Enumeration and Characterization of Human Memory T Cells by Enzyme-Linked Immunospot Assays

The enzyme-linked immunospot (ELISPOT) assay has advanced into a useful and widely applicable tool for the evaluation of T-cell responses in both humans and animal models of diseases and/or vaccine candidates. Using synthetic peptides (either individually or as overlapping peptide mixtures) or whole antigens, total lymphocyte or isolated T-cell subset responses can be assessed either after short-term stimulation (standard ELISPOT) or after their expansion during a 10-day culture (cultured ELISPOT). Both assays detect different antigen-specific immune responses allowing the analysis of effector memory T cells and central memory T cells. This paper describes the principle of ELISPOT assays and discusses their application in the evaluation of immune correlates of clinical interest with a focus on the vaccine field

Single DermaVir Immunization: Dose-Dependent Expansion of Precursor/Memory T Cells against All HIV Antigens in HIV-1 Infected Individuals

BACKGROUND: The GIHU004 study was designed to evaluate the safety and immunogenicity of three doses of DermaVir immunization in HIV-infected subjects on fully suppressive combination antiretroviral therapy (cART). METHODOLOGY/PRINCIPAL FINDINGS: This first-in-human dose escalation study was conducted with three topical DermaVir doses targeted to epidermal Langerhans cells to express fifteen HIV antigens in draining lymph nodes: 0.1 mg DNA targeted to two, 0.4 mg and 0.8 mg DNA targeted to four lymph nodes. Particularly, in the medium dose cohort 0.1 mg DNA was targeted per draining lymph node via ∼8 million Langerhans cells located in 80 cm(2) epidermis area. The 28-days study with 48-week safety follow-up evaluated HIV-specific T cell responses against Gag p17, Gag p24 and Gag p15, Tat and Rev antigens. DermaVir-associated side effects were mild, transient and not dose-dependent. Boosting of HIV-specific effector CD4(+) and CD8(+) T cells expressing IFN-gamma and IL-2 was detected against several antigens in every subject of the medium dose cohort. The striking result was the dose-dependent expansion of HIV-specific precursor/memory T cells with high proliferation capacity. In low, medium and high dose cohorts this HIV-specific T cell population increased by 325-, 136,202 and 50,759 counts after 4 weeks, and by 3,899, 9,878 and 18,382 counts after one year, respectively, compared to baseline. CONCLUSIONS/SIGNIFICANCE: Single immunization with the DermaVir candidate therapeutic vaccine was safe and immunogenic in HIV-infected individuals. Based on the potent induction of Gag, Tat and Rev-specific memory T cells, especially in the medium dose cohort, we speculate that DermaVir boost T cell responses specific to all the 15 HIV antigens expressed from the single DNA. For durable immune reactivity repeated DermaVir immunization might be required since the frequency of DermaVir-boosted HIV-specific memory T cells decreased during the 48-week follow up. TRIAL REGISTRATION: ClinicalTrial.gov NCT00712530

Standardization of cytokine flow cytometry assays

BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4(+)cytokine(+ )cells and CD8(+)cytokine(+ )cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24%) for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82%) for samples with a mean of <0.1% IFNγ + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays

Modulation of HIV immune responses in natural infection and after genetic immunization

Identification and treatment of HIV-1 infection are both urgent, in view of the continued spread of the infection. We developed easy and inexpensive serological methods for the diagnosis and follow-up of children born to HIV-1-infected mothers. A quantitative immunofluorescence method replaced the cumbersome Western blot. The possibility of titrating antibodies made discrimination between HIV-infected and noninfected children feasible. In addition, we showed that an anti-HIV-1 IgA assay is effective for early diagnosis of HIV-1-infected children, with significant and high detection in children over 6 months of age. Epitope reactivity might discriminate between different HIV-1 infected populations. Specificity of antibodies directed to conserved regions of HIV-1 was compared between patients from Argentina and Sweden. A new irnmunodominant region of gp41 was identified, against which the majority of Argentinian and Swedish patients showed reactivity. Although the epitope did not appear to be involved in functional reactivity, it may be useful as a diagnostic tool. Reactivity against peptides representing the irnmunodominant third variable region of HIV-1 gpl20 was analyzed in sera from Argentinian patients. Some peptides representing the subtype B showed low reactivities, indicating the need to identify and characterize local strains in order to design adequate diagnostic and vaccine strategies. The efficacy of HIV-DNA, a novel class of vaccines, was evaluated for induction of immune responses in humans. Asymptomatic HIV-1-infected patients were immunized with DNA constructs encoding either HIV-1 nef, rev or tat regulatory proteins. Patients were selected for having no or low antibody reactivities to the antigen encoded by the plasmid DNA used for immunization. The DNA immunization induced HIV-specific cytotoxic and proliferative cellular responses. Increased levels of cytotoxic memory cells were induced in all DNAimmunized patients. Antibody induction was of a low magnitude. This demonstrates for the first time that HIV-DNA vaccination may be capable of inducing or reinducing immune responses in HIV-infected humans. Intensive chemotherapy is capable of reducing the viral load in HIV-1 infected individuals while infected cells are still present. HIV-specific cellular immune responses were evaluated in patients who started highly active antiretroviral treatment (HAART) during or after DNA immunizations. Significant reductions in viral load and increases in CD4+ counts were observed in those patients. DNA immunization by itself did not reduce viral load. The initiation of HAART therefore appears to contribute to the induction of HIV-specific CTL responses but by itself did not cause obvious re-induction of these activities. The efficacy of a combination of plasmids encoding the HIV-1 regulatory genes (nef, rev and tat) was evaluated. The most remarkable change observed after immunization with the gene combination was the appearance of memory CTL against autologous targets infected with HIV-1. Autologous cells infected with HIV-1 will present all viral peptides and represent the in vivo situation. The possibility of enhancing HIV-specific immune responses in immunodeficient individuals is promising for continued genetic vaccine development

Enumeration and Characterization of Human Memory T Cells by Enzyme-Linked Immunospot Assays

The enzyme-linked immunospot (ELISPOT) assay has advanced into a useful and widely applicable tool for the evaluation of T-cell responses in both humans and animal models of diseases and/or vaccine candidates. Using synthetic peptides (either individually or as overlapping peptide mixtures) or whole antigens, total lymphocyte or isolated T-cell subset responses can be assessed either after short-term stimulation (standard ELISPOT) or after their expansion during a 10-day culture (cultured ELISPOT). Both assays detect different antigen-specific immune responses allowing the analysis of effector memory T cells and central memory T cells. This paper describes the principle of ELISPOT assays and discusses their application in the evaluation of immune correlates of clinical interest with a focus on the vaccine field

Approaches for monitoring of non virus-specific and virus-specific T-cell response in solid organ transplantation and their clinical applications

Opportunistic viral infections are still a major complication following solid organ transplantation. Immune monitoring may allow the identification of patients at risk of infection and, eventually, the modulation of immunosuppressive strategies. Immune monitoring can be performed using virus-specific and non virus-specific assays. This article describes and summarizes the pros and cons of the different technical approaches. Among the assays based on non virus-specific antigens, the enumeration of T-cell subsets, the quantification of cytokines and chemokines and the quantification of intracellular adenosine triphosphate following mitogen stimulation are described and their clinical applications to determine the risk for viral infection are discussed. In addition, current specific methods available for monitoring viral-specific T-cell responses are summarized, such as peptide-MHC multimer staining, intracellular cytokine staining, enzyme-linked immunospot and virus-specific IFN-γ ELISA assays, and their clinical applications to determine the individual risk for opportunistic viral infections with human cytomegalovirus, Epstein-Barr virus and polyoma BK virus are discussed. The standardization of the procedure, the choice of the antigen(s) and the criteria to define cut-off values for positive responses are needed for some of these approaches before their implementation in the clinic. Nevertheless, immune monitoring combined with virological monitoring in transplant recipients is increasingly regarded as a helpful tool to identify patients at risk of infection as well as to assess treatment efficacy

Memory T cells specific for HBV enumerated by a peptide-based cultured enzyme-linked immunospot assay in healthy HBV-vaccinated subjects

Hepatitis B vaccine is the most effective strategy to control hepatitis B virus (HBV) infection and disease. It is considered that an anti-HBs (antibodies against HBV surface antigen) titer >10 mIU/ml, measured shortly after a complete vaccination schedule, provides protection against infection. Approximately 4-10% of healthy individuals fail to respond to 3-dose vaccination. Long-term HBV-specific memory T-cell response has not been fully investigated, mainly due to the lack of a suitable assay. We quantified HBV-specific expandable memory T cells by using a cultured IFN-γ enzyme-linked immunospot (ELISPOT) assay. Cultured ELISPOT response to an overlapping peptide pool representing the complete L (large) HBV envelope polypeptide was evaluated in 41 healthy subjects vaccinated 15-20 y earlier and 5 unvaccinated. Plasma samples were tested for anti-HBs. Vaccinated subjects had significantly higher HBV-specific T-cellular response than unvaccinated (p = 0.0002). HBV-specific T-cell response was mainly mediated by CD4+ T cells. No concordance was found between cultured ELISPOT and anti-HBs data in vaccinated subjects. Thirty-one (76%) vaccinated subjects were responders (anti-HBs >10 mIU/ml). Nineteen (46%) vaccinated subjects were considered to be responders in cultured ELISPOT. Twenty-two (54%) vaccinated subjects were considered non-responders in cultured ELISPOT; 5 of them (23%) were also humoral non-responders. About 12% of healthy HBV-vaccinated subjects are both humoral and cellular non-responders. Although the prognostic value of this assay has not been established in terms of predictability for susceptibility to de-novo HBV infection, ELISPOT data suggest that these subjects may be at risk for HBV infection and disease, especially health care workers

Evaluation of EBV- and HCMV-Specific T Cell Responses in Systemic Lupus Erythematosus (SLE) Patients Using a Normalized Enzyme-Linked Immunospot (ELISPOT) Assay

Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex etiology. Opportunistic viral pathogens, such as human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV), are particularly relevant. The role of the T cell response in SLE has not been deeply studied; we investigated the role of HCMV- and EBV-specific T cell responses in SLE patients also in relation to their pharmacological immunosuppressive status. PBMCs from 70 SLE patients and 50 healthy controls were stimulated with EBV- and HCMV-specific antigens, and IFN-γ-secreting T cells were quantified. We observed that both EBV- and HCMV-specific T cell responses were significantly lower in SLE patients compared with healthy subjects. We reported decreased EBV- and HCMV-specific T cell responses among medium-high immunosuppressed patients compared to low immunosuppressed patients. Immunosuppressive level could exert a role in the control of herpesviruses reactivation, even if the immunosuppressive condition of SLE remains the driving cause of skewed virus-specific T cell response

T-lymphocyte subsets in lung transplant recipients: Association between nadir CD4 T-cell count and viral infections after transplantation

BACKGROUND: Little is known about the kinetics of T-cell subsets in lung transplant recipients (LTR) and their association with the occurrence of opportunistic infections (OI). OBJECTIVES: To analyze the kinetics of T-lymphocyte subsets in LTR and the association between nadir CD4 T-cell count and viral infections after transplantation. STUDY DESIGN: Serial measurements of peripheral blood CD4 and CD8 T-cell counts obtained during the first year post-transplantation from 83 consecutive LTR and their correlation with both viral OI and community-acquired infections post-transplantation were retrospectively analyzed. RESULTS: LTR with a nadir CD4 T-cell count 200 cells/μl (p200 cells/μl (p=0.0012 and p=0.0058, respectively). A nadir CD4 T-cell count <200 cells/μl within the first three months post-transplantation predicted a higher frequency of viral infectious episodes in BAL samples within the subsequent six month period (p=0.0066). CONCLUSIONS: Stratification of patients according to nadir CD4 T-cell count may represent a new and simple approach for early identification of patients at risk for subsequent virus infections

VS411 reduced immune activation and HIV-1 RNA levels in 28 days: randomized proof-of-concept study for antiviral-hyperactivation limiting therapeutics.

A new class of antiretrovirals, AntiViral-HyperActivation Limiting Therapeutics (AV-HALTs), has been proposed as a disease-modifying therapy to both reduce Human Immunodeficiency Virus Type 1 (HIV-1) RNA levels and the excessive immune activation now recognized as the major driver of not only the continual loss of CD4(+) T cells and progression to Acquired Immunodeficiency Syndrome (AIDS), but also of the emergence of both AIDS-defining and non-AIDS events that negatively impact upon morbidity and mortality despite successful (ie, fully suppressive) therapy. VS411, the first-in-class AV-HALT, combined low-dose, slow-release didanosine with low-dose hydroxycarbamide to accomplish both objectives with a favorable toxicity profile during short-term administration. Five dose combinations were administered as VS411 to test the AV-HALT Proof-of-Concept in HIV-1-infected subjects.Multinational, double-blind, 28-day Phase 2a dose-ranging Proof-of-Concept study of antiviral activity, immunological parameters, safety, and genotypic resistance in 58 evaluable antiretroviral-naïve HIV-1-infected adults. Randomization and allocation to study arms were carried out by a central computer system. Results were analyzed by ANOVA, Kruskal-Wallis, ANCOVA, and two-tailed paired t tests.VS411 was well-tolerated, produced significant reductions of HIV-1 RNA levels, increased CD4(+) T cell counts, and led to significant, rapid, unprecedented reductions of immune activation markers after 28 days despite incomplete viral suppression and without inhibiting HIV-1-specific immune responses. The didanosine 200 mg/HC 900 mg once-daily formulation demonstrated the greatest antiviral efficacy (HIV-1 RNA: -1.47 log(10) copies/mL; CD4(+) T cell count: +135 cells/mm(3)) and fewest adverse events.VS411 successfully established the Proof-of-Concept that AV-HALTs can combine antiviral efficacy with rapid, potentially beneficial reductions in the excessive immune system activation associated with HIV-1 disease. Rapid reductions in markers of immune system hyperactivation and cellular proliferation were obtained despite the fact that VS411 did not attain maximal suppression of HIV RNA, suggesting this effect was due to the HALT component.ITEudraCT 2007-002460-98