Abstracts

Importance of Tlr4 in the induction of innate and cellular immunity by HspE7

Chu NR, ¹ Liu H, ¹ Recktenwald A, ¹ Mizzen L, ¹ Rowse G, ¹ Siegel MI ²

1. Stressgen Biotechnologies, Victoria, Canada
2. Stressgen Biotechnologies, Collegeville, USA

Clinical responses have been observed using HspE7, an immunotherapeutic vaccine candidate that is a fusion protein containing BCG Hsp65 (Hsp65) and HPV16 E7 (E7). Preclinically HspE7 induces E7-specific cytotoxic T cells (CTL) and regresses an E7-expressing TC-1 tumour. However, both of these activities are absent in Tlr4 null (C57Bl/10 ScN) mice, even though these mice can generate E7-specific CTL using an E7 peptide plus adjuvant. To study the role of Tlr4 more extensively, innate and antibody responses to HspE7 were measured. In peritoneal macrophages from C3H/HeN (Tlr4+) and C3H/HeJ (Tlr4 mutant) mice, HspE7 induces TNF and IL-6 in macrophages, albeit the Tlr4 mutant cells produce lower amounts. Similarly, HspE7 (plus IFN-g) induces IL-12p70 in both Tlr4+ and Tlr4 mutant macrophages, but again the mutant cells produce less. Furthermore, the production of IFN-g by na•ve Tlr4 mutant splenocytes stimulated with HspE7 is also reduced compared with Tlr4+ cells.Ê As a surrogate marker for the participation of CD4+ T helper cells, induction of IgG1 and IgG2a antibody was measured in Tlr4+ and Tlr4 mutant mice immunized with HspE7. Anti-E7 antibody of both isotypes is observed in the Tlr4+ mice, but not in the Tlr4 mutant mice. These findings suggest that the cellular immune response to the E7 portion of HspE7 is Tlr4 dependent and is also consistent with the decreased innate immune responses of the HspE7-stimulated Tlr4 mutant cells. In contrast, similar levels of anti-Hsp65 IgG1 and IgG2a responses are observed in Tlr4+ and mutant mice. Interestingly, while the response to the E7 portion is strictly Tlr4 dependent, the CD4 response to Hsp65 appears to be much less so. Given that effective cellular immunity is associated only with the intact fusion protein, it appears that immunization with HspE7 utilizes both Tlr4-dependent and independent pathways.