Ebola-GP DNA Prime rAd5-GP Boost: Influence of Prime Frequency and Prime/Boost Time Interval on the Immune Response in Non-human Primates.

Heterologous prime-boost immunization regimens are a common strategy for many vaccines. DNA prime rAd5-GP boost immunization has been demonstrated to protect non-human primates against a lethal challenge of Ebola virus, a pathogen that causes fatal hemorrhagic disease in humans. This protection correlates with antibody responses and is also associated with IFNγ+ TNFα+ double positive CD8+ T-cells. In this study, we compared single DNA vs. multiple DNA prime immunizations, and short vs. long time intervals between the DNA prime and the rAd5 boost to evaluate the impact of these different prime-boost strategies on vaccine-induced humoral and cellular responses in non-human primates. We demonstrated that DNA/rAd5 prime-boost strategies can be tailored to induce either CD4+ T-cell or CD8+ T-cell dominant responses while maintaining a high magnitude antibody response. Additionally, a single DNA prime immunization generated a stable memory response that could be boosted by rAd5 3 years later. These results suggest DNA/rAd5 prime-boost provides a flexible platform that can be fine-tuned to generate desirable T-cell memory responses.

Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge.

Ebolavirus disease causes high mortality, and the current outbreak has spread unabated through West Africa. Human adenovirus type 5 vectors (rAd5) encoding ebolavirus glycoprotein (GP) generate protective immunity against acute lethal Zaire ebolavirus (EBOV) challenge in macaques, but fail to protect animals immune to Ad5, suggesting natural Ad5 exposure may limit vaccine efficacy in humans. Here we show that a chimpanzee-derived replication-defective adenovirus (ChAd) vaccine also rapidly induced uniform protection against acute lethal EBOV challenge in macaques. Because protection waned over several months, we boosted ChAd3 with modified vaccinia Ankara (MVA) and generated, for the first time, durable protection against lethal EBOV challenge.

CD8+ cellular immunity mediates rAd5 vaccine protection against Ebola virus infection of nonhuman primates.

Vaccine-induced immunity to Ebola virus infection in nonhuman primates (NHPs) is marked by potent antigen-specific cellular and humoral immune responses; however, the immune mechanism of protection remains unknown. Here we define the immune basis of protection conferred by a highly protective recombinant adenovirus virus serotype 5 (rAd5) encoding Ebola virus glycoprotein (GP) in NHPs. Passive transfer of high-titer polyclonal antibodies from vaccinated Ebola virus-immune cynomolgus macaques to naive macaques failed to confer protection against disease, suggesting a limited role of humoral immunity. In contrast, depletion of CD3(+) T cells in vivo after vaccination and immediately before challenge eliminated immunity in two vaccinated macaques, indicating a crucial requirement for T cells in this setting. The protective effect was mediated largely by CD8(+) cells, as depletion of CD8(+) cells in vivo using the cM-T807 monoclonal antibody (mAb), which does not affect CD4(+) T cell or humoral immune responses, abrogated protection in four out of five subjects. These findings indicate that CD8(+) cells have a major role in rAd5-GP-induced immune protection against Ebola virus infection in NHPs. Understanding the immunologic mechanism of Ebola virus protection will facilitate the development of vaccines for Ebola and related hemorrhagic fever viruses in humans.

Recombinant adenovirus serotype 26 (Ad26) and Ad35 vaccine vectors bypass immunity to Ad5 and protect nonhuman primates against ebolavirus challenge.

The use of adenoviruses (Ad) as vaccine vectors against a variety of pathogens has demonstrated their capacity to elicit strong antibody and cell-mediated immune responses. Adenovirus serotype C vectors, such as Ad serotype 5 (Ad5), expressing Ebolavirus (EBOV) glycoprotein (GP), protect completely after a single inoculation at a dose of 10(10) viral particles. However, the clinical application of a vaccine based on Ad5 vectors may be hampered, since impairment of Ad5 vaccine efficacy has been demonstrated for humans and nonhuman primates with high levels of preexisting immunity to the vector. Ad26 and Ad35 segregate genetically from Ad5 and exhibit lower seroprevalence in humans, making them attractive vaccine vector alternatives. In the series of studies presented, we show that Ad26 and Ad35 vectors generate robust antigen-specific cell-mediated and humoral immune responses against EBOV GP and that Ad5 immune status does not affect the generation of GP-specific immune responses by these vaccines. We demonstrate partial protection against EBOV by a single-shot Ad26 vaccine and complete protection when this vaccine is boosted by Ad35 1 month later. Increases in efficacy are paralleled by substantial increases in T- and B-cell responses to EBOV GP. These results suggest that Ad26 and Ad35 vectors warrant further development as candidate vaccines for EBOV.

Vector choice determines immunogenicity and potency of genetic vaccines against Angola Marburg virus in nonhuman primates.

The immunogenicity and durability of genetic vaccines are influenced by the composition of gene inserts and choice of delivery vector. DNA vectors are a promising vaccine approach showing efficacy when combined in prime-boost regimens with recombinant protein or viral vectors, but they have shown limited comparative efficacy as a stand-alone platform in primates, due possibly to suboptimal gene expression or cell targeting. Here, regimens using DNA plasmids modified for optimal antigen expression and recombinant adenovirus (rAd) vectors, all encoding the glycoprotein (GP) gene from Angola Marburg virus (MARV), were compared for their ability to provide immune protection against lethal MARV Angola infection. Heterologous DNA-GP/rAd5-GP prime-boost and single-modality rAd5-GP, as well as the DNA-GP-only vaccine, prevented death in all vaccinated subjects after challenge with a lethal dose of MARV Angola. The DNA/DNA vaccine induced humoral responses comparable to those induced by a single inoculation with rAd5-GP, as well as CD4(+) and CD8(+) cellular immune responses, with skewing toward CD4(+) T-cell activity against MARV GP. Vaccine regimens containing rAd-GP, alone or as a boost, exhibited cellular responses with CD8(+) T-cell dominance. Across vaccine groups, CD8(+) T-cell subset dominance comprising cells exhibiting a tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) double-positive functional phenotype was associated with an absence or low frequency of clinical symptoms, suggesting that both the magnitude and functional phenotype of CD8(+) T cells may determine vaccine efficacy against infection by MARV Angola.

Demonstration of cross-protective vaccine immunity against an emerging pathogenic Ebolavirus Species.

A major challenge in developing vaccines for emerging pathogens is their continued evolution and ability to escape human immunity. Therefore, an important goal of vaccine research is to advance vaccine candidates with sufficient breadth to respond to new outbreaks of previously undetected viruses. Ebolavirus (EBOV) vaccines have demonstrated protection against EBOV infection in nonhuman primates (NHP) and show promise in human clinical trials but immune protection occurs only with vaccines whose antigens are matched to the infectious challenge species. A 2007 hemorrhagic fever outbreak in Uganda demonstrated the existence of a new EBOV species, Bundibugyo (BEBOV), that differed from viruses covered by current vaccine candidates by up to 43% in genome sequence. To address the question of whether cross-protective immunity can be generated against this novel species, cynomolgus macaques were immunized with DNA/rAd5 vaccines expressing ZEBOV and SEBOV glycoprotein (GP) prior to lethal challenge with BEBOV. Vaccinated subjects developed robust, antigen-specific humoral and cellular immune responses against the GP from ZEBOV as well as cellular immunity against BEBOV GP, and immunized macaques were uniformly protected against lethal challenge with BEBOV. This report provides the first demonstration of vaccine-induced protective immunity against challenge with a heterologous EBOV species, and shows that Ebola vaccines capable of eliciting potent cellular immunity may provide the best strategy for eliciting cross-protection against newly emerging heterologous EBOV species.

IL-10 and IL-4 in skin allograft survival induced by T-cell depletion plus deoxyspergualin.

The mechanisms mediating T-cell depletion plus 15-deoxyspergualin (DSG)-induced prolonged allograft survival or tolerance are uncertain. The purpose of this study is to evaluate the role of IL-4 and IL-10 in prolonged allograft survival induced by T-cell depletion plus DSG. MHC mismatched skin allograft transplantation was performed, using wild-type and three separate knockout (i.e., IL-4-/-, Stat6-/-, or IL-1-/ -) mice as recipients. Induction therapy consisted of T-cell depletion and or brief course of DSG. The data demonstrate that monotherapy with T-cell-depleting mAbs or DSG prolonged skin allograft survival, compared to controls, in wild-type Balb/c recipients [median survival time (MST) = 25 and 21 vs. 10 days, p < 0.007]. T-cell depletion plus DSG further augmented skin allograft survival in wild-type animals relative to monotherapy (MST = 35 days vs. 25 and 21 days, p < 0.006 vs. mAbs or DSG only), and was equally effective in IL-4-/- and Stat6-/- recipients. In contrast, combined therapy was no better than monotherapy in IL-10-/- animals (p > 0.05). Furthermore, skin allograft survival after combined therapy was shorter in IL-10-/- versus wild-type recipients (MST 20 and 41 days, respectively, p < 0.001). IL-4-mediated signaling through Stat6 is dispensable for prolonged allograft survival induced by T-cell depletion plus DSG. In contrast, IL-10 appears to be important for prolonged allograft survival induced by combined therapy in this model.

Invariant natural killer T cells from rhesus macaque spleen and peripheral blood are phenotypically and functionally distinct populations.

BACKGROUND: Natural killer T cells (NKT) possess dual functions of innate and adaptive immune systems, controlling viral infections and regulating autoimmune diseases. Non-human primates (NHP) are penultimate models for advancing therapeutic immunoregulatory strategies for translational application in humans, though, little is known about NHP NKT cells. Here we characterized rhesus macaque NKT cells ex vivo. METHODS: The frequency, phenotype and intracellular cytokine production of V alpha 24+ 6B11+ invariant NKT (iNKT) cells were analyzed by multi-color flow cytometry. V alpha 24J alpha Q mRNA expression was analyzed by real-time RT-PCR. RESULTS: The frequencies of peripheral blood (PB) and spleen V alpha 24+ 6B11+ iNKT cells were not significantly different. The iNKT cell subset in spleen was significantly increased for CD4+ CD8+ and CD3+ CD56+ co-expression as well as intracellular interleukin-4 production, which was rarely observed in circulating PB. CONCLUSION: Spleen iNKT cells in rhesus macaques are Th2 biased and display phenotypically and functionally distinct profiles from their PB counterpart.

Expansion of CD4+CD25+ suppressive regulatory T cells from rhesus macaque peripheral blood by FN18/antihuman CD28-coated Dynal beads.

CD4(+)CD25(+) regulatory T cells (Tregs) play an important role in allograft and self-tolerance and thus have potential therapeutic application in transplantation, autoimmunity, and allergy. Although nonhuman primate (NHP) provide the most accepted preclinical models for translational studies in allograft tolerance and infectious diseases, CD4(+)CD25(+) Tregs have been rarely studied in NHP. The low frequencies of Tregs in peripheral blood will likely necessitate ex vivo expansion to enable Tregs adaptive immune therapy in NHP and humans. Tregs were isolated by magnetic and flow sorting and then stimulated weekly with antirhesus CD3 clone FN18 and antihuman CD28-coated Dynal beads plus 100 U/ml rhIL-2. Under these conditions, the Tregs were expanded 300- to 2000-fold in 4 weeks. Expanded CD4(+)CD25(+) Tregs expressed high to moderate levels of FOXP3 as well as CD95, CD62L, CD69, and CCR7 surface antigens. Expanded rhesus Tregs were anergic and suppressed the proliferation of autologous peripheral blood mononuclear cells (PBMC) in a dose-dependent fashion, and the suppression was partially reversed by anti-transforming growth factor (TGF)-beta1 neutralizing antibody (Ab). These results demonstrate that rhesus macaque suppressive regulatory CD4(+)CD25(+)FOXP3(+) Tregs can be efficiently expanded in vitro under rhesus-specific stimulation, which enables preclinical testing of Treg therapy in the NHP model.

Elevated T regulatory cells in long-term stable transplant tolerance in rhesus macaques induced by anti-CD3 immunotoxin and deoxyspergualin.

Regulatory T cells (Tregs) are implicated in immune tolerance and are variably dependent on IL-10 for in vivo function. Brief peritransplant treatment of multiple nonhuman primates (NHP) with anti-CD3 immunotoxin and deoxyspergualin has induced stable (5-10 years) rejection-free tolerance to MHC-mismatched allografts, which associated with sustained elevations in serum IL-10. In this study, we demonstrate that resting and activated PBMC from long-term tolerant NHP recipients are biased to secrete high levels of IL-10, compared with normal NHP PBMC. Although IL-10-producing CD4+ Tregs (type 1 regulatory cells (TR1)/IL-10 Tregs) were undetectable (<0.5%) in normal rhesus monkeys, 7.5 +/- 1.7% of circulating CD4+ T cells of tolerant rhesus recipients expressed IL-10. In addition to this >15-fold increase in Tr1/IL-10 Tregs, the tolerant monkeys exhibited a nearly 3-fold increase in CD4+CD25+ Tregs, 8.1 +/- 3.0% of CD4 T cells vs 2.8 +/- 1.4% in normal cohorts (p < 0.02). The frequency of CD4+CD25+IL-10+ cells was elevated 5-fold in tolerant vs normal NHP (1.8 +/- 0.9% vs 0.4 +/- 0.2%). Rhesus CD4+CD25+ Tregs exhibited a memory phenotype, and expressed high levels of Foxp3 and CTLA-4 compared with CD4+CD25- T cells. Also, NHP CD4+CD25+ Tregs proliferated poorly after activation and suppressed proliferation of CD4+CD25- effector T cells, exhibiting regulatory properties similar to rodent and human CD4+CD25+ Tregs. Of note, depletion of CD4+CD25+ Tregs restored indirect pathway antidonor responses in tolerant NHP. Our study demonstrates an expanded presence of Treg populations in tolerant NHP recipients, suggesting that these adaptations may be involved in maintenance of stable tolerance.

Tolerance induced by anti-CD3 immunotoxin plus 15-deoxyspergualin associates with donor-specific indirect pathway unresponsiveness.

Peritransplant treatment with anti-CD3 immunotoxin plus deoxyspergualin induces tolerance to kidney allografts in most rhesus macaque recipients. Tolerant recipients maintain normal function for years without evidence of chronic rejection. Indirect alloantigen presentation is implicated in chronic rejection. Accordingly, we determined if anti-CD3 immunotoxin plus deoxyspergualin induced rejection-free tolerance associates with suppression of anti-donor indirect pathway responses. Tolerant recipients exhibited an early decrease in direct anti-donor responses with recovery to baseline levels by 3 years posttransplantation. In contrast, tolerant monkeys were unresponsive to donor antigens presented by the indirect pathway. Recipients that rejected their allografts retained vigorous direct and indirect anti-donor responses. Therefore, following temporary donor-specific hyporesponsiveness, direct responses recover in tolerant recipients >1.5 years after transplantation. However, tolerant recipients tested at 1.9-4 years posttransplant are specifically unresponsive to donor antigens presented by the indirect pathway. Thus, the rejection-free state of tolerant recipients may depend on mechanisms regulating indirect pathway responsiveness.

The immune decision toward allograft tolerance in non-human primates requires early inhibition of innate immunity and induction of immune regulation.

Brief treatment of rhesus macaques with immunotoxin plus 15-deoxyspergualin has yielded exceptional numbers (54%) of stable tolerant kidney allograft recipients, surviving over 6 years without rejection or immunosuppression. An early increase in IL-10 and reduction in IFNgamma distinguished recipients that subsequently became tolerant. Furthermore, analysis suggested that this immune switch was programmed within hours of transplantation. Administering deoxyspergualin within 5 h of surgery gave a higher incidence of tolerance (76%) compared to administration >5 h before or after surgery (11%, P<0.01). Deoxyspergualin inhibits nuclear translocation of activated NF-kappaB through heat shock proteins. Lymph node biopsies from tolerant recipients showed significant reductions in cytoplasmic expression of Hsp70 and RelB and almost complete inhibition of nuclear translocation of both. The early timing effect of deoxyspergualin suggests a crucial limitation to induction of stable tolerance is activation of Hsp-dependent innate responses to damage by ischemia-reperfusion. This was supported by studies in murine kidney reperfusion injury, where deoxyspergualin given 5 h before reperfusion protected renal function and reduced levels of IL-6 and IL-12. The narrow timing window for initiating deoxyspergualin treatment suggests the innate immune system is poised to defeat allograft tolerance induction, so effective blockade of NF-kappaB-mediated innate immunity must be in place early, to enable development of a tolerogenic environment.

CD40 is expressed on ovarian cancer cells and can be utilized for targeting adenoviruses.

PURPOSE: CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed on various cell types in addition to hematopoietic cells. Recent studies show that CD40 expression is related to several carcinomas, although its role in cancer pathobiology is unknown. In this study, we demonstrate the expression of CD40 on several ovarian carcinoma cell lines and the ability of CD40 to mediate targeted adenoviral infection in vitro. EXPERIMENTAL DESIGN: CD40 expression on ovarian cancer cell lines and clinical patient samples was examined by reverse transcription-PCR and flow cytometry. To study the utilization of CD40 for gene delivery, we precomplexed a luciferase coding adenovirus (Ad), Ad5luc1, with a CD40-targeting molecule (CAR/G28). RESULTS: According to our studies, all of the examined ovarian cancer cell lines are expressing CD40. In addition, mRNA for CD40 was detected in every primary tumor sample, suggesting that CD40 is also expressed in vivo. Compared with nontargeted Ad, gene transfer was increased up to 40-fold in CD40+ cells when CD40-targeted Ad was used. Supporting the relation of targeted system to CD40, increasing the amount of targeting fusion protein results in dose response. Furthermore, blockade of CD40 receptors on cell surface decreases the infectability of CD40+ cells with CD40-targeted virus, indicating the specificity of the targeting system for CD40. CONCLUSIONS: These results suggest that CD40 is present in ovarian cancer cells and can be used for targeted gene delivery in a CAR-independent manner, circumventing the problem of the low expression levels of CAR in various cancer cells.

Rhesus monocyte-derived dendritic cells modified to over-express TGF-beta1 exhibit potent veto activity.

BACKGROUND: The tolerogenic activity of allogeneic bone marrow cells (BMCs) associates with functional inactivation of alloreactive T cells and has been attributed to a veto effect. Studies in mice and rhesus monkeys indicated that the CD8alpha molecule expressed on a subpopulation of allogeneic BMCs is necessary to induce signal transduction within the BMCs to increase veto effector molecules such as transforming growth factor (TGF)-beta1. In vitro activation of alloreactive cytotoxic T-lymphocyte precursor enhances their susceptibility to veto-mediated functional inactivation by specific alloantigen-bearing BMCs. Accordingly, we examined a hypothesis that mature rhesus monkey (Rh) monocyte-derived dendritic cells (MDDCs) modified by gene transfer to over-express active TGF-beta1 might mediate veto activity without the need to express CD8alpha. METHODS: Rh MDDCs were modified by recombinant adenovirus (Ad) transduction and characterized by phenotype and functional studies. RESULTS: Rh MDDC transduction with Ad vectors using conventional methods was remarkably inefficient. However, a single-chain anti-CD40/soluble Coxsackie and adenovirus receptor-fusion protein (G28/sCAR) permitted high-efficiency transduction of Rh MDDCs by retargeting Ad to Rh MDDC CD40. Mature Rh MDDCs that were transduced to overexpress active TGF-beta1 (AdTGF-beta1 Rh MDDC) significantly suppressed alloimmune responses in [ H]thymidine uptake mixed leukocyte reaction assays. We showed by the carboxyfluorescein succinimidyl ester dilution method that allogeneic mature AdTGF-beta1 Rh MDDCs inhibited proliferation of CD4 and CD8 responder T cells. Notably, AdTGF-beta1 Rh MDDC abrogated alloimmune responses induced by control AdGFP Rh MDDC in an antigen-specific manner. CONCLUSIONS: These results suggest that nonhuman primate mature MDDCs can be genetically engineered to function as alloantigen-specific cellular immunosuppressants, an approach that has potential to facilitate induction of allograft tolerance in vivo.