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

Abstract

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.

Figures

FIG. 1.

Binding of HPV16 L1 peptides to Kb (A) or Db (B) molecules on RMA-S cells. HPV16 L1 peptides are shown in Table 1. Stability of the MHC molecules was measured by fluorescence-activated cell sorting analysis. Note that only the Db-specific peptide L1165-173 (in panel B) induces stability similar to that of the naturally processed epitope (NP366-374).

FIG. 2.

Expression of HPV16 L1 in B6 cells after infection with recombinant HPV16 L1 MVA (MVA-L1ΔC). Western blot analysis of protein extracts of cells (infected with MVA-L1ΔC or MVA-F6 [nonrecombinant]) obtained 16 h and 4 days (4d) postinfection (pi). The specificity of the monoclonal antibody was confirmed by detection of L1 in purified VLPs obtained from recombinant baculovirus (pos). The slight difference in molecular mass between L1 protein obtained from VLPs and from recombinant MVA is due to the 34-aa C-terminal deletion in the latter (L1ΔC). The lower ca. 45-kDa band represents a cross-reacting cellular or vaccinia virus protein occurring with a different kinetics after infection by recombinant and nonrecombinant vaccinia virus (not visible 4 days after MVA-F6 infection).

FIG. 3.

CTL activity against HPV16 L1165-173. Splenocytes of C57/B6 mice immunized with buffer (PBS 1 and PBS 2) or HPV16ΔCE71-55 CVLPs (30) (CVLP 1 to 3) were restimulated with MVA-L1ΔC-infected syngeneic cells (B6) and exposed to RMA-S cells loaded with peptide L1165-173 or NP366-372 (negative control), and 51chromium was measured in the supernatant. The percentage of specific lysis is shown on the y axes.

FIG. 4.

Size distribution of the HPV16 L1ΔN10 protein. Purified VLPs (top blot) or L1ΔN10 protein (capsomeres [bottom blot]) was sedimented through a linear sucrose gradient, and 30-μl portions of individual fractions (collected from top to bottom of the tube) were analyzed by Western blotting using an L1-specific monoclonal antibody. The starting materials are shown at the left in both blots. VLPs used in this experiment consist of the C-terminally truncated L1 (HPV16 L1ΔC) (30), so the L1 molecules are slightly smaller than those in the capsomeres and show two bands due to partial degradation. VLPs are found in the bottom fractions and in the pellet (P). Sedimentation of the bulk of HPV16 L1ΔN10 protein is consistent with pentameric capsomeres. The positions of the marker proteins bovine serum albumin (4S) and catalase (11S) analyzed in parallel tubes are indicated.

FIG. 5.

Growth of C3 tumors in C57BL/6 mice after s.c. immunization with HPV16 L1 capsomeres. Mice received tumor cells and were immunized with 10 μg of capsomeres or with PBS when the tumors reached an average size of 25 mm2. Surface tumor size was measured and is indicated for the individual mice over time. Because of the size of tumors in the control group (PBS), the experiment was terminated at day 29.

FIG. 6.

CTL responses in C57BL/6 mice after immunization (i.n. or s.c.) with HPV16 capsomeres. Mice (four in each group) were immunized i.n. with PBS or HPV16 L1 capsomeres (5 or 10 μg) with or without CTB or s.c. with capsomeres (5 μg). (a) ELISPOT after two in vitro restimulations of the splenocytes. Each bar represents the number of activated T cells from one mouse. The mice were immunized i.n., except for one group that received the capsomeres s.c. The numbers (means ± SEMs) of IFN-γ-expressing T cells/104 splenocytes for the groups follow: 0.0 ± 0.0 for the PBS group (control), 8.8 ± 1.7 for the group given 10 μg of capsomeres and CTB, 12.5 ± 1.3 for the group given 10 μg of capsomeres, 10.8 ± 0.5 for the group given 5 μg of capsomeres and CTB, 16.5 ± 2.5 for the group given 5 μg of capsomeres, and 28.5 ± 3.8 for the group given 5 μg of capsomeres s.c. (b) 51Cr release assay after four in vitro restimulations. Specific lysis is shown against RMA-S cells (black circles) and RMA-S cells loaded with peptide L1165-173 (open circles). The two individual mice shown in the graphs in panel b correspond to the mice in panel a as follows (counting from left to right for each group shown in panel a): control (PBS), mouse 1 and 3; 5μg s.c., mouse 2 and 3; 10μg i.n., mouse 1 and 3; 10μg + CTB i.n., mouse 1 and 4; 5μg i.n., mouse 1 and 2; 5μg + CTB i.n., mouse 2 and 3.