Ad Vectors Research Articles

Longitudinal quantification of adenovirus neutralizing responses in Zambian mother-infant pairs: Impact of HIV-1 infection and its treatment

Vaccination offers the most cost-effective approach to limiting the adverse impact of infectious and neoplastic diseases that reduce the quality of life in sub-Saharan Africa (SSA). However, it is unclear what vaccine vectors would be most readily implementable in the setting and at what age they should be applied for maximal efficacy. Adenoviruses (Ad) and Ad-based vectors have been demonstrated to induce effective humoral and cellular immune responses in animal models and in humans. However, because immunity associated with Ad infection is lifelong, there exists a debate as to whether pre-existing immunity might decrease the efficacy of Ad vectored vaccines. In order to begin to rationally develop vaccination strategies for SSA, we have quantified neutralizing antibodies (nAb) against Ad4, Ad5, Ad7, Ad26, Ad28, Ad45 and Ad48 in 67 adult women and their infants. We are the first to define the decay kinetics of transferred maternal nAb in infants as well as the apparent initiation of de novo Ad responses. Our findings demonstrate that in Zambian adults, robust nAb responses exist against each of the Ads tested and are efficiently transferred to newborns. With few exceptions, neither the HIV-1 infection status of the mothers or the antiretroviral therapy (ART) treatment of HIV-1 disease had significant impact on maternal Ad nAb responses or their transfer to infants. However, maternal Ad nAb decays in infants to a nadir at 12 months of age such that any of the seven Ad types could function as vaccine vectors. The definition of this ‘window of opportunity’ provides important foundational data for rational design and implementation of Ad vectors in this setting.

Adenovirus-mediated transduction with Histone Deacetylase 4 ameliorates disease progression in an osteoarthritis rat model

BackgroundDownregulation of histone deacetylase-4 (HDAC4) contributes to cartilage degeneration in osteoarthritis (OA) because it promotes upregulation of runt-related transcription factor-2 (Runx-2) and osteoarthritis-related genes. The effect of HDAC4 upregulation on cartilage damage in OA remains unknown. MethodsRat chondrocytes were infected with Ad-GFP or Ad-HDAC4-GFP for 48 h, stimulated with interleukin-1β (IL-1β, 10 ng/mL) for 24 h, and then harvested for RT-qPCR. Male Sprague-Dawley rats in 3 groups were given anterior cruciate ligament transection (ACLT) or sham operation, and knee injections with different adenovirus (Ad) vectors at 48 h after surgery and every 3 weeks thereafter: ACLT+Ad-GFP (n = 17); ACLT+Ad-HDAC4-GFP (n = 20); and sham+Ad-GFP (n = 15). Three ACLT-Ad-HDAC4-GFP rats were sacrificed at different times to examine the expression of HDAC4. Two ACLT-Ad-GFP rats and two ACLT-Ad-HDAC4-GFP rats were euthanized at week-2; articular cartilage was harvested and expression of HDAC4 was determined by RT-qPCR. All other rats were euthanized at week-8. Cartilage damage and OA progression was assessed using radiography, fluorescence molecular tomography (FMT), histology, immunohistochemistry (IHC), ELISA, and RT-qPCR. ResultsOverexpression of HDAC4 in chondrocytes stimulated by IL-1β reduced the levels of Runx-2, MMP-13, and Collagen X, but increased the levels of Collagen II and Aggrecan. Upregulation of HDAC4 reduced osteophyte formation and cartilage damage, and increased articular cartilage anabolism. ConclusionHDAC4 attenuated articular cartilage damage by repression of Runx-2, MMP-13, and collagen X and induction of collagen II and ACAN in this rat model of OA. Upregulation of HDAC4 may provide chondroprotection in OA patients.

Abstract LB-269: Development of a vaccine model to track the CD8-specific response in Mauritius cynomolgus monkey

Abstract Introduction Manipulating the immune system to achieve therapeutic efficacy in cancer treatment has led to a new class of drugs that brought lasting remissions to many patients who had run out of options. The development of new immunotherapy aimed at targeting cancer cells requires the ability to monitor specifically the immune response. Although CD8 T cells play a crucial role in this process, no equivalent of the traditional T-cell-dependent antibody response (TDAR) assay, which evaluates the T-helper immune response, is currently available. Since cancer immunotherapy agents are often tested in non-tumor bearing NHP for safety assessment, the animal models currently available do not allow monitoring of potential exaggerated pharmacology and efficacy of these new drug candidates. Objectives The goal of this study was to develop an NHP vaccination model that specifically elicits a CTL response, in order to evaluate the efficacy in exacerbating the Cytotoxic T Lymphocyte (CTL) response. Experimental Procedures To reach this goal, MHC-genotyped Mauritian cynomolgus macaques (MCMs) were immunized with 3 replication incompetent recombinant adenovirus serotype 5 (Ad5) vectors, each containing the coding sequence for Gag, Nef or Pol SIV proteins. Such model allowed monitoring of the CD8 T cell activation in lieu of a tumor or live virus model. MCMs were distributed into 3 groups: one control group and 2 groups which received two intramuscular injection of the adenovirusrs spaced by 4 or 8 weeks. The immune response was monitored with blood samples taken on a weekly basis for up to 12 weeks. Blood samples were used to 1) characterize the different CD8-positive sub-populations by Tetramer staining and Immunophenotyping; 2) to correlate the immunophenotyping profile obtained with functional assays such as ex vivo recall response and IFNγ ELIspot. Results Of the three groups tested, MCM monkeys receiving 2 injections 8 weeks apart showed the more robust CD8 T cell response. This response was mainly characterized by a proliferation profile (Ki67-positive cells), the expression of activation markers at the surface of CD8 T cells and antigen specific responses of the CD8 T cells. Of the three Gag, Pol and Nef proteins, immunization with Nef gave the most robust response, as observed by the IFNγ ELIspot results and by the presence of a Nef-specific CD8-specific subpopulation observed by tetramer staining. No changes in the distribution of T, B, NK, Treg or Memory T cells was observed between the 3 groups. Conclusion The results obtained during the course of this study suggests that the described CD8-specific vaccination model using Mauritius cynomolgus macaques is a promising model to evaluate the efficacy and potential exaggerated pharmacology of new drug candidates targeting CD8 T cells. Citation Format: Richard Graveline, Morad Haida, Carolyne Dumont, Rana Samadfam, Dominic Poulin, Marie-Soleil Piche. Development of a vaccine model to track the CD8-specific response in Mauritius cynomolgus monkey [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-269.

CD40 ligand (CD40L) multi-trimer antigen fusion protein (MagaVax) as a vaccine design for high-level CD8+ T cell responses

Abstract Dendritic cells (DCs) can take up antigen via a number of routes. Of these, several studies have shown antigen uptake via the CD40 receptor generates some of the strongest CD8+ T cell responses. At the same time, stimulation through CD40 activates DCs for cross-presentation and heightened antigen presentation. Consequently, it is desirable to combine the CD40 targeting of antigen and a CD40 stimulant into a single vaccine formulation. When the CD40 stimulant is CD40 ligand (CD40L, CD154), it is important to use a multi-trimer complex that clusters CD40 in the DC membrane, thereby engaging downstream signaling pathways. Such a multi-trimer form of CD40L can be provided by fusing the extracellular domain of CD40L with the body of surfactant protein D (SPD), a self-assembling 12-chain soluble molecule with 4 trimeric “arms.” When an antigen sequence is included within the SPD arm, the entire protein complex is termed “Multi-trimer AntiGen Adjuvant Vaccine (Vax)” or “MagaVax.” Studies in mice have shown that MagaVax elicits very high levels of antigen-specific CD8+ T cells with high TCR avidity and functional activity. MagaVax can be effectively delivered as a DNA vaccine i.m. and even more effectively using an adenoviral (Ad5) vector. For a MagaVax encoding HIV-1 Gag, vaccination with Ad5-MagaVax-Gag led to complete protection (“sterilizing immunity”) from Vaccinia-Gag challenge (i.e., 7 log reduction of tissue virus). Thus far, MagaVax formulations have been made for HIV-1 Gag, Ebola, malaria, hepatitis B virus (HBV), and tumor-specific neoantigens. In all of these cases, there may be a clinical benefit for a vaccine that elicits very strong CD8+ T cell responses.

HIV-1-specific B cell immune responses in Rhesus Macaques induced by early region deleted-Ad5 vectors

Abstract As a vaccine delivery vector, Adenovirus (Ad) is recognized for its ability to prime immune responses against HIV and other infectious agents. We reported that early region 1B55k (E1B55k) deleted vector induced cellular immunity, transgene-specific antibodies and innate immune signals including chemokines, cytokines and NKG2D ligands MIC A/B in immunized mice. However, the immune priming ability of E3-E1B55k-E4orf1 and E3-E1B55k- E4orf1-4deleted Ad vectors is unknown. Here we compared HIV-specific immune responses generated by E3deleted, E3-E1B55k, E3-E1B55k-E4orf1and E3-E1B55k-E4orf1-4deleted Ad5 vectors encoding full-length single-chain HIVBalgp120 linked to the D1 and D2 domains of rhesus macaque (RM) CD4 (rhFLSC). RMs were immunized twice mucosally with each vector. Blood and rectal tissue were collected at pre and 2-week post-immunization time points. The frequency of plasmablasts (PB)/plasma cells (PC), memory B cells, and antibody secreting cells and antibody titers were assessed by flow cytometry, ELISpot and ELISA, respectively. All vectors induced increased frequencies of rectal PB, PC and rhFLSC specific memory B cells. rhFLSC specific IgA and IgG secreting cells were detected in all immunized macaques; the IgA secreting cells were significantly increased compared to pre values. This was accompanied by elevated levels of IgG, IgG1 and IgA serum antibodies. In summary all four vectors induced B cells responses while retaining replicability. Moreover, the largest deletion, E3-E1B55k-E4orf1-4, improved the transgene carrying capacity of the Ad vector while maintaining the ability to induce B cell immunity important for protective efficacy.

Replicating Ad-SIV recombinant vaccines elicit mucosal humoral immunity in rhesus macaques at both rectal and vaginal sites with potential protective efficacy

Abstract Humoral immunity, especially at mucosal sites, is important for preventing HIV acquisition. Replicating adenovirus (Ad)-SIV priming/gp120 boosting regimens have elicited mucosal immunity and protection against intrarectal SIV challenge in Rhesus macaques. Here we investigated mucosal responses in female macaques primed intranasally/orally, then intratracheally with Ad5hr-SIV(Env/gag/nef), boosted twice intramuscularly with SIV gp120, and challenged vaginally with repeated low-dose SIVmac251. Vaginal washes and rectal swabs and biopsies were obtained 3-weeks post-immunizations. gp120-specific antibodies were assayed by ELISA and memory B-cells by flow cytometry. Gp120-specific rectal memory B-cells increased post-Ad priming but waned post-boosting, whereas rectal and vaginal antibodies increased post-boosting. Compared to pre levels, rectal gp120-specific IgA (p = 0.011) and IgG (p< 0.0001) were elevated post-2nd boost; vaginal IgA/IgG antibodies differed post-1st boost (both p<0.0001) and IgG levels > IgA post-2nd boost (p=0.001). Rectal memory B cells post- 1st and 2nd Ads positively correlated with gp120-specific rectal IgA (p = 0.015, p = 0.0013, respectively) post-1st Ad, indicating vaccine targeting to mucosal effector sites. Significantly delayed SIV acquisition was not seen, but vaginal IgG post-1st Ad correlated with number of challenges (p=0.029). Vaccinated animals had lower acute viremia than controls (p = 0.054), consistent with higher vaginal gp120-specific IgG 2 weeks post-infection (p < 0.0001). Our data show that upper respiratory tract immunization with replicating Ad vectors elicits strong mucosal immunity with potential protective efficacy against rectal/genital transmission.

Genetic strategy to decrease complement activation with adenoviral therapies.

BackgroundA major obstacle to using recombinant adenoviral vectors in gene therapy is the natural ability of human adenovirus to activate the classical and alternate complement pathways. These innate immune responses contribute to hepatic adenoviral uptake following systemic delivery and enhance the humoral immune responses associated with adenoviral infection.MethodsA recombinant Ad5 vector was genetically modified to display a peptide sequence (“rH17d’”), a known inhibitor of the classical complement pathway. The replication-defective vectors Ad5.HVR2-rH17d’ and Ad5.HVR5-rH17d’ were constructed by engineering the rH17d’ peptide into the hypervariable region (HVR)-2 or HVR5 of their major capsid protein hexon. Control Ad5 vectors were created by incorporation of a 6-histidine (His6)-insert in either HVR2 or HVR5 (Ad5.HVR2-His6 and Ad5.HVR5-His6, respectively). All vectors encoded CMV promoter-controlled firefly luciferase (Luc). The four vectors were evaluated in TIB76 mouse liver cells and immunocompetent mice to compare infectivity and liver sequestration, respectively.ResultsIn vitro studies demonstrated that preincubation of all the Ad5 vectors with fresh serum significantly increased their gene transfer relative to preincubation with PBS except Ad5.HVR5-rH17d’, whose infectivity of liver cells showed no serum-mediated enhancement. In line with that, mice injected with Ad5.HVR2-rH17d’ or Ad5.HVR5-rH17d’ showed significantly lower luciferase expression levels in the liver as compared to the respective control vectors, whereas efficiency of tumor transduction by rH17d’ and His6 vectors following their intratumoral injection was similar.ConclusionsDisplaying a complement-inhibiting peptide on the Ad5 capsid surface by genetic modification of the hexon protein could be a suitable strategy for reducing Ad5 liver tropism (Ad5 sequestration by liver), which may be applicable to other gene therapy vectors with natural liver tropism.

Split tolerance permits safe Ad5-GUCY2C-PADRE vaccine-induced T-cell responses in colon cancer patients

BackgroundThe colorectal cancer antigen GUCY2C exhibits unique split tolerance, evoking antigen-specific CD8+, but not CD4+, T-cell responses that deliver anti-tumor immunity without autoimmunity in mice. Here, the cancer vaccine Ad5-GUCY2C-PADRE was evaluated in a first-in-man phase I clinical study of patients with early-stage colorectal cancer to assess its safety and immunological efficacy.MethodsTen patients with surgically-resected stage I or stage II (pN0) colon cancer received a single intramuscular injection of 1011 viral particles (vp) of Ad5-GUCY2C-PADRE. Safety assessment and immunomonitoring were carried out for 6 months following immunization. This trial employed continual monitoring of both efficacy and toxicity of subjects as joint primary outcomes.ResultsAll patients receiving Ad5-GUCY2C-PADRE completed the study and none developed adverse events greater than grade 1. Antibody responses to GUCY2C were detected in 10% of patients, while 40% exhibited GUCY2C-specific T-cell responses. GUCY2C-specific responses were exclusively CD8+ cytotoxic T cells, mimicking pre-clinical studies in mice in which GUCY2C-specific CD4+ T cells are eliminated by self-tolerance, while CD8+ T cells escape tolerance and mediate antitumor immunity. Moreover, pre-existing neutralizing antibodies (NAbs) to the Ad5 vector were associated with poor vaccine-induced responses, suggesting that Ad5 NAbs oppose GUCY2C immune responses to the vaccine in patients and supported by mouse studies.ConclusionsSplit tolerance to GUCY2C in cancer patients can be exploited to safely generate antigen-specific cytotoxic CD8+, but not autoimmune CD4+, T cells by Ad5-GUCY2C-PADRE in the absence of pre-existing NAbs to the viral vector.Trial registrationThis trial (NCT01972737) was registered at ClinicalTrials.gov on October 30th, 2013. https://clinicaltrials.gov/ct2/show/NCT01972737

Exploring the Functions of Polymers in Adenovirus-Mediated Gene Delivery: Evading Immune Response and Redirect Tropism

Adenoviruses (Ads) are promising viral carriers for gene therapy because of their unique attribution. However, clinical applications of Ad vectors are currently restricted by their immunogenicity and broad native tropism. To address these obstacles, a variety of non-immunogenic polymers are utilized to modify Ad vectors chemically or physically. In this review, we systemically discuss the functions of polymers in Ad-mediated gene delivery from two aspects, evading the host immune responses to Ads and redirecting Ad tropism. With polyethylene glycol (PEG) first in order, a variety of polymers have been developed to shield the surface of Ad vectors, and well accomplished to evade the host immune response, block CAR-dependant cellular uptake and reduce liver accumulation. Besides these functions, shielding Ad vectors with targeted polymers (including targeting ligands conjugated polymers and bio-responsive polymers) can also efficiently retarget Ad vectors to tumor tissues and reduce their distribution at non-targeted tissues. With its potentials to evade the immune response and retarget Ad vectors, modification with polymers has been generally regarded as a promising strategy to facilitate the clinical applications of Ad vectors for virotherapy.

Rodents Versus Pig Model for Assessing the Performance of Serotype Chimeric Ad5/3 Oncolytic Adenoviruses.

Oncolytic adenoviruses (Ad) are promising tools for cancer therapeutics. Most Ad-based therapies utilize species C serotypes, with Adenovirus type 5 (Ad5) most commonly employed. Prior clinical trials demonstrated low efficiency of oncolytic Ad5 vectors, mainly due to the absence of Ad5 primary receptor (Coxsackie and Adenovirus Receptor, CAR) on cancer cells. Engineering serotype chimeric vectors (Ad5/3) to utilize Adenovirus type 3 (Ad3) receptors has greatly improved their oncolytic potential. Clinical translation of these infectivity-enhanced vectors has been challenging due to a lack of replication permissive animal models. In this study, we explored pigs as a model to study the performance of fiber-modified Ad5/3 chimeric vectors. As a control, the Ad5 fiber-unmodified virus was used. We analyzed binding, gene transfer, replication, and cytolytic ability of Ad5 and Ad5/3 in various non-human cell lines (murine, hamster, canine, porcine). Among all tested cell lines only porcine cells supported active binding and replication of Ad5/3. Syrian hamster cells supported Ad5 replication but showed no evidence of productive viral replication after infection with Ad5/3 vectors. Transduction and replication ability of Ad5/3 in porcine cells outperformed Ad5, a phenomenon often observed in human cancer cell lines. Replication of Ad5 and Ad5/3 was subsequently evaluated in vivo in immunocompetent pigs. Quantitative PCR analyses 7 days post infection revealed Ad5 and Ad5/3 DNA and replication-dependent luciferase activity in the swine lungs and spleen indicating active replication in these tissues. These studies demonstrated the flaws in using Syrian hamsters for testing serotype chimeric Ad5/3 vectors. This is the first report to validate the pig as a valuable model for preclinical testing of oncolytic adenoviruses utilizing Adenovirus type 3 receptors. We hope that these data will help to foster the clinical translation of oncolytic adenoviruses including those with Ad3 retargeted tropism.

Viral vector and route of administration determine the ILC and DC profiles responsible for downstream vaccine-specific immune outcomes

This study demonstrates that route and viral vector can significantly influence the innate lymphoid cells (ILC) and dendritic cells (DC) recruited to the vaccination site, 24 h post delivery. Intranasal (i.n.) vaccination induced ST2/IL-33R+ ILC2, whilst intramuscular (i.m.) induced IL-25R+ and TSLPR+ (Thymic stromal lymphopoietin protein receptor) ILC2 subsets. However, in muscle a novel ILC subset devoid of the known ILC2 markers (IL-25R− IL-33R− TSLPR−) were found to express IL-13, unlike in lung. Different viral vectors also influenced the ILC-derived cytokines and the DC profiles at the respective vaccination sites. Both i.n. and i.m. recombinant fowlpox virus (rFPV) priming, which has been associated with induction of high avidity T cells and effective antibody differentiation exhibited low ILC2-derived IL-13, high NKp46+ ILC1/ILC3 derived IFN-γ and low IL-17A, together with enhanced CD11b+ CD103− conventional DCs (cDC). In contrast, recombinant Modified Vaccinia Ankara (rMVA) and Influenza A vector priming, which has been linked to low avidity T cells, induced opposing ILC derived-cytokine profiles and enhanced cross-presenting DCs. These observations suggested that the former ILC/DC profiles could be a predictor of a balanced cellular and humoral immune outcome. In addition, following i.n. delivery Rhinovirus (RV) and Adenovius type 5 (Ad5) vectors that induced elevated ILC2-derived IL-13, NKp46+ ILC1/ILC3-derived-IFN-γ and no IL-17A, predominantly recruited CD11b− B220+ plasmacytoid DCs (pDC). Knowing that pDC are involved in antibody differentiation, we postulate that i.n. priming with these vectors may favour induction of effective humoral immunity. Our data also revealed that vector-specific replication status and/or presence or absence of immune evasive genes can significantly alter the ILC and DC activity. Collectively, our findings suggest that understanding the route- and vector-specific ILC and DC profiles at the vaccination site may help tailor/design more efficacious viral vector-based vaccines, according to the pathogen of interest.

Abstract B052: Immunotherapy for melanoma by adenovirus-mediated full-length antibody, nivolumab

Abstract Nivolumab, a fully human, IgG4 immune checkpoint inhibitor antibody, binds PD-1 on activated immune cells to disrupt PD-1 interaction with PD-L1 and PD-L2 ligands, thereby attenuating inhibitory signals and augmenting the host antitumour response. In 2014, nivolumab was approved by FDA and has been used to treat various types of cancer such as melanoma, non-small cell lung cancer, renal cell carcinoma, and classic Hodgkin's lymphoma. However, due to the high price of nivolumab and the long cycle of treatment, many patients cannot afford and receive adequate treatment. Here, we generated a new, cheaper form of nivolumab for cancer immunotherapy, by cloning the full-length nivolumab antibody gene into two serotypes of adenovirus vectors, termed as AdHu5-Nivo and AdC68-Nivo. Ad vectors based on human serotype 5 (AdHu5) have been proved in a lot of previous studies to be safe and efficient. Compared with AdHu5, chimpanzee Ads exhibit much lower seroprevalence in human beings, which made them great alternative Ad gene vector. Until now, we have detected a high expression of nivolumab in vitro by Western blot and sandwich ELISA. In vivo studies showed that a single dose of AdHu5-Nivo or AdC68-Nivo can induce sustained nivolumab expression. The biologic activities of the two mAbs are analogous compared with commercial monoclonal antibody. To follow up we will verify their antitumor effects on both cellular level and melanoma transplant animal models. Citation Format: Xuchen Wang. Immunotherapy for melanoma by adenovirus-mediated full-length antibody, nivolumab [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B052.

A MicroRNA Derived from Adenovirus Virus-Associated RNAII Promotes Virus Infection via Posttranscriptional Gene Silencing.

The adenovirus (Ad) serotype 5 genome encodes two noncoding small RNAs (virus-associated RNAs I and II [VA-RNAI and -II]), which are approximately 160-nucleotide (nt) RNAs transcribed by RNA polymerase III. It is well known that VA-RNAI supports Ad infection via the inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. Recent studies revealed that VA-RNAs are processed into VA-RNA-derived microRNAs (miRNAs) (mivaRNAI and -II); however, we and another group recently demonstrated that mivaRNAI does not promote Ad replication. On the other hand, the roles of VA-RNAII and mivaRNAII in Ad replication have remained to be clarified. In this study, we demonstrated mivaRNAII-mediated promotion of Ad replication. Transfection with chemically synthesized 3'-mivaRNAII-138, one of the most abundant forms of mivaRNAII, significantly enhanced Ad replication, while the other species of mivaRNAII did not. We identified 8 putative target genes of 3'-mivaRNAII-138 by microarray analysis and in silico analysis. Among the 8 candidates, knockdown of the cullin 4A (CUL4A) gene, which encodes a component of the ubiquitin ligase complex, most significantly enhanced Ad replication. CUL4A expression was significantly suppressed by 3'-mivaRNAII-138 via posttranscriptional gene silencing, indicating that CUL4A is a target gene of 3'-mivaRNAII-138 and mivaRNAII functions as a viral miRNA promoting Ad infection. It has been reported that CUL4A is involved in degradation of c-Jun, which acts as a transcription factor in the Jun-N-terminal kinase (JNK) signaling cascade. Treatment with JNK inhibitors dramatically suppressed Ad replication, suggesting that mivaRNAII-mediated downregulation of CUL4A enhanced JNK signaling and thereby promoted Ad infection.IMPORTANCE Several types of viruses encode viral miRNAs which regulate host and/or viral gene expression via posttranscriptional gene silencing, leading to efficient viral infection. Adenovirus (Ad) expresses miRNAs derived from VA-RNAs (mivaRNAI and -II); however, recent studies have revealed that processing of VA-RNAI into mivaRNAI inhibits Ad replication. Conversely, we demonstrate here that mivaRNAII significantly promotes Ad replication and that mivaRNAII-mediated suppression of CUL4A expression via posttranscriptional gene silencing induces accumulation of c-Jun, leading to promotion of Ad infection. These results exhibited the significance of VA-RNAII for supporting Ad infection through a mechanism complementary to that of VA-RNAI. These observations could provide important clues toward a new perspective on host-virus interaction. Moreover, Ad is widely used as a basic framework for viral vectors and oncolytic viruses. Our findings will help to regulate Ad infection and will promote the development of novel Ad vectors and oncolytic Ad.

Tel-eVax: a genetic vaccine targeting telomerase for treatment of canine lymphoma

Backgroundwe have recently shown that Tel-eVax, a genetic vaccine targeting dog telomerase (dTERT) and based on Adenovirus (Ad)/DNA Electro-Gene-Transfer (DNA–EGT) technology can induce strong immune response and increase overall survival (OS) of dogs affected by multicentric Diffuse Large B cell Lymphoma (DLBCL) when combined to COP therapy in a double-arm study. Here, we have utilized a clinically validated device for veterinary electroporation called Vet-ePorator™, based on Cliniporator™ technology currently utilized and approved in Europe for electrochemotherapy applications and adapted to electrogenetransfer (EGT).Methods17 dogs affected by DLBCL were vaccinated using two Ad vector injections (Prime phase) followed by DNA–EGT (Boost phase) by means of a Vet-ePorator™ device and treated in the same time with a 27-week Madison Wisconsin CHOP protocol. The immune response was measured by ELISA assays using pool of peptides.ResultsNo significant adverse effects were observed. The OS of vaccine/CHOP animals was 64.5 weeks, in line with the previous study. Dogs developed antibodies against the immunizing antigen.ConclusionsTel-eVax in combination with CHOP is safe and immunogenic in lymphoma canine patients. These data confirm the therapeutic efficacy of dTERT vaccine and hold promise for the treatment of dogs affected by other cancer types. More importantly, our findings may translate to human clinical trials and represent new strategies for cancer treatment.

A Dendritic Cell-Targeted Adenoviral Vector Facilitates Adaptive Immune Response Against Human Glioma Antigen (CMV-IE) and Prolongs Survival in a Human Glioma Tumor Model

Antitumor immunotherapeutic strategies represent an especially promising set of approaches with rapid translational potential considering the dismal clinical context of high-grade gliomas. Dendritic cells (DCs) are the body's most professional antigen-presenting cells, able to recruit and activate T cells to stimulate an adaptive immune response. In this regard, specific loading of tumor-specific antigen onto dendritic cells potentially represents one of the most advanced strategies to achieve effective antitumor immunization. In this study, we developed a DC-specific adenoviral (Ad) vector, named Ad5scFvDEC205FF, targeting the DC surface receptor, DEC205. In vitro analysis shows that 60% of DCs was infected by this vector while the infectivity of other control adenoviral vectors was less than 10%, demonstrating superior infectivity on DCs. Moreover, an average of 14% of DCs were infected by Ad5scFvDEC205FF-GFP, while less than 3% of non-DCs were infected following in vivo administration, demonstrating highly selective in vivo DC infection. Importantly, vaccination with this vehicle expressing human glioma-specific antigen, Ad5scFvDEC205FF-CMV-IE, shows a prolonged survival benefit in GL261CMV-IE-implanted murine glioma models (p < 0.0007). Furthermore, when rechallenged, cancerous cells were completely rejected. In conclusion, our novel, viral-mediated, DC-based immunization approach has the significant therapeutic potential for patients with high-grade gliomas.

Antibodies against adenovirus fiber and penton base proteins inhibit adenovirus vector-mediated transduction in the liver following systemic administration

Pre-existing anti-adenovirus (Ad) neutralizing antibodies (AdNAbs) are a major barrier in clinical gene therapy using Ad vectors and oncolytic Ads; however, it has not been fully elucidated which Ad capsid protein-specific antibodies are involved in AdNAb-mediated inhibition of Ad infection in vivo. In this study, mice possessing antibodies specific for each Ad capsid protein were prepared by intramuscular electroporation of each Ad capsid protein-expressing plasmid. Ad vector-mediated hepatic transduction was efficiently inhibited by more than 100-fold in mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid. An Ad vector pre-coated with FX before administration mediated more than 100-fold lower transduction efficiencies in the liver of warfarinized mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid, compared with those in the liver of warfarinized non-immunized mice. These data suggest that anti-fiber protein and anti-penton base antibodies bind to an Ad vector even though FX has already bound to the hexon, and inhibit Ad vector-mediated transduction. This study provides important clues for the development of a novel Ad vector that can circumvent inhibition with AdNAbs.

Abstract A062: Systemic oncolytic virotherapy for prostate cancer

Abstract Oncolytic viruses hold promise as “self-amplifying” cancer therapies wherein a virally killed cell can produce thousands of new viral “drugs” that can kill more cancer cells. Oncolytic viruses may also serve to reawaken or initiate immune responses against cancers by virtue of their abilities to lyse cells, liberate cancer antigens, and provide viral DNA and proteins as immune adjuvants. Oncolytics can also be armed with cytokine or checkpoint inhibitors to amplify antitumor immune responses. Adenoviruses (Ads) are one family of oncolytic viruses. Most human studies have used human Ad serotype 5 (Ad5). Unfortunately, most patients are already immune to Ad5, increasing the likelihood that the agent will be neutralized if used as a cancer therapy. In this work, low seroprevalence Ad6 and Ad57 oncolytic viruses were tested as intravenous therapies against distant prostate cancer sites. After prostate cell infection in vitro, Ad viruses executed these cells efficiently and generated 10,000 to 100,000 progeny infectious viruses. This genome replication also amplified transgene protein production by 100 to 1,000-fold when compared to nonreplicating Ad vectors. Ad5, Ad6, and Ad57 were injected intravenously a single time in nude mice bearing human prostate tumors, and toxicity and efficacy were assessed. Ad6 and 57 produced 30-fold lower liver damage and less toxicity than Ad5. Ad6 and Ad57 significantly repressed the growth of androgen-resistant human DU145 prostate tumors and androgen-sensitive LNCaP prostate tumors and extended survival after single intravenous injection in mice. Ad6 and Ad57 E1 protein were also modified to protect normal cells from viral oncolysis while allowing the virus to amplify and kill prostate cancer cells having perturbations in p300 and pRB pathways. These and other data suggest that targeted and untargeted low seroprevalent Ad6 and Ad57 have better efficacy and safety than the benchmark oncolytic virus Ad5 for systemic therapy of advanced prostate cancer. Citation Format: Tien Nguyen, Michael A. Barry. Systemic oncolytic virotherapy for prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A062.

Immunogenicity of a Candidate Ebola Hemorrhagic Fever Vaccine in Mice Based on Controlled In Vitro Expression of Ebolavirus Glycoprotein.

Ebolavirus (EBOV) is the etiology of Ebola hemorrhagic fever (EHF). A major EHF outbreak in 2014-2015 in West Africa claimed >11,000 lives. A licensed vaccine is not available for EHF, although several vaccines have undergone clinical trials. We developed a human adenovirus (Ad) serotype 5-based candidate EHF vaccine based on controlled expression of the EBOV (Makona strain) glycoprotein (GP) as the immunogen. Two clones, AdGP72 and AdGP75, and a control Ad515 vector, were generated and tested for protein expression in vitro and immunogenicity in mice. Eight groups of mice were immunized with three doses of buffer, Ad515, AdGP72, and AdGP75, by two different dose regimens. Three different antigens (AdGP75-infected Vero E6 cell extract and two baculovirus expressed EBOV GP antigens, namely, GP alone or GP with EBOV VP40) were used to evaluate the immune response. Expression studies indicated that full-length GP was cleaved into its component subunits when expressed in mammalian cells through the Ad vectors. Moreover, in coimmunoprecipitation studies, EBOV GP was found to be associated with VP40 when expressed in baculoviruses. The candidate vaccines were immunogenic in mice, as evaluated by enzyme-linked immunosorbent assay using mammalian- or baculovirus-derived antigens. Further characterization and development of the candidate vaccines are warranted.

The role of JNK phosphorylation as a molecular target to enhance adenovirus replication, oncolysis and cancer therapeutic efficacy

ABSTRACT Oncolytic adenoviruses (Ads) are cancer selective tumoricidal agents; however their mechanism of Ad-mediated cancer cell lysis, or oncolysis, remains undefined. This report focuses upon the autophagy mediator c-JUN n-terminal kinase (JNK) and its effects upon Ad oncolysis and replication. Previously, E1b-deleted Ads have been used to treat several hundred cancer patients with limited clinical efficacy. We hypothesize that by studying the potential interactions between E1b and JNK, mechanisms to improve oncolytic Ad design and cancer therapeutic efficacy may be elucidated. To test this hypothesis, E1b was selectively deleted from the Ad genome. These studies indicated that Ads encoding E1b induced JNK phosphorylation predominately occurred via E1b-19K. The expression of another crucial Ad gene E1a was then overexpressed by the CMV promoter via the replication competent Ad vector Adhz69; these data indicated that E1A also induced JNK phosphorylation. To assess the effects of host cell JNK expression upon Ad oncolysis and replication, siRNA targeting JNK1 and JNK2 (JNK1/2) were utilized. The oncolysis and replication of the E1b-19K wild-type Ads Ad5 and Adhz63 were significantly attenuated following JNK1/2 siRNA transfection. However the oncolytic effects and replication of the E1b-19K deleted Ad Adhz60 were not altered by JNK1/2 siRNA transfection, further implicating the crucial role of E1b-19K for Ad oncolysis and replication via JNK phosphorylation. This study has demonstrated for the first time that JNK is an intriguing molecular marker associated with enhanced Ad virotherapy efficacy, influencing future Ad vector design.

Generation of the Adenovirus Vector-Mediated CRISPR/Cpf1 System and the Application for Primary Human Hepatocytes Prepared from Humanized Mice with Chimeric Liver.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 system is now widely used as a genome editing tool. CRISPR-associated endonuclease in Prevotella and Francisella 1 (Cpf1) is a recently discovered Cas endonuclease that is designable and highly specific with efficiencies comparable to those of Cas9. Here we generated the adenovirus (Ad) vector carrying an Acidaminococcus sp. Cpf1 (AsCpf1) expression cassette (Ad-AsCpf1) for the first time. Ad-AsCpf1 was applied to primary human hepatocytes prepared from humanized mice with chimeric liver in combination with the Ad vector expressing the guide RNA (gRNA) directed to the Adeno-associated virus integration site 1 (AAVS1) region. The mutation rates were estimated by T7 endonuclease I assay around 12% of insertion/deletion (indel). Furthermore, the transduced human hepatocytes were viable (ca. 60%) at two weeks post transduction. These observations suggest that the Ad vector-mediated delivery of the CRISPR/AsCpf1 system provides a useful tool for genome manipulation of human hepatocytes.