SA-4-1BBL as the immunomodulatory component of a HPV-16 E7 protein based vaccine shows robust therapeutic efficacy in a mouse cervical cancer model

Abstract

Cervical cancer is the leading cause of cancer-related deaths among women worldwide. Current prophylactic vaccines based on HPV (Human papillomavirus) late gene protein L1 are ineffective in therapeutic settings. Therefore, there is an acute need for the development of therapeutic vaccines for HPV associated cancers. The HPV E7 oncoprotein is expressed in cervical cancer and has been associated with the cellular transformation and maintenance of the transformed phenotype. As such, E7 protein represents an ideal target for the development of therapeutic subunit vaccines against cervical cancer. However, the low antigenicity of this protein may require potent adjuvants for therapeutic efficacy. We recently generated a novel chimeric form of the 4-1BBL costimulatory molecule engineered with core streptavidin (SA-4-1BBL) and demonstrated its safe and pleiotropic effects on various cells of the immune system. We herein tested the utility of SA-4-1BBL as the immunomodulatory component of HPV-16 E7 recombinant protein based therapeutic vaccine in the E7 expressing TC-1 tumor as a model of cervical cancer in mice. A single subcutaneous vaccination was effective in eradicating established tumors in approximately 70% of mice. The therapeutic efficacy of the vaccine was associated with robust primary and memory CD4(+) and CD8(+) T cell responses, Th1 cytokine response, infiltration of CD4(+) and CD8(+) T cells into the tumor, and enhanced NK cell killing. Importantly, NK cells played an important role in vaccine mediated therapy since their physical depletion compromised vaccine efficacy. Collectively, these data demonstrate the utility of SA-4-1BBL as a new class of multifunctional immunomodulator for the development of therapeutic vaccines against cancer and chronic infections.

Fig. 1

Characterization of the purified recombinant E7 protein. IMAC purified recombinant E7 protein was run on an SDS-PAGE and stained with Coomassie (A) or analyzed using antibodies against E7 in Western blots (B). Proteins of the indicated kDa used as standard size markers (M).

Fig. 2

Vaccination with E7 protein and SA-41BBL generates potent primary T cell responses. (A) In vivo endogenous CTL killing response. Naïve C57BL/6 (CD45.2⁺) mice were immunized s.c. with E7 (50 μg) + SA-4-1BBL (25 μg). Seven days post vaccination, mice received E7_49-57-pulsed syngeneic splenocytes from C57BL/6.SJL (CD45.1⁺) as targets. In vivo peptide-specific killing was assessed 2 days later, and expressed as percent lysis for each histogram (n = 3–4; ** p < 0.001 compared with E7+SA or PBS controls). (B) E7+SA-4-1BBL treated mice generate vigorous in vitro proliferative responses to the E7 protein. Splenocytes from (A) were pulsed with 5 μg of E7 protein/ml/10⁶ cells and incubated for 5 days, and proliferation was assessed by [³H] thymidine incorporation. Results are representative of three independent experiments (** p < 0.001).

Fig. 3

Vaccination with E7 protein and SA-4-1BBL eradicates established TC-1 tumors and NK cells play an important role in vaccine efficacy. (A) C57BL/6 mice were challenged with 1×10⁵ live TC-1 cells and vaccinated once s.c. on day 6 post-tumor challenge with E7 protein (50 μg) mixed with SA-4-1BBL (25 μg) or an equimolar quantity of SA (10 μg). The role of NK cells in the observed vaccine efficacy was tested by depleting these cells with i.p. injection of 500 μg anti-NK1.1 Ab/mouse one day before vaccination. *p < 0.05 for E7+SA-4-1BBL vs. NK depleted/E7+SA-4-1BBL group. ** p < 0.001 for E7+SA-41BBL vs. PBS and E7+SA groups. (B) The vaccine efficacy of E7+SA-4-1BBL is strictly dependent on 4-1BB signaling. C57BL/6 4-1BB^−/− mice were vaccinated as in (A). p < 0.05 for E7+SA-4-1BBL or E7+SA vs. PBS group.

Fig. 4

Therapeutic efficacy of E7 and SA-4-1BBL vaccine is associated with the generation of potent T cell effector and Th1 cytokine responses. (A) Long-term mice with effective therapy were boosted with E7+SA-4-1BBL and 7 days later tested for (A) E7_49-57-specific in vivo killing response (n = 3, ** p < 0.001 vs. naive control), (B) E7-specific proliferation, (C) intracellular IL-2 response (n = 3, ** p < 0.001 vs. naïve control), and (D) IFN-γ response (n = 3, * p < 0.05 vs. naive control). Naïve mice were used as controls.

Fig. 5

Vaccination with E7 protein and SA-4-1BBL results in enhanced long-term peripheral T cell memory pool and infiltration into the tumor. (A) Splenocytes from long-term tumor-challenged mice with effective immunotherapy were harvested 7 days after boosting with E7+SA-4-1BBL and subjected to phenotypic analysis using various T cell markers in multiparameter flow cytometry. Data is representative of two independent experiments. (B, C) Mice with TC-1 tumors of 3–5 mm in diameter were injected with E7+SA-4-1BBL, E7+SA, or PBS. Tumors were harvested 7 days later and subjected to analysis using flow cytometry (**p < 0.001) or confocal microscopy (C). Data are representative of 3–4 mice per treatment group per panel.

Fig. 6

Mice with effective immunotherapy generates robust NK cell killing response. Long-term mice with effective immunotherapy were boosted by s.c. vaccination with E7+SA-4-1BBL. Splenocytes were harvested 7 days later and pulsed with 5 μg of E7/ml/10⁶ cells in the presence of 50 IU IL-2 and incubated for 5 days. The viable cells were isolated using Ficoll gradient centrifugation and used as effectors against [³H] labeled YAC-1 cells as targets at various effector:target (E:T) ratios. Percent specific cytotoxicity is graphed separately for 3 experimental mice. Effector cells prepared from splenocytes harvested from unimmunized naive mice cultured under similar conditions were used as controls.