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Abstract
Ex-vivo T cell responses to vaccines and viral antigens are not always detectable by conventional functional T cell assays such as lymphoproliferation, IFN-γ ELIspot and intracellular cytokine staining (ICC) assays. A culture amplified multiparametric intracellular cytokine assay (CAMP-ICC) was developed to detect antigen-specific T cells that may be present at low frequencies, typical of those induced by DNA vaccines in humans. The assay combined methodologies of proliferation detected by carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution and cytokine expression by ICC, amplified by interleukin (IL)-15 and IL-21. The CAMP-ICC provides additional information about the functionality of CD4+ and CD8+ T cells with improved sensitivity, without loss of specificity.
The CAMP-ICC was utilised to uncover priming responses to a candidate prophylactic HIV-1 DNA vaccine. No differences were detected between placebo and active vaccine recipients either pre- or post-vaccination, strongly suggesting this vaccine was non-immunogenic.
In an attempt to uncover mechanisms of immune control of HIV-1, CAMP-ICC was utilised to compare proliferative and cytokine expression profiles to Gag between HIV-1 controllers and non-controllers. Despite performing extensive analyses few comparisons were statistically significant. These were consistent with the current literature.
To understand the developmental stages of CD4+ T cell responses to viral infection and their differentiation into long-term memory cells, individuals immunised with vaccinia virus (VV) were studied. At the peak of primary effector cell responses to VV, day 13 post-vaccination, activated effector (CD45RO+CD38+++) and naïve (CD45RO-CD38dim) CD4+ T cells were purified, mRNA extracted and microarray analysis conducted.
Surprisingly, in activated effector CD4+ compared to naïve CD4+ T cells there was a strong up-regulation of cytotoxic T-lymphocyte (CTL) associated genes, of which Granzyme K was most highly expressed. This finding was confirmed at the mRNA and protein level.
Generation of anti-viral human CD4+ T cell memory during primary immune responses is critical but poorly understood. The role of CTL associated genes in this process has not been explored. Understanding their role in the generation of effective memory may lead us closer to the development of a more effective HIV vaccine or enhance the modestly efficacious vaccines currently available for prevention of HIV-1 infection.