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Abstract: Although cryopreservation of peripheral blood mononuclear cells (PBMC) is a commonly used technique, the degree to which it affects subsequent functional studies has not been well defined. Here we demonstrate that long-term cryopreservation has detrimental effects on T cell IFN-γ responses in human immunodeficiency virus (HIV) infected individuals. Long-term cryopreservation caused marked decreases in CD4+ T cell responses to whole proteins (HIV p55 and cytomegalovirus (CMV) lysate) and HIV peptides, and more limited decreases in CD8+ T cell responses to whole proteins. These losses were more apparent in cells stored for greater than one year compared to less than six months. CD8+ T cell responses to peptides and peptide pools were well preserved. Loss of both CD4+ and CD8+ T cell responses to CMV peptide pools were minimal in HIV-negative individuals. Addition of exogenous antigen presenting cells (APC) did not restore CD4+ T cell responses to peptide stimulation and partially restored T cell IFN-γ responses to p55 protein. Overnight resting of thawed cells did not restore T cell IFN-γ responses to peptide or whole protein stimulation. A selective loss of phenotypically defined effector cells did not explain the decrement of responses, although cryopreservation did increase CD4+ T cell apoptosis, possibly contributing to the loss of responses. These data suggest that the impact of cryopreservation should be carefully considered in future vaccine and pathogenesis studies. In HIV-infected individuals short-term cryopreservation may be acceptable for measuring CD4+ and CD8+ T cell responses. Long-term cryopreservation, however, may lead to the loss of CD4+ T cell responses and mild skewing of T cell phenotypic marker expression. [Copyright &y& Elsevier]

Abstract: The Towne, human cytomegalovirus (CMV) vaccine is safe and immunogenic but has not prevented infection at doses tested to date. We administered 3000pfu Towne CMV vaccine, with or without adjuvant recombinant interleukin-12 (rhIL-12), to CMV-seronegative healthy volunteers and then measured CMV gB-specific IgG titers and CMV-specific CD4+ and CD8+ T cell proliferation and IFNγ expression after stimulation with whole viral lysate and immunodominant peptide CMV antigens. Adjuvant rhIL-12 at doses up to 2μg were well-tolerated and associated with (1) dose-related increases in peak anti-CMV gB IgG titers (though not in sustained titers), (2) dose-related increases in the weak CMV viral lysate-specific CD4+ T cell proliferation responses induced by vaccine alone after 360 days of follow-up, and (3) decreases in the very robust CMV IE-specific peak CD4+ T cell and Day 360 CD8+ T cell proliferation responses induced by the vaccine alone. Also, qualitative CD8+ T cell IFNγ responses to stimulation with the immunodominant CMV antigen, pp65, tended to occur more frequently in vaccinees who received 0.5–2.0μg rhIL-12 compared to lower dose or no rhIL-12. Thus, adjuvant IL-12 may be a promising strategy for improving antibody and T cell immune responses to a CMV vaccine. [Copyright &y& Elsevier]

Abstract: Background: Towne cytomegalovirus (CMV) vaccine is safe and immunogenic, though its protective efficacy has yet to be optimized. Objective: Describe antigen-specific T cell responses to Towne vaccination. Study design: 3000pfu Towne CMV vaccine were given to 12 CMV-seronegative volunteers. CMV-specific CD4+ and CD8+ T cell proliferation and IFNγ expression were measured by flow cytometry after stimulation with CMV lysate or peptides. Results: All vaccinees developed CD4+ and CD8+ T cell proliferation and CD4+ T cell IFNγ responses to multiple CMV antigens, but their CD8+ T cells had low or undetectable IFNγ responses to pp65 peptide pool. The seven HLA-A2+ subjects had higher CD8+ T cell proliferation and IFNγ responses to IE than pp65, and two never developed CD8+ T cell IFNγ responses to pp65. Peak CD4+ T cell IFNγ responses to CMV lysate were lower than values observed in natural CMV seropositives. Initial CD4+ and CD8+ T cell responses to lysate and pp65 waned after 12 months to levels that were lower than those in healthy CMV seropositives, while vaccinees’ CD8+ T cell responses to IE were robust and prolonged. Conclusion: Correlating CMV antigen-specific T cell responses with clinical protective efficacy may facilitate future CMV vaccine development. [Copyright &y& Elsevier]

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 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. [ABSTRACT FROM AUTHOR]