Therapeutic efficacy of a human papillomavirus type 16 E7 bacterial exotoxin fusion protein adjuvanted with CpG or GPI-0100 in a preclinical mouse model for HPV-associated disease

Abstract

Persistent human papillomavirus (HPV) infection is causally linked to the development of several human cancers, including cervical, vulvar, vaginal, anal, penile, and oropharyngeal cancers. To address the need for a therapeutic vaccine against HPV-associated diseases, here we test and compare the immunogenicity and therapeutic efficacy of a bacterial exotoxin fusion protein covalently linked to the HPV16 E7 oncoprotein adjuvanted with CpG or GPI-0100 in the C3.43 preclinical HPV16-transformed tumor model. We show that TVGV-1 protein vaccine adjuvanted with either CpG or GPI-0100 adjuvant induces a high frequency of E7-specific CD8⁺ T cells, and both adjuvants are able to assist the immune response in inducing polyfunctional cytokine-secreting lytic T cells that show therapeutic efficacy against well-established C3.43 tumors. CpG-adjuvanted TVGV-1 resulted in higher frequencies of IFNγ secreting and degranulating E7-specific T cells compared to GPI-0100-adjuvanted TVGV-1, resulting in marginally increased in vivo efficacy. Despite minor differences in immune response outcomes, we consider both CpG ODN and GPI-0100 to be promising vaccine adjuvants to increase the immunogenicity and therapeutic efficacy of the TVGV-1 protein for HPV16-driven cancers.

Keywords: CpG adjuvant; GPI-0100 adjuvant; HPV16-induced tumors; Human papillomavirus; Therapeutic vaccine.

Figure 1.. TVGV-1 vaccination induces multi-functional cytolytic and effector cytokine producing HPV-specific CD8⁺ T cells.

(A-D) Splenocytes from vaccinated mice (5/group) were stimulated with HPV16 E7(49–57) peptide and tested for the ability to express surface CD107a/CD107b, a marker for lytic degranulation (panel A), and produce the effector cytokines IFNγ (panel B), TNFα (panel C), and IL-2 (panel D). Percentage of CD8⁺ T cells expressing each marker is shown within gated CD8⁺ T cells. (E) The proportion of polyfunctional cytokine-secreting cells was analyzed through sequential flow cytometry gating and expressed as a percentage of the total HPV16 E7-specific responding CD8⁺ T cell population. IFNγ secreting cells were assessed for simultaneous secretion of TNFα and IL-2. Percentages from each mouse were averaged to generate proportions. Significance was determined by a one-way ANOVA followed by Tukey’s multiple-comparisons test.**p<0.01, ***p<0.001, ****p<0.0001, n.s., non-significant.

Figure 2.. CpG and GPI-0100 adjuvanted TVGV-1 vaccine induce HPV16 E7-specific T cells with efficient in vivo cytolytic activity.

(A) Mice (5/group) were vaccinated three times, one week apart, with 100 μg TVGV-1-C, TVGV-1-G, or TVGV-vector control. CFSE-labeled peptide-loaded target cells (equal numbers of E7_(49–57) peptide-loaded CFSE-hi and control peptide loaded CFSE-low) were injected into vaccinated or control mice 7 days after the last vaccination. Twenty hours later, splenocytes were harvested and analyzed by flow cytometry for the proportion of target cells remaining. In vivo cytotoxic activity (% in vivo lysis) is based on calculated percentage loss of the CFSE-hi target cell population. Each point represents an individual mouse. Horizontal line indicates group mean. Significance was determined by a one-way ANOVA followed by Tukey’s multiple-comparisons test. ***p<0.001, ****p<0.0001. (B) Spearman correlation analysis of E7-specific in vivo lytic activity with CD107a/b degranulation for all vaccine cohorts. High in vivo lytic activity is significantly associated with increased in vitro T cell degranulation (R²=0.6931, p<0.0001).

Figure 3.. TVGV-1 vaccination induces tumor regression in mice vaccinated therapeutically at day 3, 5, or 7 with TVGV-1-C, TVGV-1-G vaccines in the C3.43 HPV16-induced mouse tumor model.

Mice (10/group) were challenged with 1 × 10⁵ C3.43 tumor cells s.c. in the flank. At the indicated time after challenge, mice were vaccinated three times, one week apart, with 100 μg TVGV-vector control, TVGV-1-C, or TVGV-1-G. Individual tumor volumes are shown for each group. Number of tumor free mice at the end of the study is indicated in each graph.

Figure 4.. TVGV-1 vaccination increases overall survival in mice vaccinated therapeutically at day 3, 5, or 7 with TVGV-1-C, TVGV-1-G vaccines in the C3.43 HPV16-induced mouse tumor model.

Mice (10/group) were vaccinated at the indicated days post tumor challenge as described in Figure 3 and followed for up to 100 days. Mice euthanized per protocol were marked as dead the following day. Significance was calculated using the log rank (Mantel-Cox) test for survival. Overall P-values (all significant) are indicated in each graph. Differences in survival between TVGV-1-C and TVGV-1-G vaccinated groups were non-significant for all therapy cohorts.

Figure 5.. TVGV-1 therapeutic vaccination induces changes in the immune phenotype of tumor-infiltrating lymphocytes (TIL) in mice vaccinated one week post C3.43 tumor challenge.

Mice (15/group) were challenged with C3.43 tumor cells s.c. in the flank. Starting at day 7, mice were vaccinated three times, one week apart, with 100 μg TVGV-vector control, TVGV-1-C, or TVGV-1-G. Tumors from tumor-bearing mice were harvested between days 30–60 when mice were euthanized due to increasing tumor size or tumor ulceration. TIL were evaluated by flow cytometry for indicated cell populations. (A) Shown is the percentage of CD8⁺ T cell infiltration of CD45⁺ cells into tumors in treated mice. (B) Shown is the percentage of HPV16 E7_(49–57) MHC tetramer positive CD8 T cell infiltration of CD45⁺ cells in tumors of treated mice. (C) Shown is the proportion of HPV16 E7_(49–57)-tetramer positive cells within the CD8⁺ T cell subset. (D) Shown is the percentage of CD4⁺ T cell infiltration of CD45⁺ cells into tumors in treated mice. (E) Shown is the percentage of Treg (CD4⁺FoxP3⁺) cell infiltration of CD45⁺ cells into tumors of treated mice. (F) Shown is the ratio of E7-specific CD8⁺ T cells to Tregs in treated mice. Each point represents a single tumor. Horizontal line indicates group mean. *p<0.05, **p<0.01, ****p<0.0001 determined by one-way ANOVA followed by Tukey’s multiple comparisons test. n.s., non-significant.