Recombinant Ad Vectors Research Articles

Advances of Recombinant Adenoviral Vectors in Preclinical and Clinical Applications.

Adenoviruses (Ad) have the potential to induce severe infections in vulnerable patient groups. Therefore, understanding Ad biology and antiviral processes is important to comprehend the signaling cascades during an infection and to initiate appropriate diagnostic and therapeutic interventions. In addition, Ad vector-based vaccines have revealed significant potential in generating robust immune protection and recombinant Ad vectors facilitate efficient gene transfer to treat genetic diseases and are used as oncolytic viruses to treat cancer. Continuous improvements in gene delivery capacity, coupled with advancements in production methods, have enabled widespread application in cancer therapy, vaccine development, and gene therapy on a large scale. This review provides a comprehensive overview of the virus biology, and several aspects of recombinant Ad vectors, as well as the development of Ad vector, are discussed. Moreover, we focus on those Ads that were used in preclinical and clinical applications including regenerative medicine, vaccine development, genome engineering, treatment of genetic diseases, and virotherapy in tumor treatment.

An adenovirus-vectored RVF vaccine confers complete protection against lethal RVFV challenge in A129 mice.

Instruction: Rift valley fever virus (RVFV) is a mosquito-transmitted bunyavirus that causes severe disease in animals and humans. Nevertheless, there are no vaccines applied to prevent RVFV infection for human at present. Therefore, it is necessary to develop a safe and effective RVFV vaccine. Methods: We generated Ad5-GnGcopt, a replication-deficient recombinant Ad5 vector (human adenovirus serotype 5) expressing codon-optimized RVFV glycoproteins Gn and Gc, and evaluated its immunogenicity and protective efficacy in mice. Results and Discussion: Intramuscular immunization of Ad5-GnGcopt in mice induces strong and durable antibody production and robust cellular immune responses. Additionally, a single vaccination with Ad5-GnGcopt vaccination can completely protect interferon-α/β receptor-deficient A129 mice from lethal RVFV infection. Our work indicates that Ad5-GnGcopt might represent a potential vaccine candidate against RVFV. However, further research is needed, first to confirm its efficacy in a natural animal host, and ultimately escalate as a potential vaccine candidate for humans.

Oncolytic adenovirus-mediated intratumoral expression of TRAIL and CD40L enhances immunotherapy by modulating the tumor microenvironment in immunocompetent mouse models

The immune status of the tumor microenvironment is a key indicator determining the antitumor effect of immunotherapy. Oncolytic viruses directly target tumor cells or indirectly modulate the tumor microenvironment (TME) especially when properly armed. It was previously demonstrated that conditionally replicating adenovirus serotype 5 (CRAd5) encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) had outstanding antitumor effects in different human cancer cells xenograft models; however, its antitumor immune mechanism has not been evaluated in immunocompetent preclinical mouse models. We first explored the antitumor activity of CRAd5-TRAIL in several murine tumor models and found that the expression of TRAIL induced increases or activation in tumor-infiltrating T cells. To further improve the antitumor effects, mouse CD40 ligand (mCD40L) as an immune activator expressed by recombinant Ad5 vector was firstly used in combination with CRAd5-TRAIL for tumor immunotherapy. Both in vitro and in vivo studies demonstrated that mCD40L effectively activated dendritic cells (DCs), B cells, and tumor-infiltrating T cells, and also promoted tumor cell apoptosis by increasing the expression of TRAIL receptors, thereby significantly enhancing the antitumor activity of oncolytic adenoviruses in CT26 and B16 tumor-bearing models. Although affected by the restriction of oncolytic adenovirus replication in mouse cells, the combination treatment failed to completely eliminate tumor cells, our research still provided a promising strategy for oncolytic adenovirus-mediated solid tumor immunotherapy.

A recombinant adenoviral vector with a specific tropism to CD4-positive cells: a new tool for HIV-1 inhibition.

Gene therapy can be an option to overcome the side effects of chemotherapy and prevent the development of drug-resistant HIV viruses in HIV-infected patients. The need to develop a safe and efficient vector for gene transfer is always necessary and an appropriate option might be adenovirus (Ad). The use of Ad vectors in gene delivery applications is limited due to the semi-specific tropism. A strategy to overcome this tropism limitation may be the modification of the fiber protein domain involved in the viral binding to cells. Therefore, we designed an Ad5 vector with a specific tropism to CD4 + cells containing an expression system limited to HIV-infected cells. We replaced the knob region of Ad5 fiber protein with the extracellular region of the HIV-1 envelope. We also used a specific Tat-inducible promoter to express two anti-HIV-1 shRNAs. Tropism of recombinant Ad5 was assayed by a comparison of the shRNA expression level in CEM and PBMC cells (as CD4 + cells) and HEK293 cells (as CD4 cells). HIV-1 inhibition was assayed by the determination of p24 antigen in the HIV-infected CEM cells transduced with the recombinant Ad5 vector. Our results showed that the shRNA expression was significantly higher in CEM and PBMC cells than HEK293 cells when transduced with recombinant Ad5 vector. This new Ad5 vector also inhibited HIV-1 proliferation in a Tat-inducible manner. Our new recombinant Ad5 vector has a specific tropism to CD4-positive cells that can effectively suppress the HIV-1 replication.

Neutralizing antibodies to gB based CMV vaccine requires full length antigen but reduced virus neutralization on non-fibroblast cells limits vaccine efficacy in the guinea pig model

Cytomegalovirus is a leading cause of congenital disease and a vaccine is a high priority. The viral gB glycoprotein is essential for infection on all cell types. The guinea pig is the only small animal model for congenital CMV (cCMV), but requires guinea pig cytomegalovirus (GPCMV). Various GPCMV gB vaccine strategies have been investigated but not with a full length protein. Previous GPCMV gB vaccines have failed to fully protect against cCMV, with approximately 50% efficacy. In an effort to define the basis of GPCMV gB based vaccine failure, we evaluated recombinant defective Ad vectors encoding GPCMV gB full length (gBwt), or truncated protein lacking transmembrane domain (gBTMD). Both candidate vaccines evoked high anti-gB titers and neutralized virus infection on fibroblast cells but had varying weaker results on non-fibroblasts (renal epithelial and placental trophoblasts). Non-fibroblast cells are dependent upon the viral pentamer complex (PC) for endocytic pathway cell entry. In contrast, fibroblasts cells that express the viral receptor platelet derived growth factor receptor alpha (PDGFRA) to enable entry by direct cell fusion independent of the PC. Anti-gBwt sera was approximately 2-fold (renal epithelial) to 3-fold (fibroblasts) more effective at neutralizing virus compared to anti-gBTMD sera. Both gB vaccines were weakest against virus neutralization on trophoblasts. Knockout of PDGFRA cell receptor on fibroblast cells (GPKO) rendered virus dependent upon the PC pathway for cell entry and anti-gB GPCMV NA50 was more similar to epithelial cells. In a gBwt vaccine protection study, vaccination of animals significantly reduced, but did not prevent dissemination of wild type GPCMV challenge virus to target organs. Depletion of complement in vivo had limited impact on vaccine efficacy. Overall, a full length gB antigen has the potential to improve neutralizing antibody titer but fails to fully prevent virus dissemination and likely congenital infection.

Semi-quantification of antibody-dependent enhancement (ADE) in the uptake of Adenovirus serotype 5 into THP-1 cells

Replication defective recombinant Ad5 vectors (rAdV5) are extensively explored for its applications in gene therapy and vaccine delivery. Ad5 enter into monocytes and macrophages through CAR independent route as an immune complex termed as antibody-dependent enhancement (ADE). We developed an effective method for estimating the ADE of rAdV5 encoding GFP (rAdV5-GFP) into THP-1 cells, using fluorimetric semi-quantification of GFP. Initially, twenty numbers of human sera samples were screened in HeLa cells for anti-Ad5 antibody titer using neutralization assay. Uptake of rAdV5-GFP in THP-1 cells was observed only after pre-incubation with the serially diluted human sera which are attributed to ADE. The optimal dilution which showed the maximum GFP expression as per the fluorescence microscopic analysis in THP-1 cells was used for further analysis. Fluorimetric analysis of the THP-1 cell lysate showed a maximum GFP intensity of 17058 RFU, which was equivalent to the 0.397 pmoles of Alexa Fluor 488 under the same experimental condition. Similarly, immunoblot analysis of GFP in THP-1 cell lysate and HeLa cell lysate confirmed the entry of rAdV5-GFP into the cells. The assay can serve as a platform for understanding the molecular events involved in ADE for the uptake of viruses into immune cells.

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.

Endothelial cells modified by adenovirus vector containing nine copies hypoxia response elements and human vascular endothelial growth factor as the novel seed cells for bone tissue engineering.

Vascularization is one of the hotspots during the development of new therapeutic strategies for bone tissue engineering, which can alleviate hypoxic circumstance and prevent transplant failure. Vascular endothelial growth factor (VEGF) gene transfection using recombinant adenovirus (Ad) vector can effectively promote angiogenesis, but uncontrolled long-term continuous expression of VEGF brings safety concern. Here we constructed a recombinant Ad vector containing nine copies of HRE promoter and the hVEGF165 gene, which conserved the oxygen sensitivity of hypoxia-inducible factor-1/hypoxia response elements (HIF-1/HRE). After transfection into human umbilical vein endothelial cells (HUVEC), the hVEGF165 mRNA and protein levels were much higher in response to hypoxia, as revealed by RT-PCR and ELISA, respectively. Furthermore, Ad-9HRE-hVEGF165 vector effectively promoted proliferation, migration and tube formation of HUVEC under hypoxic conditions. Thus we believe that the Ad-9HRE-hVEGF165 vector can contribute to the regulation of vascularization, which may provide a new approach for a better control of the expression of hVEGF165 during bone tissue engineering.

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.

A novel capsid-modified oncolytic recombinant adenovirus type 5 for tumor-targeting gene therapy by intravenous route.

Oncolytic adenovirus (Ad)-vectored gene therapy is a promising strategy for cancer treatment. However, the lack of cancer cell selectivity or tumor tissue specificity of Ads limits their clinical application by intravenous (IV) injection. In this paper, a novel recombinant Ad5 vector was constructed carrying the capsid protein IX modified by the tumor necrosis factor related apoptosis-inducing ligand (TRAIL), which targets tumor cells bearing high levels of its receptor far above those of normal cells. Specific association of the Ad virion with TRAIL was achieved using synthetic leucine zipper-like dimerization domains (zippers). Analysis of the chemical properties of the modified recombinant Ad (rAd5pz-zTRAIL-RFP) showed that the TRAIL protein was present on the surface of purified virus particles, and it could induce apoptosis of infected cancer cells prior to expression of foreign genes. We also constructed a novel modified recombinant oncolytic Ad (rAd5pz-zTRAIL-RFP-SΔ24E1a) which showed significantly enhanced anti-tumor effects both in vitro and in vivo by linkage of TRAIL to the viral capsid. Moreover, rAd5pz-zTRAIL-RFP-SΔ24E1a showed significantly improved tumor tissue targeting and reduced liver tropism when IV injected in vivo. Thus, we successfully obtained new oncolytic Ad5 gene therapy vectors with enhanced targeting and efficacy, providing a platform for further clinical application of Ad vectors for cancer treatment.

Interaction between hexon and L4-100K determines virus rescue and growth of hexon-chimeric recombinant Ad5 vectors.

The immunogenicity of recombinant adenovirus serotype 5 (rAd5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against hexon hypervariable regions (HVRs). Preexisting immunity can be circumvented by replacing HVRs of rAd5 hexon with those derived from alternate adenovirus serotypes. However, chimeric modification of rAd5 hexon HVRs tends to cause low packaging efficiency or low proliferation of rAd5 vectors, but the related mechanism remains unclear. In this study, several Ad5-based vectors with precise replacement of HVRs with those derived from Ad37 and Ad43 were generated. We first observed that a HVR-exchanged rAd5 vector displayed a higher efficacy of the recombinant virus rescue and growth improvement compared with the rAd5 vector, although most hexon-chimeric rAd5 vectors constructed by us and other groups have proven to be nonviable or growth defective. We therefore evaluated the structural stability of the chimeric hexons and their interactions with the L4-100K chaperone. We showed that the viability of hexon-chimeric Ad5 vectors was not attributed to the structural stability of the chimeric hexon, but rather to the hexon maturation which was assisted by L4-100K. Our results suggested that the intricate interaction between hexon and L4-100K would determine the virus rescue and proliferation efficiency of hexon-chimeric rAd5 vectors.

Localization of neutralization epitopes on adenovirus fiber knob from species C.

Although potential neutralization epitopes on the fiber knob of adenovirus (AdV) serotype 2 (Ad2) and Ad5 have been revealed, few studies have been carried out to identify neutralization epitopes on the knob from a broader panel of AdV serotypes. In this study, based on sequence and structural analysis of knobs from Ad1, Ad2, Ad5 and Ad6 (all from species C), several trimeric chimeric knob proteins were expressed in Escherichia coli to identify the locations of neutralization epitopes on the knobs by analysing their reactivity with mouse and rabbit polyclonal sera raised against AdVs and human sera with natural AdV infection. The dominant neutralization epitopes were located mainly in the N-terminal part of knobs from Ad1, Ad2 and Ad5, but they seemed to be located in the C-terminal part of the Ad6 knob, with some individual differences in rabbit and human populations. Our study adds to our understanding of humoral immune responses to AdVs and will facilitate the construction of more desirable capsid-modified recombinant Ad5 vectors.

Heterologous Immunity between Adenoviruses and Hepatitis C Virus: A New Paradigm in HCV Immunity and Vaccines.

Adenoviruses (Ad) are commonly used as vectors for gene therapy and/or vaccine delivery. Recombinant Ad vectors are being tested as vaccines for many pathogens. We have made a surprising observation that peptides derived from various hepatitis C virus (HCV) antigens contain extensive regions of homology with multiple adenovirus proteins, and conclusively demonstrate that adenovirus vector can induce robust, heterologous cellular and humoral immune responses against multiple HCV antigens. Intriguingly, the induction of this cross-reactive immunity leads to significant reduction of viral loads in a recombinant vaccinia-HCV virus infected mouse model, supporting their role in antiviral immunity against HCV. Healthy human subjects with Ad-specific pre-existing immunity demonstrated cross-reactive cellular and humoral immune responses against multiple HCV antigens. These findings reveal the potential of a previously uncharacterized property of natural human adenovirus infection to dictate, modulate and/or alter the course of HCV infection upon exposure. This intrinsic property of adenovirus vectors to cross-prime HCV immunity can also be exploited to develop a prophylactic and/or therapeutic vaccine against HCV.

Improved cytotoxic T-lymphocyte immune responses to a tumor antigen by vaccines co-expressing the SLAM-associated adaptor EAT-2

The signaling lymphocytic activation molecule-associated adaptor Ewing's sarcoma's-activated transcript 2 (EAT-2) is primarily expressed in dendritic cells, macrophages and natural killer cells. Including EAT-2 in a vaccination regimen enhanced innate and adaptive immune responses toward pathogen-derived antigens, even in the face of pre-existing vaccine immunity. Herein, we investigate whether co-vaccinations with two recombinant Ad5 (rAd5) vectors, one expressing the carcinoembryonic antigen (CEA) and one expressing EAT-2, can induce more potent CEA-specific cytotoxic T lymphocyte (CTL) and antitumor activity in the therapeutic CEA-expressing MC-38 tumor model. Our results suggest that inclusion of EAT-2 significantly alters the kinetics of Th1-biasing proinflammatory cytokine and chemokine responses, and enhances anti-CEA-specific CTL responses. As a result, rAd5-EAT2-augmented rAd5-CEA vaccinations are more efficient in eliminating CEA-expressing target cells as measured by an in vivo CTL assay. Administration of rAd5-EAT2 vaccines also reduced the rate of growth of MC-38 tumor growth in vivo. Also, an increase in MC-38 tumor cell apoptosis (as measured by hematoxylin and eosin staining, active caspase-3 and granzyme B levels within the tumors) was observed. These data provide evidence that more efficient, CEA-specific effector T cells are generated by rAd5 vaccines expressing CEA, when augmented by rAd5 vaccines expressing EAT-2, and this regimen may be a promising approach for cancer immunotherapy in general.

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.

Strategies for Control of Pandemic Influenza: Active and Passive Immunization

Pandemic influenza poses a serious threat to global health and the world economy. Highly pathogenic avian influenza A virus (HPAIV) of the H5N1 subtype that has emerged since 2004, resulted in more than 430 cases in 15 countries with a 50% case-fatality rate. Novel vaccine strategies for early response include the use of attenuated vaccines/vectors administered via novel mucosal immunization routes, therapeutic anti-virals and passive immunization with virus-specific antibodies (Abs). The current influenza vaccines designed for inducing antibody (Ab) responses against viral surface antigens (i.e., hemagglutinin [HA] and neuraminidase [NA]) are limited to seasonal use because of the ability of the virus to mutate these major antigenic glycoproteins. Vaccines that target determinants conserved among influenza A viruses (IAV) to generate broad protection against infection with different influenza A subtypes (i.e., heterosubtypic immunity [HSI]) remain elusive. We have currently developed a recombinant adenovirus (Ad) vector co-encoding HA (H5 subtype) and a conserved ectodomain of matrix protein 2 (M2e) (AdH5/M2e) for induction of protective immunity to H5N1 and other subtypes. Another approach based on the use of influenza virus carrying a deletion in the nonstructural NS1 gene is being explored. Since NS1 enables the virus to disarm the host type 1 IFN response, such deletion leads to attenuation of the viruses and enhanced host antiviral response. Therefore, vaccines based on NS1 deleted viruses (DelNS1) may provide better protection than inactivated vaccines and could induce HSI to infection with different influenza virus A subtypes. Sub-lingual immunization has been found to be a safe and effective route for induction of protective immune responses in systemic and mucosal compartments including respiratory tract. We found that sublingual immunization with either AdH5/M2e or DelNS1 induces broad protective immunity to H5 viruses and other influenza virus A subtypes including H1N1. Passive immunization (the transfer of specific immunoglobulins/Abs to a previously nonimmune recipient host) could offer an alternative strategy to prevent and treat influenza virus infection and an additional therapeutic option to antiviral drugs that are limited by widespread drug resistance among influenza virus strains. Even after targeted vaccines become available, passive immunization could still have prophylactic effects and provides an additional countermeasure against influenza, especially for individuals who do not respond well to the vaccines. Attempts to develop monoclonal Abs (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV) strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We found that eggs obtained from chicken farms and supermarkets in Vietnam contain H5N1-specific immunoglobulins (IgY) that provide protection against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection with HPAIV H5N1, H5N1-specific IgY prevent disease or significantly reduce viral replication resulting in complete recovery from the disease, respectively. In addition, we generated H1N1 virusspecific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection. These results underscore the usefulness of recombinant Ad vectors encoding surface glycoprotein (HA) and conserved protein (M2e) and NS1 deleted viruses (DelNS1) as vaccine candidates for control of pre-pandemic H5N1 and newly emerging subtypes. Data on antiviral efficacy of IgY provide a proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the control of influenza outbreaks, including the potential H5N1 and current H1N1 pandemic.

Comparative Analysis of Immune Responses Induced by Vaccination With SIV Antigens by Recombinant Ad5 Vector or Plasmid DNA in Rhesus Macaques

DNA vaccines have undergone important enhancements in their design, formulation, and delivery process. Past literature supports that DNA vaccines are not as immunogenic in nonhuman primates as live vector systems. The most potent recombinant vector system for induction of cellular immune responses in macaques and humans is adenovirus serotype 5 (Ad5), an important benchmark for new vaccine development. Here, we performed a head-to-head evaluation of the Merck Ad5 SIV vaccine and an optimized electroporation (EP) delivered SIV DNA vaccine in macaques. Animals receiving the Ad5 vaccine were immunized three times, whereas the DNA-vaccinated animals were immunized up to four times based on optimized protocols. We observed significant differences in the quantity of IFNgamma responses by enzyme-linked immunosorbent spot (ELISpot), greater proliferative capacity of CD8(+) T cells, and increased polyfunctionality of both CD4(+) and CD8(+) T cells in the DNA-vaccinated group. Importantly, Ad5 immunizations failed to boost following the first immunization, whereas DNA responses were continually boosted with all four immunizations demonstrating a major advantage of these improved DNA vaccines. These optimized DNA vaccines induce very different immune phenotypes than traditional Ad5 vaccines, suggesting that they could play an important role in vaccine research and development.

Recombinant vector-induced HIV/SIV-specific CD4 + T lymphocyte responses in rhesus monkeys

The recently reported modest success of the RV144 Thai trial vaccine regimen in preventing HIV-1 acquisition has focused interest on the potential contribution to that protection of vaccine-elicited CD4 + T cell responses. We evaluated the induction of virus-specific CD4 + T cell responses in rhesus monkeys using a series of diverse vaccine vectors. We assessed both the magnitudes and functional profiles of the antigen-specific CD4 + T cells by measuring cytokine production, memory differentiation, and the expression of mucosal homing molecules. We found that DNA prime/recombinant MVA boost immunizations induced particularly high-frequency virus-specific CD4 + T cell responses with polyfunctional repertoires, and these responses were partially preserved following SHIV-89.6P challenge. The majority of the vaccine-elicited CD4 + T cells were CD28 + memory T cells that expressed low levels of β7. Neither the magnitudes nor the functional profiles of the virus-specific CD4 + T cells generated by vaccination were associated with a preservation of CD4 + T cells or control of viral replication following SHIV-89.6P challenge. Interestingly, monkeys primed with recombinant Ad5 immunogens showed a dramatic expansion of both the magnitude and polyfunctionality of the vaccine-elicited CD4 + T cell responses following envelope protein boost. These results demonstrate that vaccine strategies that include recombinant MVA or recombinant Ad5 vectors can elicit robust CD4 + T cell responses.

Immunization With a Bivalent Adenovirus-vectored Tuberculosis Vaccine Provides Markedly Improved Protection Over Its Monovalent Counterpart Against Pulmonary Tuberculosis

Recombinant virus-vectored vaccines hold great promise for tuberculosis (TB) vaccination strategies. However, there is a lack of side-by-side comparative investigations to dissect the functional differences and support the advantage of multivalent virus-vectored vaccine over its monovalent counterpart. We previously successfully developed a monovalent adenovirus (Ad)-vectored vaccine expressing Ag85a (AdAg85a) and demonstrated its superior protective efficacy in models of pulmonary TB. In this study, we have developed a bivalent Ad TB vaccine expressing Ag85a and TB10.4 antigens as a fusion protein (AdAg85a:TB10.4) and compared its T-cell-activating and immune protective efficacy with that by monovalent AdAg85a. A single intranasal (i.n.) administration of AdAg85a:TB10.4 induced robust T-cell responses toward the respective antigens within the airway lumen and spleen, although the level of Ag85a-specific T-cell responses in the airway lumen triggered by bivalent AdAg85a:TB10.4 was lower than that by its monovalent counterpart at earlier time points. Thus, a single i.n. delivery of AdAg85a:TB10.4 conferred a markedly improved and sustained level of protection in the lung against Mycobacterium tuberculosis (M.tb) challenge over that by AdAg85a or by conventional BCG immunization with similarly induced levels of protection in the spleen. Our results indicate a unique advantage of multivalent viral-vectored TB vaccines for immunization against pulmonary TB.

Construction of Adenovirus Vectors for Transcriptional Targeting

INTRODUCTIONThe balance between target and nontarget cell toxicity determines the efficacy of any therapeutic agent. Therapeutic gene transfer strategies aim to maximize gene transfer and expression in target cells. However, gene delivery systems often fail to preferentially transduce target cells in mixed cell populations. In addition, limitations in vector specificity can lead to transduction of nontarget cells, resulting in untoward toxicity, even with compartmental dosing. Thus, vector optimization is critical for the development of efficient genetic experiments. The life cycle and biology of adenovirus (Ad) infection and transgene expression in cells have been thoroughly characterized. While genetically modified Ad vectors with transductional specificity for a single cell type can be generated by direct or indirect modifications of the carboxyl terminus of the vectors’ receptor recognition site, Ad vectors can also be targeted at the level of protein expression of the transgene in the targeted cells (i.e., transcription/translation). Target-cell-specific gene expression is accomplished using tissue-specific promoters (TSPs), DNA elements that restrict expression to specific cellular subsets. The major drawback of transcriptional retargeting is that pathological change in target tissues or organs, such as degeneration or malignant transformation, can ectopically activate TSPs. In this protocol, recombinant Ad vectors that express the luciferase gene are constructed through homologous recombination in Escherichia coli. TSPs are placed in front of the luciferase gene for selective expression.