Maria A. Croyle

“Stealth” Adenoviruses Blunt Cell-Mediated and Humoral Immune Responses against the Virus and Allow for Significant Gene Expression upon Readministration in the Lung

ABSTRACT 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 of E1-deleted adenovirus vectors allows significant gene expression on readministration to liver.

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.

Development of a rapid method for the PEGylation of adenoviruses with enhanced transduction and improved stability under harsh storage conditions

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.

Nasal Delivery of an Adenovirus-Based Vaccine Bypasses Pre-Existing Immunity to the Vaccine Carrier and Improves the Immune Response in Mice

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-γ+ 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-γ+ 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-γ+ 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 ∼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-γ+ 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.

PEGylation of a Vesicular Stomatitis Virus G Pseudotyped Lentivirus Vector Prevents Inactivation in Serum

ABSTRACT 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.

PEGylated helper-dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile

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-α 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 × 1011 particles of unmodified HD-Ad expressing human alpha-1 antitrypsin (hA1AT) were rechallenged 28 days later with 8 × 1010 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 × 105 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.

Enhanced Protection against Ebola Virus Mediated by an Improved Adenovirus-Based Vaccine

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.

Emerging targets and novel approaches to ebola virus prophylaxis and treatment

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.

PEGylated adenoviruses for gene delivery to the intestinal epithelium by the oral route.

AbstractPurpose. 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 × 1012 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.

Mucosal Delivery of Adenovirus-Based Vaccine Protects against Ebola Virus Infection in Mice

BACKGROUND Mucosal vaccination can offer several advantages over conventional intramuscular immunization to protect against Ebola virus (EBOV) infection, such as immune protection at sites of viral entry into susceptible individuals, and can be administered using needle-free devices. METHODS The present study evaluated oral and nasal vaccination of mice with human adenovirus serotype 5 (Ad) expressing the Zaire ebolavirus glycoprotein (Ad-ZGP) in terms of their protection against and underlying immune responses to EBOV. RESULTS Similar to intramuscular administration, oral or nasal vaccination of mice with Ad-ZGP fully protected the mice against a lethal challenge with mouse-adapted EBOV. Both T and B cell responses developed in mice receiving oral or nasal vaccination in different body compartments, indicating qualitative improvement of the immune response after mucosal immunization, compared with intramuscular vaccination. CONCLUSIONS Overall, the breadth of the immune response noted after nasal or oral immunization, including stimulation of CD8+ T cells or effector memory T cells from the gastrointestinal tract or the lungs, was superior to that noted after intramuscular administration of the vaccine. The present study showed that adenovirus-based vaccine is effective against EBOV infection in mice after oral and nasal immunization.