Ebna1-Specific T Cell Responses During Persistent Rhesus Lcv infection and The Development of a Novel Therapeutic Prototype Vaccine for Ebv-Associated Diseases

The impact of EBV on human health is substantial, but vaccines that prevent primary EBV infections or treat EBV-associated diseases are not yet available. The Epstein-Barr nuclear antigen 1 (EBNA1) is an important target for vaccination because it is the only protein expressed in all forms of latency and in all EBV-associated malignancies. The overarching goal throughout this dissertation was to determine if EBNA1 is a suitable target for vaccine development. This was addressed in two ways. First, because an improved understanding of EBNA1-specific T cell responses benefits EBV vaccine development, we characterized responses against EBNA1 of the EBV-homologous rhesus lymphocryptovirus (rhLCV) in naturally infected rhesus macaques. We assessed frequency, phenotype, and cytokine production profiles of rhesus (rh)EBNA1-specific T cells by intracellular cytokine staining (ICS) and polychromatic flow cytometry. We found that most naturally infected animals had CD4+ and/or CD8+ T cells against rhEBNA1 and rhBZLF1, an immediate-early lytic phase antigen of rhLCV. Peptide-specific CD8+ T cells showed a more activated effector phenotype, while most peptide-specific CD4+ T cells exhibited a resting central memory phenotype. T cells were highly functional and produced various combinations of the cytokines IFN-γ, IL-2, and TNF-α. The differentiation status and functional profiles of rhEBNA1-specific T cells suggests they are not impaired by chronic exposure to low levels of antigen, and rhEBNA1-specific T cells therefore represent a viable population to target through vaccination. Similarities between our findings and the human response further validate the rhLCV model for studying chronic EBV infection and for pre-clinical vaccine development. We then asked if vaccination could expand functional rhEBNA1-specific T cells during persistent rhLCV infection. To test this, we developed two serologically distinct replication-defective adenoviral vectors that expressed chimeric rhEBNA1 constructs fused to functional and non-functional versions of Herpes Simplex Virus- glycoprotein D (HSV-gD). HSV-gD has been shown to augment T cell responses by inhibiting the immunosuppressive Herpes Virus Entry Mediator (HVEM) pathway during T cell activation. After confirmation of vaccine specificity and antigenicity in vitro, rhesus macaques were vaccinated in a prime-boost regimen, and responses in peripheral blood were measured by ICS and polychromatic flow cytometry. Importantly, we found that vaccination induced the expansion of highly functional rhEBNA1-specific CD8+ and CD4+ T cells in vivo, regardless of HSV-gD binding ability. Vaccination did not increase rhBZLF1-specific T cell responses, thus indicating that rhEBNA1-specific responses were vaccine-driven. Overall, these results serve as important proof-of-principle analyses of a therapeutic EBNA1-based vaccine regimen and demonstrate that EBNA1 is a viable target that warrants exploration in future vaccine studies

Day-4 Myeloid Dendritic Cells Pulsed with Whole Tumor Lysate Are Highly Immunogenic and Elicit Potent Anti-Tumor Responses

“Day-7” myeloid DCs are commonly used in the clinic. However, there is a strong need to develop DCs faster that have the same potent immunostimulatory capacity as “Day-7” myeloid DCs and at the same time minimizing time, labor and cost of DC preparations. Although “2 days” DCs can elicit peptide-specific responses, they have not been demonstrated to engulf, process and present complex whole tumor lysates, which could be more convenient and personalized source of tumor antigens than defined peptides. In this preclinical study, we evaluated the T-cell stimulatory capacity of Day-2, Day-4, and Day-7 cultured monocyte-derived DCs loaded with SKOV3 cell whole lysate prepared by freeze-thaw or by UVB-irradiation followed by freeze-thaw, and matured with lipopolysaccharide (LPS) and interferon (IFN)-gamma. DCs were evaluated for antigen uptake, and following maturation with LPS and IFN-gamma, DCs were assessed for expression of CD80, CD40, CD86, ICAM-1 and CCR7, production of IL-12p70 and IP-10, and induction of tumor-specific T-cell responses. Day-4 and Day-7 DCs exhibited similar phagocytic abilities, which were superior to Day-2 DCs. Mature Day-7 DCs expressed the highest CD40 and ICAM-1, but mature Day-4 DCs produced the most IL-12p70 and IP-10. Importantly, Day-4 and Day-7 DCs derived from ovarian cancer patients stimulated equally strongly tumor-specific T-cell responses. This is the first study demonstrating the highly immunogenic and strong T-cell stimulatory properties of Day-4 myeloid DCs, and provided important preclinical data for rapid development of potent whole tumor lysate-loaded DC vaccines that are applicable to many tumor types

Day-4 DCs produce the highest levels of IL-12p70 and IP-10.

<p>Day-2, Day-4, and Day-7 DCs were first pulsed with either UVBL or FTL for 16 h, and then stimulated with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) to determine (A) IL-12p70 and, (B) IP-10 production. Supernatants from the DC-tumor lysate cocultures were collected and evaluated by ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028732#s2" target="_blank">Materials and Methods</a>. The results are from 4 different normal healthy donors and are expressed as mean (pg/ml) ± standard errors.</p

Day-2, Day-4 and Day-7 DCs pulsed with UVBL or FTL mature normally following LPS and IFN-γ stimulation.

<p>Following lysate loading, DCs were stimulated with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) for 16 h. DCs were identified by HLA-DR, CD11c and one of the markers shown. Unpulsed immature DCs (iDCs) and mature (mDCs) were set up in parallel and harvested at the same time as the other tumor lysate-loaded mature DCs for analysis. (A–C) Upregulation of maturation markers CD80, CD40, CD86, ICAM-1, and CCR7 is observed on mature Day-2, Day-4 and Day-7 DCs. The open histograms represent the isotype control, while shaded histograms represent the DC markers. The solid line marks the MFI of the different markers on iDCs for each condition. Representative histogram results from 1 out of 6 donors are shown here. (D) Fold-increase in expression levels of maturation and adhesion markers on mDCs, UVBL-loaded DCs (UVBL-DC) or FTL-loaded DCs (FTL-DC) over iDC was determined by expressing the MFIs as a ratio of the DCs to unpulsed iDCs. Highly significant differences were detected in CD86 (**<i>P</i> value = 0.002), ICAM-1 (**<i>P</i> value = <0.001), and CCR7 (**<i>P</i> value = 0.002, <0.001 and 0.008, respectively; ANOVA followed by <i>post-hoc</i> testing) on unpulsed matured Day-4 DCs compared to unpulsed matured Day-7 DCs. Similar highly significant differences were detected in CD86 and CCR7 (**<i>P</i> value = 0.001, and 0.01, respectively; ANOVA followed by <i>post-hoc</i> testing) on Day-4 UVBL-DCs compared to Day-7 UVBL-DCs. CD86 and ICAM-1 (**<i>P</i> value = 0.001, and 0.01, respectively; ANOVA followed by <i>post-hoc</i> testing) were also significantly different on Day-4 FTL-DCs compared to Day-7 FTL-DCs. (E) The overall immunophenotypes of DCs loaded with UVBL or FTL were similar to unpulsed mature DCs, however Day-4 DCs loaded with UVBL consistently expressed slightly higher levels of most markers including CD80, CD86 and CCR7 relative to any other lysate-DC preparations.</p

Day-4 and Day-7 DCs, but not Day-2 DCs acquired phenotypic features of differentiated DCs.

<p>Elutriated monocytes derived from normal healthy donors were cultured for 2, 4, or 7 days in serum-free AIM-V media supplemented with recombinant GM-CSF and IL-4, and analyzed by flow cytometry at the end of the culture period. (A) Representative histograms from 1 out of 6 donors were shown here. The MFIs indicated in the histograms were derived by subtracting the MFI of the test sample from the isotype control. Day-2 DCs expressed slightly higher CD14, CD68, and CD33 markers than Day-4 and Day-7 DCs, while Day-4 DCs were similar to Day-7 DCs in immunophenotype. (B) The average MFIs of the different DC maturation markers of 6 normal healthy donors were shown here. ** <i>P</i> value = <0.001; highly significant when comparing the MFI of Day-4 or Day-7 DCs to the MFI of Day-2 DCs by Student's unpaired <i>t</i> test.</p

Day-4 DCs, but not Day-2 DCs, were as phagocytic as Day-7 DCs in actively engulfing ovarian tumor lysate.

<p>(A) Day-2, Day-4, and Day-7 DCs were cocultured with UVB lysate (UVBL) or freeze-thaw lysate (FTL) of PKH26-labeled SKOV3 cells at 37°C for 4 h or 24 h to determine active phagocytosis of tumor lysates. Representative dot plot results from 1 out of 6 donors are shown here. DCs that had engulfed PKH26-labeled tumor lysate appear as the HLA-DR⁺ PKH26⁺ double-positive population, and are expressed as the percentage of the total number of HLA-DR⁺ DCs as indicated in the upper-right quadrant of the dot plots. (B) DCs were cocultured with lysate at 4°C for 4 h or 24 h as controls to determine passive transfer of the PKH26 dye to DCs. Representative dot plot results from 1 out of 6 donors are shown here. HLA-DR⁺ PKH26⁺ double-positive DCs are expressed as the percentage of the total number of HLA-DR⁺ DCs as indicated in the upper-right quadrant of the dot plots. (C) Summary results of the percentages of Day-2, Day-4, and Day-7 DCs that have engulfed PKH26-labeled UVBL or FTL after 4 h or 24 h, at both 4°C and 37°C. Data displayed are the means ± standard errors of six independent experiments. There are no significant differences among Day-2, Day-4 and Day-7 DCs for the precentage of DCs taking up UVBL (ANOVA <i>P</i> value = 0.13) or FTL (ANOVA <i>P</i> value = 0.59) at 4 h. However, there is a significant difference for % of DCs taking up UVBL at 24 h (ANOVA <i>P</i> value = 0.02). By <i>post hoc</i> paired testing, % of Day-2 DCs taking up UVBL was significantly lower than either Day-7 or Day-4 DCs (*<i>P</i> values = 0.05 and 0.02, respectively). Highly significant differences were also observed for the uptake of FTL at 24 h (ANOVA**<i>P</i> value<0.001). The % of Day-2 DCs taking up FTL was significantly lower than either Day-7 or Day-4 DCs (**<i>P</i> values<0.001 for each). The differences between Day-4 and Day-7 DCs were insignificant for uptake of both UVBL (<i>P</i> value = 0.90) and FTL (<i>P</i> value = 0.92).</p

CAR T cells targeting BCMA and CD19 for newly diagnosed and relapsed multiple myeloma patients responding to current therapy

We conducted a phase 1 clinical trial of anti-BCMA CAR T cells (CART-BCMA) with or without anti-CD19 CAR T cells (huCART19) in multiple myeloma (MM) patients responding to third-orlater- (Phase A, N=10) or high-risk patients responding to first-line therapy (Phase B, N=20), followed by early lenalidomide or pomalidomide maintenance. We observed no high-grade CRS and only one instance of low-grade neurologic toxicity. In vivo CART-BCMA expansion was comparable to that previously observed with CART-BCMA in relapsed/refractory MM. Early maintenance therapy was safe and feasible and coincided with CAR T cell re-expansion and late-onset clinical response in some patients. Pre-treatment MM cell-surface BCMA levels were unexpectedly low despite no prior exposure to anti-BCMA therapy. Despite low disease burden and evidence of anti-MM activity in most patients, only 4 subjects converted to complete response. Outcomes with CART-BCMA + huCART19 were similar to CART-BCMA alone