The Effect of Vector Silencing during Picornavirus Vaccination against Experimental Melanoma and Glioma.

Virus vector-based vaccination against tumor-specific antigens remains a promising therapeutic approach to overcome the immune suppressive tumor microenvironment. However, the extent that the desired CD8 T cell response against the targeted tumor antigen is impacted by the CD8 T cell response against the virus vector is unclear. To address this question, we used picornavirus vaccination with Theiler's murine encephalomyelitis virus (TMEV) as our vector against tumor-expressed ovalbumin (OVA257-264) antigen in both the B16-OVA murine melanoma and GL261-quad cassette murine glioma models. Prior to vaccination, we employed vector silencing to inhibit the CD8 T cell response against the immunodominant TMEV antigen, VP2121-130. We then monitored the resulting effect on the CD8 T cell response against the targeted tumor-specific antigen, ovalbumin. We demonstrate that employing vector silencing in the context of B16-OVA melanoma does not reduce tumor burden or improve survival, while TMEV-OVA vaccination without vector silencing controls tumor burden. Meanwhile, employing vector silencing during picornavirus vaccination against the GL261-quad cassette glioma resulted in a lower frequency of tumor antigen-specific CD8 T cells. The results of this study are relevant to antigen-specific immunotherapy, in that the virus vector-specific CD8 T cell response is not competing with tumor antigen-specific CD8 T cells. Furthermore, vector silencing may have the adverse consequence of reducing the tumor antigen-specific CD8 T cell response, as demonstrated by our findings in the GL261-quad cassette model

CD8 T cell responses directed against specific epitopes can be silenced during peripheral TMEV-OVA infection.

<p>C57BL/6 mice were treated with control E7_49-57 peptide or silencing VP2_121-130 peptide. The following day, animals received an i.p. infection of TMEV-OVA. Splenocytes (N = 3 mice per group) were harvested seven days post infection and stimulated with peptide for five hours before intracellular IFNγ staining. (A) Representative flow cytometry plots and (B) average percent of CD8 T cells stimulated to secrete IFNγ are shown. Cells were gated as Viability Dye^lo, CD45^hi, and CD8α⁺. Treatment with VP2_121-130 peptide significantly reduced the VP2_121-130-specific CD8 T cell responses. Data are shown as mean±SEM. * denotes p<0.05.</p

Competition among epitope-specific CD8 T cells occurs in the central nervous system following acute infection with TMEV-OVA.

<p>Mice were administered E7_49-57 (n = 2) or VP2_121-130 (n = 4) peptide i.v. one day prior to i.c. injection of TMEV-OVA. After one week, brain-infiltrating lymphocytes were harvested and stained for surface markers and peptide:MHC tetramer. (A) Representative flow cytometry plots and (B, C) quantified peptide:MHC tetramer⁺ cells of CD8α⁺CD45^hi cells per brain demonstrate a significant decrease in CD8 T cells recognizing VP2_121-130 upon vector silencing. An increase in peptide:MHC tetramer staining for OVA_257-264 is also observed. (D) Number of total CD8 T cells infiltrating the brain was not significantly different. Data are shown as mean±SEM. * denotes p<0.05, n.s. indicates no significant difference.</p

Silencing the virus vector-specific CD8 T cell response is not necessary for the function of picornavirus vaccine against B16-OVA melanoma.

<p>C57BL/6 animals were implanted with B16-OVA tumor cells in the left leg. Six days following tumor implantation, when tumors were palpable, mice were treated with vector silencing VP2_121-130 peptide or control E7_49-57 peptide. One day following peptide treatment, mice were vaccinated i.p. with TMEV-OVA. Splenocytes (N = 3 mice per group) were harvested from B16-OVA-bearing mice seven days after vaccination. (A) Representative flow cytometry plots and (B) mean percent of IFNγ⁺CD44^hi cells show a decrease in the percentage of CD8 T cells responding to VP2_121-130 stimulation in mice administered VP2_121-130 peptide prior to vaccination. Cells were gated as CD45^hi and CD8α⁺. (C) Tumor diameter, (D) change in tumor diameter, and (E) Kaplan-Meier survival curve analysis demonstrate an improvement in mice bearing B16-OVA tumors following TMEV-OVA administration compared to wild-type TMEV, regardless of silencing peptide treatment (N = 7 mice per group). Addition of the silencing VP2_121-130 peptide does not further increase these effects. Data are shown as mean±SEM. * denotes p<0.05.</p