Presence Of Pre-existing Ad5 Immunity Research Articles

Replication deficient human adenovirus vector serotype 19a/64: Immunogenicity in mice and female cynomolgus macaques

The human adenovirus type 19a/64 (hAd19a) is a rare serotype in the human population that transduces human dendritic cells (DCs) and human muscle cells more efficiently than the well-characterized human adenovirus type 5 (hAd5).To further characterize the potential of this vector as a vaccine we designed replication deficient hAd19a, hAd5 and MVA vectors expressing a papillomavirus (PV) antigen fused to the human MHC class II associated invariant chain T cell adjuvant (hIi) and investigated their immunogenicity in vivo in mice and cynomolgus macaques.We initially showed that the hIi encoded in the hAd5 enhanced PV specific CD8+ T cell responses in mice. The T cell responses induced after hAd19a vaccination was similar to those induced by hAd5 vaccination. The hAd19a induced responses were not reduced in presence of preexisting Ad5 immunity in mice. In macaques both vaccines were equally potent at inducing CD8+ T cells after MVA boost, while the level of CD4+ T cell responses were found to be broader in hAd19a primed animals.These data demonstrate the potential of hAd19a as an alternative vector to hAd5 to elicit potent T cell responses to PV.

Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients

First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.

Abstract A29: An Ad5 [E1-, E2b-] platform carrying the TAA CEA(6D) induces CEA directed CMI responses in patients with advanced CEA-expressing colorectal cancer in a phase I/II clinical trial..

Abstract First-generation, E1-deleted adenovirus (Ad)-based vectors, although promising platforms for use as cancer vaccines, are limited by the naturally occuring or induced Ad-specific neutralizing antibodies. We hypothesized that Ad5-based vectors with deletions of the E1 and the E2b regions (the latter encoding the DNA polymerase and the preterminal protein) would, by virtue of diminished late phase viral protein expression, avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. In this translational study, we demonstrated that delivery of increasing doses of the Ad5 [E1-, E2b-]-CEA(6D) in mice resulted in CEA-specific cell-mediated immune (CMI) responses. In the follow-up human phase I/II study, escalating doses of the vaccine induced higher CEA-specific CMI responses despite the presence of pre-existing Ad5 immunity in a majority (76%) of patients. Importantly, there was minimal toxicity, and overall patient survival (54.1% at 12 months) was similar regardless of pre-existing Ad5 neutralizing antibody titers. The results demonstrate that the novel Ad5 [E1-, E2b-] gene delivery platform can both break tolerance and generate significant CMI responses to the TAA CEA in the setting of both naturally acquired Ad5-specific immunity and/or immunization-induced Ad5 immunity. Citation Format: Michael A. Morse, Elizabeth S. Gabitzsch, Younong Xu, Dua Rajesh, Susan Nguyen, Stephanie Balcaitis, Joseph P. Balint, Jr., Frank Jones. An Ad5 [E1-, E2b-] platform carrying the TAA CEA(6D) induces CEA directed CMI responses in patients with advanced CEA-expressing colorectal cancer in a phase I/II clinical trial.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A29.

Ad5 immunity after multiple safe, homologous immunizations against tumor-associated antigens with new recombinant Ad5 vector.

85 Background: The major limitation for the use of Ad5 and other vectored vaccines is the inability to be effective in the presence of pre-existing or concomitant vector immunity. An Ad5-based vector with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), by virtue of diminished late phase viral protein expression, avoids immunological clearance and induces immune responses. Methods: The Ad5 [E1-, E2b-] platform encoding tumor associated antigen(s) (TAA) such as a modified carcinoembryonic antigen (CEA(6D)), HER2 and HPV E6/E7, were evaluated for the induction of TAA specific immune responses and anti-tumor effects in murine models. In a phase I/II clinical trial, cohorts of patients (n=25 total) with advanced colorectal cancer, refractory to prior therapies, received escalating doses of Ad5 [E1-, E2b-]-CEA(6D) (109 to 1011 vp) subcutaneously every 3 weeks for 3 immunizations. CEA-specific cell mediated immunity was measured by ELISPOT. Results: In murine immunotherapy studies, mice implanted with tumors expressing TAA and subsequently treated with the Ad5 [E1-, E2b-] platform expressing that TAA had significant inhibition of tumor progression. Pre-vaccination against the TAA utilizing the Ad5 [E1-, E2b-]-TAA resulted in inhibition of tumor establishment. In a clinical trial, patients who received the highest dose of Ad5 [E1-, E2b-]-CEA(6D) exhibited the highest levels of CEA-specific CMI responses. The induction of CEA-specific CMI responses increased over the course of the 3 injections despite the presence of pre-existing Ad5 immunity in the majority (75%) of patients. There were no drug related grade 3/4 toxicities. Conclusions: The results demonstrate that the novel Ad5 [E1-, E2b-] gene delivery platform can both break tolerance and generate significant CMI responses to the TAA CEA in the setting of both naturally acquired Ad5-specific immunity and/or immunization-induced Ad5 immunity.

Vaccines expressing the innate immune modulator EAT-2 elicit potent effector memory T lymphocyte responses despite pre-existing vaccine immunity.

The mixed results from recent vaccine clinical trials targeting HIV-1 justify the need to enhance the potency of HIV-1 vaccine platforms in general. Use of first-generation recombinant adenovirus serotype 5 (rAd5) platforms failed to protect vaccinees from HIV-1 infection. One hypothesis is that the rAd5-based vaccine failed due to the presence of pre-existing Ad5 immunity in many vaccines. We recently confirmed that EAT-2-expressing rAd5 vectors uniquely activate the innate immune system and improve cellular immune responses against rAd5-expressed Ags, inclusive of HIV/Gag. In this study, we report that use of the rAd5-EAT-2 vaccine can also induce potent cellular immune responses to HIV-1 Ags despite the presence of Ad5-specific immunity. Compared to controls expressing a mutant SH2 domain form of EAT-2, Ad5 immune mice vaccinated with an rAd5-wild-type EAT-2 HIV/Gag-specific vaccine formulation significantly facilitated the induction of several arms of the innate immune system. These responses positively correlated with an improved ability of the vaccine to induce stronger effector memory T cell-biased, cellular immune responses to a coexpressed Ag despite pre-existing anti-Ad5 immunity. Moreover, inclusion of EAT-2 in the vaccine mixture improves the generation of polyfunctional cytolytic CD8(+) T cell responses as characterized by enhanced production of IFN-γ, TNF-α, cytotoxic degranulation, and increased in vivo cytolytic activity. These data suggest a new approach whereby inclusion of EAT-2 expression in stringent human vaccination applications can provide a more effective vaccine against HIV-1 specifically in Ad5 immune subjects.

Effect of the vaccine Ad5 [E1-, E2b-]-CEA(6D) on CEA-directed CMI responses in patients with advanced CEA-expressing malignancies in a phase I/II clinical trial.

2585 Background: Adenovirus (Ad) vectors are promising platforms for use as cancer vaccines because of their substantial immunogenicity; however, Ad-specific neutralizing antibodies, present due to natural infection, limit their use. We created a new vector containing additional deletions of the E2b gene and observed that this Ad5 [E1-, E2b-] vector, engineered to express carcinoembryonic antigen (CEA(6D)), could induce antitumor immune responses in mice despite pre-existing Ad5 immunity. The purpose of the current translational study was to evaluate the immunologic effect of increasing doses of Ad5 [E1-, E2b-]-CEA(6D) in mice and then to extended these pre-clinical observations into a phase I/II study. Methods: Ad5 naïve female C57Bl/6 mice, 4-6 weeks old, were immunized subcutaneously 3 times at 2 week intervals with doses of 1.4X107 viral particles (vp) up to1.4X109 vp of Ad5 [E1-, E2b-]-CEA(6D). Two weeks after the last immunization, splenocytes were harvested for immune determinations. Cohorts of patients (n=25 total) with advanced colorectal cancer, refractory to prior therapies, received escalating doses of Ad5 [E1-, E2B-]-CEA(6D) (109 to 1011 vp) subcutaneously every 3 weeks for 3 immunizations.CEA-specific cell mediated immunity was measured by ELISPOT. Results: Increasing doses of Ad5 [E1-, E2b-]-CEA(6D) in mice induced increasing CEA-specifc CMI responses. Similarly, patients who received the highest dose of Ad5 [E1-, E2b-]-CEA(6D) exhibited the highest levels of CEA-specific CMI responses. The induction of CEA-specific CMI responses increased over the course of the 3 injections despite the presence of pre-existing Ad5 immunity in the majority (75%) of patients. There were no drug related grade 3/4 toxicities.Two patients with stable disease remained so during the study. All other patients experienced progressive disease; however, 1-year survival was 54%. Conclusions: Multiple homologous doses of Ad5 [E1-, E2b-]-CEA(6D) could break tolerance and generate CEA-specific CMI responses in the setting of Ad specific immunity in colorectal cancer patients.

Induction and comparison of SIV immunity in Ad5 naïve and Ad5 immune non-human primates using an Ad5 [E1-, E2b-] based vaccine

The effectiveness of recombinant Adenovirus serotype 5 (Ad5) vectors to induce immune responses against targeted antigens has been limited by the presence of pre-existing or Ad5 vaccine induced anti-vector immunity. The Ad5 [E1-, E2b-] platform, a recombinant Ad5 with additional deletions, has been previously reported by us to induce immune responses in the presence of Ad5 immunity. In an Ad5 immune non-human primate (NHP) model, an Ad5 [E1-, E2b-] construct expressing HIV-1 Gag induced immune responses in the presence of pre-existing Ad5 immunity. In the present study we expand on these prior observations by comparing the cell mediated immune (CMI) responses induced by Ad5 [E1-, E2b-]-SIV-gag/nef in Ad5 naïve and Ad5 immune NHP. Additionally, NHP were immunized with an Ad5 [E1-, E2b-]-HIV-pol construct following two homologous administrations of Ad5 [E1-, E2b-]-SIV-gag/nef to determine if an immune response could be induced against a third antigen in the presence of vaccine induced Ad5 immunity. Positive CMI responses, as assessed by interferon-gamma (IFN-γ) secreting lymphocytes, were induced against all three antigens. These CMI responses increased over a course of multiple immunizations and the response profiles observed in Ad5 naïve and Ad5 immune NHP were similar. No influence of the major histocompatibility complex on CMI responses was observed. These data indicate that the new Ad5 [E1-, E2b-] platform based vaccine could be used for homologous vaccination regimes to induce robust CMI responses in the presence of Ad5 vector immunity.

An Ad5[E1-, E2b-]-HER2/neu vector induces immune responses and inhibits HER2/neu expressing tumor progression in Ad5 immune mice

Immunotherapy is a promising approach for the treatment of cancers. Modified Adenovirus 5 (Ad5) vectors have been used as a platform to deliver genes encoding TAA. A major obstacle to Ad5 vector immunotherapy has been the induction of vector immunity following administration or the presence of pre-existing Ad5 immunity, which results in vector mitigation. It has been reported by us that the Ad5 [E1-, E2b-] platform with unique deletions in the E1, E2b and E3 regions can induce potent cell mediated immunity (CMI) against delivered transgene products in the presence of pre-existing Ad5 immunity. Here we report the use of an Ad5 [E1-, E2b-] vector platform expressing the TAA HER2/neu as a breast cancer immunotherapeutic agent. Ad5 [E1-, E2b-]-HER2/neu induced potent CMI against HER2/neu in Ad5 naïve and Ad5 immune mice. Humoral responses were also induced and antibodies could lyse HER2/neu expressing tumor cells in the presence of complement in vitro. Ad5 [E1-, E2b-]-HER2/neu prevented establishment of HER2/neu-expressing tumors and significantly inhibited progression of established tumors in Ad5 naïve and Ad5 immune murine models. These data demonstrate that in vivo delivery of Ad5 [E1-, E2b-]-HER2/neu can induce anti-TAA immunity and inhibit progression of HER2/neu expressing cancers.

Sublingual Administration of an Adenovirus Serotype 5 (Ad5)-Based Vaccine Confirms Toll-Like Receptor Agonist Activity in the Oral Cavity and Elicits Improved Mucosal and Systemic Cell-Mediated Responses against HIV Antigens despite Preexisting Ad5 Immunity

HIV/AIDS continue to devastate populations worldwide. Recent studies suggest that vaccines that induce beneficial immune responses in the mucosal compartment may improve the efficacy of HIV vaccines. Adenovirus serotype 5 (Ad5)-based vectors remain a promising platform for the development of effective vaccines. In an effort to improve the efficacy of Ad5-based vaccines, even in the presence of preexisting Ad5 immunity, we evaluated the potential for an Ad5-based HIV vaccine to induce antigen-specific immune responses following sublingual (s.l.) administration, a route not previously tested in regard to Ad-based vaccines. s.l. vaccination with an Ad5-based HIV-Gag vaccine resulted in a significant induction of Gag-specific cytotoxic T-lymphocyte (CTL) responses in both the systemic and the mucosal compartment. We also show that s.l. immunization not only avoided preexisting Ad5 immunity but also elicited a broad repertoire of antigen-specific CTL clones. Additionally, we confirm for the first time that oral delivery of a vaccine expressing a potent Toll-like receptor (TLR) agonist can stimulate innate immune responses through induction of cytokines and chemokines and activation of NK cells, NKT cells, and macrophages in vivo. These results positively correlated with improved antigen-specific CTL responses. These results could be achieved both in Ad5-naïve mice and in mice with preexisting immunity to Ad5. The simplicity of the s.l. vaccination regimen coupled with augmentation of TLR-dependent pathways active in the oral cavity makes s.l. delivery a promising method for HIV vaccine development specifically, as well as for many other vaccine applications in general.

Anti-tumor immunotherapy despite immunity to adenovirus using a novel adenoviral vector Ad5 [E1-, E2b-]-CEA.

Adenovirus serotype 5 (Ad5) has been widely used in clinical trials because it expresses inserted transgenes robustly and augments the innate immune response. Strategies to improve Ad5 vectors that can circumvent Ad5 immunity have become a critical issue, especially for use as a cancer immunotherapeutic in which repeated immunization is required. In this study, we constructed a novel Ad5 vector with unique deletions of the viral DNA polymerase and the pre-terminal protein region (Ad5 [E1-, E2b-]). This vector contains the carcinoembryonic antigen (CEA) gene insert and is designed to induce cell-mediated immunity (CMI) against the tumor-associated target. The CEA immunogenicity and in vivo anti-tumor effects of repeated immunizations with Ad5 [E1-, E2b-]-CEA compared with those observed with current generation Ad5 [E1-]-CEA were tested in Ad5 pre-immunized mice. We report that Ad5-immune mice immunized multiple times with Ad5 [E1-, E2b-]-CEA induced CEA-specific CMI responses that were significantly increased over those detected in Ad5-immune mice immunized multiple times with a current generation Ad5 [E1-]-CEA. Ad5 immune mice bearing CEA-expressing tumors that were treated with Ad5 [E1-, E2b-]-CEA had increased anti-tumor response as compared with Ad5 [E1-]-CEA treated mice. These results demonstrate that Ad5 [E1-, E2b-]-CEA can induce CMI immune responses which result in tumor growth inhibition despite the presence of pre-existing Ad5 immunity. Multiple re-immunizations using the same vector platform are now possible with the novel Ad5 [E1-, E2b-] platform.

A preliminary and comparative evaluation of a novel Ad5 [E1-, E2b-] recombinant-based vaccine used to induce cell mediated immune responses

Adenovirus vectors have been shown to be highly effective as vaccine platforms capable of inducing both humoral and cell mediated immune (CMI) responses. An Ad serotype 5 vector containing unique deletions in the E2b region (Ad5 [E1-, E2b-]) has been reported to have several advantages over conventional Adenovirus serotype 5 (Ad5) vectors deleted in only the E1 region (Ad5 [E1-]), including increased carrying capacity and diminished viral late gene expression. Here, we evaluated a novel Ad5 [E1-, E2b-] vector utilizing the E.C7 cell line for viral packaging. Its’ effectiveness as a potential vaccine platform as compared to the currently utilized Ad5 [E1-]-based platform was assessed in both Ad5 naïve and Ad5 immune mice. We employed the HIV-1 Gag gene as the antigenic transgene expressed by the novel vector. Cellular expression of the Gag was confirmed by Western Blot analysis. Dose response studies using three intradermal immunizations of 10 7 to 10 10 virus particles (VP) of each construct revealed that immunization with 10 10 VP resulted in the maximum immunological response. Multiple immunizations of Ad naïve BALB/c mice with an Ad5 [E1-, E2b]-gag vaccine resulted in higher ELISpot CMI responses as compared to mice immunized with an Ad5 [E1-]-gag vaccine. More importantly, multiple immunizations of Ad5 immune BALB/c mice with an Ad5 [E1-, E2b]-gag vaccine resulted in significant increases in ELISpot CMI responses when compared to Ad5 immune mice vaccinated with an Ad5 [E1-]-gag vector. Preliminary studies in three Ad5 immune non-human primates (NHP) demonstrated that vaccination with Ad5 [E1-, E2b-]-gag-induced elevated levels of interferon-γ and IL-2 secreting lymphocytes as assessed by ELISpot assays. These studies indicate that the novel Ad5 [E1-, E2b-] viral vector can be utilized as a potential vaccine platform to induce elevated CMI responses as compared to current generation Ad5 [E1-] viral vectors even in the presence of pre-existing Ad5 immunity.