Expansion and diversification of virus-specific T cells following immunization of human immunodeficiency virus type 1 (HIV-1)-infected individuals with a recombinant modified vaccinia virus Ankara/HIV-1 Gag vaccine

Affordable therapeutic strategies that induce sustained control of human immunodeficiency virus type 1 (HIV-1) replication and are tailored to the developing world are urgently needed. Since CD8(+) and CD4(+) T cells are crucial to HIV-1 control, stimulation of potent cellular responses by therapeutic vaccination might be exploited to reduce antiretroviral drug exposure. However, therapeutic vaccines tested to date have shown modest immunogenicity. In this study, we performed a comprehensive analysis of the changes in virus-specific CD8(+) and CD4(+) T-cell responses occurring after vaccination of 16 HIV-1-infected individuals with a recombinant modified vaccinia virus Ankara-vectored vaccine expressing the consensus HIV-1 clade A Gag p24/p17 sequences and multiple CD8(+) T-cell epitopes during highly active antiretroviral therapy. We observed significant amplification and broadening of CD8(+) and CD4(+) gamma interferon responses to vaccine-derived epitopes in the vaccinees, without rebound viremia, but not in two unvaccinated controls followed simultaneously. Vaccine-driven CD8(+) T-cell expansions were also detected by tetramer reactivity, predominantly in the CD45RA(-) CCR7(+) or CD45RA(-) CCR7(-) compartments, and persisted for at least 1 year. Expansion was associated with a marked but transient up-regulation of CD38 and perforin within days of vaccination. Gag-specific CD8(+) and CD4(+) T-cell proliferation also increased postvaccination. These data suggest that immunization with MVA.HIVA is a feasible strategy to enhance potentially protective T-cell responses in individuals with chronic HIV-1 infection.     

Discordance between frequency of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon-producing CD4(+) T cells and HIV-1-specific lymphoproliferation in HIV-1-infected subjects with active viral replication

One hallmark of uncontrolled, chronic human immunodeficiency virus type 1 (HIV-1) infection is the absence of strong HIV-1-specific, CD4(+) T-cell-proliferative responses, yet the mechanism underlying this T helper (Th)-cell defect remains controversial. To better understand the impact of HIV-1 replication on Th-cell function, we compared the frequency of CD4(+) Th-cell responses based on production of gamma interferon to lymphoproliferative responses directed against HIV-1 proteins in HIV-1-infected subjects with active in vivo viral replication versus those on suppressed highly active antiretroviral therapy (HAART). No statistically significant differences in the frequencies of cytokine-secreting, HIV-1-specific CD4(+) T cells between the donor groups were found, despite differences in viral load and treatment status. However, HIV-1-specific lymphoproliferative responses were significantly greater in the subjects with HAART suppression than in subjects with active viral replication. Similar levels of HIV-1 RNA were measured in T-cell cultures stimulated with HIV-1 antigens regardless of donor in vivo viral loads, but only HIV-1-specific CD4(+) T cells from subjects with HAART suppression proliferated in vitro, suggesting that HIV-1 replication in vitro does not preclude HIV-1-specific lymphoproliferation. This study demonstrates a discordance between the frequency and proliferative capacity of HIV-1-specific CD4(+) T cells in subjects with ongoing in vivo viral replication and suggests that in vivo HIV-1 replication contributes to the observed defect in HIV-1-specific CD4(+) T-cell proliferation.     

Human immunodeficiency virus type 1 (HIV-1)-specific CD4+ T cells that proliferate in vitro detected in samples from most viremic subjects and inversely associated with plasma HIV-1 levels

Diminished in vitro proliferation of human immunodeficiency virus type 1 (HIV-1)-specific CD4+T cells has been associated with HIV-1 viremia and declining CD4+ T-cell counts during chronic infection. To better understand this phenomenon, we examined whether HIV-1 Gag p24 antigen-induced CD4+ T-cell proliferation might recover in vitro in a group of subjects with chronic HIV-1 viremia and no history of antiretroviral therapy (ART). We found that depletion of CD8+ cells from peripheral blood mononuclear cells (PBMC) before antigen stimulation was associated with a 6.5-fold increase in the median p24-induced CD4+ T-cell proliferative response and a 57% increase in the number of subjects with positive responses. These p24-induced CD4+ T-cell proliferative responses from CD8-depleted PBMC were associated with expansion of the numbers of p24-specific, gamma interferon (IFN-gamma)-producing CD4+ T cells. Among the 20 viremic, treatment-naive subjects studied, the only 5 subjects lacking proliferation-competent, p24-specific CD4+ T-cell responses from CD8-depleted PBMC showed plasma HIV-1 RNA levels > 100,000 copies/ml. Furthermore, both the magnitude of p24-induced CD4+ T-cell proliferative responses from CD8-depleted PBMC and the frequency of p24-specific, IFN-gamma-producing CD4+ T cells expanded from CD8-depleted PBMC were associated inversely with plasma HIV-1 RNA levels. Therefore, proliferation-competent, HIV-1-specific CD4+ T cells that might help control HIV-1 disease may persist during chronic, progressive HIV-1 disease except at very high levels of in vivo HIV-1 replication.     

The Thai phase III trial (RV144) vaccine regimen induces T cell responses that preferentially target epitopes within the V2 region of HIV-1 envelope

The Thai HIV phase III prime/boost vaccine trial (RV144) using ALVAC-HIV (vCP1521) and AIDSVAX B/E was, to our knowledge, the first to demonstrate acquisition efficacy. Vaccine-induced, cell-mediated immune responses were assessed. T cell epitope mapping studies using IFN-γ ELISPOT was performed on PBMCs from HIV-1-uninfected vaccine (n = 61) and placebo (n = 10) recipients using HIV-1 Env peptides. Positive responses were measured in 25 (41%) vaccinees and were predominantly CD4(+) T cell-mediated. Responses were targeted within the HIV Env region, with 15 of 25 (60%) of vaccinees recognizing peptides derived from the V2 region of HIV-1 Env, which includes the α(4)β(7) integrin binding site. Intracellular cytokine staining confirmed that Env responses predominated (19 of 30; 63% of vaccine recipients) and were mediated by polyfunctional effector memory CD4(+) T cells, with the majority of responders producing both IL-2 and IFN-γ (12 of 19; 63%). HIV Env Ab titers were higher in subjects with IL-2 compared with those without IL-2-secreting HIV Env-specific effector memory T cells. Proliferation assays revealed that HIV Ag-specific T cells were CD4(+), with the majority (80%) expressing CD107a. HIV-specific T cell lines obtained from vaccine recipients confirmed V2 specificity, polyfunctionality, and functional cytolytic capacity. Although the RV144 T cell responses were modest in frequency compared with humoral immune responses, the CD4(+) T cell response was directed to HIV-1 Env and more particularly the V2 region.     

Infrequent detection of HIV-1-specific, but not cytomegalovirus-specific, CD8(+) T cell responses in young HIV-1-infected infants.

Early potent combination antiretroviral therapies (ART) for HIV-1 infection can preserve or restore immune function, but control of viral replication early in infection may interfere with the development of HIV-1-specific immune responses. Using an IFN-gamma ELISPOT assay, we evaluated the breadth and intensity of HIV-1-specific CD8(+) T cell responses in 17 vertically infected infants who began ART at 1-23 mo of age. CMV-specific responses were also characterized in three infants coinfected with HIV-1 and CMV. Before ART, HIV-1-specific CD8(+) T cell responses were detected in two of 13 (15%) infants <6 mo of age. HIV-1-specific CD8(+) T cells became undetectable in these two infants after the control of viral replication. Intermittent HIV-1-specific responses were noted in six infants who did not experience durable control of viral replication. In contrast, HIV-1-specific responses were detected before ART in four of four infants >6 mo of age and became persistently undetectable in only one child. CMV-specific CD8(+) T cell responses were persistently detected in all HIV-1 and CMV coinfected infants. In conclusion, HIV-1-specific CD8(+) T cell responses were less commonly detected before therapy in young infants than in older infants. Suppression of viral replication appeared to interfere with the development and maintenance of HIV-1-specific CD8(+) T cell responses. The detection of CMV-specific responses in HIV-1 and CMV coinfected infants suggests a selective defect in the generation or maintenance of HIV-1-specific CD8(+) T cell responses. Therapeutic HIV-1 vaccine strategies in young infants may prolong the clinical benefit of ART by expanding the HIV-1-specific CD8(+) T cell pool.     

Reduced hepatitis B virus (HBV)-specific CD4+ T-cell responses in human immunodeficiency virus type 1-HBV-coinfected individuals receiving HBV-active antiretroviral therapy

Functional hepatitis B virus (HBV)-specific T cells are significantly diminished in individuals chronically infected with HBV compared to individuals with self-limiting HBV infection or those on anti-HBV therapy. In individuals infected with human immunodeficiency virus type 1 (HIV-1), coinfection with HBV is associated with an increased risk of worsening liver function following antiviral therapy and of more rapid HBV disease progression. Total HBV-specific T-cell responses in subjects with diverse genetic backgrounds were characterized by using a library of 15-mer peptides overlapping by 11 amino acids and spanning all HBV proteins. The magnitude and breadth of CD4(+) and CD8(+) T-cell responses to HBV in peripheral blood were examined by flow cytometry to detect gamma interferon production following stimulation with HBV peptide pools. Chronic HBV carriers (n = 34) were studied, including individuals never treated for HBV infection (n = 7), HBV-infected individuals receiving anti-HBV therapy (n = 13), and HIV-1-HBV-coinfected individuals receiving anti-HBV therapy (n = 14). CD4(+) and CD8(+) HBV-specific T-cell responses were more frequently detected and the CD8(+) T-cell responses were of greater magnitude and breadth in subjects on anti-HBV treatment than in untreated chronic HBV carriers. There was a significant inverse correlation between detection of a HBV-specific T-cell response and HBV viral load. HBV-specific CD4(+) and CD8(+) T-cell responses were significantly (fivefold) reduced compared with HIV-specific responses. Although, the frequency and breadth of HBV-specific CD8(+) T-cell responses were comparable in the monoinfected and HIV-1-HBV-coinfected groups, HBV-specific CD4(+) T-cell responses were significantly reduced in HIV-1-HBV-coinfected individuals. Therefore, HIV-1 infection has a significant and specific effect on HBV-specific T-cell immunity.     

Characterization of human immunodeficiency virus type 1 (HIV-1) Gag- and Gag peptide-specific CD4(+) T-cell clones from an HIV-1-seronegative donor following in vitro immunization

Substantial evidence argues that human immunodeficiency virus type 1 (HIV-1)-specific CD4(+) T cells play an important role in the control of HIV-1 replication in infected individuals. Moreover, it is increasingly clear that an HIV vaccine should elicit potent cytotoxic lymphocyte and antibody responses that will likely require an efficient CD4(+) T-cell response. Therefore, understanding and characterizing HIV-specific CD4(+) T-cell responses is an important aim. Here we describe the generation of HIV-1 Gag- and Gag peptide-specific CD4(+) T-cell clones from an HIV-1-seronegative donor by in vitro immunization with HIV-1 Gag peptides. The Gag peptides were able to induce a strong CD4(+) T-cell immune response in peripheral blood mononuclear cells from the HIV-1-seronegative donor. Six Gag peptide-specific CD4(+) T-cell clones were isolated and their epitopes were mapped. The region of p24 between amino acids 201 and 300 of Gag was defined as the immunodominant region of Gag. A new T helper epitope in the p6 protein of Gag was identified. Two clones were shown to recognize Gag peptides and processed Gag protein, while the other four clones reacted only to Gag peptides under the experimental conditions used. Functional analysis of the clones indicated that both Th1 and Th2 types of CD4(+) T cells were obtained. One clone showed direct antigen-specific cytotoxic activity. These clones represent a valuable tool for understanding the cellular immune response to HIV-1, and the study provides new insights into the HIV-1-specific CD4(+) T-cell response and the induction of an anti-Gag and -Gag peptide cellular primary immune response in vitro.     

Correlates of delayed disease progression in HIV-1-infected Kenyan children

Without treatment most HIV-1-infected children in Africa die before their third birthday (>89%) and long-term nonprogressors are rare. The mechanisms underlying nonprogression in HIV-1-infected children are not well understood. In the present study, we examined potential correlates of delayed HIV disease progression in 51 HIV-1-infected African children. Children were assigned to progression subgroups based on clinical characterization. HIV-1-specific immune responses were studied using a combination of ELISPOT assays, tetramer staining, and FACS analysis to characterize the magnitude, specificity, and functional phenotype of HIV-1-specific CD8(+) and CD4(+) T cells. Host genetic factors were examined by genotyping with sequence-specific primers. HIV-1 nef gene sequences from infecting isolates from the children were examined for potential attenuating deletions. Thymic output was measured by T cell rearrangement excision circle assays. HIV-1-specific CD8(+) T cell responses were detected in all progression groups. The most striking attribute of long-term survivor nonprogressors was the detection of HIV-1-specific CD4(+) Th responses in this group at a magnitude substantially greater than previously observed in adult long-term nonprogressors. Although long-term survivor nonprogressors had a significantly higher percentage of CD45RA(+)CD4(+) T cells, nonprogression was not associated with higher thymic output. No protective genotypes for known coreceptor polymorphisms or large sequence deletions in the nef gene associated with delayed disease progression were identified. In the absence of host genotypes and attenuating mutations in HIV-1 nef, long-term surviving children generated strong CD4(+) T cell responses to HIV-1. As HIV-1-specific helper cells support anti-HIV-1 effector responses in active disease, their presence may be important in delaying disease progression.     

Broad TCR usage in functional HIV-1-specific CD8+ T cell expansions driven by vaccination during highly active antiretroviral therapy

During chronic HIV-1 infection, continuing viral replication is associated with impaired proliferative capacity of virus-specific CD8+ T cells and with the expansion and persistence of oligoclonal T cell populations. TCR usage may significantly influence CD8+ T cell-mediated control of AIDS viruses; however, the potential to modulate the repertoire of functional virus-specific T cells by immunotherapy has not been explored. To investigate this, we analyzed the TCR Vbeta usage of CD8+ T cells populations which were expanded following vaccination with modified vaccinia virus Ankara expressing a HIV-1 gag/multiepitope immunogen (MVA.HIVA) in HIV-1-infected patients receiving highly active antiretroviral therapy. Vaccinations induced the re-expansion of HIV-1-specific CD8+ T cells and these showed broad TCR Vbeta usage which was maintained for at least 1 year in some individuals. By contrast, virus-specific CD8+ T cell populations in the same donors which failed to expand after vaccination and in unvaccinated controls were oligoclonal. Simultaneously, we observed that CD8+ T cells recognizing vaccine-derived HIV-1 epitopes displayed enhanced capacity to proliferate and to inhibit HIV-1 replication in vitro, following MVA.HIVA immunizations. Taken together, these data indicate that an attenuated viral-vectored vaccine can modulate adaptive CD8+ T cell responses to HIV-1 and improve their antiviral functional capacity. The potential therapeutic benefit of this vaccination approach warrants further investigation.     

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T(EMRA) cells in early infection are linked to control of HIV-1 viremia and predict the subsequent viral load set point

CD8(+) T cells are believed to play an important role in the control of human immunodeficiency virus type 1 (HIV-1) infection. However, despite intensive efforts, it has not been possible to consistently link the overall magnitude of the CD8(+) T-cell response with control of HIV-1. Here, we have investigated the association of different CD8(+) memory T-cell subsets responding to HIV-1 in early infection with future control of HIV-1 viremia. Our results demonstrate that both a larger proportion and an absolute number of HIV-1-specific CD8(+) CCR7(-) CD45RA(+) effector memory T cells (T(EMRA) cells) were associated with a lower future viral load set point. In contrast, a larger absolute number of HIV-1-specific CD8(+) CCR7(-) CD45RA(-) effector memory T cells (T(EM)) was not related to the viral load set point. Overall, the findings suggest that CD8(+) T(EMRA) cells have superior antiviral activity and indicate that both qualitative and quantitative aspects of the CD8(+) T-cell response need to be considered when defining the characteristics of protective immunity to HIV-1.     

Diverse repertoire of HIV-1 p24-specific,IFN-gamma-producing CD4+ T cell clones following immune reconstitution on highly active antiretroviral therapy

HIV-1 Ag-specific CD4(+) T cell proliferative responses in human subjects with advanced, untreated HIV-1 disease are often weak or undetectable. Conversely, HIV-1-specific CD4(+) T cell proliferation is occasionally detected following suppression of HIV-1 replication with highly active antiretroviral therapy (HAART). These observations suggest that unchecked HIV-1 replication may lead to depletion or dysfunction of HIV-1-specific CD4(+) T cells, and that these defects may be partially corrected by viral suppression and subsequent immune reconstitution. However, the impact of this immune reconstitution on the repertoire of HIV-1-specific CD4(+) T cells has not been thoroughly evaluated. To examine the HIV-1-specific CD4(+) T cell repertoire in this clinical setting, we established HIV-1 p24-specific CD4(+) T cell clones from a successfully HAART-treated subject whose pretreatment peripheral CD4 count was 0 cells/ micro l. Eleven different p24-specific CD4(+) T cell clonotypes were distinguished among 13 clones obtained. Most clones produced both IFN-gamma and IL-4 upon Ag stimulation. Clones targeted eight distinct epitopes that varied in their conservancy among HIV-1 strains, and responses were restricted by one of three MHC II molecules. Clones showed a range of functional avidities for both protein and peptide Ags. Additional studies confirmed that multiple HIV-1 p24-derived epitopes were targeted by IFN-gamma-producing CD4(+) cells from subjects first treated with HAART during advanced HIV-1 disease (median, 4.5 peptides/subject; range, 3-6). These results suggest that in HAART-treated subjects whose peripheral CD4(+) T cell pools were once severely depleted, the HIV-1-specific CD4(+) T cell repertoire may include a diverse array of clonotypes targeting multiple HIV-1 epitopes.     

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.     

Effects of sustained HIV-1 plasma viremia on HIV-1 Gag-specific CD4+ T cell maturation and function

An in vitro proliferative defect has been observed in HIV-1-specific CD4(+) T cells from infected subjects with high-level plasma HIV-1 viremia. To determine the mechanism of this defect, HIV-1 Gag-specific CD4(+) T cells from treated and untreated HIV-1-infected subjects were analyzed for cytokine profile, proliferative capacity, and maturation state. Unexpectedly high frequencies of HIV-1-specific, IL-2-producing CD4(+) T cells were measured in subjects with low or undetectable plasma HIV-1 loads, regardless of treatment status, and IL-2 frequencies correlated inversely with viral loads. IL-2-producing CD4(+) T cells also primarily displayed a central memory (T(Cm); CCR7(+)CD45RA(-)) maturation phenotype, whereas IFN-gamma-producing cells were mostly effector memory (T(Em), CCR7(-)CD45RA(-)). Among Gag-specific, IFN-gamma-producing CD4(+) T cells, higher T(Em) frequencies and lower T(Cm) frequencies were observed in untreated, high viral load subjects than in subjects with low viral loads. The percentage of HIV-1 Gag-specific CD4(+) T(Cm) correlated inversely with HIV-1 viral load and directly with Gag-specific CD4(+) T cell proliferation, whereas the opposite relationships were observed for HIV-1-specific CD4(+) T(Em). These results suggest that HIV-1 viremia skews Gag-specific CD4(+) T cells away from an IL-2-producing T(Cm) phenotype and toward a poorly proliferating T(Em) phenotype, which may limit the effectiveness of the HIV-1-specific immune response.     

De novo generation of escape variant-specific CD8+ T-cell responses following cytotoxic T-lymphocyte escape in chronic human immunodeficiency virus type 1 infection

Human immunodeficiency virus type 1 (HIV-1) evades CD8(+) T-cell responses through mutations within targeted epitopes, but little is known regarding its ability to generate de novo CD8(+) T-cell responses to such mutants. Here we examined gamma interferon-positive, HIV-1-specific CD8(+) T-cell responses and autologous viral sequences in an HIV-1-infected individual for more than 6 years following acute infection. Fourteen optimal HIV-1 T-cell epitopes were targeted by CD8(+) T cells, four of which underwent mutation associated with dramatic loss of the original CD8(+) response. However, following the G(357)S escape in the HLA-A11-restricted Gag(349-359) epitope and the decline of wild-type-specific CD8(+) T-cell responses, a novel CD8(+) T-cell response equal in magnitude to the original response was generated against the variant epitope. CD8(+) T cells targeting the variant epitope did not exhibit cross-reactivity against the wild-type epitope but rather utilized a distinct T-cell receptor Vbeta repertoire. Additional studies of chronically HIV-1-infected individuals expressing HLA-A11 demonstrated that the majority of the subjects targeted the G(357)S escape variant of the Gag(349-359) epitope, while the wild-type consensus sequence was significantly less frequently recognized. These data demonstrate that de novo responses against escape variants of CD8(+) T-cell epitopes can be generated in chronic HIV-1 infection and provide the rationale for developing vaccines to induce CD8(+) T-cell responses directed against both the wild-type and variant forms of CD8 epitopes to prevent the emergence of cytotoxic T-lymphocyte escape variants.     

Recognition of a defined region within p24 gag by CD8+ T cells during primary human immunodeficiency virus type 1 infection in individuals expressing protective HLA class I alleles

Human immunodeficiency virus type 1 (HIV-1)-specific immune responses during primary HIV-1 infection appear to play a critical role in determining the ultimate speed of disease progression, but little is known about the specificity of the initial HIV-1-specific CD8(+) T-cell responses in individuals expressing protective HLA class I alleles. Here we compared HIV-1-specific T-cell responses between subjects expressing the protective allele HLA-B27 or -B57 and subjects expressing nonprotective HLA alleles using a cohort of over 290 subjects identified during primary HIV-1 infection. CD8(+) T cells of individuals expressing HLA-B27 or -B57 targeted a defined region within HIV-1 p24 Gag (amino acids 240 to 272) early in infection, and responses against this region contributed over 35% to the total HIV-1-specific T-cell responses in these individuals. In contrast, this region was rarely recognized in individuals expressing HLA-B35, an HLA allele associated with rapid disease progression, or in subjects expressing neither HLA-B57/B27 nor HLA-B35 (P < 0.0001). The identification of this highly conserved region in p24 Gag targeted in primary infection specifically in individuals expressing HLA class I alleles associated with slower HIV-1 disease progression provides a rationale for vaccine design aimed at inducing responses to this region restricted by other, more common HLA class I alleles.     

Human immunodeficiency virus type 1- and cytomegalovirus-specific cytotoxic T lymphocytes can persist at high frequency for prolonged periods in the absence of circulating peripheral CD4(+) T cells

CD4(+) T cells are thought to be critical in the maintenance of virus-specific CD8(+) cytotoxic T-cell (CTL) responses. In human immunodeficiency virus type 1 (HIV-1) infection, a selective decline in HIV-1-specific CTL as the CD4(+) T-cell count decreases has been reported. Using HLA-peptide tetrameric complexes, we show the presence at high frequency of HIV-1- and cytomegalovirus-specific CD8(+) T cells when the peripheral CD4(+) T-cell count was low or zero in three HIV-1-infected patients. No direct virus-specific CD8(+)-mediated effector activity was seen in these subjects, suggesting antigen unresponsiveness, although tetramer-sorted cells could be expanded in vitro in the presence of interleukin-2 into responsive effector cells. Thus, virus-specific CD8(+) T cells can be maintained in the peripheral circulation at high frequency in the absence of circulating peripheral CD4(+) T cells, but these cells may lack direct effector activity. Strategies designed to overcome this antigen unresponsiveness may be of value in therapies for the treatment of AIDS.     

Pre-Clinical Development of BCG.HIVA(CAT), an Antibiotic-Free Selection Strain, for HIV-TB Pediatric Vaccine Vectored by Lysine Auxotroph of BCG

In the past, we proposed to develop a heterologous recombinant BCG prime-recombinant modified vaccinia virus Ankara (MVA) boost dual pediatric vaccine platform against transmission of breast milk HIV-1 and Mycobacterium tuberculosis (Mtb). In this study, we assembled an E. coli-mycobacterial shuttle plasmid pJH222.HIVA^CAT expressing HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism based on Operator-Repressor Titration (ORT) system for plasmid selection and maintenance in E. coli and lysine complementation in mycobacteria. This shuttle plasmid was electroporated into parental lysine auxotroph (safer) strain of BCG to generate vaccine BCG.HIVA^CAT. All procedures complied with Good Laboratory Practices (GLPs). We demonstrated that the episomal plasmid pJH222.HIVA^CAT was stable in vivo over a 20-week period, and genetically and phenotypically characterized the BCG.HIVA^CAT vaccine strain. The BCG.HIVA^CAT vaccine in combination with MVA.HIVA induced HIV-1- and Mtb-specific interferon γ-producing T-cell responses in newborn and adult BALB/c mice. On the other hand, when adult mice were primed with BCG.HIVA^CAT and boosted with MVA.HIVA.85A, HIV-1-specific CD8⁺ T-cells producing IFN-γ, TNF-α, IL-2 and CD107a were induced. To assess the biosafety profile of BCG.HIVA^CAT-MVA.HIVA regimen, body mass loss of newborn mice was monitored regularly throughout the vaccination experiment and no difference was observed between the vaccinated and naïve groups of animals. Thus, we demonstrated T-cell immunogenicity of a novel, safer, GLP-compatible BCG-vectored vaccine using prototype immunogen HIVA. Second generation immunogens derived from HIV-1 as well as other major pediatric pathogens can be constructed in a similar fashion to prime protective responses soon after birth.     

Induction of anti-human immunodeficiency virus type 1 (HIV-1) CD8(+) and CD4(+) T-cell reactivity by dendritic cells loaded with HIV-1 X4-infected apoptotic cells

T-cell responses to X4 strains of human immunodeficiency virus type 1 (HIV-1) are considered important in controlling progression of HIV-1 infection. We investigated the ability of dendritic cells (DC) and various forms of HIV-1 X4 antigen to induce anti-HIV-1 T-cell responses in autologous peripheral blood mononuclear cells from HIV-1-infected persons. Immature DC loaded with HIV-1 IIIB-infected, autologous, apoptotic CD8(-) cells and matured with CD40 ligand induced gamma interferon production in autologous CD8(+) and CD4(+) T cells. In contrast, mature DC loaded with HIV-1 IIIB-infected, necrotic cells or directly infected with cell-free HIV-1 IIIB were poorly immunogenic. Thus, HIV-1-infected cells undergoing apoptosis serve as a rich source of X4 antigen for CD8(+) and CD4(+) T cells by DC. This may be an important mechanism of HIV-1 immunogenicity and provides a strategy for immunotherapy of HIV-1-infected patients on combination antiretroviral therapy.     

Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses,but no correlation to viral load

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.