Immunotherapy against HPV16/18 generates potent TH1 and cytotoxic cellular immune responses

Abstract

Despite the development of highly effective prophylactic vaccines against human papillomavirus (HPV) serotypes 16 and 18, prevention of cervical dysplasia and cancer in women infected with high-risk HPV serotypes remains an unmet medical need. We report encouraging phase 1 safety, tolerability, and immunogenicity results for a therapeutic HPV16/18 candidate vaccine, VGX-3100, delivered by in vivo electroporation (EP). Eighteen women previously treated for cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) received a three-dose (intramuscular) regimen of highly engineered plasmid DNA encoding HPV16 and HPV18 E6/E7 antigens followed by EP in a dose escalation study (0.3, 1, and 3 mg per plasmid). Immunization was well tolerated with reports of mild injection site reactions and no study-related serious or grade 3 and 4 adverse events. No dose-limiting toxicity was noted, and pain was assessed by visual analog scale, with average scores decreasing from 6.2/10 to 1.4 within 10 min. Average peak interferon-γ enzyme-linked immunospot magnitudes were highest in the 3 mg cohort in comparison to the 0.3 and 1 mg cohorts, suggesting a trend toward a dose effect. Flow cytometric analysis revealed the induction of HPV-specific CD8(+) T cells that efficiently loaded granzyme B and perforin and exhibited full cytolytic functionality in all cohorts. These data indicate that VGX-3100 is capable of driving robust immune responses to antigens from high-risk HPV serotypes and could contribute to elimination of HPV-infected cells and subsequent regression of the dysplastic process.

Fig. 1

Mean VAS assessment of local pain after vaccination/EP procedure. The VAS scores shown for cohort 1 at the 10 minute time point represent only those patients whose VAS scores were greater than 2 at the 5 minute time point. For cohorts 2 and 3, the protocol was amended to collect data from all patients at the 10 minute time point.

Fig. 2

Immunoglobulin G (IgG) responses after vaccination with VGX-3100 versus time (months). (A to D) HPV16 E7 (A), HPV16 E6 (B), HPV18 E7 (C), and HPV18 E6 (D) measured by ELISA in the 0.3, 1.0, and 3.0 mg groups at entry [dose 1 (D1)], 1 week after the second immunization (D2+1), 1 week after the third immunization (D3+1), 4 weeks after the third immunization (D3+4), and 24 weeks after the third immunization (D3+24). (E) Representative HPV16 and HPV18 E7–specific seroconversion (subject 14-4) measured by Western blot at D1, D3+4, and D3+24.

Fig. 3

Robust HPV16 and HPV18 E6/E7–specific T_H1-biased cellular immune responses after vaccination with VGX-3100. (A to C) Antigen-specific IFN-γ ELISpot assays determine the number of antigen-specific IFN-γ–secreting cells in response to stimulation with HPV16 or HPV18 E6 and E7 peptide pools at D1, D2+1, D3+1, and D3+4 in the 0.3 mg (A), 1.0 mg (B), and 3.0 mg (C) groups. “Responders” by analytical criteria are designated with a bar below the subject ID. (D) Peak average responses in the 0.3, 1.0, and 3.0 mg groups.

Fig. 4

ELISpot responses to each vaccine antigen present at all doses. (A) Overall background-subtracted cellular immune responses against four vaccine antigens. (B to E) Background-subtracted antigen-specific cellular immune responses in the 0.3, 1.0, and 3.0 mg groups to (B) HPV16 E6, (C) HPV16 E7, (D) HPV18 E6, and (E) HPV18 E7.

Fig. 5

Strong memory T cell responses after vaccination with VGX-3100. (A) Memory T cell responses elicited in the responders in the 0.3, 1.0, and 3.0 mg groups, respectively. (B) Overall memory T cell responses elicited in all responders. IFN-γ ELISpot in response to stimulation with HPV16 or HPV18 E6 and E7 peptide pools in the 0.3 mg (circle), 1.0 mg (triangle), and 3.0 mg (square) groups. The baseline immune responses (Pre-Vac), peak responses (Peak-Vac), and memory responses (D3+24) of responders are shown for each dose group.

Fig. 6

IFN-γ and CTL responses by flow cytometry after vacci-nation with VGX-3100. (A) Multiparametic flow cytometry was used to detect HPV16- and HPV18-specific production of IFN-γ and regulation of CD107a, granzyme B, and perforin. Representative gating strategy and response to HPV peptide are shown. (B) HPV-specific IFN-γ synthesis increased after immunization and was detectable in both CD4⁺ and CD8⁺ T cell subsets in pre- and postimmunization samples. (C) Coexpression of CD107a, granzyme B, and perforin in CD8⁺ T cell subset in response to HPV16 and HPV18 E6/E7 was increased after immunization, suggesting the induction and expansion of CTLs.

Fig. 7

Detection of new HPV-specific functional CTL responses (quantitative killing assay). Delivery of active granzyme B from effectors to targets pulsed with HPV16 and HPV18 E6/E7 at varying E:T ratios: 10:1, 50:1, and 100:1. Whereas effector PBMCs isolated before immunization mediated little to no CTL activity in the form of granzyme B delivery (blue lines), postimmunization PBMCs were able to function as CTLs and deliver granzyme B to targets in an antigen-specific manner (red lines), suggesting an increased frequency of functional CTLs after immunization.

Fig. 8

Lytic granule loading and CTL functional quality assessment of CTLs induced by VGX-3100 (qualitative killing assay). (A) CD8⁺ T cells from pre- and post-immunization PBMCs were assessed for their ability to activate and load lytic granules upon extended antigenic stimulation. Representative gating strategy is shown. For HLA-DR, CD38, granzyme B, and perforin, red lines indicate expression levels from unstimulated samples and blue lines indicate poststimulation expression. Contour plots show stimulated pre- and postimmunization samples, including an overlay. (B) Postimmunization samples show statistically significant increases in the percentage of CD8⁺ T cells that stained positively for the activation markers HLA-DR and CD38 upon extended stimulation with HPV peptide [raw group averages of 1.19% versus 1.66% (top left); background-corrected group averages of 0.03% versus 0.49% (top right)] and also show a statistically significant increase in the number of cells costaining for granzyme B and perforin within this activated subset [group average of 32.1% versus 53.3% (bottom)]. (C) Activated CD8⁺ T cells mediate robust cytotoxicity in the form of active granzyme B delivery to HPV-stimulated target cells but not to targets incubated with irrelevant peptides. Numbers in red indicate the percentage of targets receiving active granzyme B from an effector. E:T ratio was 12.5:1, where effectors were normalized on the basis of HLA-DR and CD38 costaining of CD8⁺ T cells. (D) Data summary for CTL activity of activated CD8⁺ T cells. Preimmunization CD8⁺ T cells mediated little to no granzyme B delivery to targets stimulated with HPV peptide, whereas postimmunization PBMCs delivered a statistically significant amount when compared to preimmunization activity as well as to activity against targets incubated with irrelevant peptide.