DNA vaccines encoding altered peptide ligands for SSX2 enhance epitope-specific CD8+ T-cell immune responses

Abstract

Plasmid DNA serves as a simple and easily modifiable form of antigen delivery for vaccines. The USDA approval of DNA vaccines for several non-human diseases underscores the potential of this type of antigen delivery method as a cost-effective approach for the treatment or prevention of human diseases, including cancer. However, while DNA vaccines have demonstrated safety and immunological effect in early phase clinical trials, they have not consistently elicited robust anti-tumor responses. Hence many recent efforts have sought to increase the immunological efficacy of DNA vaccines, and we have specifically evaluated several target antigens encoded by DNA vaccine as treatments for human prostate cancer. In particular, we have focused on SSX2 as one potential target antigen, given its frequent expression in metastatic prostate cancer. We have previously identified two peptides, p41-49 and p103-111, as HLA-A2-restricted SSX2-specific epitopes. In the present study we sought to determine whether the efficacy of a DNA vaccine could be enhanced by an altered peptide ligand (APL) strategy wherein modifications were made to anchor residues of these epitopes to enhance or ablate their binding to HLA-A2. A DNA vaccine encoding APL modified to increase epitope binding elicited robust peptide-specific CD8+ T cells producing Th1 cytokines specific for each epitope. Ablation of one epitope in a DNA vaccine did not enhance immune responses to the other epitope. These results demonstrate that APL encoded by a DNA vaccine can be used to elicit increased numbers of antigen-specific T cells specific for multiple epitopes simultaneously, and suggest this could be a general approach to improve the immunogenicity of DNA vaccines encoding tumor antigens.

Keywords: APL; CTA; DNA vaccine; Prostate cancer; SSX.

Fig. 1

Modified SSX2 peptides have enhanced or reduced HLA-A2 affinity in vitro compared to native peptides. Shown is the relative HLA-A2 binding affinity of each modified SSX2 peptide as determined by mean fluorescence intensity (MFI) of HLA-A2 stabilization on T2 cells. Values were measured in triplicate, ± SD, for each peptide and normalized to the MFI of a no-antigen vehicle control. Results are representative of two independent experiments. A known HLA-A2-binding peptide derived from the influenza A matrix protein (GILGFVFTL, Flu) was used as a positive control for comparison.

Fig. 2

HHDII-DR1 mice immunized with modified peptides p41–49 and p103–111 develop increased peptide-specific immune responses. Splenocytes from individual mice immunized once with either a native SSX2 peptide, an HLA-A2 enhanced-binding peptide, or an HLA-A2 reduced-binding peptide were analyzed for the frequency of peptide-specific immune responses by IFNγ ELISPOT. Splenocytes were evaluated for responses to each of the immunizing peptides or concanavalin A (ConA) positive control. The influenza A matrix protein HLA-A2 epitope (GILGFVFTL, Flu) was evaluated as a negative control. Each colored symbol represents the frequency of peptide-specific IFNγ-secreting responses from an individual mouse for the modified p41–49 group (panel A), modified p57–65 group (panel B), modified p99–107 group (panel C), and modified p103–111 group (panel D), with n = 4–8 mice per group.

Fig. 3

HHDII-DR1 mice immunized with modified SSX2 peptides develop CTL capable of lysing peptide-pulsed target cells and the LNCaP cancer cell line. Splenocytes from HHDII-DR1 mice vaccinated with native SSX2 peptides p41–49 (panel A), p103–111 (panel C) or modified SSX2 peptides p41-AL (panel B) or p103-RF (panel D), were stimulated with the native peptide for five days and tested for specific lysis of peptide-pulsed T2 target cells (left panels), the HLA-A2⁺ LNCaP prostate cancer cell line, or the LNCaP cell line pre-incubated with an HLA-A2 blocking antibody (right panels). Shown are the means and standard deviations of percent specific lysis at three effector-to-target ratios. Data is from individual mice and representative of multiple independent experiments.

Fig. 4

HHDII-DR1 mice immunized with SSX2 plasmid DNA vaccines encoding APL develop epitope-specific immune responses at higher frequency than animals immunized with the native vaccine. (Panel A) Splenocytes from individual mice immunized with either native pTVG-SSX2 plasmid vaccine (n = 6) or pTVG-SSX2 p41-VP/p103-IP (encoding epitopes with ablated HLA-A2 binding, n = 6) were analyzed for the frequency of peptide-specific immune responses by IFNγ ELISPOT. (Panel B) Splenocytes from individual mice immunized with either native pTVG-SSX2, a plasmid encoding p41-AL (enhanced binding), a plasmid ablating p103–111 HLA-A2 binding (p103-IP), or a plasmid encoding both mutations (p41-AL/p103-IP), were analyzed for the frequency of peptide-specific immune responses by IFNγ ELISPOT (n = 5 for each treatment group). (Panel C) Splenocytes from individual mice immunized with either native pTVG-SSX2, a plasmid encoding p103-RF, or a plasmid ablating p103–111 HLA-A2 binding (p103-IP), were analyzed for the frequency of peptide-specific immune responses by IFNγ ELISPOT (n = 11 for each treatment group). (Panel D) Splenocytes from individual mice immunized with native pTVG-SSX2, a plasmid encoding p41-AL, or a plasmid encoding both p41-AL and p103-RF (p41-AL/p103-RF) were analyzed for the frequency of peptide-specific immune responses by IFNγ ELISPOT (n = 6 for each vaccine). Splenocytes were stimulated for 48 h with each of the stimulator peptides indicated or a concanavalin A positive control prior to IFNγ detection. Flu peptide was evaluated as a negative control. Each colored symbol represents the frequency of peptide-specific IFNγ-secreting responses from an individual mouse. * Indicates a significant (P < 0.05) difference in the mean number of IFNγ spot-forming units (SFU) between the 2 groups (2-tailed t test).

Fig. 5

HHDII-DR1 mice immunized with SSX2 plasmid DNA vaccines develop epitope-specific immune responses with a multi-functional phenotype. (Panel A) Splenocytes from animals (n = 3–4 animals per group) immunized with pTVG4 (control vector), pTVG-SSX2, or pTVG-SSX2 p41-AL/p103-RF (pTVG-SSX2^opt) were pooled, enriched for CD8+ T cells, and stimulated with p41–49 or p103–111 peptide overnight. The frequency of CD3+ CD8+ T cells secreting Th1 (IFNγ, TNFα, or IL-2), IL-4, IL-10, or IL-17 under each antigen-stimulating condition and for each experimental group was determined by intracellular cytokine staining. (Panel B) The CD3+ CD8+ T cells secreting Th1 cytokines (IFNγ, TNFα, or IL-2) were subdivided among those concurrently expressing one or more of these cytokines.