Ad Capsid Research Articles

774. Evaluation of Adenovirus Vectors Containing Serotype 35 Fibers for Gene Therapy of Cervical Cancer

Adenovirus vectors containing group B fibers (such as Ad5/35) are able to target tissues that are refractory to infection with commonly used Ad serotype 5 based vectors. To evaluate the utility of Ad5/35 vectors for gene transfer into tumor cells, we studied the expression of the Ad5 receptor, CAR, and the Ad5/35 receptor, CD46, on Papanicolaou (Pap) smears of normal, pre-malignant, and malignant cervical tissue (N= 45). We observed a significant correlation between the degree of malignancy and the level of CD46 expression, while in contrast, the level of CAR expression inversely correlated with the degree of malignancy. Using qRT-PCR, we showed that CAR mRNA levels were lower in malignant tissue biopsies than in histologically normal biopsies. However, there was no significant difference in CD46 mRNA levels in normal and malignant tissues, indicating that CD46 protein expression in tumors might be regulated at the post-transcriptional level. In an attempt to delineate reasons for low CAR expression in tumors, we analyzed the methylation status of CpG island present in the promoter of the CAR gene in DNA isolated from normal and malignant tumor tissue, but found the promoter unmethylated in both groups. To better predict the infectivity of Ad5/35 in tumor patients, we studied the binding of recombinant Ad fiber knob, which is the Ad capsid moiety that mediates attachment to receptors, to the surface of cells present in Pap smears. We found that binding of recombinant Ad5 and Ad35 fiber knobs mirrored the expression patterns of CAR and CD46. Finally, we cultured primary cells from cervical cancer biopsies and performed Ad5/35 transduction studies on primary cultures and single tumor cell clones derived from these cultures (N>40). Overall, the level of CAR and CD46 in cultured cells correlated well with transduction efficiency of Ad5 and Ad5/35 vectors expressing GFP. Importantly, all tumor cell cultures could be transduced at 100% efficiency with Ad5/35-GFP at low MOIs. Despite of the uniformly efficient tumor cell transduction, a conditionally replicating Ad5/35-based oncolytic vector expressing Ad E1A and TRAIL exhibited differences in tumor cell killing when analyzed in clonal tumor cell cultures. Mechanisms that confer resistance to viral oncolysis could include inefficient viral replication and/or execution of apoptosis. Genes whose expression or repression confer resistance are currently being studied using DNA array technology.

871. Surface Modification of Adenovectors with TAT- and Antp-Containing Polymers Protects Them from Neutralizing Antibodies and Increases Expression of Reporter Genes In Vitro and In Vivo

The relative inefficiency of adenovirus (Ad)-based transduction vectors in cardiovascular tissues is related to the low expression of the CAR receptor on the surface of endothelial and smooth muscle cells as well as to the rapid neutralization of Ad by anti-Ad antibodies. Polymer modification of Ad surface potentially addresses these problems by masking Ad particles from neutralizing antibodies and providing a convenient platform for the conjugation of ligands possessing transduction-facilitating properties, such as TAT and Antp peptides. Our present studies investigated the hypothesis that by surface modifying Ad using photoreactive polymers to attach either TAT or Antp increased transduction could be achieved while reducing susceptibility to neutralizing antibodies. This was studied with reporter gene transduction with surface-modified Ad vectors in cell culture and in vivo, in rat carotid arteries. A series of fluorescently labeled polyallylamine (PAA)-based photochemically activatable (either via benzophenone, or azide chemistry) thiol reactive linear polymers were custom synthesized and conjugated with TAT-SH or Antp-SH. Ad-GFP or Ad-Luc were suspended in PBS x5 and admixed with either TAT-, Antp-derivatized or unmodified polymers to obtain the final polymer/Ad ratios ranging from 1:60 to 1:10 (w/w). Cellular uptake of vectors and ensuing transgene expression in rat aortic smooth muscle cell line (A10), primary bovine aortic endothelial cells (BAEC) and human alveolar epithelial cell line (A549) were examined by the fluorescence microscopy and fluorometry. Additionally, naïve and surface- modified Ad vectors were exposed to escalating concentrations of neutralizing anti-knob antibodies (50 to 500 nM) prior to the cell infection. 6x108 pfu of either naïve or polymeric TAT-conjugated Ad-Luc were locally delivered to the balloon-injured common carotid arteries of SD rats (n=6). The transgene expression was assessed 2 days after gene delivery by bioluminescence imaging. Model experiments have shown faster kinetics of photochemical polymer attachment to the capsid proteins with azide-compared to benzophenone-containing polymers. The surface modification of Ad-GFP with TAT- and Antp-derivatized polymers resulted in 3–10-fold increase of GFP expression in the studied cell types. 15 min incubation of naïve Ad with neutralizing antibodies resulted in a 94% decrease of GFP expression in A10 cells, while the surface modification of Ad with TAT- and Antp-containing polymers bestowed protection against neutralizing antibodies in proportion with the extent of surface modification (59%, 14% and 6% inhibition for the polymer/Ad ratios of 1:60, 1:30 and 1:10, respectively). In the initial in vivo experiments local delivery of polymeric TAT- conjugated Ad resulted in 4.4-fold enhancement of luciferase activity in comparison with unmodified Ad. In conclusion, light-induced covalent conjugation of TAT- and Antp-modified PAA-based polymers to Ad capsid proteins protects adenovectors from preformed neutralizing antibodies and significantly augments gene transduction both in vitro and in vivo.

The influence of adenovirus fiber structure and function on vector development for gene therapy

The collective attributes of adenoviruses (Ads), including ease of accomplishing replication deficiency, readily achievable high titers, encoding of large expression cassettes, efficiency of gene delivery to most cell types, and well-characterized biology, have made Ads, particularly Ad serotype 5 (Ad 5), some of the most utilized vectors for gene delivery. In recent years, however, it has become apparent that additional aspects of basic Ad virology must be uncovered for this vector system to succeed in the clinic. While local gene delivery is generally efficient, the broad tropism of Ad 5 and its tendency to home to the liver after systemic administration have proved to be limitations for other therapeutic applications, such as the treatment of disseminated cancers and cardiovascular disease. This has refocused research into the biology of Ad capsid components, particularly the main tropism determinant, the fiber/penton base complex, and their influence on transduction of selected cell types in vivo.

Influence of Fiber Detargeting on Adenovirus-Mediated Innate and Adaptive Immune Activation

The major adenovirus (Ad) capsid proteins hexon, penton, and fiber influence the efficiency and tropism of gene transduction by Ad vectors. Fiber is the high-affinity receptor binding protein that serves to mediate cell attachment in vitro when using coxsackie-adenovirus receptor (CAR)-containing cell lines. This contrasts with transduction efficiency in macrophages or dendritic cells that lack high concentrations of CAR. To determine how fiber influences gene transduction and immune activation in a murine model, we have characterized Ad type 5 (Ad5) vectors with two classes of chimeric fiber, CAR binding and non-CAR binding. In a systemic infection, Ad5 fiber contributes to DNA localization and vector transduction in hepatic tissue. However, the majority of vector localization is due to Ad5 fiber-specific functions distinct from CAR binding. CAR-directed transduction occurs but at a modest level. In contrast to CAR binding vectors, the F7 and F7F41S non-CAR-binding vectors demonstrate a 2-log decrease in hepatic transduction, with a 10-fold decrease in the amount of vector DNA localizing to the hepatic tissue. To characterize the innate response to early infection using fiber chimeric vectors, intrahepatic cytokine and chemokine mRNAs were quantified 5 hours postinfection. Tumor necrosis factor alpha mRNA levels resulting from Ad5 fiber infections were elevated compared to viruses expressing serotype 7 or 41 fiber. Levels of chemokine mRNA (gamma interferon-inducible protein 10, T-cell activation gene 3, and macrophage inflammatory protein 1beta) were 10- to 20-fold higher with CAR binding vectors (Ad5 and F41T) than with non-CAR-binding vectors (F7 and F7F41S). In spite of quantitative differences in vector localization and innate activation, fiber pseudotyping did not significantly change the outcome of anti-Ad adaptive immunity. All vectors were cleared with the same kinetics as wild-type Ad5 vectors, and each induced neutralizing antibody. Although non-CAR-binding vectors were impaired in transduction by nearly 2 orders of magnitude, the level of antitransgene immunity was the same for each of the vectors. Using primary bone marrow-derived macrophages and dendritic cells, we demonstrate that transduction, induction of cytokine/chemokine, and phenotypic maturation of these antigen-presenting cells are independent of fiber content. Our data support a model where fiber-mediated hepatic localization enhances innate responses to virus infection but minimally impacts on adaptive immunity.

An adenovirus vector with a chimeric fiber incorporating stabilized single chain antibody achieves targeted gene delivery

Adenovirus (Ad) vectors are of utility for many therapeutic applications. Strategies have been developed to alter adenoviral tropism to achieve a cell-specific gene delivery capacity employing fiber modifications allowing genetic incorporation of targeting motifs. In this regard, single chain antibodies (scFv) represent potentially useful agents to achieve targeted gene transfer. However, the distinct biosynthetic pathways that scFv and Ad capsid proteins are normally routed through have thus far been problematic with respect to scFv incorporation into the Ad capsid. Utilization of stable scFv, which also maintain correct folding and thus functionality under intracellular reducing conditions, could overcome this restriction. We genetically incorporated a stable scFv into a de-knobbed, fibritin-foldon trimerized Ad fiber and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of cells. We have shown that the scFv employed in this study retains functionality and that stabilizing the targeting molecule, per se, is critical to allow retention of antigen recognition in the adenovirus capsid-incorporated context.

28. Adenovirus Targeting Via Leucine Zipper Peptide-Mediated Ligand Attachment

Gene therapy vectors based on human adenovirus (Ad) serotypes 2 and 5 continue to show increasing promise as gene therapy delivery vehicles. Initial gene therapy efforts, however, revealed that the clinical benefit of these gene delivery modalities was dependent on localization of the therapeutic agent. Therefore, a fundamental requirement for somatic cell gene therapy is targetable gene delivery vectors. Ad vectors incorporating genetic modifications have provided gains in vector efficiency; however, specific vector targeting to specific cell-surface molecules is still required. For cell-specific targeting, the use of antibodies or single chain antibody fragments (scFv) remains a very attractive concept made possible by the definition of disease-specific membrane antigens and a wide selection of cognate antibody molecules. However, a barrier to the development of Ad vectors capsid-incorporated scFvs occurs when the antibody requires posttranslational modifications not available to Ad capsid proteins due to their cytoplasmic routing during virion assembly. To address this problem, we have developed a targeting strategy that uses a secreted scFv that undergoes the requisite posttranslational modification (disulfide bond formation) in the endoplasmic reticulum and is trafficked for secretion. Formation of the vector/ligand targeting complex is achieved via genetic tagging of the Ad capsid and scFv with synthetic leucine zipper-like dimerization domains (Zippers) that have been optimized for structural and biological compatibility with the Ad capsid and the secreted ligand. Importantly, we have designed our Zipper domains in pairs, such that only compatible heterodimers can associate, thereby eliminating the possibility of aggregate formation. We have incorporated Zippers into the C-terminus of Ad5 fiber and a knob-deleted chimeric fiber molecule, fiber-fibritin/11F (11F). The ligand is comprised of a scFv fused with the kappa-chain IgG signal peptide and a Zipper peptide. Our results show that Zipper domain-containing fiber or 11F molecules trimerize and associate with the virion penton base protein and that these vectors can be amplified. Further, we demonstrate that peptide Zippers can be genetically fused to scFv without decreasing ligand recognition of antigen. Most important, we show that Zipper-tagged virions and ligands can self-associate and form targeting complexes capable of binding to target receptors. This approach to targeting ligand design solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.

975. Immunization with an Adenovirus Vector Expressing a P. aeruginosa OprF Epitope in the Capsid Enables Boosting of Anti-Epitope Immune Responses and Protects Against Pulmonary Infection with P. aeruginosa

Top of pageAbstract Pseudomonas aeruginosa causes chronic infections of the respiratory tract, particularly in cystic fibrosis (CF). The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and our prior studies have shown that an adenovirus (Ad) vector expressing a secreted form of OprF (AdOprF), evokes protective immunity against P. aeruginosa. The present study is focused on development of a 2nd generation clinical vaccine candidate (AdOprF.RGD.Epi8) by two innovations to AdOprF to enhance the immunogenicity: (1) genetic modification of the Ad hexon to incorporate an epitope (Epi8) from OprF; and (2) genetic modification of the Ad fiber to incorporate an RGD sequence to enhance gene delivery to antigen presenting cells. We first evaluated the immunogenic properties of an Ad vector in which the Ad hexon protein was modified to include the 14-mer OprF epitope Epi8 in the hexon (AdZ.Epi8), with focus on the hypothesis that immune responses against epitopes that are part of the Ad capsid could be augmented with repeat administration. To evaluate the protective effect of immunization with AdZ.Epi8 against pulmonary infection with P. aeruginosa, C57Bl/6 mice, immunized with either AdZ.Epi8 or AdZ were challenged with a lethal dose of agar-encapsulated P. aeruginosa (laboratory strain PAO1 or two isolates of P. aeruginosa derived from patients with CF). Unimmunized mice or mice infected with AdZ died within the first 3 days. In contrast, more than 60% of the mice immunized with AdZ.Epi8 survived more than 2 wk (p 0.1, all comparisons). Similar results were observed in the CD4 Epi8-specific IL-4 as well as CD4 and CD8 Epi8-specific |[gamma]|-interferon cellular responses, suggesting that the addition of an OprF epitope to the Ad capsid enables boosting of the anti-OprF response. Finally, we evaluated the immunogenic properties of the combined AdOprF.RGD.Epi8 vector. Mice received AdOprF.RGD.Epi8, AdOprF, AdNull, (2|[times]|1011pu) or PBS and the anti-OprF antibody titers were determined at 2 and 4 wk. No anti-OprF antibodies were detected in the AdNull or PBS group. The mean titer for AdOprF was 5710 and 8602 at 2 and 4 wk, respectively, vs 11841 and 10632 for the AdOprFRGD.Epi8 injected mice, suggesting a faster and more robust induction of the anti-OprF humoral response with AdOprFRGD.Epi8. We conclude that the capsid modifications in AdOprF.RGD.Epi8 vector make it a more effective candidate vector for vaccination against P. aeruginosa.

411. Trapping of Adenovirus Vectors in Blood

In an attempt to circumvent the limitations of Adenovirus (Ad) vectors derived from group C serotype Ad5 we recently developed vectors based on group B serotype Ad11. These vectors are benefited by the low prevalence of Ad11-neutralizing antibodies in humans, and the CD46-dependent tropism of Ad11. CD46 is expressed at high levels in cell types including hematopoietic stem cells, dendritic cells and metastatic tumor cells. Following systemic intravenous delivery of an Ad5 vector, a chimeric Ad5 vector possessing the CD46 interacting Ad11 fiber (Ad5/11), and an Ad11 vector to CD46 transgenic mice we discovered that the kinetics of vector clearance from blood are different. Using real time quantitative PCR to detect viral genomes we found that the number of Ad11 vector genomes in mouse serum at 3 minutes post tail vein injection was 2 orders of magnitude lower than for Ad5 and Ad5/11 vectors, but at 15 and 120 minutes post injection the number of vector genomes was comparable between Ad5, Ad5/11 and Ad11. To investigate the differences between the numbers of vector genomes in serum at 3 minutes post injection the association of vector genomes with blood cells was also monitored by real time quantitative PCR at all time points. The number of vector genomes associated with blood cells was more than one order of magnitude higher for Ad11 than Ad5 or Ad5/11 at all time points tested with the highest difference seen at 3 minutes post injection (|[sim]|2 orders of magnitude higher). To study which type of blood cells associate with Ad11 vectors, CD46 transgenic mice were injected in the tail vein with 3H-[thymidine] labeled Ad vector. Individual cell types were separated from blood collected at 3 minutes post injection and the highest levels of tritium labeled Ad11 were associated with the platelet fraction. Subsequent analyses revealed that Ad5, Ad5/11 and Ad11 vectors associate with platelets within minutes after intravenous injection but that Ad11 vectors associate at higher levels. Electron microscopic analysis of platelets isolated 3 minutes after injection of Ad5, Ad5/11 or Ad11 vectors revealed that Ad particles were associated with the platelet surface and structures of the surface-connected canalicular system. We also noticed that Ad11 vectors associate with mononuclear cells more efficiently than Ad5/11 vectors. When taken together, the observations that Ad11 associates with platelets and mononuclear cells more efficiently than Ad5/11 suggests that capsid proteins other than fiber may mediate blood cell interactions. We are currently investigating the mechanism(s) by which Ad capsids and blood cells interact. The roles of the serotype specific (Ad5 or Ad11) capsid proteins hexon, penton and fiber in Ad/blood cell interactions will be presented. Our aim is to develop new Ad vectors that are not trapped in blood upon systemic intravascular administration.

407. Complement Blockade Prevents Several Innate Toxicities Rapidly Induced after Intravenous Adenovirus (Ad) Vector Administration

Toxicities resulting from Ad capsid interactions with the innate immune system present numerous challenges for adenoviral gene transfer in vivo. In 2004: Mol. Ther. 10(6):1140-2, we demonstrated that Ads can initiate the human complement pathway via activation of the alternative complement pathway, and that the Ad capsid can directly bind to human C3 protein. After initial pathogen engagement by proteins of the classical and/or alternative complement pathways, C3 acts as a pivotal protein, initiating the subsequent activation of the common complement pathway. Activated complement proteins interact with Kupffer cells, platelets, and endothelial cells, and modulate several innate and adaptive immune response systems. Since several of these same systems have been implicated with Ad associated toxicities, we hypothesized that these toxicities may be significantly diminished by pre-emptive complement system blockade.

1130. A Single-Component CD40-Targeted Adenoviral Vector for Efficient Transduction and Activation of Cutaneous Dendritic Cells

Dendritic cell (DC)-based tumor vaccination usually involves the administration of ex vivo generated autologous DC. Transduction of DC by viral vectors in vivo has been proposed as a more standardized, and easily clinically applicable approach. Previously, we have employed retargeting approaches to route adenoviral (Ad) vectors via the CD40 pathway as a means to circumvent the paucity of the Ad5 virus's primary receptor (CAR), on the surface of DC and to selectively target DC in situ. CD40-targeted Ad-mediated transduction of DC has been successfully realized through the use of bispecific adapter proteins. With these CD40-targeted Ad complexes we have shown an enhanced and more selective transduction as well as a simultaneous CD40-dependent maturation of DC. However, these approaches required the production of several components followed by physical association to form targeted vector complexes. Evidently for those techniques the presence of an excess of free targeting ligands in analyzed samples leaves open questions about the causal component of DC activation and the ability of targeting ligands as integral components of viral particles to induce this process with similar efficiency. Direct incorporation of the TNF-like domain of human CD40L into the Ad capsid, preserving the targeting ability of the genetically modified Ad vector, now allows us to answer these questions and offers a single-component targeted vector for direct clinical application. In the present study, we evaluate the ability of this vector to deliver transgenes in a stringent human substrate system. We report the capacity of this CD40-targeted vector to infect, with high efficiency, cutaneous DC resident in human skin explants, while simultaneously inducing their activation and maturation. This latest generation of single-component, fully targeted vectors should make feasible the clinical testing of in vivo DC-targeted vaccines.

1088. Antigen Epitopes Expressed in Different Adenovirus Capsid Proteins Induce Different Levels of Epitope-Specific Immunity

Top of pageAbstract The immune response against adenovirus gene transfer vectors (Ad) is directed against the capsid proteins and transgene expressed by the vector. Incorporation of pathogen-derived epitopes into capsid proteins may be a useful strategy to evoke an anti-pathogen immunity and may complement the anti-transgene immune response. Our previous data (Worgall et al Mol Ther 2004; 9:S261) demonstrate that incorporating an epitope of the outer membrane protein of Pseudomonas aeruginosa into the hexon of the adenovirus capsid provides protection against pulmonary challenge by P. aeruginosa in experimental animals. In this study, we compare the immune response against an epitope incorporated into different capsid proteins (hexon, fiber, penton base and protein IX) and determine if the immune response against the epitopes will be proportional to the number of copies of that protein in the capsid. To test this hypothesis, we used the major antigenic site HA from the hemagglutinin protein of influenza virus type A as model epitope. Replication deficient Ad vectors were engineered expressing the HA sequence YPYDVPDYAGG on the surface of the adenoviral virion as part of following 4 capsid proteins: in HVR5 loop of hexon protein (Ad.Hexon-HA), in HI loop of fiber protein (Ad.Fiber-HA), in RGD loop of penton protein (Ad.Penton-HA), and attached to the C-terminus of Protein IX (Ad.pIX-HA). The HA epitope is present at various copy numbers in each virion: hexon, 720; protein IX 240; penton base 60; and fiber 36. Using an anti-HA antibody, in vitro studies demonstrated that the modified capsid proteins were incorporated into virions and express the HA-sequences in the expected capsid proteins of each recombinant vector. CBA mice were immunized by the intramuscular route with the same dose of adenoviral vectors (5|[times]|109 particle units) and serum anti-HA titer was evaluated at 4 wk by ELISA against purified recombinant HIS-tagged HA fusion protein. Titers against HA in Ad.Fiber-HA immunized mice were three times higher compared to Ad.Hexon-HA immunized mice (555|[plusmn]|40 vs 162|[plusmn]|36, p IgG2b for Ad.Fiber-HA and Ad-Hexon-HA. In contrast to the variable response to a specific antigen inserted into different locations of the capsid, anti-Ad capsid titer were the same for all 4 HA modified vectors and also for an AdNull vector with the wild type capsid delivered by the same dose and route. We conclude that incorporation of an epitope into the fiber gene is the most potent strategy to elucidate an anti-epitope response, at least for this model system . The fact that immune response was independent of the dose of the epitope suggested that antigen position in the capsid and its recognition by B cells is the critical component of the response to the epitope incorporated into the Ad capsid.

873. Adenovirus Polypeptide IIIa as a Novel Locale for Incorporation of Heterologous Peptides

Top of pageAbstract The utility of the present generation of recombinant adenovirus (Ad) vectors for gene therapy applications could potentially be improved by restricting broad native viral tropism to selected cell types. In order to achieve cell-specific targeting of Ad vectors, we have proposed modification of viral tropism via genetic engineering of the viral coat proteins to include targeting ligands. To this end, major Ad capsid proteins fiber, hexon, penton and the minor capsid protein polypeptide IX (pIX) have been exploited as platforms for the presentation of ligand peptides. While each of these capsid locales may be of utility, additional capsid locales for the presentation of heterologous peptides should be investigated. The adenovirus capsid protein pIIIa is a 570 amino acid protein present in the virion as a 63.5 kDa monomer. Structural studies indicate that sixty copies of pIIIa are present in the Ad5 virion, with two pIIIa monomers positioned along each of the thirty icosahedral edges. Further, cryo-electron microscopy studies indicate that the bulk of pIIIa is localized to the outer surface of the virion. Based on these considerations, we hypothesized that the minor capsid protein pIIIa could be exploited as a novel locale for ligand incorporation. In this study, we engineered recombinant Ad vector genomes encoding pIIIa modified to incorporate a 6-Histidine tag or FLAG octapeptide/6-Histidine at the N-terminus. Both pIIIa Ad variants were rescued, indicating that the presence of these non-native pIIIa proteins does not significantly disturb viral replication and/or assembly. Further, by using the relevant specific antibodies, we have confirmed that FLAG- and 6His-containing pIIIa chimeric capsomers are incorporated into virions, and are accessible for binding. These findings suggest that pIIIa may possess properties consistent with its employment in Ad capsid modification strategies and may be a promising locale for incorporation of targeting moieties.

1108. Ad5 Penton Base Recombinant Protein: Intracellular Trafficking & Implications for Gene Delivery

A growing interest in utilizing viral capsid proteins for non-viral delivery has led us to investigate how these proteins contribute to viral trafficking. For example, we and others have developed recombinant adenovirus (Ad) capsid proteins, such as the penton base, into non-viral vectors. The penton base facilitates viral infection by binding cell surface integrins and triggering receptor-mediated endocytosis. Previous studies have shown that Ad serotype 5 (Ad5) escapes the endosome at an early stage of cell entry, and hijacks dynein motors to translocate along intact microtubules for trans-cytoplasmic migration toward the nuclear periphery. Those findings suggest that one or all of the three major Ad5 capsid proteins (penton base, fiber, and/or hexon) interact with the cytoskeletal machinery to mediate intracellular trafficking of the virus during the early stages of infection.

1090. Adenovirus Fiber Incorporates an Immunodominant MHC Class I Restricted Epitope Peptide

Top of pageAbstract Adenovirus has been extensively investigated as one of the most promising gene delivery vectors. One of the major limitations to its application is the generation of a strong CD8+ cytotoxic T lymphocyte (CTL) response. However, analysis of the viral specific cytotoxic T lymphocyte (CTL) response and the correlation with the elimination of vector and transgene has not been possible due to the lack of a specific method to detect viral antigen-induced CTLs. To identify Adenovirus specific CTLs generated after administration of recombinant adenovirus vectors, we utilized two MHC binding peptide prediction programs, BIMAS and SYFPEITHI, to predict epitope peptides from adenovirus hexon, penton and fiber proteins that are processed and presented by the mouse MHC class I H2-Kb antigen. To test the specificity of the predicted peptides, the specific CTL activity was determined by an in vivo CTL assay and an IFN|[gamma]| ELISPOT assay. C57BL/6 (B6; H2b) mice were i.v. injected with 8|[times]|108i.u of wild type Ad5 through the tail vein. Eight days later, spleen cells from naive B6 mice were labeled with high (2uM; CFSEHi) concentration of CFSE and then pulsed with predicated peptides, respectively, before co-transferred to the wild type Ad5 primed B6 mice (recipient) with identical number of unpulsed CFSELo (0.2uM) cells for in vivo CTL assay. The known immunodominant E1B peptide (AA192|[ndash]|200 VNIRNCCYI ) of Ad was used as a positive control and human flu M1 peptide (AA58|[ndash]|66 GILGFVFTL) was used as a negative control. After six hours, spleen cells and liver mononuclear cells (MNCs) were recovered from recipient mice and analyzed by flow cytometry and by the IFN|[gamma]| ELISPOT assay. In the spleen of immunized B6 mice, 85.9|[plusmn]|13% of fiber peptide (AA391|[ndash]|399 VGNKNNDKL) pulsed CFSEHi target cells were specifically lysed, compared to 98.2|[plusmn]|5% of E1B positive control peptide pulsed cells and no specific lysis to M1 negative control peptide pulsed cells. There were 202|[plusmn]|18 spot forming cells (SFC) per 106 liver MNCs in immunized mice that were responsive to fiber peptide (AA391|[ndash]|399 VGNKNNDKL) and 468|[plusmn]|87 SFC/106 liver MNCs specific for E1B peptide, compared to 10|[plusmn]|10 SFC/106 liver MNCs specific for M1 peptide. There were no SFCs specific for other predicted peptides and no specific killing in target cells pulsed with other peptides (data not shown). To further confirm the immunogenicity of the fiber peptide in the context of E1-deleted Ad vectors, B6 mice were injected with 109i.u of AdLacZ and then analyzed by ELISPOT assay. There were 109|[plusmn]|23/106 liver MNCs specific this fiber peptide, whereas no SFCs specific for E1B peptide or M1 peptide. These results indicated that the fiber peptide is a strong immunogenic peptide and the in vivo CTL assay could be used as a sensitive method to measure Ad-specific CTL response to any viral vectors that derived from Ad2 or Ad5 virus. These findings will provide us with a powerful tool for assessing immune response to Ad capsid in the context of gene therapy.

994. E1 Adenovirus Gene Transfer Vectors Inhibit HIV-1 Replication in Human Alveolar Macrophages

E1 adenovirus (Ad) vector infection of human alveolar macrophages (AM) inhibits HIV-1 replication independently of any transgene (Rice et al, Am J Respir Cell Mol Biol 2002; 27: 214-219) when the AM are infected with HIV-1 in vitro. We have previously shown that deletions of E4, Ad DNA polymerase or preterminal protein had no effect on the ability of Ad to block HIV-1 replication, empty Ad capsids do not block HIV-1 replication, and Ad acts, in part, by suppressing transcription from the HIV-1 long terminal repeats (LTR). The inhibition of HIV-1 replication by Ad is dose-dependent. We hypothesized that Ad would cause a similar dose-dependent inhibition of endogenous HIV-1 replication in AM obtained from HIV-1+ individuals. To address this question, we examined the shape of the dose-response curve for submaximal Ad inhibition of HIV-1 replication in AM. AM (6|[times]|105 cells/well) from a normal individual were infected with AdNull (0 to 2.5 |[times]| 104 pu/cell) or treated with azidothymidine (AZT, 10 |[mu]|g/ml), prior to infection with HIV-1 (103 TCID50/well). Conditioned media was collected twice weekly, and HIV-1 replication was quantified by measuring p24 in the conditioned media by ELISA. On day 16 post-infection, at a time of linear increases in p24, indicating active HIV-1 replication, p24 levels were (ng/ml) No Ad: 19.4; Ad doses in pu/cell: 102: 17.2 ; 5|[times]|102: 12.5; 103: 5.2; 5|[times]|103: 0.72; 104 0.11; 2.5|[times]|104: 0.04; AZT: 0.04. We compared these results with Ad effects on HIV-1 spontaneously elaborated from AM recovered from an HIV-1 positive individual. AM (8 |[times]| 105 cells/well) were infected with AdNull (0 to 2.5 |[times]| 104 pu/cell) or treated with AZT. p24 levels in the culture increased linearly over 4 days. On day 4 post-infection with Ad, p24 levels were No Ad: 44.2; 102: 32.4 ; 5|[times]|102: 24.7; 103: 10.7; 5|[times]|103: 1.70; 104: 0.27; 2.5|[times]|104: 0.08; AZT: 0.08. The dose-response for inhibitory effects of Ad on HIV-1 replication was similar under both conditions over several logs of Ad doses. In a separate experiment, Ad inhibition of HIV-1 replication in AM obtained from another HIV+ individual was evaluated as a function of Ad dose in half-log increments spanning the range from 102 |[minus]|3|[times]|104 pu/cell and yielding a range of p24 levels on day 4 ranging from 1.1-26.2. When the percent inhibition of HIV-1 replication was plotted as a function of log [Ad dose], the relationship was nearly linear, R2=0.96 over a 2 log range of doses. These data indicate that Ad inhibits endogenous HIV-1 replication in AM with a dose-response relationship similar to that observed when normal AM are infected with HIV-1 in vitro.

1091. Analysis of T-Cell Responses to Helper-Dependent Serotype 5-Based Capsid-Modified Adenovirus (HD.5/35) or First-Generation Serotype 11- Based (Ad11) Vectors

Top of pageAbstract Cellular immunity to adenovirus serotype 5 (Ad5) is an obstacle for Ad5-based gene therapy. We asked if this could be overcome by either a helper-dependent capsid-modified Ad5 vector possessing the Ad35 fiber (HD.5/35), or a first-generation vector completely derived from Ad serotype 11 (Ad11). Ad35 and Ad11 fiber containing Ad vectors efficiently transduce dendritic cells (DC) in vitro and are being evaluated for immunotherapy strategies. We transduced human peripheral blood mononuclear cells (PBMC) from 11 donors (including cancer patients) with GFP expressing Ad11 or HD.5/35 vectors, or first generation Ad5, Ad5/35 or Ad5/11 vectors as controls. Regardless of the vector used, all donors showed IFN-|[gamma]| secreting cells, detected by ELISpot assays (200|[ndash]|400 spots in 1|[times]|10e6 PBMC). The Ad-specific T-cells against the HD vector indicates a response to Ad capsid epitopes from the incoming particles. It was speculated that this could interfere with responses to a vaccination or therapy antigen expressed from Ad vectors. We evaluated this in vivo and in vitro. For in vivo studies, we injected C57Bl/6 mice transgenic for CD46 (the receptor for Ad5/35) with Ad vectors expressing the model antigen hepatitis B virus e antigen (Ad5.HBeAg, Ad5/35.HBeAg and HD.5/35.HBeAg). Fourteen days later, their splenocytes were mixed with splenocytes from na|[iuml]|ve mice in vitro pulsed with HBeAg recombinant protein, or transduced with the same or control Ad vectors. A strong anti-Ad T cell response was detected by an IFN-|[gamma]| ELISpot assay irrespective of the vector used. The frequency of HBeAg specific T cells was similar with all the vectors tested, and at least two orders of magnitude lower than that to the Ad vectors. For the in vitro human studies, we used the HD.5/35.Her2.Fc vector. It expresses a target antigen in breast cancer, Her-2/neu. We stimulated twice 5|[times]|10e7 PBMC from a normal donor with 5|[times]|10e6 transduced and matured DC. The T cell line was then stimulated with DC pulsed with a library of 168 15mer-overlapping peptides covering the extracellular domain (ECD) of Her2/neu, or transduced with a control vector (HD.5/35.GFP), or pulsed with an Ad5 hexon-derived peptide or other control peptides in an IFN-|[gamma]| ELISpot plate. A strong T cell response against Ad transduced DC and against the hexon epitope was observed. No ECD specific T cell was detected. In conclusion, i) we found a strong T cell response to Ad11 vectors, which was surprising considering previous studies from our lab and others that showed a low serum-prevalence of neutralizing anti-Ad11 antibodies in humans; this would indicate either pre-existing T-cell immunity to Ad11 or, most likely, T-cell cross-reactivity between Ad serotypes; ii) the use of HD vectors did not prevent induction of anti Ad T cells in na|[iuml]|ve animals, and the incoming Ad particles activated memory T cell responses in in vitro human studies; iii) we could not detect strong antigen specific immune responses in our experiments. Then, these studies support the idea that cellular immunity, directed to the Ad capsid epitopes, is an important problem to be solved for antigen specific vaccination/immunotherapy purposes.

CryoEM Structure at 9 Å Resolution of an Adenovirus Vector Targeted to Hematopoietic Cells

We report a sub-nanometer resolution cryo-electron microscopy (cryoEM) structural analysis of an adenoviral vector, Ad35F, comprised of an adenovirus type 5 (Ad5) capsid pseudo-typed with an Ad35 fiber. This vector transduces human hematopoietic cells via association of its fiber protein with CD46, a member of the complement regulatory protein family. Major advances in data acquisition and image processing allowed a significant improvement in resolution compared to earlier structures. Analysis of the cryoEM density was enhanced by docking the crystal structures of both the hexon and penton base capsid proteins. CryoEM density was observed for hexon residues missing from the crystal structure that include hypervariable regions and the epitope of a neutralizing monoclonal antibody. Within the penton base, density was observed for the integrin-binding RGD loop missing from the crystal structure and for the flexible beta ribbon of the variable loop on the side of the penton base. The Ad35 fiber is flexible, consistent with the sequence insert in the third beta-spiral repeat. On the inner capsid surface density is revealed at the base of the hexons and below the penton base. A revised model is presented for protein IX within the virion. Well-defined density was assigned to a conserved domain in the N terminus of protein IX required for incorporation into the virion. For the C-terminal domain of protein IX two alternate conformations are proposed, either binding on the capsid surface or extending away from the capsid. This model is consistent with the tolerance of the C terminus for inserted ligands and its potential use in vector retargeting. This structural study increases our knowledge of Ad capsid assembly, antibody neutralization mechanisms, and may aid further improvements in gene delivery to important human cell types.

Identification of Sites in Adenovirus Hexon for Foreign Peptide Incorporation

Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His6) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His6 epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His6 epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His6 epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His6 epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His6 epitopes did not appear to mediate His6-dependent viral infection, as assessed in two His6 artificial receptor systems. Our study provided valuable information for studies involving hexon modification.

Transfer of the full-length dystrophin-coding sequence into muscle cells by a dual high-capacity hybrid viral vector with site-specific integration ability.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, making it a potential target for gene therapy. There is, however, a scarcity of vectors that can accommodate the 14-kb DMD cDNA and permanently genetically correct muscle tissue in vivo or proliferating myogenic progenitors in vitro for use in autologous transplantation. Here, a dual high-capacity adenovirus-adeno-associated virus (hcAd/AAV) vector with two full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements is presented. These vectors are generated from input linear monomeric DNA molecules consisting of the Ad origin of replication and packaging signal followed by the recently identified AAV DNA integration efficiency element (p5IEE), the transgene(s) of interest, and the AAV inverted terminal repeat (ITR). After infection of producer cells with a helper Ad vector, the Ad DNA replication machinery, in concert with the AAV ITR-dependent dimerization, leads to the assembly of vector genomes with a tail-to-tail configuration that are efficiently amplified and packaged into Ad capsids. These dual hcAd/AAV hybrid vectors were used to express the dystrophin-coding sequence in rat cardiomyocytes in vitro and to restore dystrophin synthesis in the muscle tissues of mdx mice in vivo. Introduction into human cells of chimeric genomes, which contain a structure reminiscent of AAV proviral DNA, resulted in AAV Rep-dependent targeted DNA integration into the AAVS1 locus on chromosome 19. Dual hcAd/AAV hybrid vectors may thus be particularly useful to develop safe treatment modalities for diseases such as DMD that rely on efficient transfer and stable expression of large genes.

Adenovirus Protein VI Mediates Membrane Disruption following Capsid Disassembly

In contrast to enveloped viruses, the mechanisms involved in membrane penetration by nonenveloped viruses are not as well understood. In these studies, we determined the relationship between adenovirus (Ad) capsid disassembly and the development of membrane lytic activity. Exposure to low pH or heating induced conformational changes in wild-type Ad but not in temperature-sensitive Ad (ts1) particles that fail to escape the early endosome. Wild-type Ad but not ts1 particles permeabilized model membranes (liposomes) and facilitated the cytosolic delivery of a ribotoxin. Alterations in wild-type Ad capsids were associated with the exposure of a pH-independent membrane lytic factor. Unexpectedly, this factor was identified as protein VI, a 22-kDa cement protein located beneath the peripentonal hexons in the viral capsid. Recombinant protein VI and preprotein VI, but not a deletion mutant lacking an N-terminal amphipathic alpha-helix, possessed membrane lytic activity similar to partially disassembled virions. A new model of Ad entry is proposed based on our present observations of capsid disassembly and membrane penetration.