Matthew M. Halpert

Evaluations for In Vitro Correlates of Immunogenicity of Inactivated Influenza A H5, H7 and H9 Vaccines in Humans

Background Serum antibody responses in humans to inactivated influenza A (H5N1), (H9N2) and A (H7) vaccines have been varied but frequently low, particularly for subunit vaccines without adjuvant despite hemagglutinin (HA) concentrations expected to induce good responses. Design To help understand the low responses to subunit vaccines, we evaluated influenza A (H5N1), (H9N2), (H7N7) vaccines and 2009 pandemic (H1N1) vaccines for antigen uptake, processing and presentation by dendritic cells to T cells, conformation of vaccine HA in antibody binding assays and gel analyses, HA titers with different red blood cells, and vaccine morphology in electron micrographs (EM). Results Antigen uptake, processing and presentation of H5, H7, H9 and H1 vaccine preparations evaluated in humans appeared normal. No differences were detected in antibody interactions with vaccine and matched virus; although H7 trimer was not detected in western blots, no abnormalities in the conformation of the HA antigens were identified. The lowest HA titers for the vaccines were <1∶4 for the H7 vaccine and 1∶661 for an H9 vaccine; these vaccines induced the fewest antibody responses. A (H1N1) vaccines were the most immunogenic in humans; intact virus and virus pieces were prominent in EM. A good immunogenic A (H9N2) vaccine contained primarily particles of viral membrane with external HA and NA. A (H5N1) vaccines intermediate in immunogenicity were mostly indistinct structural units with stellates; the least immunogenic A (H7N7) vaccine contained mostly small 5 to 20 nm structures. Summary Antigen uptake, processing and presentation to human T cells and conformation of the HA appeared normal for each inactivated influenza A vaccine. Low HA titer was associated with low immunogenicity and presence of particles or split virus pieces was associated with higher immunogenicity.

Chemo-immunotherapy mediates durable cure of orthotopic Kras(G12D)/p53(-/-) pancreatic ductal adenocarcinoma.

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States, exhibiting a five-year overall survival (OS) of only 7% despite aggressive standard of care. Recent advances in immunotherapy suggest potential application of immune-based treatment approaches to PDAC. To explore this concept further, we treated orthotopically established K-rasG12D/p53−/− PDAC tumors with gemcitabine and a cell-based vaccine previously shown to generate durable cell-mediated (TH1) immunity. Tumor progression was monitored by IVIS. The results indicated that the combination of chemotherapy and dendritic cell (DC) vaccination was effective in eliminating tumor, preventing metastasis and recurrence, and significantly enhancing OS. No animal that received the combination therapy relapsed, while mice that received gemcitabine-only or vaccine-only regimens relapsed and progressed. Analysis of circulating PBMC demonstrated that mice receiving the combination therapy exhibited significantly elevated levels of CD8+IFNγ+CCR7+NK1.1+ T-cells with significantly reduced levels of exhausted GITR+CD8+ T-cells after the cessation of treatment. Retro-orbital tumor re-challenge of surviving animals at six-months post-treatment demonstrated durable antitumor immunity only among mice that had received the combination therapy. CD8+ splenocytes derived from surviving mice that had received the combination therapy were sorted into NK1.1pos and NK1.1neg populations and adoptively transferred into naive recipients. Transfer of only 1,500 CD8+NK1.1pos T-cells was sufficient to mediate tumor rejection whereas transfer of 1,500 CD8+NK1.1neg T-cells imparted only minimal effects. The data suggest that addition of a TH1 DC vaccine regimen as an adjuvant to existing therapies can mediate eradication of tumors and offer durable protection against PDAC.

Generation of functional CLL-specific cord blood CTL using CD40-ligated CLL APC.

Though remissions have been observed following allo-HSCT for the treatment of CLL, many CLL patients are ineligible for transplant due to the lack of HLA-compatible donors. The use of umbilical cord blood (UCB) permits transplantation of many patients who lack HLA-compatible donors due to reduced requirements for stringent HLA matching between graft and recipient; however, disease relapse remains a concern with this modality. The generation of CLL-specific CTL from UCB T-cells, primed and expanded against the leukemic clone, might enhance the GVL effect and improve outcomes with UCB transplantation. Here we report the generation of functional, CLL-specific CTL using CD40-ligated CLL cells to prime partially-HLA matched UCB T-cells. Functionality and specificity were demonstrated by immune synapse assay, IFN-γ ELISpot, multi-parametric intracellular cytokine flow cytometry, and 51Cr release assay. The use of patient-specific, non-CLL controls demonstrated the generation of both alloantigen and CLL-specific responses. Subsequently, we developed a clinically-applicable procedure permitting separation of alloreactive CTL from leukemia-specific CTL. Leukemia-specific CTL were able to mediate in vivo killing of CLL in humanized mice without concurrent or subsequent development of xenoGVHD. Our results demonstrate that generation of CLL-specific effectors from UCB is feasible and practical, and the results support further exploration of this strategy as a treatment modality for CLL.

Modeling Dendritic Cell Vaccination for Influenza Prophylaxis: Potential Applications for Niche Populations

BACKGROUND Cancer patients can exhibit negligible responses to prophylactic vaccinations, including influenza vaccination. To help address this issue, we developed in vitro and in vivo models of dendritic cell (DC) immunotherapy for the prevention of influenza virus infection. METHODS Human cord blood (CB)-derived or mouse splenocyte-derived DCs were loaded with purified recombinant hemagglutinin (rHA). T-cell responses to HA-loaded CB-derived DCs were determined by ELISpot. Protective efficacy was determined by vaccination of BALB/c mice with a single injection of 10(6) autologous DCs. DC migration to peripheral lymphoid organs was verified by carboxyfluorescein succinimidyl ester staining, and HA-specific antibody titers were determined by enzyme-linked immunosorbent assay. Mice were then challenged intranasally with BALB/c-adapted A/New Caledonia influenza virus derived from four consecutive lung pool passages. Antigen-presenting cell (APC) dysfunction was modeled using the MAFIA transgenic system, in which the Csf1r promoter conditionally drives AP20178-inducible Fas. RESULTS CB-derived human DCs were able to generate de novo T-cell responses against rHA, as determined by a system of rigorous controls. Mice vaccinated intraperitoneally developed HA titers detectable at serum dilutions of >1:1000. HA seroconverters survived virus challenge, whereas unvaccinated controls and vaccinated nonseroconverters lost weight and died. Furthermore, use of a model of APC-specific immunosuppression revealed that DC vaccination could generate HA-specific antibody titers under conditions in which protein vaccination could not. CONCLUSIONS The model demonstrates that DC immunotherapy for the prevention of influenza is feasible, and studies are underway to determine whether populations of immunosuppressed individuals might ultimately benefit from the procedure.

AIMp1 Potentiates TH1 Polarization and Is Critical for Effective Antitumor and Antiviral Immunity

Dendritic cells (DCs) must integrate a broad array of environmental cues to exact control over downstream immune responses including TH polarization. The multienzyme aminoacyl-tRNA synthetase complex component AIMp1/p43 responds to cellular stress and exerts pro-inflammatory functions; however, a role for DC-expressed AIMp1 in TH polarization has not previously been shown. Here, we demonstrate that the absence of AIMp1 in bone marrow-derived DC (BMDC) significantly impairs cytokine and costimulatory molecule expression, p38 MAPK signaling, and TH1 polarization of cocultured T-cells while significantly dysregulating immune-related gene expression. These deficits resulted in significantly compromised BMDC vaccine-mediated protection against melanoma. AIMp1 within the host was also critical for innate and adaptive antiviral immunity against influenza virus infection in vivo. Cancer patients with AIMp1 expression levels in the highest tertiles exhibited a 70% survival advantage at 15-year postdiagnosis as determined by bioinformatics analysis of nearly 9,000 primary human tumor samples in The Cancer Genome Atlas database. These data establish the importance of AIMp1 for the effective governance of antitumor and antiviral immune responses.

Genetic Adjuvantation of a Cell-Based Therapeutic Vaccine for Amelioration of Chagasic Cardiomyopathy

ABSTRACT Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a leading cause of heart disease (“chagasic cardiomyopathy”) in Latin America, disproportionately affecting people in resource-poor areas. The efficacy of currently approved pharmaceutical treatments is limited mainly to acute infection, and there are no effective treatments for the chronic phase of the disease. Preclinical models of Chagas disease have demonstrated that antigen-specific CD8+ gamma interferon (IFN-γ)-positive T-cell responses are essential for reducing parasite burdens, increasing survival, and decreasing cardiac pathology in both the acute and chronic phases of Chagas disease. In the present study, we developed a genetically adjuvanted, dendritic cell-based immunotherapeutic for acute Chagas disease in an attempt to delay or prevent the cardiac complications that eventually result from chronic T. cruzi infection. Dendritic cells transduced with the adjuvant, an adenoviral vector encoding a dominant negative isoform of Src homology region 2 domain-containing tyrosine phosphatase 1 (SHP-1) along with the T. cruzi Tc24 antigen and trans-sialidase antigen 1 (TSA1), induced significant numbers of antigen-specific CD8+ IFN-γ-positive cells following injection into BALB/c mice. A vaccine platform transduced with the adenoviral vector and loaded in tandem with the recombinant protein reduced parasite burdens by 76% to >99% in comparison to a variety of different controls and significantly reduced cardiac pathology in a BALB/c mouse model of live Chagas disease. Although no statistical differences in overall survival rates among cohorts were observed, the data suggest that immunotherapeutic strategies for the treatment of acute Chagas disease are feasible and that this approach may warrant further study.

Abstract 1564: Partial immunovesiculectomy by Th-1 dendritic cell vaccination: Implications for immunotherapy of solid tumors

The efficient and specific manner by which the mammalian immune system identifies and eradicates target antigen has stood as a testament to both its power and potential to similarly eradicate neoplastic self. In spite of this undeniable potential, the recalcitrance of the immune system to directed manipulation has been formidable, and the manner by which self-directed cellular (Th-1) immunity might be reproducibly promulgated has not yet been elucidated. Recent basic advances in the understanding of dendritic cell (DC) maturation, Th-1 polarization, T-cell homing, and plasmacytoid DC biology might allow promulgation of self-directed cellular immunity provided that all important aspects of such promulgation have been identified and are properly implemented. To investigate this hypothesis, seminal vesicle (SV), was chosen as a model organ system in the wild type mouse. In this system, SV serves as a proxy tumor from an immunological perspective: antigenically distinguishable from other organ systems yet comprised entirely of self tissue antigens and stringently protected from immunological recognition by mechanisms of central and peripheral tolerance. Equivalent class I and class II antigenic environments were provided to spleen-derived DC by electroporation with SV mRNA and incubation with SV lysate. DC were matured with a full complement of inflammatory cytokines. DC were applied i.p. in the vicinity of the SV so as to have access to the specific lymphatics that drain the area. Plasmacytoid recruitment and IFN-γ secretion was induced by local i.p. administration of particulate imiquimod. Two months post-vaccination, SV and other organs were harvested and examined for pathological changes by HE 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1564. doi:1538-7445.AM2012-1564

Abstract LB-132: The potential role of the multienzyme aminoacyl-tRNA synthetase complex in Th-1 immunity: implications for cancer immunotherapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Recent advances in dendritic cell (DC)-based cancer vaccines have highlighted the potential utility of arming the immune system to target neoplastic self. A strong, yet controlled, TH1 response has typically been the goal of DC-based protocols, as it is cell-based adaptive immunity that possesses the ability to most-efficiently destroy the nascent tumor or occult micrometastases. Nonetheless, many DC-intrinsic factors have impeded the development of a vaccine strong enough to impart meaningful clinical results, and there remains a need for further study of the parameters required for maximal, targeted DC activity. Previously, we have shown that contemporaneous loading of DC with overlapping MHC class I and MHC class II peptide antigens leads to an enhanced TH1 response when compared to canonical DC stimulation as assayed by release of IL-12, sCTLA-4, proliferation of CD8+ CD25+, INF-γ release, and specific killing of target both in vitro and in vivo. These and other data have lead to the hypothesis that DC possess a sensor mechanism capable of discerning amino acid sequence similarities between peptides bound by MHC class I and class II and that tRNA synthetases may serve as critical components of the putative recognition machinery due to their inherent ability to recognize and distinguish between individual amino acid side chains. Here we report that the aminoacyl-tRNA synthetase complex (MACS), a large molecular complex comprised of at least nine aminoacyl-tRNA synthetases, possess interesting characteristics that might link it to this phenomenon. Components of the MACS complex, including the p43 structural subunit, were found in DC exosomes, the contents of which mimic those of the late endosome, a known cross-presentation compartment. MARS (methionyl-tRNA synthetase), a component of the MACS, as well as non-MACS components VARS (valyl-tRNA synthetase) and GARS (glycyl-tRNA synthetase) were found within exosomes as well. UVC cross-linking of exosomal extracts resulted in the incorporation of individual tRNA-synthetase isoforms into a very large complex of indeterminate size. p43 was differentially released from the MACS in response to the loading of DC only with matched class I (GFP mRNA) and class II (recombinant GFP protein) determinants. siRNA knockdown of MACS structural component p38 was able to ablate the ability of doubly-loaded DC to enhance the generation of CD8+CD25+ cells as well as the ability of these cells to secrete IFN-γ. Further, siRNA knockdown of p38 abrogated the release of p43 from DC loaded with matched class I and II determinants. With an enhanced understanding of these regulatory phenomena, the development of small molecule TH1 agonists and/or immunotherapeutic protocols with enhanced clinical efficacy become realistic and achievable goals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-132. doi:1538-7445.AM2012-LB-132