Characterization of HCV-specific Patr class II restricted CD4+ T cell responses in an acutely infected chimpanzee

Resolution of hepatitis C virus (HCV) infection is associated with strong and sustained virus-specific CD4+ T cell responses. In this study, we investigated the evolution of functional T cell responses during acute infection of a chimpanzee and the longevity of these lymphocytes in blood and liver after resolution of infection. Viremia increased through the first 3 weeks of infection and then remained stable until the onset of T cell responses at weeks 6 and 8 postinfection. CD4+ T cells targeting nonstructural HCV proteins were detected in proliferation assays by week 6 postinfection, but they failed to produce interferon gamma (IFN-gamma). HCV-specific CD4+ and CD8+ T cells with the ability to produce IFN-gamma appeared at week 8 when a rapid 10-fold reduction in plasma viremia was first observed. This cytokine response persisted through to week 24 when infection apparently resolved. T cell lines targeting 3 CD4+ T cell epitopes and 1 CD8+ T cell epitope were derived from liver and their Patr major histocompatibility complex (MHC) restriction elements were identified. In retrospective studies performed on cryopreserved peripheral blood mononuclear cells (PBMCs) collected at various timepoints after infection, the onset of an IFN-gamma response measured against the class II restricted epitopes correlated with viral clearance. In conclusion, the characterization of the HCV epitopes and MHC class II restriction elements described here will facilitate a detailed comparison of CD4+ T cell function in animals with resolved and persistent infections.     

Induction of CD4(+) T cell-dependent CD8(+) type 1 responses in humans by a malaria DNA vaccine

We assessed immunogenicity of a malaria DNA vaccine administered by needle i.m. or needleless jet injection [i.m. or i.m./intradermally (i.d.)] in 14 volunteers. Antigen-specific IFN-gamma responses were detected by enzyme-linked immunospot (ELISPOT) assays in all subjects to multiple 9- to 23-aa peptides containing class I and/or class II restricted epitopes, and were dependent on both CD8(+) and CD4(+) T cells. Overall, frequency of response was significantly greater after i.m. jet injection. CD8(+)-dependent cytotoxic T lymphocytes (CTL) were detected in 8/14 volunteers. Demonstration in humans of elicitation of the class I restricted IFN-gamma responses we believe necessary for protection against the liver stage of malaria parasites brings us closer to an effective malaria vaccine.     

A comparison of standard immunogenicity assays for monitoring HIV type 1 gag-specific T cell responses in Ad5 HIV Type 1 gag vaccinated human subjects

Currently, there are numerous candidate HIV vaccines aimed at inducing T-cell mediated immune responses against HIV. To assess the immunogenicity of such vaccines, a reliable T cell assay must be utilized and typically one of the following assays is chosen for this purpose: bulk culture CTL, MHC I tetramer staining, IFN-gamma ELISPOT, or IFN-gamma intracellular cytokine staining. In this paper we report a comparison of the T cell responses detected by each assay in a large cohort of healthy normal volunteers vaccinated with adenovirus serotype 5 expressing HIV gag. Using stringently validated formats of each of these assays and pools of overlapping HIV gag peptides, we demonstrate that there is a high degree of correlation between all four of the common T cell assays, but inherent differences in the sensitivity of each assay to detect responders. In this study, the ELISPOT assay is shown to have the greatest sensitivity in detecting vaccine responses, while the ICS assay, although less sensitive, has the advantage of providing additional information on the phenotype of the responding cells.     

HIV subtypes induce distinct profiles of HIV-specific CD8(+) T cell responses

CD8(+) T cells play an important role in controlling HIV infection and qualitative differences in HIV-specific CD8(+) responses may determine the degree of immune control. We studied 56 HIV-infected, ARV-naive Ugandans and examined the role of subtypes in modulating their HIV-specific T cell responses. Gag-specific responses were readily detectable in our study population. Interestingly, we found significantly decreased Gag-specific cytolysis (as measured by CD107 expression) in subtype D (n = 21) compared to subtype A (n = 35) HIV infection. Sequence analyses within identified epitopes suggest patterns of conservation that are subtype specific. We conclude that HIV subtypes may promote distinct profiles of T cells responses and immune control.     

Association between HIV Type 1-specific T cell responses and CD4+ T cell counts or CD4+:CD8+ T cell ratios in HIV Type 1 subtype B infection in China

CD4+ T cell counts and CD4+:CD8+ T cell ratios represent key determinants of HIV disease progression and infectivity. However, the relationship between the HIV-1-specific cytotoxic T lymphocyte (CTL) response and these determinants has not been elucidated for all HIV-1B and HIV-1C proteins. In the present study, virusspecific T cell responses to HIV-1B and HIV-1C proteins were analyzed with interferon gamma (IFN-gamma) enzyme- linked immunospot (ELISpot) assays using synthetic overlapping peptides corresponding to naturally occurring HIV-1B and HIV-1C consensus sequences. For Gag/Gag p24/Gag p17, a correlation between T cell responses and CD4+ T cell count in HIV-1 clade B and clade C was seen: elevated T cell response resulted in higher CD4+ T cell production. A statistically significant correlation between the Pol-specific T cell response and CD4+ T cell counts was also found in HIV-1 subtype C. For all HIV-1B and HIV-1C proteins, a correlation between the HIV-1-specific T cell response and CD4+:CD8+ T cell ratios was found for Tat and Pol proteins. CD4+ T cell counts in patients with Tat and/or Rev T cell response were higher than in patients without Tat and/or Rev T cell response. We suggest that this correlation within HIV-1B and HIV-1C Gag p24/Gag p17 responses makes the Gag p24/Gag p17 region a potential vaccine candidate and that HIV-1-specific CTL epitopes toward Pol are important in controlling HIV-1 infection; we emphasize that future vaccination strategies should include these early antigens, Tat and Rev.     

HIV type 1 vaccine-induced T cell memory and cytotoxic T lymphocyte responses in HIV type 1-uninfected volunteers

T cell memory to human immunodeficiency virus type 1 (HIV-1) antigens and anti-HIV-1 cytotoxic T lymphocyte (CTL) activity were assessed after administration of live canarypox virus (ALVAC) expressing HIV-1 env, gag, and protease (vCP205) vaccine given alone, vCP205 given with SF-2 recombinant gp120 (rgp120) vaccine, and placebos at 0, 1, 3, and 6 months. Healthy, HIV-1-uninfected subjects reporting high-risk and low-risk behavior for HIV-1 were enrolled. Anti-HIV-1 Env CD8(+) CTLs (HIV-1(MN) and/or HIV-1 subtype B and C primary isolate sequences) were detected in 12 (60%) and anti-HIV-1 Gag CD8(+) CTLs in 7 (35%) of the 20 vCP205 vaccine recipients tested by CTL assay 3.5 months after the final immunization. Fourteen days after the fourth immunization, lymphocyte proliferation in response to HIV-1 Gag antigen was detected in 14 (48%) of 29 vCP205 vaccine recipients, but secreted cytokine levels to HIV-1 Gag antigen were not above unstimulated levels. Coadministration of SF-2 rgp120 vaccine with vCP205 vaccine enhanced lymphocyte proliferation in response to HIV-1 envelope glycoprotein and broadened the envelope-stimulated cytokine secretion pattern, so that it consisted of both Th1 and Th2 cytokines compared with only interferon gamma (IFN-gamma) after vCP205 vaccine given alone. There was a possible association between HIV-1 envelope glycoprotein-stimulated interleukin 2 secretion and CD8(+) CTLs against HIV-1 envelope glycoprotein, and an inverse relation between lymphocyte proliferation and CTLs against HIV-1 Gag antigens. Thus, a durable anti-HIV-1 CD8(+) CTL response was detected after immunization with the live canarypox virus vaccine and preexisting helper T cell memory responses did not necessarily predict later CD8(+) CTL activity.     

Enhancement of HIV type 1 antigen-specific CD4+ T cell memory in subjects with chronic HIV type 1 infection receiving an HIV type 1 immunogen

We examined HIV-1 specific memory helper T immune responses in chronically HIV-infected subjects who received an immune-based therapy (HIV-1 immunogen, Remune). Subjects in this study exhibited significant increases (p < 0.05) in the frequency of helper T memory cells expressing interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in response to HIV-1 antigens in vitro. The frequencies of HIV-specific memory T cells increased after successive immunizations and exhibited a correlation with the standard tritiated thymidine incorporation lymphocyte proliferation assay (r = 0.72, p < 0.0008). These results support the notion that HIV-specific memory immune responses can be stimulated in subjects with chronic HIV infection. Further investigations are warranted to determine whether the induction of such responses is associated with virologic control.     

Enhancement of human immunodeficiency virus type 1-specific CD4 and CD8 T cell responses in chronically infected persons after temporary treatment interruption

Immunologic and virologic outcomes of treatment interruption were compared for 5 chronically human immunodeficiency virus (HIV)-infected persons who have maintained antiretroviral therapy-mediated virus suppression, as compared with 5 untreated controls. After a median interruption of 55 days of therapy accompanied by rebound of virus, reinitiated therapy in 4 of 5 subjects resulted in suppression of 98.86% of plasma virus load by 21-33 days and no significant decrease in CD4 T cell percentage from baseline. Increased T helper responses against HIV-1 p24 antigen (P=. 014) and interferon-gamma-secreting CD8 T cell responses against HIV-1 Env (P=.004) were present during interruption of therapy and after reinitiation of treatment. The remaining subject whose treatment was interrupted did not resume treatment and continued to have a low virus load (<1080 HIV-1 RNA copies/mL) and persistent antiviral cell-mediated responses. In summary, cellular immunity against autologous HIV-1 has the potential to be acutely augmented in association with temporary treatment interruption in chronically infected persons.     

New CD4+ and CD8+ T cell responses induced in chronically HIV type-1-infected patients after immunizations with an HIV type 1 lipopeptide vaccine

We showed that an anti-HIV lipopeptide vaccine injected to HIV-uninfected volunteers was well tolerated and able to induce a specific CD4(+) and CD8(+) T cell responses. The same vaccine was injected in HIV-1 chronically infected patients controlled by HAART to evaluate its immunogenicity. In this trial, 24 patients were immunized three times with a mixture of six lipopeptides (Nef 66-97, Nef 117-147, Nef 182-205, Gag 183-214, Gag 253-284, and Env 303-335) at 0, 3, and 6 weeks. We studied the HIV-1-specific CD4(+) T cell proliferative responses. The IFN-gamma secretion by activated CD8(+) T cells was evaluated, using an ex vivo ELISpot assay and 60 CD8(+) T cell epitopes derived from the vaccine. Before immunization (W0), anti-HIV CD4(+) T cell responses to Gag, Nef, and Env large peptides were detected in 7/23 (30%) analyzable patients. After three injections, 17/23 (74%) patients had a proliferative response and 16 of them induced new specific CD4(+) T cell responses. At W0, CD8(+) T cell responses to HIV-1 epitopes were detected in 6/23 (26%) patients. After vaccination, 16/23 (70%) patients showed CD8(+) T cell responses and 13 of these patients induced new T cell responses to 25 different HIV-1 epitopes. These HIV-1 epitopes were detected in patients with various HLA class I molecules (HLA-A2, -A3/A11, -A24, -B7 superfamily, -B8), as found in the majority of the white population. Lipopeptides induce new anti-HIV T cell responses in vaccinated infected patients and could be used as a new immunotherapy strategy. The majority of these responders induced specific new CD4(+) and CD8(+) T cell responses.     

Lymphocyte kinetics and precursor frequency-dependent recovery of CD4(+)CD45RA(+)CD62L(+) naive T cells following triple-drug therapy for HIV type 1 infection

New therapeutic regimens have dramatically altered morbidity and mortality attributed to HIV-1 infection. Changes in lymphocyte subsets after treatment may mirror salutary clinical changes. Over 4 months we analyzed lymphocyte subsets in 20 patients starting new HIV-1 therapy. Absolute numbers of lymphocytes, CD4+ T cells, CD8+ T cells, and B cells increased significantly by 4 months, but CD8+ T cell and B cell increases were restricted to late-stage patients. Subset analysis revealed that the magnitude of recovering naive-phenotype CD4+ T cells (slope) correlated with the number of these cells present at baseline, equaling or exceeding the memory-phenotype slope within days if these naive cells were abundant at baseline. Five of 10 patients in whom naive-phenotype CD4+ T cells were absent at baseline partially repopulated these cells by 4 months. These findings have important implications for the origin and mechanisms of renewal of naive-phenotype CD4+ T cells following effective treatment for HIV-1 infection.     

The Role of CD4(+) and CD8(+) T Cells in Controlling HIV Infection

Presently, it is thought that virus-specific T cells play a major role in restricting lentiviral replication and determining the rate of disease progression in humans. However, it remains unclear why this restriction fails in the majority of infected individuals. The major exception is a rare subgroup of HIV-infected long-term nonprogressors (LTNPs) who have been infected for approximately 20 years yet maintain normal CD4⁺ T-cell counts and less than 50 copies of viral RNA/mL of plasma. Although virus-specific cellular (CD4⁺ and CD8⁺ T lymphocytes) immune responses have been shown to exert some degree of in vivo control of HIV replication, the precise correlates of protective immunity differentiating LTNPs from patients with progressive disease remain unknown. A greater understanding of the components and magnitude of an effective immune response to HIV is an important step toward the development of effective vaccines and immunotherapies.     

Consistent subtype-specific anti-HIV type 1 T lymphocyte responses in Indian subjects recently infected with HIV type 1

Anti-HIV-1-specific T cell responses in early HIV-1 infection have been found to be important in deciding the course of disease progression. But there are few data concerning nonsubtype B HIV infection. HIV-1 subtype C is the most prevalent subtype in India. HIV-1 Gag-specific T cell responses in 12 Indian subjects with recent HIV-1 infection were characterized by an ELISpot assay at two consecutive visits and their correlation with plasma viral load and CD4(+) T lymphocyte counts was studied. Ten of the 12 subjects demonstrated T cell responses to either one or both Gag B and C peptides, on at least one visit. Five of 10 responders showed a consistent response (response at both visits): 4 exhibited a Gag C-specific consistent response and 1 showed a consistent response to Gag B. The remaining five patients, showing response at only one of the two visits, were considered inconsistent responders. None of the individuals showed a consistent response to both B and C Gag peptides. Marginally significant correlation was observed between consistency of the response and lower plasma viral load (p = 0.062). The subtype-specific Gag C response was also found to be correlated with lower viral load as compared with the response to Gag B (r = -0.336, p = 0.054 for subtype C and r = -0.234, p = 0.13 for subtype B). The data suggest that the patients exhibiting consistent subtype-specific responses to HIV-1 Gag might have better control of viral replication in early HIV infection.     

Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad integrated antibody and CD4(+) and CD8(+) T cell responses in humans

NY-ESO-1 is a "cancer-testis" antigen expressed in many cancers. ISCOMATRIX is a saponin-based adjuvant that induces antibody and T cell responses. We performed a placebo-controlled clinical trial evaluating the safety and immunogenicity of recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant. Forty-six evaluable patients with resected NY-ESO-1-positive tumors received three doses of vaccine intramuscularly at monthly intervals. The vaccine was well tolerated. We observed high-titer antibody responses, strong delayed-type hypersensitivity reactions, and circulating CD8(+) and CD4(+) T cells specific for a broad range of NY-ESO-1 epitopes, including known and previously unknown epitopes. In an unplanned analysis, vaccinated patients appeared to have superior clinical outcomes to those treated with placebo or protein alone. The vaccine is safe and highly potent immunologically.     

HIV type 1-infected dendritic cells induce apoptotic death in infected and uninfected primary CD4 T lymphocytes

In addition to their essential role in adaptive immunity, dendritic cells (DCs) participate in innate immunity. In the context of measles virus (MV) or cytomegalovirus infections, they develop cytotoxic functions that may contribute in vivo to the elimination of virus-infected cells, but that also kill infected and noninfected T lymphocytes. Because the human immunodeficiency virus (HIV) induces T cell depletion through mechanisms that are still obscure, we investigated its ability to trigger DC cytotoxicity. When incubated with HIV, monocyte-derived DCs induced apoptosis in MDA-231 cells, which are sensitive to MV-induced DC cytotoxicity, and in uninfected as well as HIV-infected H9 CD4+ T cell lines. This apoptosis was inhibited by a mixture of FasL, TRAIL, TNF-alpha, and TWEAK inhibitors. Indeed, HIV infection induced or enhanced sensitivity to TRAIL, TNF-alpha, and TWEAK in H9 cells. Moreover, dendritic cells incubated with HIV-1 BAL or a wildtype HIV-1 isolate induced apoptosis in autologous primary CD4+ T lymphocytes, infected or not with a wild-type HIV-1 isolate. Therefore, induction of DC cytotoxicity by HIV may be relevant to in vivo HIV infection. Induction of cytotoxicity in DCs by HIV might contribute to HIV-associated T cell depletion through induction of apoptosis, especially in the early stages of infection. It may also contribute to elimination of infected cells in vivo, thereby enhancing cross-presentation of HIV by DCs. Therefore this new cytotoxic function of DCs may play an important role in innate and adaptive immunity during HIV infection.     

Short communication: CD8(+) T cell polyfunctionality profiles in progressive and nonprogressive pediatric HIV type 1 infection

Pediatric HIV-1 infection is characterized by rapid disease progression and without antiretroviral therapy (ART), more than 50% of infected children die by the age of 2 years. However, a small subset of infected children progresses slowly to disease in the absence of ART. This study aimed to identify functional characteristics of HIV-1-specific T cell responses that distinguish children with rapid and slow disease progression. Fifteen perinatally HIV-infected children (eight rapid and seven slow progressors) were longitudinally studied to monitor T cell polyfunctionality. HIV-1-specific interferon (IFN)-γ(+) CD8(+) T cell responses gradually increased over time but did not differ between slow and rapid progressors. However, polyfunctional HIV-1-specific CD8(+) T cell responses, as assessed by the expression of four functions (IFN-γ, CD107a, TNF-α, MIP-1β), were higher in slow compared to rapid progressors (p=0.05) early in infection, and was associated with slower subsequent disease progression. These data suggest that the quality of the HIV-specific CD8(+) T cell response is associated with the control of disease in children as has been shown in adult infection.     

Dendritic cell amplification of HIV type 1-specific CD8+ T cell responses in exposed,seronegative heterosexual women

In the Heterosexual AIDS Transmission Study (HATS), the frequency of high-risk sexual activity and viral load in the seropositive partner were shown to correlate with HIV-1 transmission. However, these parameters could not account for the status of some exposed, seronegative (ESN) individuals who remained uninfected despite years of exposure. To test the hypothesis that antiviral immune responses are a correlate of nontransmission in this cohort, we developed two sensitive methods for assessing HIV-1-specific humoral and cell-mediated responses. To quantify T cell responses, autologous mature dendritic cells (DCs) were used as antigen-presenting cells to elicit HIV-1-specific IFN-gamma production by ELISPOT. Antibody responses to HIV-1 gp120 were assessed by combination immunoprecipitation-Western blot (IP-WB). Previous studies of this cohort, using limiting dilution analysis, did not reveal HIV-1-specific cytotoxic T lymphocyte activity. However, when autologous DCs were used to present HIV-1 antigens, T cells from three of eight ESN women (38%) responded by producing IFN-gamma. T cells from three of four seropositive partners responded to HIV-1 antigens, whereas five negative controls did not. The use of DCs as antigen-presenting cells increased sensitivity by 2- to 30-fold relative to standard ELISPOT. Using IP-WB, low levels of gp120-reactive antibodies were detected in plasma from 1 of 14 ESN women. These results support the hypothesis that HIV-1-specific T cell responses play a role in immune surveillance in this cohort of North American serodiscordant couples. This report also demonstrates the ability of dendritic cells to reveal T cell responses that might be overlooked by other methods.     

Impaired antigen-specific CD4(+) T lymphocyte responses in cavitary tuberculosis

The clinical outcome in tuberculosis is determined by the host-pathogen interaction. Successful immune responses result in granulomas and curtailment of disease, whilst cavitation indicates a failing immune response. We sought to investigate the mechanisms involved in these processes. Fourteen patients with confirmed pulmonary tuberculosis underwent bronchoalveolar lavage with washings from areas of cavitation, or pulmonary infiltrates and also from the contra-lateral radiologically normal lung. Flow cytometry was utilised to determine both the leukocyte populations and the Mycobacterium antigen-specific T cell component of the response. Cavitation was associated with local neutrophilia and relative lymphopenia, whereas lymphocytosis and lower levels of granulocytes were detected from areas of pulmonary infiltrates and also from radiologically unaffected lobes. The Mycobacterium-specific T cell response from cavitary sites was significantly lower when compared to the radiologically normal lobes in the same individuals (p=0.003). By contrast, the Mycobacterium-specific T cell responses from areas of infiltrates were remarkably similar to paired responses from the radiologically unaffected lung (p=0.45). These results confirm the selective accumulation of Mycobacterium-specific lymphocytes in the lung in general though they also demonstrate that this is diminished in cavities. It remains unclear whether neutrophilia is a cause, or a result of cavitation in pulmonary tuberculosis.     

Expression of the dengue virus type 2 NS5 protein in a CD4(+) T cell line inhibits HIV replication

Human immunodeficiency virus (HIV) load is suppressed during dengue virus infection. The NS5A phosphoprotein of GB virus C (a related flavivirus) inhibits HIV replication in vitro. To determine whether the dengue virus NS5 protein inhibits HIV replication, CD4(+) T cell lines expressing this protein were generated. HIV replication in dengue virus NS5-expressing cells decreased by >90% compared with control cells ([Formula: see text]), and this was mediated in part by decreased HIV coreceptor (CXCR4) expression and increased production of SDF-1. Thus, the dengue virus NS5 protein inhibits HIV replication in vitro, potentially explaining the reduction in HIV load observed during acute dengue virus infection.     

Relationship of CD4+ T cell counts and HIV type 1 viral loads in untreated, infected adolescents. Adolescent Medicine HIV/AIDS Research Network

The REACH Project (Reaching for Excellence in Adolescent Care and Health) of the Adolescent Medicine HIV/AIDS Research Network was designed as a study of an adolescent cohort composed of HIV-1-infected and -uninfected subjects. The goal of the analysis presented was to examine the relationship of CD4+ T cell counts and HIV-1 plasma viral loads in adolescents. The CD4+ T cell counts of 84 HIV+ subjects who were 13 to 19 years of age were measured at the clinical sites, using ACTG standardized techniques. HIV-1 viral loads in frozen plasma were determined by the NASBA/NucliSens assay at a central laboratory. Past and current treatment with antiretroviral drugs was determined by medical record abstraction and interview data. The slope of the line generated by regressing log10 HIV-1 RNA (copies/ml) versus CD4+ T cell counts of REACH subjects who are antiretroviral drug naive was negative and significantly different than zero. A negative association has also been reported for antiretroviral drug-naive, adult males in the Pittsburgh Men's Study, a component of MACS (Pitt-MACS) (Mellors J, et al.: Science 1996;272:1167). These data show that in adolescents, as in adults, HIV-1 RNA concentrations are correlated with corresponding absolute CD4+ T cell count. The slopes of the lines generated with data from each cohort were different (p = 0.003). In addition to age, there are sex and racial differences in the makeup of the two cohorts. Any or all of these differences may affect the slopes of the lines.     

Oligoclonal expansion of CD4(+)CD28(-) T lymphocytes in recipients of allogeneic hematopoietic cell grafts and identification of the same T cell clones within both CD4(+)CD28(+) and CD4(+)CD28(-) T cell subsets.

Recipients of allogeneic bone marrow grafts have clonally expanded CD8(+)CD28(-) T lymphocytes during the early period after transplantation, which leads to skewing of T cell receptor (TCR) repertoires. Here, we have addressed the question of whether clonal expansion of CD28(-) T cells is also observed in CD4(+) T lymphocytes after human allogeneic hematopoietic cell transplantation. We found that the fraction of T cells lacking CD28 expression in the CD4(+) subset was increased after transplantation, and expanded CD4(+)CD28(-) T lymphocytes carrying certain TCRBV subfamilies showed limited TCR diversity. In order to further study the ontogeny of CD4(+)CD28(-) T cells, we analyzed the complementarity-determining region 3 (CDR3) of the TCR-beta chain of CD4(+)CD28(+) and CD4(+)CD28(-) cells. We identified the same T cell clones within both CD4(+)CD28(-) and CD4(+)CD28(+) T cell subsets. These results suggest that both subsets are phenotypic variants of the same T cell lineage.