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Abstract In this report we test the hypothesis that long-term virus-induced alterations in CYP occur from changes initiated by the virus that may not be related to the immune response. Enzyme activity, protein expression and mRNA of CYP3A2, a correlate of human CYP3A4, and CYP2C11, responsive to inflammatory mediators, were assessed 0.25, 1, 4, and 14 days after administration of several different recombinant adenoviruses at a dose of 5.7 × 1012 virus particles (vp)/kg to male Sprague Dawley rats. Wild type adenovirus, containing all viral genes, suppressed CYP3A2 and 2C11 activity by 37% and 39%, respectively within six hours. Levels fell to 67% (CYP3A2) and 79% (CYP2C11) of control by 14 days (p ≤ 0.01). Helper-dependent adenovirus, with all viral genes removed, suppressed CYP3A2 (43%) and CYP2C11 (55%) within six hours. CYP3A2 remained significantly suppressed (47%, 14 days, p ≤ 0.01) while CYP2C11 returned to baseline at this time. CYP3A2 and 2C11 were reduced by 45 and 42% respectively 6 hours after treatment with PEGylated adenovirus, which has a low immunological profile (p ≤ 0.05). CYP3A2 remained suppressed (34%, p ≤ 0.05) for 14 days while CYP2C11 recovered. Inactivated virus suppressed CYP3A2 activity by 25–50% for 14 days (p ≤ 0.05). CYP2C11 was affected similar manner but recovered by day 14. Microarray and in vitro studies suggest that changes in cellular signaling pathways initiated early in virus infection contribute to changes in CYP.

COVID-19 vaccines consisting of mRNA lipid nanoparticles (LNPs) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein antigen protected millions of people from severe disease; however, they must be stored frozen prior to use. The objective of this study was to evaluate the compatibility and stability of mRNA LNPs within a polymer-based film matrix. An optimized formulation of polymer base, glycerol, surfactants, and PEGylated lipid that prevents damage to the LNP due to physical changes during the film-forming process (osmotic stress, surface tension, spatial stress, and water loss) was identified. Surfactants added to LNP stock prior to mixing with other film components contributed to this effect. Formulations prepared at pH ≥ 8.5 extended transfection efficiency beyond 4 weeks at 4°C when combined with known nucleic acid stabilizers. mRNA LNPs were most stable in films when manufactured in an environment of ∼50% relative humidity. The optimized formulation offers 16-week stability at 4°C., Competing Interests: M.A.C. serves as co-founder of and scientific advisor for Jurata Thin Film and holds several patents on film-based stabilization technology and equity in Jurata Thin Film. An international patent application (PCT/US2023/075045) has been filed related to this work., (© 2024 The Author(s).)

Plasmids are essential source material for production of biological drugs, vaccines and vectors for gene therapy. They are commonly formulated as frozen solutions. Considering the cost associated with maintenance of cold chain conditions during storage and transport, there is a significant need for alternative methods for stabilization of plasmids at ambient temperature. The objective of these studies was to identify a film-based formulation that preserved transfection efficiency of plasmids at 25 °C. A model plasmid, pAAVlacZ, was used for these studies. Transfection efficiency and agarose gel electrophoresis were utilized to assess bioactivity and changes in physical conformation of plasmid during storage. An amino acid, capable of sustaining a positive charge while supporting an alkaline environment within the film matrix, preserved transfection efficiency for 9 months at 25 °C. Addition of sugar and a plasticizer to the formulation preserved the plasmid in an amorphous state and improved handling properties of the film. The manner in which excipients were incorporated into bulk formulations and environmental humidity in which films were stored significantly impacted transfection efficiency of plasmid in the rehydrated solution. Taken together, these results suggest that plasmids can be stored for extended periods of time without refrigeration within a film matrix., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This article reports on an American Society of Pharmacology and Therapeutics, Division of Drug Metabolism and Disposition symposium held at Experimental Biology on April 2, 2022, in Philadelphia. As of July 2022, over 500 million people have been infected with SARS-CoV-2 (the virus causing COVID-19) and over 12 billion vaccine doses have been administered. Clinically significant interactions between viral infections and hepatic drug metabolism were first recognized over 40 years ago during a cluster of pediatric theophylline toxicity cases attributed to reduced hepatic drug metabolism amid an influenza B outbreak. Today, a substantive body of research supports that the activated innate immune response generally decreases hepatic cytochrome P450 activity. The interactions extend to drug transporters and other organs and have the potential to impact drug absorption, distribution, metabolism, and excretion (ADME). Based on this knowledge, altered ADME is predicted with SARS-CoV-2 infection or vaccination. The report begins with a clinical case exploring the possibility of SARS-CoV-2 vaccination increasing clozapine levels. This is followed by discussions of how SARS-CoV-2 infection or vaccines alter the metabolism and disposition of complex drugs, such as nanoparticles and biologics and small molecule therapies. The review concludes with a discussion of the effects of viral infections on placental amino acid transport and their potential to impact fetal development. The session improved our understanding of the impact of emerging viral infections and vaccine technologies on drug metabolism and disposition, which will help mitigate drug toxicity and improve drug and vaccine safety and effectiveness. SIGNIFICANCE STATEMENT: Altered pharmacokinetics of small molecule and complex molecule drugs and fetal brain distribution of amino acids following SARS-CoV-2 infection or immunization are possible. The proposed mechanisms involve decreased liver cytochrome P450 metabolism of small molecules, enhanced innate immune system metabolism of complex molecules, and altered placental and fetal blood-brain barrier amino acid transport, respectively. Future research is needed to understand the effects of these interactions on adverse drug responses, drug and vaccine safety, and effectiveness and fetal neurodevelopment., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Adeno-associated virus (AAV) vectors are stored and shipped frozen which poses logistic and economic barriers for global access to these therapeutics. To address this issue, we developed a method to stabilize AAV serotype 9 (AAV9) in a film matrix that can be stored at ambient temperature and administered by systemic injection., Methods: AAV9 expressing the luciferase transgene was mixed with formulations, poured into molds and films dried under aseptic conditions. Films were packaged in individual particle-free bags with foil overlays and stored at various temperatures under controlled humidity. Recovery of AAV9 from films was determined by serial dilution of rehydrated film in media and infection of HeLa RC32 cells. Luciferase expression was compared to that of films rehydrated immediately after drying. Biodistribution of vector was determined by in vivo imaging and quantitative real-time PCR. Residual moisture in films was determined by Karl Fischer titration., Results: AAV9 embedded within a film matrix and stored at 4 °C for 5 months retained 100% of initial titer. High and low viscosity formulations maintained 90 and 85% of initial titer after 6 months at 25 °C respectively. AAV was not detected after 4 months in a Standard Control Formulation under the same conditions. Biodistribution and transgene expression of AAV stored in film at 25 or 4 °C were as robust as vector stored at -80 °C in a Standard Control Formulation., Conclusions: These results suggest that storage of AAV in a film matrix facilitates easy transport of vector to remote sites without compromising in vivo performance., (© 2022. The Author(s).)

Thermostability of vaccines and biologic drugs are key to increasing global access to a variety of life-saving agents. In this report, we characterize interactions between a novel zwitterionic surfactant and adenovirus serotype 5 which allow the virus to remain stable at room temperature in a thin film matrix. Complexity of the adenovirus capsid and the polydispersity of the surfactant required use of a variety of techniques to achieve this goal. The CMC of the surfactant in Tris buffer (pH 6.5) was estimated to be 0.7-1.17 × 10 -4  M by the pyrene 1:3 ratio method. TEM images depict micelle formation around virus capsids. An estimated Kd of the virus-surfactant interaction of 2.25 × 10 -9  M was determined by isothermal titration calorimetry. Associated data suggest that this interaction may be thermodynamically favorable and entropically driven. A competitive saturation study and TEM images indicate that the surfactant also binds to hexon proteins on the virus capsid. Taken together, these data support the working hypothesis that the surfactant is capable of forming micelles in the solid and liquid state and that it forms a protective coating around the virus by binding to hexon proteins on the virus capsid during the film forming process., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: Infectious disease emergencies like the 2013-2016 Ebola epidemic and the 2009 influenza and current SARS-CoV-2 pandemics illustrate that vaccines are now given to diverse populations with preexisting pathologies requiring pharmacological management. Many natural biomolecules (steroid hormones, fatty acids, vitamins) and ~60% of prescribed medications are processed by hepatic cytochrome P450 (CYP) 3A4. The objective of this work was to determine the impact of infection and vaccines on drug metabolism., Methods: The impact of an adenovirus-based vaccine expressing Ebola glycoprotein (AdEBO) and H1N1 and H3N2 influenza viruses on hepatic CYP 3A4 and associated nuclear receptors was evaluated in human hepatocytes (HC-04 cells) and in mice., Results: CYP3A activity was suppressed by 55% in mice 24 h after administration of mouse-adapted H1N1, while ˂10% activity remained in HC-04 cells after infection with H1N1 and H3N2 due to global suppression of cellular translation capacity, indicated by reduction (70%, H1N1, 56%, H3N2) of phosphorylated eukaryotic translation initiation factor 4e (eIF4E). AdEBO suppressed CYP3A activity in vivo (44%) and in vitro (26%) 24 hours after infection., Conclusion: As the clinical evaluation of vaccines for SARS-CoV-2 and other global pathogens rise, studies to evaluate the impact of new vaccines and emerging pathogens on CYP3A4 and other metabolic enzymes are warranted to avoid therapeutic failures that could further compromise the public health during infectious disease emergencies.

A novel, thin-film platform that preserves live viruses, bacteria, antibodies, and enzymes without refrigeration for extended periods of time is described. Studies with recombinant adenovirus in an optimized formulation that supports recovery of live virus through 16 freeze-thaw cycles revealed that production of an amorphous solid with a glass transition above room temperature and nitrogen-hydrogen bonding between virus and film components are critical determinants of stability. Administration of live influenza virus in the optimized film by the sublingual and buccal routes induced antibody-mediated immune responses as good as or better than those achieved by intramuscular injection. This work introduces the possibility of improving global access to a variety of medicines by offering a technology capable of reducing costs of production, distribution, and supply chain maintenance., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Landmark studies describing the effect of microbial infection on the expression and activity of hepatic CYP3A used bacterial lipopolysaccharide as a model antigen. Our efforts to determine whether these findings were translatable to viral infections led us to observations suggesting that engagement of integrin receptors is key in the initiation of processes responsible for changes in hepatic CYP3A4 during infection and inflammation. Studies outlined in this article were designed to evaluate whether engagement of integrins, receptors commonly used by a variety of microbes to enter cellular targets, is vital in the regulation of CYP3A in the presence and absence of virus infection. Mice infected with a recombinant adenovirus (AdlacZ) experienced a 70% reduction in hepatic CYP3A catalytic activity. Infection with a mutant virus with integrin-binding arginine-glycine-aspartic acid (RGD) sequences deleted from the penton base protein of the virus capsid (AdΔRGD) did not alter CYP3A activity. CYP3A mRNA and protein levels in AdlacZ-treated animals were also suppressed, whereas those of mice given AdΔRGD were not significantly different from uninfected control mice. Silencing of the integrinβ-subunit reverted adenovirus-mediated CYP3A4 suppression in vitro. Silencing of theα-subunit did not. Suppression of integrin subunits had a profound effect on nuclear receptors pregnane X receptor and constitutive androstane receptor, whereas retinoid X receptorαwas largely unaffected. To our knowledge, this is the first time that extracellular receptors, like integrins, have been indicated in the regulation of CYP3A. This finding has several implications owing to the important role of integrins in normal physiologic process and in many disease states., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

As the Ebola outbreak in West Africa continues and cases appear in the United States and other countries, the need for long-lasting vaccines to preserve global health is imminent. Here, we evaluate the long-term efficacy of a respiratory and sublingual (SL) adenovirus-based vaccine in non-human primates in two phases. In the first, a single respiratory dose of 1.4×10(9) infectious virus particles (ivp)/kg of Ad-CAGoptZGP induced strong Ebola glycoprotein (GP) specific CD8+ and CD4+ T cell responses and Ebola GP-specific antibodies in systemic and mucosal compartments and was partially (67%) protective from challenge 62 days after immunization. The same dose given by the SL route induced Ebola GP-specific CD8+ T cell responses similar to that of intramuscular (IM) injection, however, the Ebola GP-specific antibody response was low. All primates succumbed to infection. Three primates were then given the vaccine in a formulation that improved the immune response to Ebola in rodents. Three primates were immunized with 2.0×10(10) ivp/kg of vaccine by the SL route. Diverse populations of polyfunctional Ebola GP-specific CD4+ and CD8+ T cells and significant anti-Ebola GP antibodies were present in samples collected 150 days after respiratory immunization. The formulated vaccine was fully protective against challenge 21 weeks after immunization. While diverse populations of Ebola GP-specific CD4+ T cells were produced after SL immunization, antibodies were not neutralizing and the vaccine was unprotective. To our knowledge, this is the first time that durable protection from a single dose respiratory adenovirus-based Ebola vaccine has been demonstrated in primates.

The severity and longevity of the current Ebola outbreak highlight the need for a fast-acting yet long-lasting vaccine for at-risk populations (medical personnel and rural villagers) where repeated prime-boost regimens are not feasible. While recombinant adenovirus (rAd)-based vaccines have conferred full protection against multiple strains of Ebola after a single immunization, their efficacy is impaired by pre-existing immunity (PEI) to adenovirus. To address this important issue, a panel of formulations was evaluated by an in vitro assay for their ability to protect rAd from neutralization. An amphiphilic polymer (F16, FW ∼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50). In vivo performance of rAd in F16 was compared with unformulated virus, virus modified with poly(ethylene) glycol (PEG), and virus incorporated into poly(lactic-co-glycolic) acid (PLGA) polymeric beads. Histochemical analysis of lung tissue revealed that F16 promoted strong levels of transgene expression in naive mice and those that were exposed to adenovirus in the nasal cavity 28 days prior to immunization. Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations. These effects were not compromised by PEI. Data from formulations that provided partial protection from challenge consistently identified specific immunological requirements necessary for protection. This approach may be useful for development of formulations for other vaccine platforms that also employ ubiquitous pathogens as carriers like the influenza virus.

HC-04 cells were evaluated as an in vitro model for mechanistic study of changes in the function of hepatic CYP3A during virus infection. Similar to in vivo observations, infection with a first generation recombinant adenovirus significantly inhibited CYP3A4 catalytic activity in an isoform-specific manner. Virus (MOI 100) significantly reduced expression of the retinoid X receptor (RXR) by 30% 96 hours after infection. Cytoplasmic concentrations of the pregnane X receptor (PXR) were reduced by 50%, whereas the amount of the constitutive androstane receptor (CAR) in the nuclear fraction doubled with respect to uninfected controls. Hepatocyte nuclear factor 4α (HNF-4α) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) were also reduced by ∼70% during infection. Virus suppressed CYP3A4 activity in the presence of the PXR agonist rifampicin and did not affect CYP3A4 activity in the presence of the CAR agonist CITCO [6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime], suggesting that virus-induced modification of PXR may be responsible for observed changes in hepatic CYP3A4. The HC-04 cell line is easy to maintain, and CYP3A4 in these cells was responsive to known inducers and suppressors. Dexamethasone (200 μM) and phenobarbital (500 μM) increased activity by 230 and 124%, whereas ketoconazole (10 μM) and lipopolysaccharide (LPS) (10 μg/ml) reduced activity by 90 and 92%, respectively. This suggests that HC-04 cells can be a valuable tool for mechanistic study of drug metabolism during infection and for routine toxicological screening of novel compounds prior to use in the clinic., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Ebola is a highly virulent pathogen causing severe hemorrhagic fever with a high case fatality rate in humans and non-human primates (NHPs). Although safe and effective vaccines or other medicinal agents to block Ebola infection are currently unavailable, a significant effort has been put forth to identify several promising candidates for the treatment and prevention of Ebola hemorrhagic fever. Among these, recombinant adenovirus-based vectors have been identified as potent vaccine candidates, with some affording both pre- and post-exposure protection from the virus. Recently, Investigational New Drug (IND) applications have been approved by the US Food and Drug Administration (FDA) and phase I clinical trials have been initiated for two small-molecule therapeutics: anti-sense phosphorodiamidate morpholino oligomers (PMOs: AVI-6002, AVI-6003) and lipid nanoparticle/small interfering RNA (LNP/siRNA: TKM-Ebola). These potential alternatives to vector-based vaccines require multiple doses to achieve therapeutic efficacy, which is not ideal with regard to patient compliance and outbreak scenarios. These concerns have fueled a quest for even better vaccination and treatment strategies. Here, we summarize recent advances in vaccines or post-exposure therapeutics for prevention of Ebola hemorrhagic fever. The utility of novel pharmaceutical approaches to refine and overcome barriers associated with the most promising therapeutic platforms are also discussed.

Pre-existing immunity (PEI) to human adenovirus serotype 5 (Ad5) worldwide is the primary limitation to routine clinical use of Ad5-based vectors in immunization platforms. Using systemic and mucosal PEI induction models in rodents (mice and guinea pigs), we assessed the influence of PEI on the type of adaptive immune response elicited by an Ad5-based vaccine for Ebola with respect to immunization route. Splenocytes isolated from vaccinated animals revealed that immunization by the same route in which PEI was induced significantly compromised Ebola Zaire glycoprotein (ZGP)-specific IFN-γ+ CD8+ T cells and ZGP-specific multifunctional CD8+ T cell populations. ZGP-specific IgG1 antibody levels were also significantly reduced and a sharp increase in serum anti-Ad5 neutralizing antibody (NAB) titers were noted following immunization. These immune parameters correlated with poor survival after lethal challenge with rodent-adapted Ebola Zaire virus (ZEBOV). Although the number of IFN-γ+ CD8+ T cells was reduced in animals given the vaccine by a different route from that used for PEI induction, the multifunctional CD8+ T cell response was not compromised. Survival rates in these groups were higher than when PEI was induced by the same route as immunization. These results suggest that antigen-specific multifunctional CD8(+) T cell and Th2 type antibody responses compromised by PEI to Ad5 are required for protection from Ebola. They also illustrate that methods for induction of PEI used in preclinical studies must be carefully evaluated for successful development of novel Ad5-based vaccines.

Marburg and Ebola hemorrhagic fevers have been described as the most virulent viral diseases known to man due to associative lethality rates of up to 90%. Death can occur within days to weeks of exposure and there is currently no licensed vaccine or therapeutic. Recent evidence suggests an important role for antiviral T cells in conferring protection, but little detailed analysis of this response as driven by a protective vaccine has been reported. We developed a synthetic polyvalent-filovirus DNA vaccine against Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SUDV). Preclinical efficacy studies were performed in guinea pigs and mice using rodent-adapted viruses, whereas murine T-cell responses were extensively analyzed using a novel modified assay described herein. Vaccination was highly potent, elicited robust neutralizing antibodies, and completely protected against MARV and ZEBOV challenge. Comprehensive T-cell analysis revealed cytotoxic T lymphocytes (CTLs) of great magnitude, epitopic breadth, and Th1-type marker expression. This model provides an important preclinical tool for studying protective immune correlates that could be applied to existing platforms. Data herein support further evaluation of this enhanced gene-based approach in nonhuman primate studies for in depth analyses of T-cell epitopes in understanding protective efficacy.

Sublingual (SL) delivery, a noninvasive immunization method that bypasses the intestinal tract for direct entry into the circulation, was evaluated with an adenovirus (Ad5)-based vaccine for Ebola. Mice and guinea pigs were immunized via the intramuscular (IM), nasal (IN), oral (PO) and SL routes. SL immunization elicited strong transgene expression in and attracted CD11c(+) antigen presenting cells to the mucosa. A SL dose of 1 × 10⁸ infectious particles induced Ebola Zaire glycoprotein (ZGP)-specific IFN-γ⁺ T cells in spleen, bronchoalveolar lavage, mesenteric lymph nodes and submandibular lymph nodes (SMLN) of naive mice in a manner similar to the same dose given IN. Ex vivo CFSE and in vivo cytotoxic T lymphocyte (CTL) assays confirmed that SL immunization elicits a notable population of effector memory CD8+ T cells and strong CTL responses in spleen and SMLN. SL immunization induced significant ZGP-specific Th1 and Th2 type responses unaffected by pre-existing immunity (PEI) that protected mice and guinea pigs from lethal challenge. SL delivery protected more mice with PEI to Ad5 than IM injection. SL immunization also reduced systemic anti-Ad5 T and B cell responses in naive mice and those with PEI, suggesting that secondary immunizations could be highly effective for both populations.

The immune response to recombinant adenoviruses is the most significant impediment to their clinical use for immunization. We test the hypothesis that specific virus-antibody combinations dictate the type of immune response generated against the adenovirus and its transgene cassette under certain physiological conditions while minimizing vector-induced toxicity. In vitro and in vivo assays were used to characterize the transduction efficiency, the T and B cell responses to the encoded transgene, and the toxicity of 1 × 10(11) adenovirus particles mixed with different concentrations of neutralizing antibodies. Complexes formed at concentrations of 500 to 0.05 times the 50% neutralizing dose (ND(50)) elicited strong virus- and transgene-specific T cell responses. The 0.05-ND(50) formulation elicited measurable anti-transgene antibodies that were similar to those of virus alone (P = 0.07). This preparation also elicited very strong transgene-specific memory T cell responses (28.6 ± 5.2% proliferation versus 7.7 ± 1.4% for virus alone). Preexisting immunity significantly reduced all responses elicited by these formulations. Although lower concentrations (0.005 and 0.0005 ND(50)) of antibody did not improve cellular and humoral responses in naïve animals, they did promote strong cellular (0.005 ND(50)) and humoral (0.0005 ND(50)) responses in mice with preexisting immunity. Some virus-antibody complexes may improve the potency of adenovirus-based vaccines in naïve individuals, while others can sway the immune response in those with preexisting immunity. Additional studies with these and other virus-antibody ratios may be useful to predict and model the type of immune responses generated against a transgene in those with different levels of exposure to adenovirus.

Clinically relevant doses of helper-dependent adenoviruses (HDAds) provoke the host response against capsid proteins in primates and rodents. To determine if PEGylation truly affects this, baboons and mice were given either HDAd or PEG-HDAd expressing beta-galactosidase at 5 × 10¹¹ or 3 × 10¹² virus particles per kilogram (vp/kg) by iv infusion. Serum cytokines and blood chemistries were assessed for 96 h. PEG-HDAd reduced IL-6 6-fold in mice and 3-fold in the primate. This vector reduced IL-12 by 50% in both animal models. PEGylation reduced serum transaminases by approximately 50% at each dose in the primate and the mouse. PEGylation did not alter hepatic transduction efficiency in the mouse but did reduce transduction efficiency in the liver and the spleen of primates. Unmodified and PEGylated virus suppressed hepatic CYP3A activity in both animal models. PEGylation doubled the half-life (t(½)) of the virus in the mouse and cut plasma clearance (CL) in half without affecting the half-life in primates. These results suggest that there are notable species-specific differences in the biodistribution of and response to PEG-modified vectors which may be linked to differences in binding properties to coagulation factors, receptor density and tissue architecture in the liver.

Ebolavirus is a highly infectious pathogen with a case fatality rate as high as 90%. Currently there is a lack of licensed Ebolavirus vaccines as well as pre- and post-exposure treatments. Recent increases in the frequency of natural human Ebolavirus infections and its potential use as a bioterrorism agent makes vaccine development a priority for many nations. Significant progress has been made in understanding the pathogenesis of Ebolavirus infection and several promising vaccine candidates were shown to be successful in protecting NHPs against lethal infection. These include replication-deficient adenovirus vectors, replication-competent VSV, HPIV-3 vectors and virus-like particle preparations. Recent advances in the generation of effective post-exposure immunization strategies highlight the possibility of developing a single dose vaccine that will confer full protection in humans following Ebolavirus exposure. Post-exposure protection is particularly important in outbreak and biodefense settings, as well as clinical and laboratory settings in the case of accidental exposure.

A nasal adenovirus-based vaccine is under development. To determine if aggregation occurs during vaccination, infectious titer (limiting dilution) and capsid integrity (dynamic light scattering) were assessed after extrusion of a model vector from two intranasal delivery devices. Preparations of 2.5x10(12) and 1.25x10(11) virus particles (vp)/ml were studied. Virus aggregated ( approximately 10%) in the multi-dose vessel. Virus titer dropped by one log. Virus in the unit-dose device aggregated ( approximately 1%). Titer remained unchanged. Aggregation was concentration dependent. Formulations prevented aggregation during actuation, freeze-thaw and long-term storage. The device, formulation and dose may significantly influence aggregation and potency of any nasal adenovirus 5-based vaccine., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Covalent modification with polyethylene glycol (PEG), a non-toxic polymer used in food, cosmetic and pharmaceutical preparations for over 60 years, can profoundly influence the pharmacokinetic, pharmacologic and toxciologic profile of protein and peptide-based therapeutics. This review summarizes the history of PEGylation and PEG chemistry and highlights the value of this technology in the context of the design and development of recombinant viruses for gene transfer, vaccination and diagnostic purposes. Specific emphasis is placed on the application of this technology to the adenovirus, the most potent viral vector with the most highly characterized toxicity profile to date, in several animal models.

Virus infections are on the rise. Although the first description of CYP expression during virus infection was recorded 50 years ago, mechanistic studies of this phenomenon only began to appear in the last decade due to breakthroughs in molecular biology, genomic and transgenic technology. This review describes the relationship(s) among CYP-mediated drug metabolism, virus infection and the immune response and evaluates in vitro and in vivo models for mechanistic studies. The first studies that assessed CYP expression during infection focused on inflammatory mediators and the innate immune response at early time points. Recent studies assessing virus infection and its effect on hepatic CYP expression noted more long-term effects. An obvious approach toward understanding how viruses affect hepatic CYP3A expression and function would be to assess key regulators of CYP during infection. Improvements in techniques to identify post-translational modifications of CYP and systems that focus on virus-receptor interactions which allow subtraction and addition of immunological and regulatory elements that drive CYP will demonstrate that long-term changes in drug metabolism start from the time the virus enters the circulation, are reinforced by virus binding to cellular targets and further solidified by changes in cellular processes long after the virus is cleared.

Modern cancer therapy combines recombinant viruses with traditional chemotherapeutic agents that are metabolized by hepatic cytochrome P450 3A4 (CYP3A4). A single dose of recombinant adenovirus (Ad) expressing beta-galactosidase (AdlacZ) significantly alters CYP3A2, the correlate of CYP3A4, in rats for 14 days. Recombinant adenovirus expressing human p53 (Adp53) also suppresses CYP3A2. Plasma clearance of docetaxel (DTX) in animals given AdlacZ (3.38+/-0.22 l h(-1) kg(-1)) was significantly lower than that of those given DTX alone (7.35+/-1.22 l h(-1) kg(-1), P

Background: The Ebola virus is transmitted by direct contact with bodily fluids of infected individuals, eliciting death rates as high as 90% among infected humans. Currently, replication defective adenovirus-based Ebola vaccine is being studied in a phase I clinical trial. Another Ebola vaccine, based on an attenuated vesicular stomatitis virus has shown efficacy in post-exposure treatment of nonhuman primates to Ebola infection. In this report, we modified the common recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZEBOV glycoprotein sequence from a CMV promoter (Ad-CMVZGP). The immune response elicited by this improved expression cassette vector (Ad-CAGoptZGP) and its ability to afford protection against lethal ZEBOV challenge in mice was compared to the standard Ad-CMVZGP vector., Methodology/principal Findings: Ad-CMVZGP was previously shown to protect mice, guinea pigs and nonhuman primates from an otherwise lethal challenge of Zaire ebolavirus. The antigenic expression cassette of this vector was improved through codon optimization, inclusion of a consensus Kozak sequence and reconfiguration of a CAG promoter (Ad-CAGoptZGP). Expression of GP from Ad-CAGoptZGP was substantially higher than from Ad-CMVZGP. Ad-CAGoptZGP significantly improved T and B cell responses at doses 10 to 100-fold lower than that needed with Ad-CMVZGP. Additionally, Ad-CAGoptZGP afforded full protections in mice against lethal challenge at a dose 100 times lower than the dose required for Ad-CMVZGP. Finally, Ad-CAGoptZGP induced full protection to mice when given 30 minutes post-challenge., Conclusions/significance: We describe an improved adenovirus-based Ebola vaccine capable of affording post-exposure protection against lethal challenge in mice. The molecular modifications of the new improved vaccine also translated in the induction of significantly enhanced immune responses and complete protection at a dose 100 times lower than with the previous generation adenovirus-based Ebola vaccine. Understanding and improving the molecular components of adenovirus-based vaccines can produce potent, optimized product, useful for vaccination and post-exposure therapy.

Introduction: Doses of 2 x 10(12) virus particles/kilogram (vp/kg) and higher of recombinant human adenovirus serotype 5 (HAdV-5) given via the tail vein induce significant toxicity and mortality in the rat. This was not observed when doses of 5.7 x 10(12) vp/kg were given through a surgically implanted jugular catheter. Here we assess how the manner by which HAdV-5 is introduced into the systemic circulation affects biodistribution, transgene expression, toxicity and mortality 0.25, 1, and 4 days after treatment in the rat. Animals were given 5.7 x 10(12) vp/kg of HAdV-5 expressing beta-galactosidase or saline through a jugular catheter or by direct tail vein injection., Results: All animals survived after jugular vein dosing. Tail vein injection of HAdV-5 increased the mortality rate to 42% (p< or =0.01). All deaths occurred within 4 h. Animals dosed through the jugular vein had significantly higher levels of transgene expression in the liver and spleen and significantly more viral genomes in these tissues and kidney and lung within the first 24 h of viral infection compared to those dosed by tail vein injection (p< or =0.01). There was no significant difference between the groups thereafter. Samples from animals that died contained even higher levels of viral genomes and serum transaminases were elevated on average by a factor of 4 at the time of death. There was no significant difference between the two dosing methods with respect to changes in hepatic cytochrome P450 expression and activity throughout the study., Conclusion: These findings suggest that the method of systemic administration should be carefully considered when assessing toxicity data and other parameters at early time points after virus administration in the rat and possibly other animal models.

In this report we test the hypothesis that long-term virus-induced alterations in CYP occur from changes initiated by the virus that may not be related to the immune response. Enzyme activity, protein expression and mRNA of CYP3A2, a correlate of human CYP3A4, and CYP2C11, responsive to inflammatory mediators, were assessed 0.25, 1, 4, and 14 days after administration of several different recombinant adenoviruses at a dose of 5.7 x 1012 virus particles (vp)/kg to male Sprague Dawley rats. Wild type adenovirus, containing all viral genes, suppressed CYP3A2 and 2C11 activity by 37% and 39%, respectively within six hours. Levels fell to 67% (CYP3A2) and 79% (CYP2C11) of control by 14 days (p

Inactivated viruses are important tools for vaccine development and gene transfer. 8-Methoxypsoralen (8-MOP) and long-wavelength ultraviolet irradiation (LWUVI) inactivates many viruses. Toxicity limits its use in animals and humans. Toxicological and photosensitizing properties of riboflavin make it suitable for virus inactivation in preparations for biological use. Viruses expressing beta-galactosidase were mixed with either 8-MOP (1.5mM) or riboflavin (50 microM) and exposed to LWUVI (365 nm) for 2 h. Virus activity was determined by limiting dilution. The half-life of the adenovirus preparation treated with 8-MOP was 8.28 ns(-1) and 36.5 ns(-1) after treatment with riboflavin. Despite the difference in half-life, both preparations were completely inactivated within 45 min. In contrast, the half-lives for adeno-associated virus (AAV) preparations were similar (63 ns(-1) 8-MOP vs. 67 ns(-1) riboflavin). Each AAV preparation was fully inactivated within 90 min. The half-life of lentivirus was 193.4 ns(-1) after treatment with 8-MOP and 208 ns(-1) after exposure to riboflavin. Virus treated with riboflavin was inactivated within 20 min. Virus exposed to 8-MOP was inactivated in 90 min. DNA and RNA viruses can be inactivated by riboflavin and LWUVI and used in physiological systems sensitive to other photochemicals.

Pre-existing immunity to human adenovirus serotype 5 (Ad5) is common in the general population. Bypassing pre-existing immunity could maximize Ad5 vaccine efficacy. Vaccination by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route with Ad5 expressing Ebola Zaire glycoprotein (Ad5-ZGP) fully protected naïve mice against lethal challenge with Ebola. In the presence of pre-existing immunity, only mice vaccinated I.N. survived. The frequency of IFN-gamma+ CD8+ T cells was reduced by 80% and by 15% in animals vaccinated by the I.M. and P.O. routes respectively. Neutralizing antibodies could not be detected in serum from either treatment group. Pre-existing immunity did not compromise the frequency of IFN-gamma+ CD8+ T cells (3.9+/-1% naïve vs. 3.6+/-1% pre-existing immunity, PEI) nor anti-Ebola neutralizing antibody (NAB, 40+/-10 reciprocal dilution, both groups). The number of INF-gamma+ CD8+ cells detected in bronchioalveolar lavage fluid (BAL) after I.N. immunization was not compromised by pre-existing immunity to Ad5 (146+/-14, naïve vs. 120+/-16 SFC/million MNCs, PEI). However, pre-existing immunity reduced NAB levels in BAL by approximately 25% in this group. To improve the immune response after oral vaccination, the Ad5-based vaccine was PEGylated. Mice given the modified vaccine did not survive challenge and had reduced levels of IFN-gamma+ CD8+ T cells 10 days after administration (0.3+/-0.3% PEG vs. 1.7+/-0.5% unmodified). PEGylation did increase NAB levels 2-fold. These results provide some insight about the degree of T and B cell mediated immunity necessary for protection against Ebola virus and suggest that modification of the virus capsid can influence the type of immune response elicited by an Ad5-based vaccine.

Recombinant adenovirus (Ad) significantly alters hepatic cytochrome P450 (CYP). Because changes in renal function can alter hepatic CYP, the effect of Ad on renal CYPs 4A1, 4A2, 4F1, and 2E1 was evaluated. Male Sprague-Dawley rats were given one of six intravenous doses (5.7x10(6)-5.7x10(12) viral particles/kg [VP/kg]) of Ad expressing beta-galactosidase or saline. CYP protein, activity, gene expression, and serum creatinine (SCr) were evaluated 0.25, 1, 4, and 14 days later. Doses of 5.7x10(11) and 5.7x10(12) VP/kg increased CYP4A protein within 24 hr by 35 and 48%, respectively (p<0.05). A similar trend was observed on day 4. CYP4A1 mRNA doubled 6 hr after doses of 5.7x10(10)-10(12) VP/kg (p<0.01). Similar effects were observed 1 day after each dose tested. CYP4A2 gene expression was 20% above control 1 day after treatment with 5.7x10(10)-10(12) VP/kg and remained high through day 14. CYP4F1 expression was unaffected by all doses (p=0.08). CYP2E1 activity and gene expression were significantly suppressed 24 hr after administration of all doses and began to normalize by day 14 (p<0.01). SCr was significantly reduced (approximately 50%) throughout the study for doses at and below 5.7x10(11) VP/kg. SCr was increased by a factor of 3 by 5.7x10(12) VP/kg and glomerular filtration was significantly reduced (p<0.01). This suggests that changes in renal CYP and corresponding arachidonic acid metabolites may play a role in the documented toxicity associated with the systemic administration of recombinant Ad.

Background: Systemic administration of a first-generation adenovirus expressing E. coli beta-galactosidase (AdlacZ) alters expression and function of two hepatic drug-metabolizing enzymes, cytochrome P450 (CYP) 3A2 and 2C11, for 14 days. The objective of these studies was to determine how the transgene cassette influences CYP expression and function., Methods: Sprague-Dawley rats were given 5.7 x 10(12) viral particles (vp)/kg of either: AdlacZ, Ad expressing murine erythropoietin (Epo), Ad without a transgene (Null), or phosphate-buffered saline (Vehicle). Hepatic CYP protein expression, activity, mRNA and alanine aminotransferase (ALT) levels were analyzed 0.25, 1, 4, and 14 days following a single intravenous injection., Results: Administration of Epo did not alter CYP3A2 activity, but induced RNA levels by a factor of 2 at 4 and 14 days (P< or =0.01). This vector suppressed CYP2C11 activity levels by 45% at 1 day (P< or =0.05) and RNA levels throughout the study period (P< or =0.05). The Null vector suppressed CYP3A2 activity by 36, 63, 34, and 45% at 0.25, 1, 4 and 14 days, respectively (P< or =0.05). CYP2C11 activity was suppressed 1 day after administration (41%) and RNA levels were suppressed at 6 h (53%) and 1 day (36%, P< or =0.05). In contrast, AdlacZ suppressed both CYP3A2 and 2C11 at all time points., Conclusions: The immunogenic and biological nature of the transgene cassette can influence changes in CYP3A2, but not the 2C11 isoform. The shift in transcription and translation of protein for maintenance of physiologic homeostasis to production of viral proteins and transgene product and their associated toxicity during viral infection may explain our observations., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Adeno-associated virus (AAV), capable of producing significant, long-term transgene expression, is one of the least toxic vectors employed in pre-clinical and clinical studies of gene transfer. One limitation is generation of neutralizing antibodies against viral capsids, blocking gene expression after readministration. AAV2 capsids were modified with poly(ethylene) glycols (PEGs) activated by cyanuric chloride (CCPEG), succinimidyl succinate (SSPEG) and tresyl chloride (TMPEG). SSPEG and TMPEG conjugation did not compromise gene transfer to the liver and muscle and improved gene expression in the lung 5 fold. Transduction efficiency of CCPEG-AAV was impeded in all tissues by aggregation. TMPEG afforded the best protection from neutralization in vitro and in vivo. Gene expression in mice immunized against unmodified AAV was reduced by a factor of 10 from that of naïve animals after intramuscular rechallenge with PEGylated AAV but was not significantly different from naïve mice after intravenous readministration (p=0.08). Gene expression was markedly reduced in muscle after two doses of PEGylated AAV. In contrast, mice given two intravenous doses of TMPEG-AAV had significantly higher transgene levels than naïve animals 14 days after rechallenge (p=0.001). This technology could promote successful readministration of vector in the clinic and marked expression in patients with anti-AAV antibodies.

Transgene expression from helper-dependent adenoviral (HD-Ad) vectors is effective and long lasting, but not permanent. Their use is also limited by the host response against capsid proteins that precludes successful gene expression upon readministration. In this report, we test the hypothesis that PEGylation of HD-Ad reduces its toxicity and promotes transgene expression upon readministration. PEGylation did not compromise transduction efficiency in vitro and in vivo and reduced peak serum IL-6 levels two-fold. IL-12 and TNF-alpha levels were reduced three- and seven-fold, respectively. Thrombocytopenia was not detected in mice treated with the PEGylated vector. Serum transaminases were not significantly elevated in mice treated with either vector. Mice immunized with 1 x 10(11) particles of unmodified HD-Ad expressing human alpha-1 antitrypsin (hA1AT) were rechallenged 28 days later with 8 x 10(10) particles of unmodified or PEG-conjugated vector expressing beta-galactosidase. Trace levels of beta-galactosidase (52.23+/-19.2 pg/mg protein) were detected in liver homogenates of mice that received two doses of unmodified HD-Ad. Mice rechallenged with PEGylated HD-Ad produced significant levels of beta-galactosidase (5.1+/-0.4 x 10(5) pg/mg protein, P=0.0001). This suggests that PEGylation of HD-Ad vectors may be appropriate for their safe and efficient use in the clinic.

Recombinant adenovirus (Ad) serotype 5 is a vector commonly used for gene delivery. Although this vector has a natural tropism for the liver, there is a limited understanding of how Ad administration affects one of the primary hepatic processes, drug metabolism. The effects of systemic administration of a model recombinant adenoviral vector on two hepatic cytochrome P450 (P450) enzymes, CYP3A2 and 2C11, were investigated. Sprague-Dawley rats were treated with one of six vector doses, ranging from 5.7 x 10(6) to 5.7 x 10(12) virus particles (vp)/kg. Hepatic P450 protein expression, catalytic activity, and mRNA levels were measured over 14 days. Ad administration (5.7 x 10(10)-5.7 x 10(12) vp/kg) reduced CYP3A2 over the duration of the study. Six hours after administration of 5.7 x 10(12) vp/kg, CYP3A2 activity and mRNA levels were suppressed by 45 and 65%, respectively (P < or = 0.01). This continued throughout the study with levels dropping to 36 and 45% of controls by 14 days, respectively (P < or = 0.01). A similar trend was detected for CYP2C11 within this dosing range. Administration of 5.7 x 10(6), 5.7 x 10(8), and 5.7 x 10(9) vp/kg of Ad significantly increased both CYP2C11 protein expression by 86, 71, and 107% and activity 110, 118, and 53%, respectively, above those of animals treated with saline (P < or = 0.01). These results clearly indicate that a single dose of adenovirus significantly alters key drug metabolizing enzymes for an extended period of time and should be investigated further in the context of the design and implementation of clinical trial protocols.

One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing Escherichia coli beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo.

Purpose: Adenoviruses are being developed for diseases of the gastrointestinal tract. Several in vitro assays were used to predict stability of PEGylated adenovirus along the GI tract and determine in vivo gene transfer after oral administration., Methods: Recombinant adenovirus was modified with monomethoxypoly(ethylene) glycols activated by cyanuric chloride, succinimidyl succinate, and tresyl chloride. Transduction efficiency was assessed on Caco-2 cells. In vitro stability of viruses in simulated gastric fluid, pancreatic fluid, and bile was assessed by serial dilution on 293 cells. Transduction efficiency in vivo was determined by oral administration of 1 x 10(12) particles of unmodified or PEGylated virus to fasted Sprague-Dawley rats., Results: Titers of unmodified virus declined to undetectable levels after 40 min in simulated gastric fluid while the infectious titer of the modified vectors did not change for 3 h. Similar results were seen with simulated pancreatic fluid. PEGylation also enhanced adenoviral transduction efficiency in Caco-2 cells by a factor of 20. PEGylation enhanced adenovirus transduction efficiency 10- to 40-fold in vivo in intestinal segments that do not express significant amounts of adenovirus receptors (jejunum, colon) with transgene expression located in the crypt regions., Conclusions: PEGylated adenoviruses are suitable gene delivery vehicles for oral administration.

Systemic administration of adenoviral vectors leads to activation of innate and antigen-specific immunity. In an attempt to diminish T and B cell-specific immune responses to E1-deleted adenoviral vectors, capsid proteins were modified with various activated monomethoxypolyethylene glycols (MPEGs). The impact of this modification was studied in a murine model of liver-directed gene transfer in which an E1-deleted adenovirus expressing the lacZ gene was given intravenously. The efficiency of vector transduction of hepatocytes in vivo was not compromised by any of the polymer chemistries. PEGylation of the virus, however, diminished the activation of cytotoxic T lymphocytes and helper T cells of the type 1 subset (Th1 cells) against native viral antigens; neutralizing antibodies to native virus were also diminished. PEGylation prolonged transgene expression and allowed partial readministration with native virus or with a virus PEGylated with a heterologous chemical moiety. Apparently, modification of the capsid leads to a shift in antigenic epitopes because vector readministration was not possible when the immunizing vector had been modified by the same PEGylation chemistry used to modify the second vector. In light of these results, the concept of improving the performance of adenoviral vectors through modification of the capsid with PEG shows promise.

Despite remarkable progress in the development of both viral and non-viral gene delivery vectors for cystic fibrosis therapy, low efficiency of gene transfer to the airway epithelium is a major obstacle to clinical application. Here we develop formulations that enhance cellular absorption of adenoviral vectors. We selected excipients from a panel of pharmaceutically acceptable com-pounds known to enhance drug absorption. Transduction efficiency of the virus in the presence of each ingredient was assessed in vitro and in vivo. Mannitol and chitosan substantially enhanced transduction efficiency in vitro and augmented expression in vivo by 4 and 8 log units, respectively. The most successful formulation (a blend of sucrose, mannitol, and Pluronic F68) transduced 100% of an A549 cell population in vitro and produced areas of intense gene expression in both large and small airways in vivo with minimal toxicity. Dose response studies also indicate that when placed in this formulation, the viral dose can be lowered by 1/2 log while maintaining superior levels of transgene expression. This formulation also enhanced the physical stability of the virus. No significant loss in titer was detected from a lyophilized formulation after storage at 25 degrees C for 30 days.

Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy.

PEGylation is the covalent attachment of activated monomethoxy poly(ethylene) glycols (MPEGs) to free lysine groups of therapeutic proteins. This technology has enhanced the physical stability of proteins and ablated humoral immune responses generated against them. In this study, adenoviral vectors were modified with MPEGs activated by cyanuric chloride, succinimidyl succinate, and tresyl chloride. Under proper buffering conditions, reactions were complete within 2 hr. Transduction efficiency of PEGylated adenoviruses was not compromised by neutralizing antibodies to native adenovirus in vitro. These preparations retained titers that were significantly greater than those of the unconjugated virus after storage at 42, 25, 4, and -20 degrees C. Stability profiles of PEGylated preparations at -20 degrees C suggest that glycerol could be eliminated from formulations without significant loss of viral titer. PEGylated adenoviruses produced a two- to threefold increase in transduction in the lung when administered by intratracheal injection and a fivefold increase in transduction in the liver when administered intravenously.

Purpose: In general, the intestinal epithelium is quite refractory to viral and non-viral methods of gene transfer. In this report, various cyclodextrin formulations were tested for their ability to enhance adenoviral transduction efficiency in two models of the intestinal epithelium: differentiated Caco-2 cells and rat jejunum., Methods: Transduction efficiency of replication-deficient adenovirus type 5 vectors encoded with either the E. coli beta-galactosidase or the jellyfish green fluorescent protein gene was assessed by X-gal staining or visualization of fluorescence 48 hours after infection. In vivo experiments were performed using an intestinal loop ligation technique., Results: Several formulations of neutral and positively charged beta cyclodextrins significantly enhanced adenoviral-mediated gene transfer in the selected models. The cyclodextrin formulations studied increased adenoviral transduction in the intestine by enhancing both viral binding and internalization. Viral binding was significantly increased on cell membranes treated with positively charged cyclodextrins, as seen with confocal microscopy and rhodamine-labeled virus. Permeability studies and TEER readings revealed that the most successful formulations gently disrupt cell membranes. This enhances internalization of viral particles and results in increased levels of gene expression., Conclusions: These formulations can be of value in gene transfer to cells and tissues in which adenoviral infection is limited due to a lack of fiber and alpha(v) integrin receptors. They are simple to prepare and do not affect the ability of the virus to transduce target cells.

Recently, replication-deficient adenoviruses have received increasing attention as vector for gene delivery and as potential vaccine carriers. With the increased use of the vector in vivo and in clinical trails, the demand for a safe, rapid, and cost effective purification process has been heightened. In this report, a simple and efficient method for the purification of large quantities of live adenoviral vectors was developed. The process involved the replacement of cesium chloride (CsCl) gradients with sucrose gradients. Ultracentrifugation times were reduced and the desalting step eliminated, decreasing total preparation time by 15 hr. A 20-80% linear sucrose gradient provided optimal recovery of infectious viral particles and positioning of the viral band in the gradient. Purification with this gradient system produced a preparation containing 1.39 x 10(14) lac-forming units (lfu)/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the process also removed all associated cellular proteins from the preparation. Studies have shown that direct lyophilization of the vector in sucrose after purification produces a product containing 1.4 x 10(12) lfu/ml. Minimal degradation was seen in the lyophilized preparation. A viral concentration of 6 x 10(11) lfu/ml was detected in the product after 150 days in storage at -20 degrees C. This approach will not only simplify the preparation of adenoviral vectors for in vivo studies and clinical trials, but will facilitate production of stable adenoviral formulations for oral gene delivery.

This report identifies formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and cooling rates. Adenovirus type 5 containing the lacZ marker gene was suspended in combinations of trehalose, sorbitol, sucrose, mannitol, glycine, CaCl2, and gelatin. X-gal stains of 293 cells were used to determine the lac-forming units (lfu)/ml of each preparation before and after treatments. Phosphate-buffered solutions (except those containing sucrose or trehalose) demonstrated a drop of 3 pH units upon freezing regardless of cryoprotectant used. Tris-buffered solutions demonstrated a variation in pH which was dependent upon chosen cryoprotectant, with 1 M trehalose exhibiting no change and a 5% mannitol/10 mM CaCl2 combination showing a 3-unit drop in pH. 4-[2-Hydroxyethyl]-1-piperazine ethanesulfonic acid (HEPES)-buffered solutions showed little change in initial pH when frozen regardless of cryoprotectant chosen. In solution, adenovirus was not affected by incubation for 24 hr in buffers ranging from pH 4 to 8. However, when the solutions were frozen, the number of remaining infectious virions was dependent upon the final pH of the suspending medium. Cryoprotectant solutions that significantly maintained viral stability during a single freeze--thaw cycle were 0.5 M sucrose, 0.5 M trehalose, and 10% sorbitol/0.4% gelatin. Long-term stability studies were performed at 4 degrees C with lyophilized sorbital/gelatin and sucrose preparations. Both formulations provided adequate stability for the adenovirus, with 2.6 and 5.6 x 10(11) lfu/ml detected 150 days after drying, respectively.

Adenoviral vectors are being developed for oral delivery of therapeutic genes to the intestine. Initial studies in the rat using mucolytics and direct application of adenovirus encoded with the interleukin-1 receptor antagonist gene to the jejunum produced limited gene expression. The goal of this study was to determine the role of integrins in adenovirus-mediated gene delivery to the intestinal epithelium. Integrins are involved in cellular differentiation and tight junction formation and mediate adenoviral internalization. Results from Caco-2 and IEC-18 cells suggest that, as enterocytes differentiate, cell-surface integrin expression decreases. Pretreatment of Caco-2 cells with RGD peptides reduced adenoviral transduction efficiency by 80% in undifferentiated cells and 20% in differentiated cells. Both differentiated and undifferentiated IEC-18 cells showed a 70% drop in transduction when pretreated with the peptide. Infection inhibition studies with monoclonal antibodies further suggest that alpha(v)beta3 and alpha6beta1 integrins play significant roles in adenoviral internalization in the intestine. Expression of integrins in cell culture models of the intestine correlated with in vivo expression in intestinal segments. These results indicate that the ileum is a prime target for efficient adenovirus-mediated gene transfer in the rat. To enhance transduction in differentiated enterocytes (probable targets for oral gene delivery), Caco-2 cells were treated with interleukin-1beta (a cytokine known to increase integrin expression) prior to administration of the virus. Transduction efficiency increased four-fold.

Purpose: Adenoviral (Ad) vectors have been used as efficient tools for gene therapy in various tissues, whereas in some differentiated epithelium transduction efficiency is almost abolished., Methods: Caco-2 cell monolayers were chosen as an in vitro model for the differentiated intestinal epithelium. Fluorescence-labeled adenoviral particles were used for binding studies to cell surfaces. Internalization receptors for adenoviral uptake were detected by a fluorescence-labeled vitronectin antibody. Gene expression was studied by using the beta-galactosidase reporter gene. All experiments were done on undifferentiated and differentiated Caco-2 cells. Furthermore, adenoviral particles were allowed to bind to differentiated Caco-2 monolayers followed by a trypsinization step that disintegrates the monolayers and result in a cell suspension. Gene expression was tested after reseeding the cells into dishes., Results: The results from adenoviral binding studies, vitronectin immunofluorescence detection and gene expression are in good agreement and indicate that virion binding as well as the expression of internalization receptors almost disappear in fully differentiated cells. Nonetheless, adenoviral binding to differentiated monolayers seems to be sufficient to cause up to 53% gene expression, but only if internalization of the vector can be induced by disintegrating the monolayers and releasing free vitronectin receptors., Conclusions: These findings indicate that gene transfer to the intestinal epithelium utilizing adenoviral vectors is poor and ineffective, because of the lack of sufficient internalization receptors. If these receptors can be exposed in differentiated epithelium, transduction can be made more efficient. Alternatively, a viral vector must be developed whose uptake mechanism is independent of integrin receptor expression like the enteral virus Ad40, or Ad5 could be conjugated to ligands that trigger viral internalization by receptor-mediated endocytosis.