Recombinant Listeria Vaccines Containing PEST Sequences Are Potent Immune Adjuvants for the Tumor-Associated Antigen Human Papillomavirus-16 E7

Abstract

Previous work in our laboratory has established that the fusion of tumor-associated antigens to a truncated form of the Listeria monocytogenes virulence factor listeriolysin O (LLO) enhances the immunogenicity and antitumor efficacy of the tumor antigen when delivered by Listeria or by vaccinia. LLO contains a PEST sequence at the NH 2 terminus. These sequences, which are found in eukaryotic proteins with a short cellular half-life, target proteins for degradation in the ubiquitin-proteosome pathway. To investigate whether the enhanced immunogenicity conferred by LLO is due to the PEST sequence, we constructed new Listeria recombinants that expressed the HPV-16 E7 antigen fused to LLO, which either contained or had been deleted of this sequence. We then compared the antitumor efficacy of this set of vectors and found that Listeria expressing the fusion protein LLO-E7 or PEST-E7 were effective at regressing established macroscopic HPV-16 immortalized tumors in syngeneic mice. In contrast, Listeria recombinants expressing E7 alone or E7 fused to LLO from which the PEST sequence had been genetically removed could only slow tumor growth. Because CD8 ؉ T cell epitopes are generated in the ubiquitin-proteosome pathway, we also investigated the ability of the vaccines to induce E7-specific CD8 ؉ T cells in the spleen and to generate E7-specific tumor-infiltrating lymphocytes. A strong correlation was observed between CD8 ؉ T-cell induction and tumor homing and the antitumor efficacy of the Listeria-E7 vaccines. These findings suggest a strategy for the augmentation of tumor antigen-based immunotherapeutic strategies that may be broadly applicable.

Fusion to Listeriolysin O and Delivery by Listeria monocytogenes Enhances the Immunogenicity of HER-2/neu and Reveals Subdominant Epitopes in the FVB/N Mouse

“…However, subsequent to these earlier experiments, we found that fusion of target Ags to either LLO or a PEST-like sequence contained in LLO resulted in an increased antitumor response (31). The exact reason for this enhancement in immunogenicity of fused Ags is not yet clear, but we hypothesize that these fused Ags are more readily degraded by the proteasome and subsequently targeted to the class I pathway.…”

“…However, we believe that LLO is doing more to increase the immunogenicity of target Ags than simply increasing the degradation and subsequent presentation of these Ags. We compared vaccines where the tumor Ag is fused to LLO vs fusion to PEST and showed that both elicited similar antitumor immune responses (31). However, even fusing the Ag to a LLO molecule from which the PEST sequence has been deleted resulted in better antitumor efficacy than using the Ag alone, suggesting that LLO may have an adjuvant effect quite apart from enhancing Ag processing.…”

Harnessing Listeria monocytogenes to target tumors

“…Vaccination with LM-LLO-E7 strongly eradicated E7-expressing tumors, while LM-E7 or LM-LLO-E7 (lacking PEST) did not, despite the presence of strong immune responses to E7 in all three vaccine studies. 48,49 Our vaccine studies showed that immunization with LM-LLO was as effective as LM-LLO-Mage-b against 4T1 metastases, despite the presence of strong Mage-b responses in the LM-LLO-Mage-b vaccinated mice. 70 Also, while LM-LLO was highly effective against 4T1 metastases in vivo, LM-OVA was not (unpublished results).…”

“…48 Interestingly, vaccination with LM-E7 or LM-LLO-E7 missing the PEST (a sequence rich in proline, glutamic acid, serine and threonine) in the amino terminus of LLO, only induced slower growth of TC-1 tumors, despite strong immune responses to E7. 48,49 The Lm-LLO-E7 vaccine was also tested for its ability to break tolerance to a self-tumor antigen using a mouse that was transgenic for HPV-16, E6 and E7. 50 These mice develop autochthonous thyroid tumors, as the transgene is under the control of the thyroglobulin promoter.…”

“…49,84 This vaccine designated as LM-LLO-E7, has been tested in fifteen cancer patients (doses 10 9 to 10 10 CFU) with advanced carcinoma of the cervix in a Phase I/II clinical trial. All patients had a history of failed surgery, chemotherapy and/or radiation.…”