A simple and efficient method for the monitoring of antigen-specific T cell responses using peptide pool arrays in a modified ELISpot assay.

In this study, we describe a simple and efficient method for both the monitoring of antigen-specific CD4 and CD8 T cell responses as well as the identification of novel CD4 and CD8 T cell epitopes using a modified ELISpot assay and pools of 20mer peptides. We have demonstrated that pools containing as many as 64 20mer peptides may be used to screen for CD4 and CD8 T cell responses to HPV16 L1, E1, and E7 in mice. Using arrays of pools of overlapping 20mer peptides, we have identified novel CD4 and CD8 epitopes in both HPV16L1 and HPV16E1 which are presented in Balb/c mice. We have further shown that the use of 20mer peptides is equivalent to using minimal 9mer epitopes for the stimulation of CD8 T cell responses in our assay. While our experiments are conducted in mice, the use of peptide pool arrays allows for the identification of epitope-specific responses using far fewer cells than is required for testing a panel of overlapping peptides individually, making this strategy particularly useful in clinical settings where immune cells may be limiting.

Enhanced type I immune response to a hepatitis B DNA vaccine by formulation with calcium- or aluminum phosphate.

DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. When compared with conventional vaccines, however, DNA vaccines often induce lower antibody titers. We have now found that formulation of a DNA vaccine encoding hepatitis B surface antigen with calcium- or aluminum phosphate adjuvants can increase antibody titers by 10-100-fold and decrease the immunogenic dose of DNA by 10-fold. Furthermore, boosting an HBs protein-primed response with the adjuvanted DNA vaccine resulted in a dramatic increase in the HBs-specific IgG2a response reflecting a shift towards a TH1 response. The mechanism by which aluminum phosphate exerts its adjuvant effect is not through increased expression of HBsAg in vivo; rather, the adjuvant appears to increase the number and affinity of HBs peptide antigen-specific IFN-gamma and IL-2 secreting T cells.

Targeting of HIV-1 antigens for rapid intracellular degradation enhances cytotoxic T lymphocyte (CTL) recognition and the induction of de novo CTL responses in vivo after immunization.

CD8+ cytotoxic T lymphocytes (CTLs) have the ability to recognize and eliminate virally infected cells before new virions are produced within that cell. Therefore, a rapid and vigorous CD8+ CTL response, induced by vaccination, can, in principle, prevent disseminated infection in vaccinated individuals who are exposed to the relevant virus. There has thus been interest in novel vaccine strategies that will enhance the induction of CD8+ CTLs. In this study, we have tested the hypothesis that targeting an antigen to undergo more efficient processing by the class I processing pathway will elicit a more vigorous CD8+ CTL response against that antigen. Targeting a type I transmembrane protein, the HIV-1 envelope (env) protein, for expression in the cytoplasm, rather than allowing its normal co-translational translocation into the endoplasmic reticulum, sensitized target cells expressing this mutant more rapidly for lysis by an env-specific CTL clone. Additionally, a greatly enhanced de novo env-specific CTL response was induced in vivo after immunization of mice with recombinant vaccinia vectors expressing the cytoplasmic env mutant. Similarly, targeting a cytoplasmic protein, HIV-1 nef, to undergo rapid cytoplasmic degradation induced a greatly enhanced de novo nef-specific CD8+ CTL response in vivo after immunization of mice with either recombinant vaccinia vectors or DNA expression plasmids expressing the degradation targeted nef mutant. The targeting of viral antigens for rapid cytoplasmic degradation represents a novel and highly effective vaccine strategy for the induction of enhanced de novo CTL responses in vivo.

Transporter-independent processing of HIV-1 envelope protein for recognition by CD8+ T cells.

CD8+ cytolytic T lymphocytes (CTL) identify virally infected cells by recognizing processed viral antigen in association with class I major histocompatibility complex (MHC) molecules on infected cells. Processing begins in the cytosol with the generation of peptides, possibly by a protease complex with MHC-encoded subunits, known as the proteasome. Transport of the resulting cytosolic peptides into the endoplasmic reticulum for association with class I molecules is essential and probably involves a heterodimer of the MHC-encoded proteins, Tap-1 and Tap-2. The site of processing of viral envelope proteins is uncertain. These proteins are not present in the cytosol because of cotranslational translocation into the endoplasmic reticulum. We show here that the HIV-1 envelope (env) protein is processed in infected cells by a novel Tap-1/Tap-2-independent pathway that seems to be localized to the endoplasmic reticulum.