ALBERT

Description: Adoptive transfer of ex vivo expanded CMV-specific T cells is an effective approach, and an attractive alternative to using anti-virals to manage CMV infection for HSCT recipients. We recently published a robust approach to expanding CMV-specific CTL based on infection of autologous EBV-LCL with the attenuated poxvirus, Modified Vaccinia Ankara (MVA), expressing CMV pp65, pp150, and IE1 proteins. This approach causes vigorous, up to 500fold expansions in as little as 12–14 days of memory CD8+ T cells specific for these immunodominant antigens. In order to improve the specificity of the expanded T cells, a method was sought to derive effective antigen presenting cells (APC) that avoided the use of EBV-LCL. Of equal importance is to develop an expansion approach that avoids the need to involve virally infected APC in developing a clinical product. Our preliminary observation is that rMVA can infect PBMC in vitro, causing high levels of expression of recombinant CMV antigens. To be permissible for high level expression from rMVA, fresh PBMC were treated with different combinations of single-stranded CpG-containing phosphorothioate backbone oligonucleotides (ODN). A three-day incubation with a combination of two ODN (ODN # 2006 and 2216) which are known to stimulate both plasmacytoid dendritic and B-cells were found to reproducibly generate a highly rMVA infectable population of PBMC. In all five healthy CMV-positive donors tested, CpG ODN treated autologous PBMC, infected with recombinant rMVA, elicited a 20-fold average expansion of CMV-specific CD8+ T cells, in 10 days. Several different rMVA expressing CMV genes were evaluated, including a novel vector expressing the UL44 gene product, an immunodominant target of the host cellular immune response. The expanded T cell populations showed minimal alloreactivity, and exhibited high levels of CMV-specific MHC Class I tetramer binding, epitope-specific cytokine production, and cytotoxic activity. The availability of a source of autologous professional APC that can be used after only 3 days of priming, enhances the attractiveness of using rMVA for adoptive immunotherapy for HSCT recipients or donor vaccination.

Description: CMV infection is an important complication of patient recovery from transplantation, and affects a wide variety of individuals including newborns and HIV patients with advanced disease. An effective CMV vaccine for patients who have already acquired an infection has yet to successfully incorporate an antigenic repertoire capable of eliciting a cellular immune response. To address this problem, we have developed a vaccine candidate derived from modified vaccinia Ankara (MVA) that expresses three immunodominant antigens (pp65, IE1, IE2) from CMV, we have termed CMV-MVA. While other antigens are also immunologically recognized to varying degrees, the evidence for these three antigens to be involved in protective immune responses in a majority of CMV-infected patients is compelling and justifies their inclusion into a vaccine to prevent viremia and control infection. MVA has an extensive history of successful delivery into rodents, Rhesus macaques, and other non-human primates, and more recently as a clinical vaccine in cancer patients and HIV patients in a state of immunosuppression. CMV-MVA is engineered with a bacterial marker to track its purification, which can be removed by recombination, a requirement for clinical development. The novelty of this vaccine is the fusion of the two largest and adjacent protein-coding exons from the immediate-early (IE) region of CMV, their successful expression as a fusion protein in MVA, and robust immunogenicity in both primary and memory response models. The advantages of this approach include placement of all vaccine antigens in one vector, and diminishing the dose of virus needed to attain sufficient immunity simultaneously against all of the included antigens. Evaluation of the immunogenicity of the viral vaccine in transgenic HLA mouse models (A2, B7, A11) shows that it can stimulate primary immunity against all three antigens in both the CD4+ and CD8+ T cell subsets. Evaluation using human PBMC from CMV-positive donors shows robust stimulation of existing CMV-specific T cells in both the CD4+ and CD8+ T cell subset. These results extend to both healthy volunteers and patients within 6 months of receiving hematopoietic cell transplant (HCT). Evaluating PBMC from transplant recipients in all three risk categories (D+/R+,D+/R−, D−R+), we found an equivalently strong recognition of both antigens, in some cases more vigorous than in the PBMC of healthy adults. This candidate vaccine is being developed in partnership with the NCI as a therapeutic for HCT recipients. Strategies of vaccine delivery include vaccinating the transplant donor, and/or the recipient at day 90 or later, if warranted clinically and with sufficient evidence of safety. The ongoing evaluation of a DNA vaccine against CMV suggests a worthwhile strategy of combining MVA with a plasmid DNA vaccine. Our preliminary studies using DNA prime and MVA boost in Rhesus macaques show it to be a more powerful CMV vaccine regimen than either component given separately. Evidence for the capacity of CMV-MVA to modify viremia through immunologic mechanisms from both clinical and monkey studies will be presented.

Description: In the era of preemptive antiviral therapy, cytomegalovirus (CMV) still remains the cause of major health complications, profound defects in immune reconstitution, and significant morbidity in post-transplant recovery of immune-compromised HCT recipients. Substituting antivirals with a vaccine that harnesses the native immune response to CMV may improve outcomes for HCT recipients. Modified Vaccinia Ankara (MVA), a vaccinia virus being investigated as a smallpox vaccine by the Defense Department, is a safe and robust delivery system to treat or prevent a wide range of diseases including HIV and cancer. Replication-defective MVA is safe, well tolerated and strongly immunogenic when given to HCT recipients or AIDS patients. We developed a multiple-antigen recombinant MVA with genes encoding 3 immunodominant CMV proteins: UL83 (pp65), UL123 (IE1-exon4), and UL122 (IE2-exon5) (CMV-MVA-Triplex), and established its pre-clinical safety and immunogenicity using humanized HLA transgenic mouse models and human PBMC from CMV-seropositive healthy volunteers and HCT recipients. Defining safety, persistence of the virus, maximum tolerated dose and immunogenicity of CMV-MVA-Triplex in healthy volunteers is a critical first step in its clinical development for HCT recipients as required by the FDA. In a Phase I trial (NCT01941056), these endpoints were evaluated in 24 healthy volunteers (age: 18-60), with or without prior immunity to CMV and vaccinia. Three escalating dose levels (DL) were administered intramuscularly (DL1=10xE7; DL2=5x10E7; DL3=5x10E8 pfu/dose) in 8 subjects/DL, with a booster injection 28 days later, and follow up for 1 year. As of July 2015, all 24 planned volunteers were enrolled, vaccinated and followed for at least 4 months. Vaccinations at all DL were well-tolerated, with only a few expected injection reactions and no SAE or dose limiting toxicities. Immunogenicity of the vaccine was evaluated by measuring the levels of the CD137 T-cell surface marker representing functional activation of PBL harvested from vaccinees and stimulated 24 hours with full-length pp65, IE-1 and IE2 overlapping peptide libraries, or direct measurement of CMV-specific T-cells using HLA multimers. CMV-MVA-Triplex induced robust expansion of pp65-, IE1- and IE2-specific CD8 and CD4 T-cells in vaccinated CMV-seropositives, at each DL (Cf. plot showing geometric mean with upper lower/limits of the pp65 T-cell levels for DL2 cohort). HLA multimers identified CMV-specific T-cells whose expansion closely followed CD137-activated CMV-specific T-cells in vaccinees with common HLA alleles which have a corresponding known CMV-CTL epitope (data to be presented). A statistical analysis performed using generalized estimated equations indicated that the post-vaccination levels of pp65-, IE1- or IE2-specific CD8 and CD4 T-cells were significantly increased, with p-values ranging from 3x10-5 to 0.025. For example, the pre-/post-vaccination median pp65-reactive CD4+ CD137+ T-cells rose from 1.3 to 4.4 cells/µL (p=3x10-5); and pp65-reactive CD8+ CD137+ T-cells rose from a median of 0.22 to 3.1 cells/µL (p=0.003). Importantly, robust immunity was detected in CMV-seronegatives (as shown in the plot for UPN 14 and 18), as well as in subjects who had received smallpox vaccinations. Elevated frequencies of CMV-specific CD4 and CD8 T cells for all 3 antigens plateaued after day 56, but in most cases remained elevated up to one year post-vaccination (data to be presented). Circulating MVA vector in blood was assessed by real-time PCR post-injection and showed only minimal residual vector DNA [10-30 gc/mL] in just 2 vaccinees in the DL3 cohort that disappeared within 3 months. These results provide evidence that CMV-seropositive HCT recipients, whether they receive stem cell product from CMV-seropositive or -seronegative donors could respond to CMV-MVA-Triplex by generating protective CMV-specific immunity. CMV-MVA-Triplex is the first vaccine against CMV that uses a recombinant MVA incorporating multiple CMV antigens, developed for HCT recipients, who are at risk for CMV reactivation. The safety and marked immunogenicity of CMV-MVA-Triplex in this Phase I trial, warrant testing of the vaccine in the HCT setting. A Phase 2 multicenter, placebo-controlled trial to assess CMV-MVA-Triplex in CMV seropositive recipients, receiving HCT from matched related or unrelated donors will start in Fall 2015. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Diamond: Helocyte, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Description: Reconstitution of adaptive T-cell responses to human cytomegalovirus (CMV) is critical to protection from CMV disease following hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). However, there is an incomplete understanding of which CMV antigens and epitopes are most crucial to providing protective responses. The functional status of cytotoxic T-lymphocyte (CTL) populations recognizing cytomegalovirus IE-1 and pp65 polypeptides was investigated in PBMC from either HSCT or SOT recipients. Our previous finding of differing levels of degranulation between CMV IE1 and pp65/pp50 specific T-cells was complicated by the possibility that differences were epitope and/or HLA-specific. We generalized the approach using a combined flow-based CD107a/b degranulation/mobilization and intracellular cytokine (ICC) assays using peptide libraries as antigens. These assays indicated that a significantly higher proportion of pp65-specific CTLs were in a more mature functional state compared to IE-1-specific CTLs. Degranulation/multicytokine ICC assays also indicated that a significantly higher proportion of the pp65-specific versus IE-1-specific CTLs secreted both IFN-γ and TNF-α, in addition to possessing greater cytotoxic potential. These results support our earlier findings of functional differences between CTLs recognizing individual epitopes within the IE-1 and pp65 antigens in HSCT recipients, and extend them to a broader array of HLA-restricted responses to those antigens. A report that a subset of HIV-1 specific CTLs capable of producing both IFN-γ and TNF-α was associated with improved cytotoxic activity prompted us to investigate whether degranulation, a functional correlate of cytotoxicity, was positively associated with dual cytokine production and predicted differences between IE1 and pp65-specific CD8+ T-cells. A higher proportion of pp65-specific compared to IE1-specific T-cells were present in the trifunctional IFN-γ+,TNF-α+, CD107+ population (p=0.008) in HSCT recipients. We have extended these findings to investigate the role of donor CMV status in terms of functional maturity of CMV-specific T cell response in transplant recipients. T cell maturation/function may act as a mechanistic correlate to the survival advantage of recipients receiving a stem-cell graft from CMV sero-positive donors. These principles have also been applied to investigations of a high risk population of sero-negative recipients of a sero-positive liver allograft. Data from this study will also be reviewed in the context of the model of trifunctional T cells being indicative of enhanced protective capacity against CMV disease and associated with survival.

Description: Background: Cytomegalovirus (CMV) infection causes significant complications in recipients of hematopoietic cell transplant (HCT). Preemptive anti-viral therapy limits CMV viremia and disease, though its use is associated with toxicity, delayed immune reconstitution, and risk of late-onset CMV disease. We developed a novel CMV vaccine (CMVPepVax) based on the HLA-A*0201 pp65495-503 CD8+T-cell epitope (NLVPMVATV) fused with the Tetanus toxin P2 epitope (tt830-843:QYIKANSKFIGITE). The vaccine was formulated with PF03512676 (1.0 mg), a synthetic single-stranded phosphorothioate DNA containing CpG motifs provided by Pfizer Inc. In a Phase 1b healthy volunteer trial, CMVPepVax was found to be safe and immunogenic [LaRosa et al. 2012]. We report on clinical results demonstrating reliable safety, reduced CMV reactivation and chronic (c)GVHD in HCT recipients injected with CMVPepVax compared to randomized observational HCT recipients. Objectives: The primary study objective was safety, with secondary objectives including immunologic responses of HCT recipients to CMVPepVax measured by pentamer assay, prevention of CMV reactivation, and clinical outcome. Patients and methods: Eligible recipients (R+) wereCMV seropositive, HLA-A*0201, between 18-75 years (y) receiving allogeneic T-replete HCT for hematologic malignancy from matched-related (MRD) or 8/8 and 7/8 matched unrelated donors (MUD), excluding those with acute leukemia not in remission. Patients were reassessed on day (d)28 for eligibility and randomized unless they failed to engraft, experienced pre-d28 CMV reactivation or grade 3-4 acute (a)GVHD, received prednisone 〉1mg/kg/day within 7d of immunization, or had ongoing non-hematologic toxicity (CTCAE 〉=grade 3). Randomization was stratified by donor (D) CMV serostatus D- vs D+. Patients randomized to the vaccine arm (VA) received CMVPepVax on d28 and d56. Patients randomized to the observation arm (OA) were followed as per standard of care. All patients were monitored for safety/toxicity, CMV-related events, and immune recovery. Results: As of July 2014, of 36 planned patients, 30 patients were enrolled and followed for at least 100d. The median age was 50y (range: 20-76y), with balanced gender accrual. All patients received peripheral blood stem cell grafts, while hematologic diagnosis, pre-HCT conditioning regimens, and GVHD prophylaxis were similar in the VA vs OA. Vaccinations were well tolerated in all 14 patients, and no patient met the safety stopping rule per-protocol. Of three VA patients who developed serious adverse events (SAE), 2 were unrelated and only one was probable (Grade 1 fever), while 8 patients experienced SAE in the OA. The frequency and severity of unrelated AE was similar between arms across all organ systems. None of 6 monitored vaccine recipients developed anti-dsDNA antibodies. In 9 VA patients who reached d180 without reactivation, increases in CMVpp65 pentamer levels after 1st and 2ndinjections were compared to 10 OA patients, using generalized estimating equations. The model indicates an average 6.5fold positive vaccine effect (p=0.02). While this pilot trial was not powered to detect reduced CMV reactivation, we observed a ~5-fold lower rate of CMV reactivation in the VA (1/14 =7%) compared to the OA (6/16=37%, p=0.09) by d100. aGVHD occurred post-randomization in 6/16 (grade II; 5, III: 1) subjects on the OA, and in 2/14 subjects (grade II: 2) on the VA (p = 0.2). An unexpected and striking finding was differing rates of cGVHD in patients who reached d180 in the VA (1/11) vs. OA (7/12, p=0.03). Noteworthy is that the vaccine formulation is more likely to stimulate TH1 responses, yet TH2 immunity is mechanistically associated with cGVHD. An interesting, but unexplained difference in relapse was observed in the OA (3/16) vs VA (0/14) patients, though the observation period is short. Recent observations relating CMV reactivation and reduced relapse have driven strong interest in a mechanistic understanding of the phenomena observed in this trial related to CMV vaccine or the adjuvant, PF03512676. Conclusions: Trial results indicate safety of injecting CMVPepVax in HCT recipients on d28 and d56, no increase in aGVHD, and reduced CMV reactivation associated with vaccine-stimulated immunity. Compelling observations of reduced cGVHD and relapse will be further tested in a recently NIH-funded multi-institution expanded accrual phase 2 trial. Disclosures Merson: Pfizer Inc.: Employment.

Description: Epitope vaccines have shown promise for inducing cellular immune responses in animal models of infectious disease. In cases where cellular immunity was augmented, peptide vaccines composed of covalently linked minimal cytotoxic T-lymphocyte (CTL) and T-helper (TH) epitopes generally showed the most efficacy. To address a clinical vaccine strategy for cytomegalovirus (CMV) in the context of HCT (hematopoietic cell transplantation), we observed that linking the synthetically derived pan-DR epitope peptide (PADRE) or one of several tetanus TH epitopes to the immunodominant human leukocyte antigen (HLA) A*0201–restricted CTL epitope from CMV-pp65 to create a fusion peptide caused robust cytotoxic cellular immune responses in HLA A*0201/Kbtransgenic mice. Significantly, the fusion peptides are immunogenic when administered in saline solution by either subcutaneous or intranasal routes. CpG-containing single-stranded DNA (ss-oligodeoxynucleotide [ODN]) added to the fusion peptides dramatically up-regulated immune recognition by either route. Notably, target cells that either expressed full-length pp65 protein from vaccinia viruses or were sensitized with the CTL epitope encoded in the vaccine were recognized by splenic effectors from immunized animals. Visualization of murine peptide–specific CTL by flow cytometry was accomplished using an HLA A*0201 tetramer complexed with the pp65495-503 CTL epitope. TH-CTL epitope fusion peptides in combination with CpG ss-ODN represent a new strategy for parenteral or mucosal delivery of vaccines in a safe and effective manner that has applicability for control or prophylaxis of infectious disease, especially in situations such as vaccination of donors or recipients of HCT, where highly inflammatory adjuvants are not desired.

Description: Human CMV continues to be a major risk factor in patients undergoing allogeneic hematopoietic stem cell (HSCT) and solid organ transplant (SOT), despite advancement of antiviral therapy. CMV tegument protein pp65 and CMV immediate early gene product IE1 are both considered immunodominant targets of cell-mediated immunity (CMI) and potentially capable of controlling CMV infection. Similar to healthy adults, CMV tegument protein pp65 and IE1 are frequently recognized by human CD4 and CD8 T cells in HCT and SOT recipients. To better assess their role in host defense, we have constructed a novel attenuated poxvirus (MVA) transfer vector named pZWIIA and generated a recombinant MVA (rMVA) expressing both full-length pp65 and exon4 of IE1 (pp65-IE1-MVA) at high levels, followed by the genetic removal of the bacterial marker gene used to distinguish recombinant forms during the screening process. Immunogenicity evaluation indicates that pp65-IE1-MVA not only can induce robust primary CMI to both antigens in HLA A2.1, B7 and A11 transgenic mice, but also can stimulate vigorous expansion of memory T lymphocyte responses to pp65 and IE1 in PBMC of CMV-positive donors. The recent discovery that additional early antigens of CMV are also well-recognized by healthy adults prompted us to include the IE2 antigen in the vaccine. Evaluation of in vivo immunogenicity of the vaccine expressing IE2 in both transgenic mice and in vitro antigenicity in human peripheral blood lymphocytes of HSCT and SOT recipients will be presented. A novel overlapping peptide library spanning full-length IE2 has been constructed in our laboratory, and used in conjunction with the vaccine expressing IE2, along with IE1 and pp65. Since MVA has a foreign gene capacity of over 30 kilobases (Kb), we have developed a strategy to maximize coverage of haplotypes through insertion of 6 additional antigens into novel insertion sites of the virus, for a total of just 12.0 Kb of the CMV genome. The selected antigens cover 83% of the population, while representing over 22% of the combined CD4 and CD8 T cell repertoire in seropositives. Our studies of the recognition of these antigens expressed in MVA and monitored with our own synthesized over-lapping peptide libraries in both HSCT and SOT recipients will be presented. These properties make the MVA-based vaccine ideal for the dual role of priming and boosting CMV-specific T cell immunity as a means to control CMV disease in recipients of HCT or SOT. pZWIIA alone or in combination with other MVA transfer vectors can be used to generate MVA based multiple-antigen vaccine which have application in vaccine development for a wide spectrum of infectious diseases and cancer.

Description: Abstract 953 Wilms' tumor antigen (WT1) is expressed in many solid tumors and hematologic malignancies and serves a physiological role in the maintenance of the tumorigenic phenotype. Immunologic recognition of WT1 has been observed in patients with myeloid and lymphoid leukemia, solid tumors, and even in healthy adults. However, little is known about WT1 expression or WT1-specific immune responses in patients with non-Hodgkin lymphoma (NHL). Current therapeutic modalities for NHL patients are predominantly based on the highly successful application of the anti-CD20 mAb (Rituximab™) or radioactive derivatives (Zevalin™ or Bexxar™) combined with standard chemotherapy. Recent approaches have focused on generating NHL-specific idiotypes as an example of personalized medicine. However, harnessing a T cell response to malignant B or T cells in NHL patients has not succeeded, despite its attractiveness as a universal therapeutic option. We developed a detection system for WT1-specific T cells that is HLA independent by constructing a peptide library of 15mers (11 residues overlapping) corresponding to full length WT1 antigen. We analyzed peripheral blood mononuclear cells (PBMC) after priming with the peptide library for immune responses to the WT1 antigen detectable by flow cytometry based on the CD137 activation marker. Sixty-three NHL patients and 30 healthy donors (HD) were recruited under IRB-approved protocols. The cohort included patients who received allogeneic stem cell transplantation (alloHCT: n=42) or chemotherapy/autologous HCT (n=21). We observed that WT1-specific ex vivo CD8+ T cell responses were significantly greater in our NHL patient cohort (median 0.035%) compared to our randomly chosen HD cohort (0%, p=0.0013)(Figure 1). These WT1-specific CD8+CD137+ T cells co-expressed CD45RA and CD57 surface markers, consistent with them being a TEMRA (Terminal Effector Memory, RA+) population of highly differentiated T cells. Twenty-four of 43 patients (55.8%) with high/intermediate-grade NHL had a positive CD8+ T cell response (CD8+CD137+ ≥0.05%) to the WT1 antigen compared to four of 20 patients (20%) with low-grade NHL (p=0.011). None of the other clinical variables such as patient age, treatment modality (alloHCT vs. autoHCT or chemotherapy), disease status (CR vs. non-CR), prior acute GVHD (only in alloHCT, grade 0-I vs. II–IV) were significantly associated (p≤0.05) with WT1-specific CD8+ T-cell response in NHL patients when we conducted a multivariate analysis. PBMC from one HD and 12 NHL patients were expanded using in vitro stimulation (IVS) with the WT1 peptide library. All PBMC samples expanded in culture after 10–14 days by single IVS (median 7-fold expansion). We observed significantly higher IFN-γ responses in CD8+ T cells when IVS cells were re-stimulated with the WT1 library (median=3.0%, range: 0.08–5.5%) compared with the medium control (median=0.39%, range: 0.05–1.7%, p=0.002). Remarkably, using standard in vitro techniques, the expanded PBMC were cytotoxic to WT1-peptide library-loaded T2 cells and autologous EBV-transformed B-cell lines derived from patients enrolled in the study. These results challenge the notion that central tolerance mechanisms eliminate high affinity WT1-specific T cells that kill tumor cells. In order to distinguish whether WT1-specific T cells are maintained in NHL patients because of an aberration of tolerance mechanisms or the presence of WT1 antigen as a stimulator, we used sensitive real time quantitative PCR (RT-qPCR) methods to detect transcripts in LN from NHL patients and HD. We succeeded in detecting WT1 mRNA transcripts in diseased lymph node (LN) tissues of NHL patients utilizing RT-qPCR technology. There was significantly greater WT1 mRNA in NHL LN (n=6) with an average copy number ratio of 39 (WT1/c-ABL × 104) compared with 0.66 in non-disease LN obtained from individuals without a cancer diagnosis (n=7, p

Description: Key PointsFirst in human trial of Triplex vaccine shows safety and expansion of durable CMV-specific T cells with potential for viremia control. Triplex is immunogenic in both CMV-seronegative and -seropositive healthy adults with or without previous smallpox vaccination.