Survivin is an intracellular tumor-associated antigen that is broadly expressed in a large variety of tumors and also in tumor associated endothelial cells but mostly absent in differentiated tissues. Naked DNA vaccines targeting survivin have been shown to induce T cell as well as humoral immune responses in mice. However, the lack of epitope-specific CD8+ T cell detection and modest tumor protection observed highlight the need for further improvements to develop effective survivin DNA vaccination approaches. Here, the efficacy of a human survivin DNA vaccine delivered by intradermal electroporation (EP) was tested. The CD8+ T cell epitope surv(20-28) restricted to H-2 Db was identified based on in-silico epitope prediction algorithms and binding to MHC class I molecules. Intradermal DNA EP of mice with a human survivin encoding plasmid generated CD8+ cytotoxic T lymphocyte (CTL) responses cross-reactive with the mouse epitope surv(20-28), as determined by intracellular IFN-gamma staining, suggesting that self-tolerance has been broken. Survivin-specific CTLs displayed an activated effector phenotype as determined by CD44 and CD107 up-regulation. Vaccinated mice displayed specific cytotoxic activity against B16 and peptide-pulsed RMA-S cells in vitro as well as against surv(20-28) peptide-pulsed target cells in vivo. Importantly, intradermal EP with a survivin DNA vaccine suppressed angiogenesis in vivo and elicited protection against highly aggressive syngeneic B16 melanoma tumor challenge. We conclude that intradermal EP is an attractive method for delivering a survivin DNA vaccine that should be explored also in clinical studies.

To study DNA vaccination directed against human HER-2 in the HHD mouse Tg strain, we created a novel HER-2-expressing syngeneic tumor transplantation model. We found that a DNA vaccine encoding the full length HER-2 DNA protected HHD mice from HER-2(+) tumor challenge by a CTL independent mechanism. A more efficient approach to induce HLA-A2 restricted CTLs, through immunization with a multi-epitope DNA vaccine expressing the HLA-A2 restricted HER-2 369-377, 435-443 and 689-697 epitopes, resulted in high numbers of peptide specific T cells but failed to induce tumor protection. Subsequently we discovered that HER-2 transfected tumor cells down-regulated MHC class I antigen expression and exhibited a series of defects in the antigen processing pathway which impaired the capacity to produce and display MHC class I peptide-ligands to specific CTLs. Our data demonstrate that HER-2 transfection is associated with defects in the MHC class I presentation pathway, which may be the underlying mechanism behind the inability of CTLs to recognize tumors in this HLA-A2 transgenic model. As defective MHC class I presentation may be a common characteristic of HER-2 expressing tumors, vaccines targeting HER-2 should aim at inducing an integrated immune response where also CD4(+) T cells and antibodies are important components.

Summary The composition of lymphocyte subsets in the lung has been found to be compartment‐specific. To characterize the effect of age, weanling, young adult and adult rats were studied in control conditions and after a single intratracheal dose of the Toll‐like receptor 2/6 (TLR2/6) agonist macrophage activating lipopeptide‐2 (MALP‐2). In all age groups, T, B and natural killer (NK) cells increased dramatically in the epithelium and lamina propria of the bronchi. Male adult rats were found to have responded to MALP‐2 to a much greater extent than females when lymphocyte subsets were counted in the epithelium and the lamina propria. In a second series of experiments the time kinetics of regulatory T‐cell (Treg) subsets and dendritic cells (DCs) in the lung was studied after local stimulation with MALP‐2. Different time‐dependent patterns were found in the Treg subsets CD4+ CD25+, CD4+ CD25+ neuropilin 1+ and CD4+ CD25+ Foxp3+ cells. Neutrophils and DCs also showed different patterns. Thus, the local application of a TLR agonist increased the number of lymphocyte subsets in a compartment‐specific pattern. However, data should not be generalized or extrapolated from one age group, sex or lymphocyte subpopulation to another.

The ability of heat shock proteins (HSPs) to increase the potency of protein-and DNA-based vaccines has been previously reported. We have constructed several plasmid-based vectors encoding chimeric proteins containing prostate-specific antigen (PSA) fused to Mycobacterium tuberculosis hsp70, M. bovis hsp65, Escherichia coli DnaK (hsp70), or human hsp70. Immunizing mice with these plasmids induced CD8 + cytotoxic T lymphocytes (CTLs) specific to human PSA and protected mice from a subsequent subcutaneous challenge with PSA-expressing tumors. We did not observe a significant difference either in the levels of PSAspecific CTLs or in protection against tumor challenge in mice immunized with plasmids expressing PSA-HSP chimeric proteins, as compared to mice receiving a conventional PSA-expressing DNA plasmid. Our data indicate that using HSPs as fusion partners for tumorspecific antigens does not always result in the enhancement of antigen-specific CTL responses when applied in the form of DNA vaccines.