Younong Xu

Novel Adenovirus type 5 vaccine platform induces cellular immunity against HIV-1 Gag, Pol, Nef despite the presence of Ad5 immunity

Recombinant Adenovirus serotype 5 (Ad5) vectors have been used as vaccine platforms in numerous animal and human clinical studies. The immune response induced by Ad5 vaccines can be mitigated due to pre-existing Ad5 immunity. We previously reported the use of a novel Ad5 platform to induce cellular immune responses (CMI) against HIV-1 Gag in Ad5 hyper immune mice. Here, the effectiveness of the Ad5 [E1-, E2b-] vaccine platform was evaluated using a triad mixture of HIV-1 Gag, Pol, and Nef as antigenic transgenes. Broad CMI was induced following vaccination with the HIV-1 expressing vectors in Ad5 naïve and Ad5 immunized mice. A mixture of the three vaccines induced CMI against each transgene product even in the presence of hyper Ad5 immunity. These studies revealed that CMI responses to immunization with Ad5 [E1-, E2b-]-gag, Ad5 [E1-, E2b-]-pol or Ad5 [E1-, E2b-]-nef vectors were transgene specific and did not induce CMI responses against irrelevant antigens such as carcinoembryonic antigen (CEA), herpes simplex virus glycoprotein B (HSV), cytomegalovirus (CMV) or influenza virus antigens. We are evaluating this recombinant triad viral vector as an HIV-1 vaccine in a non-human primate model and the data indicate that the vaccine is worthy of clinical evaluation.

miR-1269 promotes metastasis and forms a positive feedback loop with TGF-β

As patient survival drops precipitously from early-stage cancers to late-stage and metastatic cancers, microRNAs that promote relapse and metastasis can serve as prognostic and predictive markers as well as therapeutic targets for chemoprevention. Here we show that miR-1269a promotes colorectal cancer (CRC) metastasis and forms a positive feedback loop with TGF-β signaling. miR-1269a is upregulated in late-stage CRCs, and long-term monitoring of 100 stage II CRC patients revealed that miR-1269a expression in their surgically removed primary tumors is strongly associated with risk of CRC relapse and metastasis. Consistent with clinical observations, miR-1269a significantly increases the ability of CRC cells to invade and metastasize in vivo. TGF-β activates miR-1269 via Sox4, while miR-1269a enhances TGF-β signaling by targeting Smad7 and HOXD10, hence forming a positive feedback loop. Our findings suggest that miR-1269a is a potential marker to inform adjuvant chemotherapy decisions for CRC patients and a potential therapeutic target to deter metastasis.

A non-oncogenic HPV 16 E6/E7 vaccine enhances treatment of HPV expressing tumors

Human papillomaviruses (HPVs) are the causative factor for >90% of cervical cancers and 25% of head and neck cancers. The incidence of HPV positive (+) head and neck squamous cell carcinomas has greatly increased in the last 30 years. E6 and E7 are the two key viral oncoproteins that induce and propagate cellular transformation. An immune response generated during cisplatin/radiation therapy improves tumor clearance of HPV(+) cancers. Augmenting this induced response during therapy with an adenoviral HPV16 E6/E7 vaccine improves long-term survival in pre-clinical models. Here, we describe the generation of an HPV16 E6/E7 construct, which contains mutations that render E6/E7 non-oncogenic, while preserving antigenicity. These mutations do not allow E6/E7 to degrade p53, pRb, PTPN13, or activate telomerase. Non-oncogenic E6/E7 (E6Δ/E7Δ) expressed as a stable integrant, or in the [E1-, E2b-] adenovirus, lacks the ability to transform human cells while retaining the ability to induce an HPV-specific immune response. Moreover, E6Δ/E7Δ plus chemotherapy/radiation statistically enhances clearance of established HPV(+) cancer in vivo.

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.

Prevention of influenza virus shedding and protection from lethal H1N1 challenge using a consensus 2009 H1N1 HA and NA adenovirus vector vaccine.

Vaccines against emerging pathogens such as the 2009 H1N1 pandemic virus can benefit from current technologies such as rapid genomic sequencing to construct the most biologically relevant vaccine. A novel platform (Ad5 [E1-, E2b-]) has been utilized to induce immune responses to various antigenic targets. We employed this vector platform to express hemagglutinin (HA) and neuraminidase (NA) genes from 2009 H1N1 pandemic viruses. Inserts were consensuses sequences designed from viral isolate sequences and the vaccine was rapidly constructed and produced. Vaccination induced H1N1 immune responses in mice, which afforded protection from lethal virus challenge. In ferrets, vaccination protected from disease development and significantly reduced viral titers in nasal washes. H1N1 cell mediated immunity as well as antibody induction correlated with the prevention of disease symptoms and reduction of virus replication. The Ad5 [E1-, E2b-] should be evaluated for the rapid development of effective vaccines against infectious diseases.

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 tumor associated antigens (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.

Induction and comparison of SIV immunity in Ad5 naive 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.

Control of SIV infection and subsequent induction of pandemic H1N1 immunity in rhesus macaques using an Ad5 [E1-, E2b-] vector platform

Anti-vector immunity mitigates immune responses induced by recombinant adenovirus vector vaccines, limiting their prime-boost capabilities. We have developed a novel gene delivery and expression platform (Ad5 [E1-, E2b-]) that induces immune responses despite pre-existing and/or developed concomitant Ad5 immunity. In the present study, we evaluated if this new Ad5 platform could overcome the adverse condition of pre-existing Ad5 immunity to induce effective immune responses in prime-boost immunization regimens against two different infectious diseases in the same animal. Ad5 immune rhesus macaques (RM) were immunized multiple times with the Ad5 [E1-, E2b-] platform expressing antigens from simian immunodeficiency virus (SIV). Immunized RM developed cell-mediated immunity against SIV antigens Gag, Pol, Nef and Env as well as antibody against Env. Vaccinated and vector control RMs were challenged intra-rectally with homologous SIVmac239. During a 7-week follow-up, there was perturbation of SIV load in some immunized RM. At 7 weeks post-challenge, eight immunized animals (53%) did not have detectable SIV, compared to two RM controls (13%) (P<0.02; log-rank Mantel-Cox test). There was no correlation of protective MHC contributing to infection control. The RM without detectable circulating SIV, now hyper immune to Ad5, were then vaccinated with the same Ad5 [E1-, E2b-] platform expressing H1N1 influenza hemagglutinin (HA). Thirty days post Ad5 [E1-, E2b-]-HA vaccination, significant levels of influenza neutralizing antibody were induced in all animals that increased after an Ad5 [E1-, E2b-]-HA homologous boost. These data demonstrate the versatility of this new vector platform to immunize against two separate disease targets in the same animal despite the presence of immunity against the delivery platform, permitting homologous repeat immunizations with an Ad5 gene delivery platform.

Novel adenoviral serotype 5 based immunotherapeutic induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients

Naturally occurring or induced Ad-specific neutralizing antibodies impeded the activity of recombinant Ad5-based vectors with E1 deletions. An improved Ad vector with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, with significantly 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. In the present phase I/II study, cohorts of patients with advanced metastatic colorectal cancer (mCRC) were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). The carcinoembryonic antigen (CEA) transgene employed contains a modification (CAP1-6D) designed to enhance CTL stimulation. CEA-specific CMI responses were observed despite the presence of pre-existing Ad5 immunity in a majority (61.3%) of patients. Long-term follow-up in some of the patients reveled a waning of the induced CEA directed immune responses at around 6 months. Importantly, there was minimal toxicity, and overall patient survival (48% at 12 months) was similar regardless of pre-existing Ad5 neutralizing antibody titers. Our patient demographics, albeit limited in size, we similar with previously published studies of patients with chemotherapy-refractory mCRC. Of particular interest is the observation that treated mCRC patients in our study exhibited favorable survival probability. 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..

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.