AAV-vectored expression of monospecific or bispecific monoclonal antibodies protects mice from lethal Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa poses a significant threat to immunocompromised individuals and those with cystic fibrosis. Treatment relies on antibiotics, but persistent infections occur due to intrinsic and acquired resistance of P. aeruginosa towards multiple classes of antibiotics. To date, there are no licensed vaccines for this pathogen, prompting the urgent need for novel treatment approaches to combat P. aeruginosa infection and persistence. Here we validated AAV vectored immunoprophylaxis as a strategy to generate long-term plasma and mucosal expression of highly protective monoclonal antibodies (mAbs) targeting the exopolysaccharide Psl (Cam-003) and the PcrV (V2L2MD) component of the type-III secretion system injectosome either as single mAbs or together as a bispecific mAb (MEDI3902) in a mouse model. When administered intramuscularly, AAV-αPcrV, AAV-αPsl, and AAV-MEDI3902 significantly protected mice challenged intranasally with a lethal dose of P. aeruginosa strains PAO1 and PA14 and reduced bacterial burden and dissemination to other organs. While all AAV-mAbs provided protection, AAV-αPcrV and AAV-MEDI3902 provided 100% and 87.5% protection from a lethal challenge with 4.47 × 107 CFU PAO1 and 87.5% and 75% protection from a lethal challenge with 3 × 107 CFU PA14, respectively. Serum concentrations of MEDI3902 were ~10× lower than that of αPcrV, but mice treated with this vector showed a greater reduction in bacterial dissemination to the liver, lung, spleen, and blood compared to other AAV-mAbs. These results support further investigation into the use of AAV vectored immunoprophylaxis to prevent and treat P. aeruginosa infections and other bacterial pathogens of public health concern for which current treatment strategies are limited.

Analysis of the impact of pluronic acid on the thermal stability and infectivity of AAV6.2FF.

BACKGROUND: The advancement of AAV vectors into clinical testing has accelerated rapidly over the past two decades. While many of the AAV vectors being utilized in clinical trials are derived from natural serotypes, engineered serotypes are progressing toward clinical translation due to their enhanced tissue tropism and immune evasive properties. However, novel AAV vectors require formulation and stability testing to determine optimal storage conditions prior to their use in a clinical setting. RESULTS: Here, we evaluated the thermal stability of AAV6.2FF, a rationally engineered capsid with strong tropism for lung and muscle, in two different buffer formulations; phosphate buffered saline (PBS), or PBS supplemented with 0.001% non-ionic surfactant Pluronic F68 (PF-68). Aliquots of AAV6.2FF vector encoding the firefly luciferase reporter gene (AAV6.2FF-ffLuc) were incubated at temperatures ranging from -20°C to 55°C for varying periods of time and the impact on infectivity and particle integrity evaluated. Additionally, the impact of several rounds of freeze-thaw treatments on the infectivity of AAV6.2FF was investigated. Vector infectivity was measured by quantifying firefly luciferase expression in HEK 293 cells and AAV particle integrity was measured by qPCR quantification of encapsidated viral DNA. CONCLUSIONS: Our data demonstrate that formulating AAV6.2FF in PBS containing 0.001% PF-68 leads to increased stability and particle integrity at temperatures between -20℃ to 21℃ and protection against the destructive effects of freeze-thaw. Finally, AAV6.2FF-GFP formulated in PBS supplemented with 0.001% PF-68 displayed higher transduction efficiency in vivo in murine lung epithelial cells following intranasal administration than vector buffered in PBS alone further demonstrating the beneficial properties of PF-68.

Mucosal Vaccination with a Newcastle Disease Virus-Vectored Vaccine Reduces Viral Loads in SARS-CoV-2-Infected Cynomolgus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged following an outbreak of unexplained viral illness in China in late 2019. Since then, it has spread globally causing a pandemic that has resulted in millions of deaths and has had enormous economic and social consequences. The emergence of SARS-CoV-2 saw the rapid and widespread development of a number of vaccine candidates worldwide, and this never-before-seen pace of vaccine development led to several candidates progressing immediately through clinical trials. Many countries have now approved vaccines for emergency use, with large-scale vaccination programs ongoing. Despite these successes, there remains a need for ongoing pre-clinical and clinical development of vaccine candidates against SARS-CoV-2, as well as vaccines that can elicit strong mucosal immune responses. Here, we report on the efficacy of a Newcastle disease virus-vectored vaccine candidate expressing SARS-CoV-2 spike protein (NDV-FLS) administered to cynomolgus macaques. Macaques given two doses of the vaccine via respiratory immunization developed robust immune responses and had reduced viral RNA levels in nasal swabs and in the lower airway. Our data indicate that NDV-FLS administered mucosally provides significant protection against SARS-CoV-2 infection, resulting in reduced viral burden and disease manifestation, and should be considered as a viable candidate for clinical development.

A promoterless AAV6.2FF-based lung gene editing platform for the correction of surfactant protein B deficiency.

Surfactant protein B (SP-B) deficiency is a rare genetic disease that causes fatal respiratory failure within the first year of life. Currently, the only corrective treatment is lung transplantation. Here, we co-transduced the murine lung with adeno-associated virus 6.2FF (AAV6.2FF) vectors encoding a SaCas9-guide RNA nuclease or donor template to mediate insertion of promoterless reporter genes or the (murine) Sftpb gene in frame with the endogenous surfactant protein C (SP-C) gene, without disrupting SP-C expression. Intranasal administration of 3 × 1011 vg donor template and 1 × 1011 vg nuclease consistently edited approximately 6% of lung epithelial cells. Frequency of gene insertion increased in a dose-dependent manner, reaching 20%-25% editing efficiency with the highest donor template and nuclease doses tested. We next evaluated whether this promoterless gene editing platform could extend survival in the conditional SP-B knockout mouse model. Administration of 1 × 1012 vg SP-B-donor template and 5 × 1011 vg nuclease significantly extended median survival (p = 0.0034) from 5 days in the untreated off doxycycline group to 16 days in the donor AAV and nuclease group, with one gene-edited mouse living 243 days off doxycycline. This AAV6.2FF-based gene editing platform has the potential to correct SP-B deficiency, as well as other disorders of alveolar type II cells.

AAV-Vectored Expression of Marburg Virus-Neutralizing Antibody MR191 Provides Complete Protection From Challenge in a Guinea Pig Model.

Although there are no approved countermeasures available to prevent or treat disease caused by Marburg virus (MARV), potently neutralizing monoclonal antibodies (mAbs) derived from B cells of human survivors have been identified. One such mAb, MR191, has been shown to provide complete protection against MARV in nonhuman primates. We previously demonstrated that prophylactic administration of an adeno-associated virus (AAV) expressing MR191 protected mice from MARV. Here, we modified the AAV-MR191 coding sequence to enhance efficacy and reevaluated protection in a guinea pig model. Remarkably, 4 different variants of AAV-MR191 provided complete protection against MARV, despite administration 90 days prior to challenge. Based on superior expression kinetics, AAV-MR191-io2, was selected for evaluation in a dose-reduction experiment. The highest dose provided 100% protection, while a lower dose provided ∼88% protection. These data confirm the efficacy of AAV-mediated expression of MR191 and support the further development of this promising MARV countermeasure.

BTN3A3 evasion promotes the zoonotic potential of influenza A viruses.

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Antibody-based protection against respiratory syncytial virus in mice and their offspring through vectored immunoprophylaxis.

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections, with potential lower respiratory tract infections, which can be particularly problematic in infants and the elderly. There are no approved vaccines for RSV. The current standard of care for high-risk individuals is monthly administration of palivizumab, a humanized murine monoclonal antibody (mAb) targeting the RSV fusion protein. Adeno-associated virus (AAV)-mediated expression of mAbs has previously led to sustained expression of therapeutic concentrations of mAbs in several animal models, representing an alternative to repetitive passive administration. Intramuscular (IM) administration of AAV6.2FF expressing RSV antibodies, palivizumab or hRSV90, resulted in high concentrations of human (h)IgG1 mAbs in the serum and at various mucosal surfaces, while intranasal administration limited hIgG expression to the respiratory tract. IM administration of AAV6.2FF-hRSV90 or AAV6.2FF-palivizumab in a murine model provided sterilizing immunity against challenge with RSV A2. Evidence of maternal passive transfer of vectorized hRSV90 was detected in both murine and ovine models, with circulating mAbs providing sterilizing immunity in mouse progeny. Finally, addition of a "kill switch" comprised of LoxP sites flanking the mAb genes resulted in diminished serum hIgG after AAV-DJ-mediated delivery of Cre recombinase to the same muscle group that was originally transduced with the AAV-mAb vector. The ability of this AAV-mAb system to mediate robust, sustained mAb expression for maternal transfer to progeny in murine and ovine models emphasizes the potential of this platform for use as an alternative prophylactic vaccine for protection against neonatal infections, particularly in high-risk infants.

AAV-mediated delivery of actoxumab and bezlotoxumab results in serum and mucosal antibody concentrations that provide protection from C. difficile toxin challenge.

Clostridium difficile is the leading cause of antibiotic-associated nosocomial diarrhea in the developed world. When the host-associated colon microbiome is disrupted by the ingestion of antibiotics, C. difficile spores can germinate, resulting in infection. C. difficile secretes enterotoxin A (TcdA) and cytotoxin B (TcdB) that are responsible for disease pathology. Treatment options are limited as the bacterium demonstrates resistance to many antibiotics, and even with antibacterial therapies, recurrences of C. difficile are common. Actotoxumab and bezlotoxumab are human monoclonal antibodies that bind and neutralize TcdA and TcdB, respectively. In 2016, the US food and drug administration (FDA) approved bezlotoxumab for use in the prevention of C. difficile infection recurrence. To ensure the long-term expression of antibodies, gene therapy can be used. Here, adeno-associated virus (AAV)6.2FF, a novel triple mutant of AAV6, was engineered to express either actotoxumab or bezlotoxumab in mice and hamsters. Both antibodies expressed at greater than 90 µg/mL in the serum and were detected at mucosal surfaces in both models. Hundred percent of mice given AAV6.2FF-actoxumab survived a lethal dose of TcdA. This proof of concept study demonstrates that AAV-mediated expression of C. difficile toxin antibodies is a viable approach for the prevention of recurrent C. difficile infections.

AAV-Vectored Expression of the Vascular Normalizing Agents 3TSR and Fc3TSR, and the Anti-Angiogenic Bevacizumab Extends Survival in a Murine Model of End-Stage Epithelial Ovarian Carcinoma.

Epithelial ovarian cancer is the deadliest gynecological malignancy. The lack of effective treatments highlights the need for novel therapeutic interventions. The aim of this study was to investigate whether sustained adeno-associated virus (AAV) vector-mediated expression of vascular normalizing agents 3TSR and Fc3TSR and the antiangiogenic monoclonal antibody, Bevacizumab, with or without oncolytic virus treatment would improve survival in an orthotopic syngeneic mouse model of epithelial ovarian carcinoma. AAV vectors were administered 40 days post-tumor implantation and combined with oncolytic avian orthoavulavirus-1 (AOaV-1) 20 days later, at the peak of AAV-transgene expression, to ascertain whether survival could be extended. Flow cytometry conducted on blood samples, taken at an acute time point post-AOaV-1 administration (36 h), revealed a significant increase in activated NK cells in the blood of all mice that received AOaV-1. T cell analysis revealed a significant increase in CD8+ tumor specific T cells in the blood of AAV-Bevacizumab+AOaV-1 treated mice compared to control mice 10 days post AOaV-1 administration. Immunohistochemical staining of primary tumors harvested from a subset of mice euthanized 90 days post tumor implantation, when mice typically have large primary tumors, secondary peritoneal lesions, and extensive ascites fluid production, revealed that AAV-3TSR, AAV-Fc3TSR+AOaV-1, or AAV-Bevacizumab+AOaV-1 treated mice had significantly more tumor-infiltrating CD8+ T cells than PBS controls. Despite AAV-mediated transgene expression waning faster in tumor-bearing mice than in non-tumor bearing mice, all three of the AAV therapies significantly extended survival compared to control mice; with AAV-Bevacizumab performing the best in this model. However, combining AAV therapies with a single dose of AOaV-1 did not lead to significant extensions in survival compared to AAV therapies on their own, suggesting that additional doses of AOaV-1 may be required to improve efficacy in this model. These results suggest that vectorizing anti-angiogenic and vascular normalizing agents is a viable therapeutic option that warrants further investigation, including optimizing combination therapies.

Generation and Characterization of a SARS-CoV-2-Susceptible Mouse Model Using Adeno-Associated Virus (AAV6.2FF)-Mediated Respiratory Delivery of the Human ACE2 Gene.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19) that has caused a pandemic with millions of human infections. There continues to be a pressing need to develop potential therapies and vaccines to inhibit SARS-CoV-2 infection to mitigate the ongoing pandemic. Epidemiological data from the current pandemic indicates that there may be sex-dependent differences in disease outcomes. To investigate these differences, we proposed to use common small animal species that are frequently used to model disease with viruses. However, common laboratory strains of mice are not readily infected by SARS-CoV-2 because of differences in the angiotensin-converting enzyme 2 (ACE2), the cellular receptor for the virus. To overcome this limitation, we transduced common laboratory accessible strains of mice of different sexes and age groups with a novel a triple AAV6 mutant, termed AAV6.2FF, encoding either human ACE2 or luciferase via intranasal administration to promote expression in the lung and nasal turbinates. Infection of AAV-hACE2-transduced mice with SARS-CoV-2 resulted in high viral titers in the lungs and nasal turbinates, establishment of an IgM and IgG antibody response, and modulation of lung and nasal turbinate cytokine profiles. There were insignificant differences in infection characteristics between age groups and sex-related differences; however, there were significant strain-related differences between BALB/c vs. C57BL/6 mice. We show that AAV-hACE2-transduced mice are a useful for determining immune responses and for potential evaluation of SARS-CoV-2 vaccines and antiviral therapies, and this study serves as a model for the utility of this approach to rapidly develop small-animal models for emerging viruses.

Downregulation of Cell Surface Major Histocompatibility Complex Class I Expression Is Mediated by the Left-End Transcription Unit of Fowl Adenovirus 9.

Major histocompatibility complex class I (MHC-I) molecules play a critical role in the host's antiviral response by presenting virus-derived antigenic peptides to cytotoxic T lymphocytes (CTLs), enabling the clearance of virus-infected cells. Human adenoviruses evade CTL-mediated cell lysis, in part, by interfering directly with the MHC-I antigen presentation pathway through the expression of E3-19K, which binds both MHC-I and the transporter associated with antigen processing protein and sequestering MHC-I within the endoplasmic reticulum. Fowl adenoviruses have no homologues of E3-19K. Here, we show that representative virus isolates of the species Fowl aviadenovirus C, Fowl aviadenovirus D, and Fowl aviadenovirus E downregulate the cell surface expression of MHC-I in chicken hepatoma cells, resulting in 71%, 11%, and 14% of the baseline expression level, respectively, at 12 h post-infection. Furthermore, this work reports that FAdV-9 downregulates cell surface MHC-I through a minimum of two separate mechanisms-a lysosomal-independent mechanism that requires the presence of the fowl adenovirus early 1 (FE1) transcription unit located within the left terminal genomic region between nts 1 and 6131 and a lysosomal-dependent mechanism that does not require the presence of FE1. These results establish a new functional role for the FE1 transcription unit in immune evasion. These studies provide important new information about the immune evasion of FAdVs and will enhance our understanding of the pathogenesis of inclusion body hepatitis and advance the progress made in next-generation FAdV-based vectors.

Intranasal vaccination with a Newcastle disease virus-vectored vaccine protects hamsters from SARS-CoV-2 infection and disease.

The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19). Worldwide efforts are being made to develop vaccines to mitigate this pandemic. We engineered two recombinant Newcastle disease virus (NDV) vectors expressing either the full-length SARS-CoV-2 spike protein (NDV-FLS) or a version with a 19 amino acid deletion at the carboxy terminus (NDV-Δ19S). Hamsters receiving two doses (prime-boost) of NDV-FLS developed a robust SARS-CoV-2-neutralizing antibody response, with elimination of infectious virus in the lungs and minimal lung pathology at five days post-challenge. Single-dose vaccination with NDV-FLS significantly reduced SARS-CoV-2 replication in the lungs but only mildly decreased lung inflammation. NDV-Δ19S-treated hamsters had a moderate decrease in SARS-CoV-2 titers in lungs and presented with severe microscopic lesions, suggesting that truncation of the spike protein was a less effective strategy. In summary, NDV-vectored vaccines represent a viable option for protection against COVID-19.

Safety and Tolerability of the Adeno-Associated Virus Vector, AAV6.2FF, Expressing a Monoclonal Antibody in Murine and Ovine Animal Models.

Adeno-associated virus (AAV) vector mediated expression of therapeutic monoclonal antibodies is an alternative strategy to traditional vaccination to generate immunity in immunosuppressed or immunosenescent individuals. In this study, we vectorized a human monoclonal antibody (31C2) directed against the spike protein of SARS-CoV-2 and determined the safety profile of this AAV vector in mice and sheep as a large animal model. In both studies, plasma biochemical parameters and hematology were comparable to untreated controls. Except for mild myositis at the site of injection, none of the major organs revealed any signs of toxicity. AAV-mediated human IgG expression increased steadily throughout the 28-day study in sheep, resulting in peak concentrations of 21.4-46.7 µg/ mL, demonstrating practical scale up from rodent to large animal models. This alternative approach to immunity is worth further exploration after this demonstration of safety, tolerability, and scalability in a large animal model.

A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens.

More than 100 million people have been infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Common laboratory mice are not susceptible to wild-type SARS-CoV-2 infection, challenging the development and testing of effective interventions. Here, we describe the development and testing of a mouse model for SARS-CoV-2 infection based on transduction of the respiratory tract of laboratory mice with an adeno-associated virus vector (AAV6) expressing human ACE-2 (AAV6.2FF-hACE2). We validated this model using a previously described synthetic DNA vaccine plasmid, INO-4800 (pS). Intranasal instillation of AAV6.2FF-hACE2 resulted in robust hACE2 expression in the respiratory tract. pS induced robust cellular and humoral responses. Vaccinated animals were challenged with 105 TCID50 SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) and euthanized four days post-challenge to assess viral load. One immunization resulted in 50% protection and two immunizations were completely protective. Overall, the AAV6.2FF-hACE2 mouse transduction model represents an easily accessible, genetically diverse mouse model for wild-type SARS-CoV-2 infection and preclinical evaluation of potential interventions.

Characterization of a fowl adenovirus 9 (FAdV-9) early promoter and its application in generating dual expression FAdV-9s.

The CMV immediate early promoter from the EGFP expression plasmid pEGFP-N1 was replaced with the very left end of the fowl adenovirus 9 (FAdV-9) genome (ntds 73-574) to demonstrate and delineate the promoter function of this sequence. Expression of an EGFP ORF which replaced ORF1 and ORF2 demonstrated that the native promoter can drive down stream foreign gene expression. Replacement of ORF1 and ORF2 with a bicistronic cassette, incorporating a 493 bp IRES from an Ontario strain of avian encephalomyelitis virus (AEV) separating an EGFP ORF and mCherry ORF allowed for expression of both ORFs from a recombinant FAdV. These results provide an additional platform for multivalent vaccines development based on a native FAdV-9 promoter and an avian virus IRES.

A lung tropic AAV vector improves survival in a mouse model of surfactant B deficiency.

Surfactant protein B (SP-B) deficiency is an autosomal recessive disorder that impairs surfactant homeostasis and manifests as lethal respiratory distress. A compelling argument exists for gene therapy to treat this disease, as de novo protein synthesis of SP-B in alveolar type 2 epithelial cells is required for proper surfactant production. Here we report a rationally designed adeno-associated virus (AAV) 6 capsid that demonstrates efficiency in lung epithelial cell transduction based on imaging and flow cytometry analysis. Intratracheal administration of this vector delivering murine or human proSFTPB cDNA into SP-B deficient mice restores surfactant homeostasis, prevents lung injury, and improves lung physiology. Untreated SP-B deficient mice develop fatal respiratory distress within two days. Gene therapy results in an improvement in median survival to greater than 200 days. This vector also transduces human lung tissue, demonstrating its potential for clinical translation against this lethal disease.

Fowl adenovirus 9 ORF19, a lipase homolog, is nonessential for virus replication and is suitable for foreign gene expression.

Fowl adenovirus 9 (FAdV-9) has one of the largest genomes (45 kb) so far sequenced from all adenoviruses studied. Genus-specific genes located within the early (E) regions at the right and left ends of the viral genome have unknown functions except for ORF8 (Gam-1 gene), ORF22 and ORF1 (dUTPase gene). ORF19, located at the right end of the genome (nts 34,220-36,443), is predicted to encode a lipase protein and its homologs are also found in all FAdV genomes so far sequenced. The role of ORF19 in virus replication and virulence is unknown. To study ORF19 and explore its potential as a locus for foreign gene insertion, we generated one ORF19-deleted mutant virus (rFAdV-9Δ19-SwaI) and three FAdV-9Δ19-based recombinant viruses replacing ORF19 as follows: rFAdV-9Δ19-CAT and enhanced-green fluorescent protein (EGFP) cassette (CMV promoter-EGFP-poly A) in a rightward (rFAdV-9Δ19-EGFP-R) and leftward orientation (rFAdV-9Δ19-EGFP-L). All recombinant viruses were stable after three passages. In chicken hepatoma cells, rFAdV-9Δ19-SwaI, rFAdV-9Δ19-CAT and rFAdV-9Δ19-EGFP-R replicated at titers similar to that of the wild-type virus, whilst rFAdV-9Δ19-EGFP-L replicated at a much lower titer. Interestingly, FAdV-9Δ19-SwaI replicated at higher titers in cells and in embryonated eggs, respectively than those of wild-type and recombinant viruses. These observations suggest ORF19 is nonessential for replication and can be used as a novel cloning site for engineering FAdV-9-based recombinant viruses and rFAdV-9Δ19-SwaI could be used to determine its role for virus replication in vivo.

Fowl Adenovirus 4 (FAdV-4)-Based Infectious Clone for Vaccine Vector Development and Viral Gene Function Studies.

Fowl adenovirus 4 (FAdV-4) is associated with economically important poultry diseases. Recent studies of fully sequenced genomes of FAdV-4 isolates suggest potential genomic regions associated with virulence and amenable for manipulation and vector development. Direct manipulation of viral genomes is cumbersome, as opposed to that of infectious clones-viral genomes cloned into plasmid or cosmid vectors. In this work, we generated an infectious clone, pFAdV-4 ON1, containing the entire viral genome of a nonpathogenic FAdV-4 (ON1 isolate). pFAdV-4 ON1 was used for targeted deletion of open reading frames (ORFs) 16 and 17 and replacement with the enhanced green fluorescence protein (EGFP) expression cassette to generate recombinant viruses. These viruses were viable, and EGFP was expressed in infected cells. Their replication, however, was significantly reduced with respect to that of the wild-type virus. These observations suggest the potential utility of FAdV-4 as a vaccine vector and the importance of ORFs 16 and 17 for virus replication at wild-type levels. To our knowledge, this is the first report of an infectious clone based on the FAdV-4 genome, and our results demonstrate its utility for studies of virulence determinants and as a platform for either vaccine or gene delivery vectors.

Generation and characterization of a fowl adenovirus 9 dual-site expression vector.

Fowl adenoviruses (FAdVs) are widely considered as excellent platforms for vaccine development and gene therapy. We improved on our right-end partial TR-2 deleted or a left-end 2.3 kb deleted vectors by developing a single, dual-site delivery vector. We demonstrated that, in addition to ORF11, the right end ORF17 is also dispensable. To further improve the capacity and flexibility of the FAdV-9 based vector system, we generated an infectious recombinant FAdV-9 dual-site expression clone lacking 1.9 kb of the left end and replaced with mCherry under the control of a native promoter, and 3.6 kb of the right-end replaced with an EGFP expression cassette. Five intermediate FAdmid clones were successfully constructed: a) pFAdV-9Δ0-2RED (mCherry replacing the left end 2.2 kb ORF0 to 2); b) pFAdV-9RED (mCherry replacing the left end 1.9 kb ORF1 to 2); c) pFAdV-9Δ17 (deletion of ORF17 and 393 bp downstream untranslated region); d) pFAdV-9GFP (EGFP expression cassette replacing the right end 3.6 kb) and e) pFAdV-9Dual (both mCherry in the left end and the EGFP expression cassette in the right end of our vector). Our novel FAdV-9 dual-site vaccine vector, produced infectious virus and expressed either one or both mCherry and EGFP.

Fowl Aviadenovirus 9 dUTPase Plays a Role in Regulation of the Host Immune Response.

Fowl aviadenoviruses (FAdVs) are distributed worldwide in poultry farms. Some FAdVs are the causative agents of inclusion body hepatitis and hydropericardium syndrome that cause significant economic losses to the poultry industry. In contrast with human adenovirus, the study of the molecular biology of FAdV is still far behind. We previously showed that FAdV-9 open reading frame 1 (ORF1) is a dUTPase enzyme that contributes to the upregulation of type I interferons and is not required for virus replication in vitro. In the present study, we compared virus replication in vivo and the host immune response in chickens orally inoculated with a dUTPase knockout virus (ORF1stop), the rescued version of ORF1stop (resORF1), and wtFAdV-9. Our data showed that replication of ORF1stop was delayed on days 1 and 3 postinoculation compared with wtFAdV-9, as evidenced by significantly less virus shedding in feces and lower viral loads in tissues. Moreover, we found that there was a significant difference in the induction of cytokine gene mRNA expression in tissues and IgG antibody responses in ORF1stop versus wtFAdV-9-infected chickens, suggesting that ORF1 plays some roles in modulating the host immune response. Our study provides useful data on the mechanism of the host immune response against FAdV infection.