Characterization of SARS-CoV-specific memory T cells from recovered individuals 4 years after infection.

SARS-CoV infection of human results in antigen-specific cellular and humoral immune responses. However, it is critical to determine whether SARS-CoV-specific memory T cells can persist for long periods of time. In this study, we analyzed the cellular immune response from 21 SARS-recovered individuals who had been diagnosed with SARS in 2003 by using ELISA, CBA, ELISpot and multiparameter flow cytometry assays.

Replication-defective adenovirus vectors with multiple deletions do not induce measurable vector-specific T cells in human trials.

The magnitude and character of adenovirus serotype 5 (Ad5)-specific T cells were determined in volunteers with and without preexisting neutralizing antibodies (NAs) to Ad5 who received replication-defective Ad5 (rAd5)-based human immunodeficiency virus vaccines. There was no correlation between T-cell responses and NAs to Ad5. There was no increase in magnitude or activation state of Ad5-specific CD4(+) T cells at time points where antibodies to Ad5 and T-cell responses to the recombinant gene products could be measured.

Adenovirus serotype 5 infects human dendritic cells via a coxsackievirus-adenovirus receptor-independent receptor pathway mediated by lactoferrin and DC-SIGN.

The coxsackievirus-adenovirus receptor (CAR) is the described primary receptor for adenovirus serotype 5 (Ad5), a common human pathogen that has been exploited as a viral vector for gene therapy and vaccination. This study showed that monocytes and dendritic cells (DCs), such as freshly isolated human blood myeloid DCs, plasmacytoid DCs and monocyte-derived DCs, are susceptible to recombinant Ad5 (rAd5) infection despite their lack of CAR expression. Langerhans cells and dermal DCs from skin expressed CAR, but blocking CAR only partly decreased rAd5 infection, together suggesting that other receptor pathways mediate viral entry of these cells.

Early depletion of Mycobacterium tuberculosis-specific T helper 1 cell responses after HIV-1 infection.

Background: The acid-fast bacillus Mycobacterium tuberculosis is often the first manifestation of acquired immunodeficiency syndrome in patients infected with human immunodeficiency virus (HIV). This study was conducted to better understand the mechanism underlying M. tuberculosis-specific pathogenicity early after onset of HIV infection.

Emerging concepts in the immunopathogenesis of AIDS.

There is an intense interplay between HIV and the immune system, and the literature is replete with studies describing various immunological phenomena associated with HIV infection. Many of these phenomena seem too broad in scope to be attributable either to HIV-infected cells or to the HIV-specific immune response. Recently, a more fundamental understanding of how HIV affects various T cells and T cell compartments has emerged.

Biodistribution and toxicological safety of adenovirus type 5 and type 35 vectored vaccines against human immunodeficiency virus-1 (HIV-1), Ebola, or Marburg are similar despite differing adenovirus serotype vector, manufacturer's construct, or gene inserts.

The Vaccine Research Center has developed vaccine candidates for different diseases/infectious agents (including HIV-1, Ebola, and Marburg viruses) built on an adenovirus vector platform, based on adenovirus type 5 or 35. To support clinical development of each vaccine candidate, pre-clinical studies were performed in rabbits to determine where in the body they biodistribute and how rapidly they clear, and to screen for potential toxicities (intrinsic and immunotoxicities). The vaccines biodistribute only to spleen, liver (Ad5 only), and/or iliac lymph node (Ad35 only) and otherwise remain in the site of injection muscle and overlying subcutis.

A SARS DNA vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a Phase I clinical trial.

Background: The severe acute respiratory syndrome (SARS) virus is a member of the Coronaviridae (CoV) family that first appeared in the Guangdong Province of China in 2002 and was recognized as an emerging infectious disease in March 2003. Over 8000 cases and 900 deaths occurred during the epidemic. We report the safety and immunogenicity of a SARS DNA vaccine in a Phase I human study.

Optimizing peptide matrices for identifying T-cell antigens.

Mapping T-cell epitopes for a pathogen or vaccine requires a complex method for screening hundreds to thousands of peptides with a limited amount of donor sample. We describe an optimized deconvolution process by which peptides are pooled in a matrix format to minimize the number of tests required to identify peptide epitopes. Four peptide pool matrices were constructed to deconvolute the HIV-specific T-cell response in three HIV-infected individuals.

Isolation and quantitation of HIV in peripheral blood.

Quantitation of replication-competent human immunodeficiency virus (HIV) in peripheral blood of infected individuals is critical for investigations of HIV pathogenesis and therapy. In this unit, the basic protocol determines the HIV titer in seropositive blood by measuring the tissue culture infectious dose (TCID) by an end-point dilution method. A second basic protocol utilizes the PHA-stimulated T cell blasts (activated T cells) in co-culture with PBMC as described in the first basic protocol for the short-term growth of HIV in vitro.

Immune activation driven by CTLA-4 blockade augments viral replication at mucosal sites in simian immunodeficiency virus infection.

The importance of chronic immune activation in progression to AIDS has been inferred by correlative studies in HIV-infected individuals and in nonhuman primate models of SIV infection. Using the SIV(mac251) macaque model, we directly address the impact of immune activation by inhibiting CTLA-4, an immunoregulatory molecule expressed on activated T cells and a subset of regulatory T cells. We found that CTLA-4 blockade significantly increased T cell activation and viral replication in primary SIV(mac251) infection, particularly at mucosal sites, and increased IDO expression and activity.

Polyfunctional T cell responses are a hallmark of HIV-2 infection.

HIV-2 is distinguished clinically and immunologically from HIV-1 infection by delayed disease progression and maintenance of HIV-specific CD4(+) T cell help in most infected subjects. Thus, HIV-2 provides a unique natural human model in which to investigate correlates of immune protection against HIV disease progression. Here, we report a detailed assessment of the HIV-2-specific CD4(+) and CD8(+) T cell response compared to HIV-1, using polychromatic flow cytometry to assess the quality of the HIV-specific T cell response by measuring IFN-gamma, IL-2, TNF-alpha, MIP-1beta, and CD107a mobilization (degranulation) simultaneously following Gag peptide stimulation.

A West Nile virus DNA vaccine induces neutralizing antibody in healthy adults during a phase 1 clinical trial.

Background: West Nile virus (WNV) is a mosquito-borne flavivirus that can cause severe meningitis and encephalitis in infected individuals. We report the safety and immunogenicity of a WNV DNA vaccine in its first phase 1 human study.

Methods: A single-plasmid DNA vaccine encoding the premembrane and the envelope glycoproteins of the NY99 strain of WNV was evaluated in an open-label study in 15 healthy adults.

Immunisation with BCG and recombinant MVA85A induces long-lasting, polyfunctional Mycobacterium tuberculosis-specific CD4+ memory T lymphocyte populations.

In the search for effective vaccines against intracellular pathogens such as HIV, tuberculosis and malaria, recombinant viral vectors are increasingly being used to boost previously primed T cell responses. Published data have shown prime-boost vaccination with BCG-MVA85A (modified vaccinia virus Ankara expressing antigen 85A) to be highly immunogenic in humans as measured by ex vivo IFN-gamma ELISPOT. Here, we used polychromatic flow cytometry to investigate the phenotypic and functional profile of these vaccine-induced Mycobacterium tuberculosis (M.

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.

Persistent memory CD4+ and CD8+ T-cell responses in recovered severe acute respiratory syndrome (SARS) patients to SARS coronavirus M antigen.

The membrane (M) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is a major glycoprotein with multiple biological functions. In this study, we found that memory T cells against M protein were persistent in recovered SARS patients by detecting gamma interferon (IFN-gamma) production using ELISA and ELISpot assays. Flow cytometric analysis showed that both CD4+ and CD8+ T cells were involved in cellular responses to SARS-CoV M antigen.

Myeloid and plasmacytoid dendritic cells are susceptible to recombinant adenovirus vectors and stimulate polyfunctional memory T cell responses.

Although replication-incompetent recombinant adenovirus (rAd) type 5 is a potent vaccine vector for stimulating T and B cell responses, high seroprevalence of adenovirus type 5 (Ad5) within human populations may limit its clinical utility. Therefore, alternative adenovirus serotypes have been studied as vaccine vectors. In this study, we characterized the ability of rAd5 and rAd35 to infect and induce maturation of human CD11c(+) myeloid dendritic cells (MDCs) and CD123(+) plasmacytoid dendritic cells (PDCs), and their ability to stimulate Ag-specific T cells.

Immunization with vaccinia virus induces polyfunctional and phenotypically distinctive CD8(+) T cell responses.

Vaccinia virus immunization provides lifelong protection against smallpox, but the mechanisms of this exquisite protection are unknown. We used polychromatic flow cytometry to characterize the functional and phenotypic profile of CD8(+) T cells induced by vaccinia virus immunization in a comparative vaccine trial of modified vaccinia virus Ankara (MVA) versus Dryvax immunization in which protection was assessed against subsequent Dryvax challenge. Vaccinia virus-specific CD8(+) T cells induced by both MVA and Dryvax were highly polyfunctional; they degranulated and produced interferon gamma, interleukin 2, macrophage inflammatory protein 1beta, and tumor necrosis factor alpha after antigenic stimulation.

SIV-specific CD8+ T cells express high levels of PD1 and cytokines but have impaired proliferative capacity in acute and chronic SIVmac251 infection.

Programmed death-1 (PD-1) is a critical mediator of virus-specific CD8+ T-cell exhaustion. Here, we examined the expression of PD-1 on simian immunodeficiency virus (SIV)-specific CD8+ T cells and its possible involvement in regulation of cytokine production, proliferation, and survival of these cells. The majority of SIV-specific CD8+ T cells expressed a PD-1(high) phenotype, independent of their differentiation status, in all tissues tested.

Phase I clinical evaluation of a six-plasmid multiclade HIV-1 DNA candidate vaccine.

Needle-free delivery of a six-plasmid HIV-1 DNA vaccine encoding EnvA, EnvB, EnvC, and subtype B Gag, Pol, and Nef underwent open-label evaluation in 15 subjects; 14 completed the 0, 1, 2 month vaccination schedule. T cell responses to HIV-specific peptide pools were detected by intracellular cytokine staining of CD4(+) [13/14 (93%)] and CD8(+) [5/14 (36%)], and by ELISpot in 11/14 (79%). Ten of 14 (71%) had ELISA antibody responses to Env proteins.

Keratinocyte growth factor augments immune reconstitution after autologous hematopoietic progenitor cell transplantation in rhesus macaques.

Opportunistic infections contribute to morbidity and mortality after peripheral blood progenitor cell (PBPC) transplantation and are related to a deficient T-cell compartment. Accelerated T-cell reconstitution may therefore be clinically beneficent. Keratinocyte growth factor (KGF) has been shown to protect thymic epithelial cells in mice.

Interleukin-15 but not interleukin-7 abrogates vaccine-induced decrease in virus level in simian immunodeficiency virus mac251-infected macaques.

The loss of CD4(+) T cells and the impairment of CD8(+) T cell function in HIV infection suggest that pharmacological treatment with IL-7 and IL-15, cytokines that increase the homeostatic proliferation of T cells and improve effector function, may be beneficial. However, these cytokines could also have a detrimental effect in HIV-1-infected individuals, because both cytokines increase HIV replication in vitro. We assessed the impact of IL-7 and IL-15 treatment on viral replication and the immunogenicity of live poxvirus vaccines in SIV(mac251)-infected macaques (Macaca mulatta).

Safety and immunogenicity of a Gag-Pol candidate HIV-1 DNA vaccine administered by a needle-free device in HIV-1-seronegative subjects.

Objective: To evaluate the safety and immunogenicity of a candidate HIV DNA vaccine administered using a needle-free device.

Design: In this phase 1, dose escalation, double-blind, placebo-controlled clinical trial, 21 healthy adults were randomized to receive placebo or 0.5, 1.

Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation.

The role of CD4+ T cells in the control of persistent viral infections beyond the provision of cognate help remains unclear. We used polychromatic flow cytometry to evaluate the production of the cytokines interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2, the chemokine macrophage inflammatory protein (MIP)-1beta, and surface mobilization of the degranulation marker CD107a by CD4+ T cells in response to stimulation with cytomegalovirus (CMV)-specific major histocompatibility complex class II peptide epitopes. Surface expression of CD45RO, CD27, and CD57 on responding cells was used to classify CD4+ T cell maturation.

Safety, immunogenicity and efficacy of modified vaccinia Ankara (MVA) against Dryvax challenge in vaccinia-naïve and vaccinia-immune individuals.

Modified vaccinia Ankara (MVA) was evaluated as an alternative to Dryvax in vaccinia-naïve and vaccinia-immune adult volunteers. Subjects received intramuscular MVA or placebo followed by Dryvax challenge at 3 months. Two or more doses of MVA prior to Dryvax reduced severity of lesion formation, decreased magnitude and duration of viral shedding, and augmented post-Dryvax vaccinia-specific CD8(+) T cell responses and extracellular enveloped virus protein-specific antibody responses.

Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 DNA candidate vaccine.

Background: Gene-based vaccine delivery is an important strategy in the development of a preventive vaccine for acquired immunodeficiency syndrome (AIDS). Vaccine Research Center (VRC) 004 is the first phase 1 dose-escalation study of a multiclade HIV-1 DNA vaccine.

Methods: VRC-HIVDNA009-00-VP is a 4-plasmid mixture encoding subtype B Gag-Pol-Nef fusion protein and modified envelope (Env) constructs from subtypes A, B, and C.