Ad Serotype Research Articles

A pseudotyped adenovirus serotype 5 vector with serotype 49 fiber knob is an effective vector for vaccine and gene therapy applications

Adenoviruses (Ad) have demonstrated significant success as replication-deficient (RD) viral vectored vaccines, as well as broad potential across gene therapy and cancer therapy. Ad vectors transduce human cells via direct interactions between the viral fiber knob and cell surface receptors, with secondary cellular integrin interactions. Ad receptor usage is diverse across the extensive phylogeny. Commonly studied human Ad serotype 5 (Ad5), and chimpanzee Ad-derived vector “ChAdOx1” in licensed ChAdOx1 nCoV-19 vaccine, both form primary interactions with the Coxsackie and Adenovirus Receptor (CAR), which is expressed on human epithelial cells and erythrocytes. CAR usage is suboptimal for targeted gene delivery to cells with low/negative CAR expression, including human dendritic cells (DCs) and vascular smooth muscle cells (VSMCs). We evaluated the performance of a RD Ad5 vector pseudotyped with the fiber knob of human Ad serotype 49, termed Ad5/49K vector. Ad5/49K demonstrated superior transduction of murine and human DCs over Ad5, which translated into significantly increased T cell immunogenicity when evaluated in a mouse cancer vaccine model using 5T4 tumour-associated antigen. Additionally, Ad5/49K exhibited enhanced transduction of primary human VSMCs. These data highlight the potential of Ad5/49K vector for both vascular gene therapy applications and as a potent vaccine vector.

Development of adenoviral vectors that transduce Purkinje cells and other cerebellar cell-types in the cerebellum of a humanized mouse model

Viral vector gene therapy has immense promise for treating central nervous system (CNS) disorders. Although adeno-associated virus vectors (AAVs) have had success, their small packaging capacity limits their utility to treat the root cause of many CNS disorders. Adenoviral vectors (Ad) have tremendous potential for CNS gene therapy approaches. Currently, the most common vectors utilize the Group C Ad5 serotype capsid proteins, which rely on the Coxsackievirus-Adenovirus receptor (CAR) to infect cells. However, these Ad5 vectors are unable to transduce many neuronal cell types that are dysfunctional in many CNS disorders. The human CD46 (hCD46) receptor is widely expressed throughout the human CNS and is the primary attachment receptor for many Ad serotypes. Therefore, to overcome the current limitations of Ad vectors to treat CNS disorders, we created chimeric first generation Ad vectors that utilize the hCD46 receptor. Using a "humanized" hCD46 mouse model, we demonstrate these Ad vectors transduce cerebellar cell types, including Purkinje cells, that are refractory to Ad5 transduction. Since Ad vector transduction properties are dependent on their capsid proteins, these chimeric first generation Ad vectors open new avenues for high-capacity helper-dependent adenovirus (HdAd) gene therapy approaches for cerebellar disorders and multiple neurological disorders.

Development of oncolytic and gene therapy vectors based on adenovirus serotype 4 as an alternative to adenovirus serotype 5.

Adenoviral vectors are among the most frequently used vectors for gene therapy and cancer treatment. Most vectors are derived from human adenovirus (Ad) serotype 5 despite limited applicability caused by pre-existing immunity and unfavorable liver tropism, whereas the other more than 100 known human serotypes remain largely unused. Here, we screened a library of human Ad types and identified Ad4 as a promising candidate vector. Reporter-gene-expressing viruses representative of the natural human Ad diversity were used to transduce an array of muscle cell lines and two- or three-dimensional tumor cultures. The time-course of transgene expression was monitored by fluorescence or luminescence measurements. To generate replication-deficient Ad4 vector genomes, successive homologous recombination was applied. Ad4, 17 and 50 transduced human cardiomyocytes more efficiently than Ad5, whereas Ad37 was found to be superior in rhabdomyocytes. Despite its moderate transduction efficiency, Ad4 showed efficient and long-lasting gene expression in papillomavirus (HPV) positive tumor organoids. Therefore, we aimed to harness the potential of Ad4 for improved muscle transduction or oncolytic virotherapy of HPV-positive tumors. We deleted the E1 and E3 transcription units to produce first generation Ad vectors for gene therapy. The E1- and E1/E3-deleted vectors were replication-competent in HEK293 cells stably expressing E1 but not in the other cell lines tested. Furthermore, we show that the Ad5 E1 transcription unit can complement the replication of E1-deleted Ad4 vectors. Our Ad4-based gene therapy vector platform contributes to the development of improved Ad vectors based on non-canonical serotypes for a broad range of applications.

Mucosal vaccination with an adenoviral vector vaccine protects against pulmonary breast cancer metastasis by robust induction of tissue-resident memory T-cells in the lung

Abstract The presence of tissue-resident memory T-cells (TRM) in solid tumors positively correlates with an improved overall prognosis and responsiveness to checkpoint inhibitor therapy. Rationally designed vaccine approaches capable of inducing antitumor TRM at cancer sites are needed to harness the full potential of localized immunity in tumor therapy. Our project aims to establish tumor antigen-specific lung TRM by mucosal vaccination with an adenoviral (Ad) vaccine to prevent and treat lung metastasis in the murine 4T1 breast cancer model. We employ Ad serotype 5 vectors encoding for 4T1 tumor-associated antigens from Mage-b and MuLV gp70 for intranasal vaccination, supplemented with an Interleukin-1β-encoding vector to specifically boost lung TRM induction. Indeed, particularly if Ad-IL-1β was co-delivered, mucosal vaccination established high numbers of tumor antigen-specific CD69 +CD103 +lung TRM as shown by phenotypic and functional analyses in combination with intravascular staining. Vaccine efficacy was evaluated in a 4T1 pseudometastasis model mimicking a prophylactic vaccination against lung metastasis in breast cancer patients. Local immunization led to a significant protection against pulmonary metastasis and improved survival compared to non-vaccinated and, notably, intramuscularly immunized mice. By using FTY720 to inhibit T-cell circulation upon tumor challenge, we confirmed that TRM are crucial contributors to the vaccine efficacy. In conclusion, induction of tumor-antigen specific lung TRM are a key point in local protection against pulmonary metastasis. Ongoing studies analyze the therapeutic use of this vaccination strategy as well as its potential in combination with immune checkpoint inhibition. Supported by grants from 'Anschubfinanzierung (ELAN) im IZKF' and 'Doktor Robert Pfleger-Stiftung'.

Treatment of Human Pancreatic Cancers Following Local and Systemic Administration of Oncolytic Adenovirus Serotype 35.

Oncolytic adenoviruses (Ads) (OAds) are gaining attention as an effective remedy for pancreatic cancer. Most OAds are based on human Ad serotype 5 (Ad5) (OAd5); however, two major drawbacks of OAd5 have been reported. Expression of coxsackievirus-adenovirus receptor, a primary infection receptor of Ad5, is often decreased on malignant tumor cells, including pancreatic cancers. More than 60% of adults have neutralizing antibodies against Ad5. Previously, we developed an OAd composed of Ad serotype 35 (Ad35) (OAd35). Ad35 recognizes CD46, which is often up-regulated on pancreatic cancers. In addition, only 20% or fewer adults have anti-Ad35 neutralizing antibodies. We examined the tumor cell lysis activities of OAd35 in the four human pancreatic cancer cell lines in the presence and absence of human serum. The tumor growth suppression effects of OAd35 after local and systemic administration were evaluated in nude mice bearing human pancreatic tumors. OAd35 showed higher levels of tumor cell lysis activities than OAd5 in the human pancreatic cancer cell lines AsPC-1 and BxPC-3. Although the in vitro tumor cell lysis activities of OAd5 against MIA PaCa-2 and PANC-1 cells were strongly attenuated in the presence of human serum, OAd35 mediated comparable levels of tumor cell lysis in the presence and absence of human serum. Systemic administration of OAd5 did not mediate significant growth inhibition against the subcutaneous BxPC-3 tumor. On the other hand, OAd35 significantly suppressed tumor growth. OAd35 would be suitable as an alternative anticancer agent for pancreatic cancer.

Role of Fiber Shaft Length in Tumor Targeting with Ad5/3 Vectors.

Desmoglein 2 (DSG2) is overexpressed in many epithelial cancers and therefore represents a target receptor for oncolytic viruses, including Ad5/3-based viruses. For most Ad serotypes, the receptor-binding fiber is composed of tail, shaft, and knob domains. Here, we investigated the role of the fiber shaft in Ad5/3 tumor transduction in vitro and in human DSG2-transgenic mice carrying human DSG2high tumors. DSG2tg mice express DSG2 in a pattern similar to humans. We constructed Ad5/3L (with the "long" Ad5 shaft) and Ad5/3S (with the "short" Ad3 shaft) expressing GFP or luciferase. In in vitro studies we found that coagulation factor X, which is known to mediate undesired hepatocyte transduction of Ad5, enhances the transduction of Ad5/3(L), but not the transduction of Ad5/3(S). We therefore hypothesized that Ad5/3(S) would target DSG2high tumors while sparing the liver after intravenous injection. In vivo imaging studies for luciferase and analysis of luciferase activity in isolated organs, showed that Ad5/3(L) vectors efficiently transduced DSG2high tumors and liver but not normal epithelial tissues after intravenous injection. Ad5/3(S) showed minimal liver transduction, however it failed to transduce DSG2high tumors. Further modifications of the Ad5/3(S) capsid are required to compensate for the lower infectivity of Ad5/3(S) vectors.

Persistent Antigen Harbored by Alveolar Macrophages Enhances the Maintenance of Lung-Resident Memory CD8+ T Cells.

Lung tissue-resident memory T cells are crucial mediators of cellular immunity against respiratory viruses; however, their gradual decline hinders the development of T cell-based vaccines against respiratory pathogens. Recently, studies using adenovirus (Ad)-based vaccine vectors have shown that the number of protective lung-resident CD8+ TRMs can be maintained long term. In this article, we show that immunization of mice with a replication-deficient Ad serotype 5 expressing influenza (A/Puerto Rico/8/34) nucleoprotein (AdNP) generates a long-lived lung TRM pool that is transcriptionally indistinct from those generated during a primary influenza infection. In addition, we demonstrate that CD4+ T cells contribute to the long-term maintenance of AdNP-induced CD8+ TRMs. Using a lineage tracing approach, we identify alveolar macrophages as a cell source of persistent NP Ag after immunization with AdNP. Importantly, depletion of alveolar macrophages after AdNP immunization resulted in significantly reduced numbers of NP-specific CD8+ TRMs in the lungs and airways. Combined, our results provide further insight to the mechanisms governing the enhanced longevity of Ag-specific CD8+ lung TRMs observed after immunization with recombinant Ad.

Human Adenovirus Serotype 3 Infection Modulates the Biogenesis and Composition of Lung Cell-Derived Extracellular Vesicles.

Adenovirus (Ad) is a major causal agent of acute respiratory infections. However, they are a powerful delivery system for gene therapy and vaccines. Some Ad serotypes antagonize the immune system leading to meningitis, conjunctivitis, gastroenteritis, and/or acute hemorrhagic cystitis. Studies have shown that the release of small, membrane-derived extracellular vesicles (EVs) may offer a mechanism by which viruses can enter cells via receptor-independent entry and how they influence disease pathogenesis and/or host protection considering their existence in almost all bodily fluids. We proposed that Ad3 could alter EV biogenesis, composition, and trafficking and may stimulate various immune responses in vitro. In the present study, we evaluated the impact of in vitro infection with Ad3 vector on EV biogenesis and composition in the human adenocarcinoma lung epithelial cell line A549. Cells were infected in an exosome-free media at different multiplicity of infections (MOIs) and time points. The cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorometric calcein-AM. EVs were isolated via ultracentrifugation. Isolated EV proteins were quantified and evaluated via nanoparticle tracking, transmission electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting assays. The cell viability significantly decreased with an increase in MOI and incubation time. A significant increase in particle mean sizes, concentrations, and total EV protein content was detected at higher MOIs when compared to uninfected cells (control group). A549 cell-derived EVs revealed the presence of TSG101, tetraspanins CD9 and CD63, and heat shock proteins 70 and 100 with significantly elevated levels of Rab5, 7, and 35 at higher MOIs (300, 750, and 1500) when compared to the controls. Our findings suggested Ad3 could modulate EV biogenesis, composition, and trafficking which could impact infection pathogenesis and disease progression. This study might suggest EVs could be diagnostic and therapeutic advancement to Ad infections and other related viral infections. However, further investigation is warranted to explore the underlying mechanism(s).

Integrin αvβ3 and disulfide bonds play important roles in NGR-retargeted adenovirus transduction efficiency

AimsAdenoviruses that have CNGRCVSGCAGRC peptide inserted into fiber (AdFNGR) or hexon (AdHNGR) protein, respectively, showed increased transduction of endothelial cells. In this study we investigated if cysteines within the CNGRCVSGCAGRC sequence inserted into Ad serotype 5 Ad5 fiber or hexon protein form disulfide bond(s) and whether they play a role in retargeting potential of AdFNGR and AdHNGR. MethodsTransduction efficiency of adenoviruses was done by counting infected cells under the microscope. Adenovirus attachment and internalization were measured by qPCR. Flow cytometry was used to evaluate the expression of CD13 and integrins. Gene knockdown was achieved by transfection of small interfering RNA. Mass spectrometry was used for determining disulfide bonds in adenovirus fiber and hexon protein. Molecular modeling was use to predict interaction of CNGRCVSGCAGRC peptide and CD13. Key findingsAdFNGR and AdHNGR attach better to CD13 and/or αvβ3 integrin-positive cells than Adwt. Reducing disulfide bonds using DTT decreased transduction efficiency and attachment of both AdFNGR and AdHNGR. Cysteins from CNGRCVSGCAGRC peptide within AdHNGR do not form disulfide bonds. Knockdown of αvβ3 integrin reduced increased transduction efficiency of both AdFNGR and AdHNGR, while CD13 knockdown had no effect, indicating that retargeting properties of these viruses rely mainly on αvβ3 integrin expression. SignificanceInsertion site of NGR-containing peptides as well as NGR flanking residues are critical for receptor binding affinity/specificity and transduction efficiency of NGR retargeted adenoviral vectors.

The infectivity of progeny adenovirus in the presence of neutralizing antibody.

Human adenoviruses (Ads), common pathogens that cause upper respiratory and gastrointestinal infections, are blocked by neutralizing antibodies (nAbs). However, Ads are not fully eliminated even in hosts with nAbs. In this study, we assessed the infectivity of progeny Ad serotype 5 (Ad5) in the presence of nAb. The infectivity of Ad5 was evaluated according to the expression of the Ad genome and reporter gene. Infection by wild-type Ad5 and Ad5 vector continued to increase until 3 days after infection even in the presence of nAb. We established an assay for determining the infection levels of progeny Ad5 using a sorting system with magnetic beads and observed little difference in progeny Ad5 counts in the presence and absence of nAb 1 day after infection. Moreover, progeny Ad5 in the presence of nAb more effectively infected coxsackievirus and adenovirus receptor (CAR)-positive cells than CAR-negative cells. We investigated the function of fiber proteins, which are the binding partners of CAR, during secondary infection, observing that fibre proteins spread from infected cells to adjacent cells in a CAR-dependent manner. In conclusion, this study revealed that progeny Ad5 could infect cells even in the presence of nAb, differing from the common features of the Ad5 infection cycle. Our findings may be useful for developing new therapeutic agents against Ad infection.

En Guard! The Interactions between Adenoviruses and the DNA Damage Response.

Virus–host cell interactions include several skirmishes between the virus and its host, and the DNA damage response (DDR) network is one of their important battlegrounds. Although some aspects of the DDR are exploited by adenovirus (Ad) to improve virus replication, especially at the early phase of infection, a large body of evidence demonstrates that Ad devotes many of its proteins, including E1B-55K, E4orf3, E4orf4, E4orf6, and core protein VII, and utilizes varied mechanisms to inhibit the DDR. These findings indicate that the DDR would strongly restrict Ad replication if allowed to function efficiently. Various Ad serotypes inactivate DNA damage sensors, including the Mre11-Rad50-Nbs1 (MRN) complex, DNA-dependent protein kinase (DNA-PK), and Poly (ADP-ribose) polymerase 1 (PARP-1). As a result, these viruses inhibit signaling via DDR transducers, such as the ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases, to downstream effectors. The different Ad serotypes utilize both shared and distinct mechanisms to inhibit various branches of the DDR. The aim of this review is to understand the interactions between Ad proteins and the DDR and to appreciate how these interactions contribute to viral replication.

Characterization of a Species E Adenovirus Vector as a Zika virus vaccine

The development of a safe and efficacious Zika virus (ZIKV) vaccine remains a global health priority. In our previous work, we developed an Adenovirus vectored ZIKV vaccine using a low-seroprevalent human Adenovirus type 4 (Ad4-prM-E) and compared it to an Ad5 vector (Ad5-prM-E). We found that vaccination with Ad4-prM-E leads to the development of a strong anti-ZIKV T-cell response without eliciting significant anti-ZIKV antibodies, while vaccination with Ad5-prM-E leads to the development of both anti-ZIKV antibody and T-cell responses in C57BL/6 mice. However, both vectors conferred protection against ZIKV infection in a lethal challenge model. Here we continued to characterize the T-cell biased immune response observed in Ad4 immunized mice. Vaccination of BALB/c mice resulted in immune correlates similar to C57BL/6 mice, confirming that this response is not mouse strain-specific. Vaccination with an Ad4 expressing an influenza hemagglutinin (HA) protein resulted in anti-HA T-cell responses without the development of significant anti-HA antibodies, indicating this unique response is specific to the Ad4 serotype rather than the transgene expressed. Co-administration of a UV inactivated Ad4 vector with the Ad5-prM-E vaccine led to a significant reduction in anti-ZIKV antibody development suggesting that this serotype-specific immune profile is capsid-dependent. These results highlight the serotype-specific immune profiles elicited by different Adenovirus vector types and emphasize the importance of continued characterization of these alternative Ad serotypes.

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.

Evaluation of adenovirus 19a as a novel vector for mucosal vaccination against influenza A viruses

Since preexisting immunity and enhanced infection rates in a clinical trial of an HIV vaccine have raised some concerns on adenovirus (Ad) serotype 5-based vaccines, we evaluated the subgroup D adenovirus serotype Ad19a for its suitability as novel viral vector vaccine against mucosal infections. In BALB/c mice, we compared the immunogenicity and efficacy of E1/E3-deleted Ad19a vectors encoding the influenza A virus (IAV)-derived antigens hemagglutinin (HA) and nucleoprotein (NP) to the most commonly used Ad5 vectors. The adenoviral vectors were applied intranasally and induced detectable antigen-specific T cell responses in the lung and in the spleen as well as robust antibody responses. A prior DNA immunization significantly improved the immunogenicity of both vectors and resulted in full protection against a lethal infection with a heterologous H3N2 virus. Nevertheless, the Ad5-based vectors were slightly superior in reducing viral replication in the lung which corresponded to higher NP-specific T cell responses measured in the lungs.

A novel fiber chimeric conditionally replicative adenovirus-Ad5/F35 for tumor therapy

ABSTRACTSignificant progress has been made in the diagnosis and treatment of cancer; however, significant challenges remain. Conditionally replicating adenoviruses (CRAds), which not only kill cancer cells, but also serve as vectors to express therapeutic genes, are a novel and effective method to treat cancer. However, most adenoviruses are Ad5, which infect cells through the coxsackie and adenovirus receptor (CAR). The transduction efficacy of Ad5 is restricted because of the absent or low expression of CAR on several cancer cells. Ad serotype 35 has a different tropism pattern to Ad5. Ad35 attaches to cells via a non-CAR receptor, CD46, which is expressed widely on most tumor cells. Thus, chimeric adenoviral vectors consisting of the knob and shaft of Ad35 combined with Ad5 have been constructed. The chimeric fiber adenoviral vectors can transduce CAR-positive and CAR-negative cell lines. In this review, we explore the application of the novel fiber chimeric conditionally replicative adenovirus-Ad5/F35 in tumor therapy in terms of safety, mechanism, transduction efficacy, and antitumor effect.

Development of Novel Vaccines Against Infectious Diseases Based on Chimpanzee Adenoviral Vector.

Vaccination is considered to be the most effective method of preventing infectious or other diseases. Adenovirus (Ad) is one the most promising vectors in vaccine research and development. It can induce not only potent humoral but also cellular immune responses, and has therefore been widely applied in basic and translational studies. Chimpanzee Ad is a rare serotype circulating in humans. This circumvents the problem of preexisting immunity to human Ad serotypes, enhancing Chimpanzee Ad prospects in vaccine development. Here we describe experimental procedures used to generate a new generation of rabies vaccine based on a chimpanzee Ad vector, which can be extended in the development of novel vaccines against other infectious diseases.

A Recombinant Chimeric Ad5/3 Vector Expressing a Multistage Plasmodium Antigen Induces Protective Immunity in Mice Using Heterologous Prime-Boost Immunization Regimens.

An ideal malaria vaccine should target several stages of the parasite life cycle and induce antiparasite and antidisease immunity. We have reported a Plasmodium yoelii chimeric multistage recombinant protein (P. yoelii linear peptide chimera/recombinant modular chimera), engineered to express several autologous T cell epitopes and sequences derived from the circumsporozoite protein and the merozoite surface protein 1. This chimeric protein elicits protective immunity, mediated by CD4(+) T cells and neutralizing Abs. However, experimental evidence, from pre-erythrocytic vaccine candidates and irradiated sporozoites, has shown that CD8(+) T cells play a significant role in protection. Recombinant viral vectors have been used as a vaccine platform to elicit effective CD8(+) T cell responses. The human adenovirus (Ad) serotype 5 has been tested in malaria vaccine clinical trials with excellent safety profile. Nevertheless, a major concern for the use of Ad5 is the high prevalence of anti-vector neutralizing Abs in humans, hampering its immunogenicity. To minimize the impact of anti-vector pre-existing immunity, we developed a chimeric Ad5/3 vector in which the knob region of Ad5 was replaced with that of Ad3, conferring partial resistance to anti-Ad5 neutralizing Abs. Furthermore, we implemented heterologous Ad/protein immunization regimens that include a single immunization with recombinant Ad vectors. Our data show that immunization with the recombinant Ad5/3 vector induces protective efficacy indistinguishable from that elicited by Ad5. Our study also demonstrates that the dose of the Ad vectors has an impact on the memory profile and protective efficacy. The results support further studies with Ad5/3 for malaria vaccine development.

13. Functional Role of Adenovirus Penton in Modulating In Vivo Properties of Liver-Targeted and Liver-Detargeted Adenovirus Variants

Pharmacokinetic studies of adenovirus (Ad) vectors after intravascular delivery demonstrate that the majority of an administered virus dose is rapidly sequestered from the circulation by the liver. The molecular determinants that target Ad to the liver have been found on each of the virus capsid proteins, including hexon, penton, and fiber. The penton protein in the majority of Ad serotypes comprises a hypervariable loop containing an RGD motif that has been shown to interact with a number of cellular integrins upon virus entry into the cell. The binding of penton to integrins triggers virus internalization and relaxes the virus structure for subsequent endosomal disassembly. However, RGD motif interaction with integrins on tissue macrophages leads to production of inflammatory cytokines and significantly contributes to systemic Ad toxicity. Despite its importance for the virus entry into the cell, the penton is not well employed in the development of therapeutic Ad vectors. Therefore, in this study we evaluated strategies for modulating Ad penton interactions with cellular integrins. Ad vectors were created bearing substitutions of the RGD motif with mimetic non-RGD-containing peptides derived from extracellular proteins laminin 1 and laminin 3. These peptides are interacting with a different subset of integrins compared to those binging penton RGD motif. Unlike wild type (WT) Ad5, these penton-modified vectors were able to utilize β4 integrin for cell entry. The in vivo studies demonstrated that penton-modified Ads cannot activate a full inflammatory cascade and their in vivo toxicity, therefore, is dramatically reduced. Although the penton-mutated vectors were infecting a set of cancer cell lines in vitro and hepatocytes in vivo with a similar efficacy as the WT Ad5, interactions of virus with Kupffer cells and virus accumulation in the liver after intravascular vector delivery remain similar for penton-mutated and WT Ad vector. We also developed Ad vectors containing mutations abrogating virus interactions with all types of liver cells. Additionally to penton modifications, we introduced a T425A mutation in the hexon hyper variable loop HVR7 to completely abrogate virus interaction with blood coagulation FX and prevent transduction of hepatocytes. Furthermore, a set of mutations in the hexon HVR1 region were introduced to prevent Ad uptake by Kupffer cells. Due to penton modifications these vectors had very low toxicity and combinations of three mutations diverted the vector away from the liver. These data demonstrate that RGD loop modification represents a useful approach for introducing beneficial properties to the Ad vectors for greatly reducing their systemic toxicity and enable liver de-targeting. CryoEM analyses are currently underway to identify the structural differences in the capsid between the penton mutants and the wild type Ad.

Epithelial Junction Opener Improves Oncolytic Adenovirus Therapy in Mouse Tumor Models.

A central resistance mechanism in solid tumors is the maintenance of epithelial junctions between malignant cells that prevent drug penetration into the tumor. Human adenoviruses (Ads) have evolved mechanisms to breach epithelial barriers. For example, during Ad serotype 3 (Ad3) infection of epithelial tumor cells, massive amounts of subviral penton-dodecahedral particles (PtDd) are produced and released from infected cells to trigger the transient opening of epithelial junctions, thus facilitating lateral virus spread. We show here that an Ad3 mutant that is disabled for PtDd production is significantly less effective in killing of epithelial human xenograft tumors than the wild-type Ad3 virus. Intratumoral spread and therapeutic effect of the Ad3 mutant was enhanced by co-administration of a small recombinant protein (JO; produced in Escherichia coli) that incorporated the minimal junction opening domains of PtDd. We then demonstrated that co-administration of JO with replication-competent Ads that do not produce PtDd (Ad5, Ad35) resulted in greater attenuation of tumor growth than virus injection alone. Furthermore, we genetically modified a conditionally replicating Ad5-based oncolytic Ad (Ad5Δ24) to express a secreted form of JO upon replication in tumor cells. The JO-expressing virus had a significantly greater antitumor effect than the unmodified AdΔ24 version. Our findings indicate that epithelial junctions limit the efficacy of oncolytic Ads and that this problem can be address by co-injection or expression of JO. JO has also the potential for improving cancer therapy with other types of oncolytic viruses.