An effective AIDS vaccine based on live attenuated vesicular stomatitis virus recombinants.

We developed an AIDS vaccine based on attenuated VSV vectors expressing env and gag genes and tested it in rhesus monkeys. Boosting was accomplished using vectors with glycoproteins from different VSV serotypes. Animals were challenged with a pathogenic AIDS virus (SHIV89.6P). Control monkeys showed a severe loss of CD4+ T cells and high viral loads, and 7/8 progressed to AIDS with an average time of 148 days. All seven vaccinees were initially infected with SHIV89.6P but have remained healthy for up to 14 months after challenge with low or undetectable viral loads. Protection from AIDS was highly significant (p = 0.001). VSV vectors are promising candidates for human AIDS vaccine trials because they propagate to high titers and can be delivered without injection.

Direct measurement of CD4+ and CD8+ T-cell responses to CMV in HIV-1-infected subjects.

Data from murine models of chronic viral infection suggest that CD4+ T-cell responses to viral pathogens are important in sustaining the number and/or function of CD8+ cytotoxic T-cell (CTL) effectors. In this study, we used cytokine flow cytometry (CFC), staining with HLA-A*0201-peptide tetramers, and peptide stimulation with epitopic peptides to study functional CD4+ and CD8+ T-cell responses to cytomegalovirus (CMV) in human subjects coinfected with CMV and the human immunodeficiency virus, type 1 (HIV-1). We show that strong CD4+ and CD8+ T-cell responses to CMV antigens are sustained over time in HIV-1-infected individuals. Those who maintain a strong CD4+ T-cell response to CMV are also likely to maintain higher frequencies of CD8+ T cells capable of binding to HLA-A*0201-CMV pp65 (A2-pp65) tetramers as well as responses to pp65 peptide stimulation with effector cytokine production. These data support the hypothesis that declines in frequencies of CD4+ T-cell responses to CMV are associated with an inability to sustain high levels of CMV-specific CD8+ T-cell responses in HIV-1-infected subjects. These declines may precede the onset of CMV-associated end organ disease.

HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function.

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells.

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.

Direct measurement of CD8+ T cell responses in macaques infected with simian immunodeficiency virus.

The simian immunodeficiency virus (SIV) macaque model system has been used extensively to study AIDS pathogenesis and to test candidate vaccines for their ability to protect against homologous or heterologous challenge with pathogenic SIV or SHIV. Recent studies suggest that stimulation of HIV-1-specific CTL responses is important for effective vaccination against HIV-1. While quantitative measurements of SIV-specific cytotoxic T lymphocyte (CTL) responses have been facilitated by the use of tetrameric peptide complexes, this technique is currently limited to the study of Mamu-A*01-positive rhesus macaques. Furthermore, very few SIV-specific CTL epitopes have been identified, and there is limited identification of other MHC alleles in macaques. In this study, cytokine flow cytometry (CFC) was used to quantify SIV-specific CD8+ antigen-reactive T cells in macaques infected with SIV. We found a strong correlation (r = 0.96, P < 0.001) between CD8+ antigen-reactive T cells stained with the Mamu-A*01 p11C, C-M tetramer and production of intracellular TNF-alpha in the CFC assay. Furthermore, the CFC assay was used to identify a novel SIV-specific CTL epitope in Envelope (SIV Env, a.a. 486-494, sequence AEVAELYRL). The use of the CFC assay facilitates the study of antigen-reactive T cell responses in SIV infection and vaccination.

Strong human immunodeficiency virus (HIV)-specific CD4+ T cell responses in a cohort of chronically infected patients are associated with interruptions in anti-HIV chemotherapy.

Virus-specific CD4+ T-helper cell function is important in controlling human immunodeficiency virus (HIV) infection but is impaired in patients with progressive HIV disease. It has been reported that after highly active antiretroviral therapy (HAART), HIV-specific lymphoproliferative responses remain absent, whereas responses to non-HIV microbial antigens are restored. However, in analyzing immune responses in a cohort of chronically infected adults on HAART, we observed strong HIV-specific CD4+ T cell responses of Th-1 phenotype in 11 of 22 patients. The magnitude and frequency of HIV-specific lymphoproliferative responses was strongly associated with previous interruptions in HAART (P=.001). In contrast, the magnitude of CD8+ T cell responses to HIV Gag, Pol, Env, and Nef was similar in patients who had and those who had not interrupted HAART. We conclude that (1) a significant proportion of chronically HIV-infected patients on HAART can generate strong HIV-specific CD4+ and CD8+ T cell immunity and (2) transient interruptions in antiviral treatment may prime or boost HIV-specific CD4+ T-helper responses.

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.

High frequency of cytomegalovirus-specific cytotoxic T-effector cells in HLA-A*0201-positive subjects during multiple viral coinfections.

How the cellular immune response copes with diverse antigenic competition is poorly understood. Responses of virus-specific cytotoxic T lymphocytes (CTL) were examined longitudinally in an individual coinfected with human immunodeficiency virus type 1 (HIV-1), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CTL responses to all 3 viruses were quantified by limiting dilution analysis and staining with HLA-A*0201 tetrameric complexes folded with HIV-1, EBV, and CMV peptides. A predominance of CMV-pp65-specific CTL was found, with a much lower frequency of CTL to HIV-1 Gag and Pol and to EBV-BMLF1 and LMP2. The high frequency of CMV-specific CTL, compared with HIV-1- and EBV-specific CTL, was confirmed in an additional 16 HLA-A*0201-positive virus-coinfected subjects. Therefore, the human immune system can mount CTL responses to multiple viral antigens simultaneously, albeit with different strengths.

Simian immunodeficiency virus-specific cytotoxic T lymphocytes and protection against challenge in rhesus macaques immunized with a live attenuated simian immunodeficiency virus vaccine.

In this study, we examined the role of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes (CTLs) in macaques immunized with an attenuated strain of simian immunodeficiency virus (SIVmac239Deltanef) in protection against pathogenic challenge with SIVmac251. Our results indicate that attenuated SIVmac239Deltanef can elicit specific CTL precursor cells (CTLp), but no correlation was observed between breadth or strength of CTLp response to structural proteins SIV-Env, -Gamg or -Pol (as measured by limiting dilution assay) and protection against infection. In one animal, we longitudinally followed the SIV-Gag-specific response to an MHC class I Mamu-A*01-restricted epitope p11C, C-M using a tetrameric MHC/peptide complex reagent. A low frequency of SIV p11C, C-M peptide-specific tetramer-reactive cells was present at the time of challenge but could be expanded in vitro. Surprisingly, the low level of Mamu-A*01/p11C, C-M-specific CTLs induced through attenuated SIVmac239Deltanef vaccination increased in the absence of detectable SIVmac251 or SIVmac239Deltanef proviral DNA. Overall, our results suggest that protection against infection in this model can be achieved through more than one mechanism, with SIV-specific CTLs being important in controlling SIVmac239Deltanef viral replication postchallenge.

HIV-1-specific immune responses in subjects who temporarily contain virus replication after discontinuation of highly active antiretroviral therapy.

Therapeutic intervention with highly active antiretroviral therapy (HAART) can lead to suppression of HIV-1 plasma viremia to undetectable levels for 3 or more years. However, adherence to complex drug regimens can prove problematic, and subjects may temporarily discontinue HAART for variable periods. We studied 6 HIV-1-infected individuals who stopped therapy. Off HAART, levels of viremia were suppressed to fewer than 500 copies/mL in 2 subjects for more than 12 and more than 24 months, respectively, and in 1 subject for 4 months on 1 occasion. Three subjects failed to contain plasma viremia. Broad and strong HIV-1-specific immune responses were detected in subjects with prolonged suppression of viral replication. This longitudinal study suggests that containment of HIV-1 replication to low or undetectable levels after discontinuation of HAART is associated with strong virus-specific immune responses. Boosting of HIV-1-specific immune responses should be considered as an adjunctive treatment strategy for HIV-1-infected individuals on HAART.

Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells.

Dendritic cells (DCs) are potent antigen-presenting cells that initiate protective T-cell immunity in mice. To study the immunogenicity of DCs in humans, we injected 9 healthy subjects subcutaneously with a control injection of autologous monocyte-derived, mature DCs, followed 4-6 weeks later by DCs pulsed with keyhole limpet hemocyanin (KLH), HLA-A*0201-positive restricted influenza matrix peptide (MP), and tetanus toxoid (TT). Four more subjects received these antigens without DCs. Injection of unpulsed DCs, or antigens alone, failed to immunize. Priming of CD4(+) T cells to KLH was observed in all 9 subjects injected with KLH-pulsed DCs, and boosting of TT-specific T-cell immunity was seen in 5 of 6 subjects injected with TT-pulsed DCs. Injection of antigen-pulsed DCs led to a severalfold increase in freshly isolated MP-specific, IFN-gamma-secreting CD8(+) T cells in all 6 HLA-A*0201-positive subjects, as early as 7 days after injection. When T cells were boosted in culture, there was an increase in MHC tetramer-binding cells and cytotoxic T cells after DC vaccination. These data provide the first controlled evidence of the immunogenicity of DCs in humans, and demonstrate that a single injection of mature DCs rapidly expands T-cell immunity.

Comparison of restimulation methods to elicit SIV specific cytotoxic T-lymphocytes (CTL) in vitro: Staphylococcal enterotoxin B (SEB) provides a novel method for the quantification of SIV specific CTL precursors.

Most investigators believe that an effective HIV-1 vaccine will have to induce high levels of HIV-1 specific cytotoxic T-cells (CTL). The macaque SIV challenge/protection model system has been used to test candidate vaccines, but quantitative immunogenicity measurements are difficult due to technical limitations of the assays available. The quantification of SIV specific CTLp is crucial to understanding correlates of immunity for these vaccines, but are difficult to measure. We have compared various methods to quantify SIV specific CTLp, and describe a novel method of SIV specific CTL in vitro stimulation using the superantigen Staphylococcal enterotoxin B (SEB). SEB can stimulate high levels of CTLp in vitro, and provides an alternative method to induce SIV specific CTL.