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Ex vivo analysis of CMV pp65-specific CD8+ T cells in circulation before and after immunotherapy.

Patient schematic.

DC vaccination enhances the proportion of CMV pp65-specific CD8+ T cells with multiple effector functions

Patient weight distribution and weight-based ATCT infusion counts.

Median survival for glioblastoma (GBM) remains ex vivo analyses from this study found no change in the capacity of CMV pp65-specific T cells to gain multiple effector functions or polyfunctionality, which has been associated with superior antitumor efficacy. Previous studies have shown that dendritic cells (DC) could further enhance tumor-specific CD8+ T-cell polyfunctionality in vivo when administered as a vaccine. Therefore, we hypothesized that vaccination with CMV pp65 RNA-loaded DCs would enhance the frequency of polyfunctional CMV pp65-specific CD8+ T cells after ATCT. Here, we report prospective results of a pilot trial in which 22 patients with newly diagnosed GBM were initially enrolled, of which 17 patients were randomized to receive CMV pp65-specific T cells with CMV-DC vaccination (CMV-ATCT-DC) or saline (CMV-ATCT-saline). Patients who received CMV-ATCT-DC vaccination experienced a significant increase in the overall frequencies of IFNγ+, TNFα+, and CCL3+ polyfunctional, CMV-specific CD8+ T cells. These increases in polyfunctional CMV-specific CD8+ T cells correlated (R = 0.7371, P = 0.0369) with overall survival, although we cannot conclude this was causally related. Our data implicate polyfunctional T-cell responses as a potential biomarker for effective antitumor immunotherapy and support a formal assessment of this combination approach in a larger randomized study.Significance: A randomized pilot trial in patients with GBM implicates polyfunctional T-cell responses as a biomarker for effective antitumor immunotherapy. Cancer Res; 78(1); 256–64. ©2017 AACR.

We recently completed a Phase I evaluation of a bivalent, virus-like replicon particle (VRP) vaccine (AVX601) expressing three CMV proteins in CMV seronegative adults. One VRP component expressed the pp65 and IE1 genes as a polyprotein and the second expressed gB. AVX601 was well-tolerated, with no significant safety issues identified. Analysis of the immune responses by IFN-γ ELISPOT and CMV neutralization showed the vaccine to be highly immunogenic. In this study, multiparameter flow cytometry was used to further characterize the AVX601-induced T cell responses. Overall, the CD8 T cell responses to all 3 antigens were of higher magnitude than the CD4 T cell responses. The highest levels of CD8 and CD4 antigen-specific responses detected were 0.97% and 0.17% for pp65, 0.12% and 0.10% for IE1, and 0.18% and 0.08% for gB. Antigen-specific T cells were analyzed for their ability to produce IFNγ, TNFα and/or IL-2 after CMV peptide stimulation. Polyfunctional subsets of both CD4 and CD8 lineages specific for all 3 antigens were detected at all time points tested. The maximum % of CD8 T cells positive for 2 or 3 cytokines at any time point studied was 0.58% for pp65, 0.03% for IE1 and 0.07% for gB, and for CD4 T cells was 0.12% for pp65, 0.06% for IE1 and 0.05% for gB. These data demonstrate that this novel alphavirus replicon particle vaccine for CMV elicited polyfunctional CD4 and CD8 T cells to all 3 CMV immunogens.

An RNA replicon vector system derived from an attenuated strain of VEE virus was used to produce virus-like replicon particles (VRP) expressing SARS-CoV genes. We previously showed in mouse studies that complete protection was conferred by SARS S VRP and partial protection with SARS M VRP. In this study, two assays for cellular immunity were used to characterize the T cell phenotypes and cytokine profiles induced after SARS VRP vaccination. Following SARS M, N, S or E VRP immunization of BALB/c mice, splenocytes were harvested for antigen specific ICS and ELISPOTs. The SARS S VRP induced both CD4 and CD8 responses with the latter being the dominant component as measured by ICS for IFN-γ or TNF-α. Polyfunctional CD8 cells secreting both IFN-γ and TNF-α were also detected. Following SARS N VRP immunization, T cells secreting IFN-γ or TNF-α were readily detected with cells secreting both predominately identified as CD4 cells. Th1 cytokine secretion was not detected in cells from SARS M VRP inoculated mice. SARS E VRP elicited a robust CD8 response as shown by cytokine expression in cells stimulated with a 9mer peptide. IL-4 was not detectable after any SARS VRP vaccination. The IFN-γ ELISPOT results paralleled those from ICS, indicating that the SARS VRP vaccines induced Th1 biased responses, although the cytokines elicited differed depending on the SARS gene expressed. This study was supported by NIH grant #UC1-AI62582

We previously evaluated the immunogenicity and efficacy of a virus-like replicon particle (VRP) vaccine expressing the SARS CoV S glycoprotein in mice. SARS S VRP were highly immunogenic and conferred protection upon challenge. To extend that work we have evaluated the immune responses induced by SARS S VRP in macaques immunized at 0, 4 and 24 weeks. PBMC were assayed at two week intervals by ELISPOT and ICS. Post prime, both CD4 and CD8 T cells were found that secrete IFN- γ, IL-2 and TNF- α. The percentage of CD4 cells secreting IFN- γ, IL-2 and TNF- α was substantially more than the percentage of CD4 cells secreting IL-4 post prime indicating a Th1 bias. Post boost, the number of CD4 cells secreting all measured cytokines were equivalent indicating that the SARS S VRP induced a balanced Th1/Th2 response after 2 inoculations. By week 8 a measured decline in the percentage of cytokine-secreting CD8 cells was observed. There were no significant differences in the numbers of T cells secreting cytokines with 2 different VRP dosages given as measured by ICS, however a dose response was seen by ELISPOT. Neutralizing antibodies (NAb) to SARS CoV were measured in sera following post prime and boost inoculations. Three of 4 animals in the low dose group had measurable NAb post boost (GMT = 101) whereas all 4 animals in the high dose group had serum NAb after two doses (GMT = 190). These data provide support to further development of a SARS S VRP vaccine. This study was supported by NIH grant #UC1-AI62582