Polio vaccines, SV40 and human tumours, an update on false positive and false negative results.

Simian virus 40 (SV40) has been detected in different human tumours in numerous laboratories. The detection of SV40 in human tumours has been linked to the administration of SV40-contaminated polio vaccines from 1954 until 1963. Many of these reports linked SV40 to human mesothelioma. Some studies have failed to detect SV40 in human tumours and this has caused a controversy. Here we review the current literature. Moreover, we present evidence showing how differences in the sensitivities of methodologies can lead to a very different interpretation of the same study. The same 20 mesothelioma specimens all tested negative, 2/20 tested positive or 7/20 tested positive for SV40 Tag by simply changing the detection method on the same immuno-precipitation/western blot membranes. These results provide a simple explanation for some of the apparent discordant results reported in the literature.

Eradication of established tumors by vaccination with Venezuelan equine encephalitis virus replicon particles delivering human papillomavirus 16 E7 RNA.

The etiological role of human papillomaviruses (HPV) in cervical and other cancers suggests that therapeutic vaccines directed against requisite viral antigens may eradicate tumors or their precursors. A Venezuelan equine encephalitis (VEE) alphavirus vector delivering the HPV16 E7 RNA was evaluated for antitumor efficacy using a murine E7+ tumor model. Vaccination with VEE replicon particles expressing E7 (E7-VRP) induced class I-restricted CD8+ T-cell responses as determined by IFN-gamma enzyme-linked immunospot (ELISPOT), tetramer, and cytotoxicity assays. E7-VRP vaccination prevented tumor development in all of the mice and effectively eliminated 7-day established tumors in 67% of tumor-bearing mice. The induction of protective T-cell responses was dependent on CD8+, but not CD4+ T cells. Long-lasting T-cell memory responses developed in E7-VRP-vaccinated mice as determined by complete protection from tumor challenge 3 months after the final vaccination. These promising results highlight the potent CD8+ T-cell-mediated antitumor effects elicited by VEE replicon-based vectors and support their further development toward clinical testing against cervical intraepithelial neoplasia or carcinoma.

Human T cell responses to endogenously presented HLA-A*0201 restricted peptides of Simian virus 40 large T antigen.

Presence of the simian virus 40 (SV40) has recently been demonstrated in a relatively high percentage of human mesotheliomas and it is associated with the development of these malignancies in pleural cavities. Therefore, we have initiated a study to identify candidate peptides presented by the human HLA-A*0201 molecule for vaccination approaches against SV40 and monitoring of SV40 directed human immune responses. Initial screening of SV40 large T (Tag) domains required for transformation of cells for HLA-A*0201 binding motifs revealed ten possible binding peptides. Screening of these candidate peptides showed that seven of the ten peptides could bind and stabilize HLA-A*0201 molecules. In an in vitro immunization assay the two peptides with the highest binding affinity for HLA-A*0201, Tag aa 396-405 and aa 577-585, were tested for their ability to induce peptide specific cytotoxic T cells in two healthy donors. One donor developed cytotoxic T cells against Tag aa 396-405 and in T cell cultures of both donors Tag aa 577-585 specific T cells were initiated. The T cells against Tag aa 577-585 not only recognized and killed peptide pulsed cells, but, most importantly, SV40 transformed human mesothelial cells. This is the first demonstration of the induction of SV40 specific human cytotoxic T lymphocytes that recognize endogenously processed peptides from SV40. This peptide identification study opens the possibility to investigate immune responses against SV40 in mesothelioma patients and in individuals exposed to SV40.

Pharmacokinetic differences between a T cell-tolerizing and a T cell-activating peptide.

Vaccination with a peptide representing a CTL epitope from the human papillomavirus (HPV)16 E7 protein induces a specific CTL response that prevents the outgrowth of HPV16 E7-expressing tumors. In contrast, vaccination with a peptide encoding an adenovirus type 5 (Ad5) E1A CTL epitope results in CTL tolerance and enhanced growth of an Ad5 E1A-expressing tumor. It is unclear why these peptides induce such opposite effects. To determine whether a difference in pharmacokinetics can explain the functional contrasts, tritiated Ad5 E1A and HPV16 E7 peptides were injected into mice. Results show that the tolerizing peptide spread through the body 16 times faster than the activating peptide and was cleared at least 2 times faster. The HPV16 E7 peptide kinetics correlated with the kinetics of HPV16 E7-specific CTL induction. In contrast, Ad5 E1A peptide injection resulted in physical deletion of preexisting Ad5 E1A-specific CTLs within 24 h after injection. This tolerization occurred at the time when the peptide reached its maximum peptide concentration in the organs. These data suggest that ubiquitous expression of the tolerizing Ad5 E1A peptide within a short period of time causes activation-induced cell death of Ad5 E1A-specific CTLs. Therefore, information on the pharmacokinetics of peptides is vital for the safety and efficacy of peptide-based vaccines.

Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNA vaccine.

Loss of immunogenic epitopes by tumors has urged the development of vaccines against multiple epitopes. Recombinant DNA technologies have opened the possibility to develop multiepitope vaccines in a relatively rapid and efficient way. We have constructed four naked DNA-based multiepitope vaccines, containing CTL, Th cell, and B cell epitopes of the human papillomavirus type 16. Here we show that gene gun-mediated vaccination with an epitope-based DNA vaccine protects 100% of the vaccinated mice against a lethal tumor challenge. The addition of spacers between the epitopes was crucial for the epitope-induced tumor protection, as the same DNA construct without spacers was significantly less effective and only protected 50% of the mice. When tested for therapeutic potential, only the epitope construct with defined spacers significantly reduced the size of established tumors, but failed to induce tumor regression. Only after targeting the vaccine-encoded protein to the protein degradation pathway by linking it to ubiquitin, the vaccine-induced T cell-mediated eradication of 100% of 7-day established tumors in mice. The finding that defined flanking sequences around epitopes and protein targeting dramatically increased the efficacy of epitope string DNA vaccines against established tumors will be of importance for the further development of multiepitope DNA vaccines toward clinical application.