Human papillomavirus type 16 L1 capsomeres induce L1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6 mice.

We analyzed capsomeres of human papillomavirus type 16 (HPV16) consisting of the L1 major structural protein for their ability to trigger a cytotoxic T-cell (CTL) response. To this end, we immunized C57BL/6 mice and used the L1(165-173) peptide for ex vivo restimulation of splenocytes prior to analysis ((51)Cr release assay and enzyme-linked immunospot assay [ELISPOT]). This peptide was identified in this study as a D(b)-restricted naturally processed CTL epitope by HPV16 L1 sequence analysis, major histocompatibility complex class I binding, and (51)Cr release assays following immunization of C57BL/6 mice with HPV16 L1 virus-like particles (VLPs). HPV16 L1 capsomeres were obtained by purification of HPV16 L1 lacking 10 N-terminal amino acids after expression in Escherichia coli as a glutathione S-transferase fusion protein (GST-HPV16 L1 Delta N10). Sedimentation analysis revealed that the majority of the purified protein consisted of pentameric capsomeres, and assembled particles were not observed in minor contaminating higher-molecular-weight material. Subcutaneous (s.c.) as well as intranasal immunization of C57BL/6 mice with HPV16 L1 capsomeres triggered an L1-specific CTL response in a dose-dependent manner as measured by ELISPOT and (51)Cr release assay. Significant reduction of contaminating bacterial endotoxin (lipopolysaccharide) from the capsomere preparation did not diminish the immunogenicity. Antibody responses (serum and vaginal) were less robust under the experimental conditions employed. In addition, s.c. vaccination with HPV16 L1 capsomeres induced regression of established tumors expressing L1 determinants (C3 tumor cells). Our data demonstrate that capsomeres are potent inducers of CTL responses similar to completely assembled T=7 VLPs. This result is of potential relevance for the development of (combined prophylactic and therapeutic) HPV-specific vaccines, since capsomeres can be produced easily and also can be modified to incorporate heterologous sequences such as early HPV proteins.

Chimeric virus-like particles of the human papillomavirus type 16 (HVP 16) as a prophylactic and therapeutic vaccine

Infection by certain human papillomaviruses (HPV), most notably HPV types 16 and 18, is the mejor risk factor cervical cancer. Worldwide, this disease represents the second most frequent malignant tumor in women; thus, there is urgent need for efficient therapy and prevention. The natural history of cervical cancer and its precursors (cervical intraepithelial neoplasias), as well as animal experiments, strogly suggest that immune system controls both the primary infection (by neutralizing antibodies directed against the major structural protein L1) and the progression o the disease (via cytotoxic T cell specific for the viral oncoproteins expressed in transformed cells, e.g., E7). By the expression of an HPV 16 L1E7 fusion proteins, we have generated chimeric virus-like particles (CVLP). Immunization of mice with CVLPs induces neutralizing antibodies directed against L1 virus-like particles (devoid of the E7 portion) and E7-specific t cells as measured in vitro. Vaccinated animals are protected against tumor growth followings inoculation of syngeneic HPV 16-transformed cells. In addition, we observed a therapeutic effect of vaccination on pre-existing tumors. This data allowed us to concelude that CVLPs are suitable for prevention and therapy of HPV infection. A vaccine based on HPV 16 L1E7 CVLPs is currently under development