected cells) to stimulate memory (19, 34, 59, 69) or even primary (13, 14, 33, 63, 66, 67) CD8⁺ T cells in vitro. In vivo, SIV-specific CD8⁺ and CD4⁺ T-cell responses were induced in macaques using DC expressing SIV antigen (63). Finally, Lu and Andrieu and Lu et al. (36, 37) showed that DC pulsed with chemically inactivated autologous virus specifically stimulated HIV-specific immune responses in vitro and in vivo in cells of HIV-1-seropositive individuals.Recently, we (47, 48, 61) and others (9, 15, 22, 28, 40, 54, 57) have shown that transfection with mRNA is more effective than mRNA lipofection, peptide pulsing, or viral transduction to generate primary (65) and memory (57) responses. Furthermore, we demonstrated that DC from treatment-naïve HIV-1-seropositive subjects can efficiently be transfected with HIV gag and env mRNA, derived either from consensus subtype B or autologous viral or proviral HIV, and that these DC readily trigger autologous CD4⁺ and CD8⁺ T cells to release IFN-γ and IL-2 in a short-term ex vivo enzyme-linked immunospot (ELISPOT) assay (60).Our previous study (60) considered only the direct ex vivo immune responses of untreated HIV-1-seropositive persons, who have, by definition, a rather damaged immune system (42). Therefore, with the ultimate aim to develop an immunotherapy based on DC, we decided to evaluate the responses of treatment-naïve and HAART-treated HIV-1-seropositive persons after 1 week of stimulation with electroporated DC. Besides IFN-γ production, other parameters were also evaluated, such as a series of other cytokines, measured in various ways (by ELISPOT, microbead assay, and intracellular cytometry), and the potential influence of regulatory T cells (Treg) on the response. Finally, because HIV escapes very easily from the immune system, we also investigated if it is possible to use autologous proviral gag mRNA and to broaden the immune response.     

Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.     

Random T-cell receptor recruitment in human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells from genetically identical twins infected with the same HIV-1 strain

Human immunodeficiency virus type 1 (HIV-1) cytotoxic T-lymphocyte escape mutations represent both a major reason for loss of HIV immune control and a considerable challenge for HIV-1 vaccine design. Previous data suggest that initial HIV-1-specific CD8⁺ T-cell responses are determined largely by viral and host genetics, but the mechanisms influencing the subsequent viral evolution are unclear. Here, we show a random recruitment of T-cell receptor (TCR) alpha and beta clonotypes of the initial HIV-1-specific CD8⁺ T cells during primary infection in two genetically identical twins infected simultaneously with the same virus, suggesting that stochastic TCR recruitment of HIV-1-specific CD8⁺ T cells contributes to the diverse and unpredictable HIV-1 sequence evolution.