Recombinant fiber-1 protein of fowl adenovirus serotype 4 induces high levels of neutralizing antibodies in immunized chickens.

Virulent fowl adenovirus serotype 4 (FAdV-4) causes hydropericardium syndrome (HPS) with high mortality in chickens, leading to significant economic losses to the poultry industry. The development of an effective vaccine is essential for successful disease control. Here, we produced recombinant fiber-1 protein of FAdV-4, isolated from a Japanese HPS outbreak strain, JP/LVP-1/96, using a baculovirus expression system and evaluated its immunogenicity and protective efficacy. Recombinant fiber-1 protein induced high levels of neutralizing antibodies in immunized chickens, which were maintained for a minimum of 10 weeks. After being challenged with the virulent FAdV-4 strain JP/LVP-1/96, the immunized chickens did not exhibit clinical signs of infection or histopathological changes, there was a significant reduction in the viral load in various organs and total serum proteins, and albumin levels did not decline. These results suggest that the recombinant fiber-1 protein produced in this study can serve as a subunit vaccine to control HPS in chickens.

Recombinant Fowl aviadenovirus E (FAdV-E) penton base vaccination fails to confer protection against inclusion body hepatitis (IBH) in chickens.

Inclusion body hepatitis (IBH) is a metabolic disease affecting chickens, associated with different serotypes of fowl adenovirus (FAdV). Experimentally tested vaccines against IBH include several capsid-based subunit vaccines, but not the penton base protein. In the present study, specific pathogen-free chickens were vaccinated with recombinant penton base expressed from each of two different FAdV serotypes (FAdV-7 and FAdV-8b), followed by challenge with a virulent IBH-causing strain. No protection was observed with either vaccine, possibly due to the low immunogenicity of each protein and their inability to induce neutralizing antibodies in the host.

MAGI-1 PDZ2 Domain Blockade Averts Adenovirus Infection via Enhanced Proteolysis of the Apical Coxsackievirus and Adenovirus Receptor.

Adenoviruses (AdVs) are etiological agents of gastrointestinal, heart, eye, and respiratory tract infections that can be lethal for immunosuppressed people. Many AdVs use the coxsackievirus and adenovirus receptor (CAR) as a primary receptor. The CAR isoform resulting from alternative splicing that includes the eighth exon, CAREx8, localizes to the apical surface of polarized epithelial cells and is responsible for the initiation of AdV infection. We have shown that the membrane level of CAREx8 is tightly regulated by two MAGI-1 PDZ domains, PDZ2 and PDZ4, resulting in increased or decreased AdV transduction, respectively. We hypothesized that targeting the interactions between the MAGI-1 PDZ2 domain and CAREx8 would decrease the apical CAREx8 expression level and prevent AdV infection. Decoy peptides that target MAGI-1 PDZ2 were synthesized (TAT-E6 and TAT-NET1). PDZ2 binding peptides decreased CAREx8 expression and reduced AdV transduction. CAREx8 degradation was triggered by the activation of the regulated intramembrane proteolysis (RIP) pathway through a disintegrin and metalloproteinase (ADAM17) and γ-secretase. Further analysis revealed that ADAM17 interacts directly with the MAGI-1 PDZ3 domain, and blocking the PDZ2 domain enhanced the accessibility of ADAM17 to the substrate (CAREx8). Finally, we validated the efficacy of TAT-PDZ2 peptides in protecting the epithelia from AdV transduction in vivo using a novel transgenic animal model. Our data suggest that TAT-PDZ2 binding peptides are novel anti-AdV molecules that act by enhanced RIP of CAREx8 and decreased AdV entry. This strategy has additional translational potential for targeting other viral receptors that have PDZ binding domains, such as the angiotensin-converting enzyme 2 receptor. IMPORTANCE Adenovirus is a common threat in immunosuppressed populations and military recruits. There are no currently approved treatments/prophylactic agents that protect from most AdV infections. Here, we developed peptide-based small molecules that can suppress AdV infection of polarized epithelia by targeting the AdV receptor, coxsackievirus and adenovirus receptor (CAREx8). The newly discovered peptides target a specific PDZ domain of the CAREx8-interacting protein MAGI-1 and decrease AdV transduction in multiple polarized epithelial models. Peptide-induced CAREx8 degradation is triggered by extracellular domain (ECD) shedding through ADAM17 followed by γ-secretase-mediated nuclear translocation of the C-terminal domain. The enhanced shedding of the CAREx8 ECD further protected the epithelium from AdV infection. Taken together, these novel molecules protect the epithelium from AdV infection. This approach may be applicable to the development of novel antiviral molecules against other viruses that use a receptor with a PDZ binding domain.

Highly soluble and stable 'insertion domain' of the capsid penton base protein provides complete protection against infections caused by fowl adenoviruses.

In the current study, we have evaluated the protective efficacy of the 'insertion domain' which is commonly found in the capsid penton base protein of many adenoviruses. Using the 'insertion domain' of the penton base protein of a representative fowl adenovirus, fowl adenovirus serotype 4 (FAdV-4), we find that the 'insertion domain' can readily be expressed in a soluble form in the bacterial system, and can be purified in sufficient quantities through simple chromatographic methods. We demonstrate that the 'insertion domain', when employed as a subunit vaccine candidate, provides complete protection against hydropericardium syndrome, caused by FAdV-4, in chickens. The data presented here indicate that the protein, adjuvanted with Montanide™ ISA71 VG, provides complete protection in chickens against a lethal FAdV-4 challenge after administration of two doses (100 µg of the protein per dose) two weeks apart (the first dose at the 7th day of life and a booster dose at the age of 21 days). Furthermore, the purified protein can be stored at low temperatures without any observable loss in the protein integrity up to one year, tested so far. Due to the conserved nature of the 'insertion domain' across the penton base protein of fowl adenoviruses, it is suggested that homologous insertion domains could be employed as highly stable and cost-effective subunit vaccine candidates against infections caused by respective fowl adenoviruses.

An inactivated novel chimeric FAdV-4 containing fiber of FAdV-8b provides full protection against hepatitis-hydropericardium syndrome and inclusion body hepatitis.

Fowl adenovirus serotype 4 (FAdV-4) and FAdV-8b are causative agents of hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH), respectively. HHS and IBH co-infections were often reported in clinical, yet there are no commercially available bivalent vaccines for prevention and control of both FAdV-4 and -8b. In the present study, a chimeric FAdV-4 was firstly generated by substituting fiber-1 of FAdV-4 with fiber of FAdV-8b. The chimeric virus, rFAdV-4-fiber/8b, exhibited similar replication ability in vitro and pathogenicity in vivo to the parental wild type FAdV-4. A single dosage of vaccination with the inactivated rFAdV-4-fiber/8b induced high antibody titers against fiber-2 of FAdV-4 and fiber of FAdV-8b and provided full protection against FAdV-4 and -8b challenge. These results demonstrated that fiber of FAdV-8b could replace the role of fiber-1 of FAdV-4 in the process of viral infection, and rFAdV-4-fiber/8b could be used to make a potential bivalent vaccine for the control and prevention of HHS and IBH.

Fiber-1, Not Fiber-2, Directly Mediates the Infection of the Pathogenic Serotype 4 Fowl Adenovirus via Its Shaft and Knob Domains.

Recently, the disease of hepatitis-hydropericardium syndrome (HPS) caused by serotype 4 fowl adenovirus (FAdV-4) has spread widely and resulted in huge economic losses to the poultry industry. Although the genome of FAdV-4 has two fiber genes (fiber-1 and fiber-2), the exact role of the genes in the infection of FAdV-4 is barely known. In this study, through superinfection resistance analysis and an interfering assay, we found that fiber-1, but not fiber-2, was the key factor for directly triggering the infection of FAdV-4. The truncation analysis further revealed that both of the shaft and knob domains of fiber-1 were required for the infection. Moreover, the sera against the knob domain were able to block FAdV-4 infection, and the knob-containing fusion protein provided efficient protection against the lethal challenge of FAdV-4 in chickens. All the data demonstrated the significant roles of fiber-1 and its knob domain in directly mediating the infection of FAdV-4, which established a foundation for identifying the receptor of FAdV-4 and developing efficient vaccines against FAdV-4.IMPORTANCE Among 12 serotypes of fowl adenovirus (FAdV), FAdV-1, FAdV-4, and FAdV-10 all carry two fiber genes (i.e., fiber-1 and fiber-2), whereas other serotypes have only one. As important viral surface proteins, the fibers play vital roles in the infection and pathogenesis of FAdV. However, the importance of the fibers to the infection and pathogenesis of FAdV may be different from each other. Recent studies reveal that fiber-2 is identified as a determinant of virulence, but which fiber triggers the infection of FAdV-4 remains unknown. In this study, fiber-1 was identified as a key factor for directly mediating the infection of FAdV-4 through its shaft and knob domains, whereas fiber-2 did not play a role in triggering FAdV-4 infection. The results suggest that fiber-1 and its knob domain may serve as a target for identifying the receptor of FAdV-4 and developing efficient drugs or vaccines against FAdV-4.

Low circulation of Influenza A and coinfection with SARS-CoV-2 among other respiratory viruses during the COVID-19 pandemic in a region of southern Brazil.

With the arrival of coronavirus disease 2019 (COVID-19) in Brazil in February 2020, several preventive measures were taken by the population aiming to avoid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection including the use of masks, social distancing, and frequent hand washing then, these measures may have contributed to preventing infection also by other respiratory viruses. Our goal was to determine the frequencies of Influenza A and B viruses (FLUAV/FLUBV), human mastadenovirus C (HAdV-C), Enterovirus 68 (EV-68), and rhinovirus (RV) besides SARS-CoV-2 among hospitalized patients suspect of COVID-19 with cases of acute respiratory disease syndrome (ARDS) in the period of March to December 2020 and to detect possible coinfections among them. Nucleic acid detection was performed using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) in respiratory samples using naso-oropharyngeal swabs and bronchoalveolar lavage. A total of 418 samples of the 987 analyzed (42.3%) were positive for SARS-CoV-2, 16 (1.62%) samples were positive for FLUAV, no sample was positive for FLUBV or EV-68, 67 (6.78%) samples were positive for HAdV-C, 55 samples were positive for RV 1/2 (26.3%) and 37 for RV 2/2 (13.6%). Coinfections were also detected, including a triple coinfection with SARS-CoV-2, FLUAV, and HAdV-C. In the present work, a very low frequency of FLUV was reported among hospitalized patients with ARDS compared to the past years, probably due to preventive measures taken to avoid COVID-19 and the high influenza vaccination coverage in the region in which this study was performed.

A novel fiber-2-edited live attenuated vaccine candidate against the highly pathogenic serotype 4 fowl adenovirus.

Recently, the outbreaks of hydropericardium-hepatitis syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry globally. Although several inactivated or subunit vaccines have been developed against FAdV-4, live-attenuated vaccines for FAdV-4 are rarely reported. In this study, a recombinant virus FA4-EGFP expressing EGFP-Fiber-2 fusion protein was generated by the CRISPR/Cas9 technique. Although FA4-EGFP shows slightly lower replication ability than the wild type (WT) FAdV-4, FA4-EGFP was significantly attenuated in vivo compared with the WT FAdV-4. Chickens infected with FA4-EGFP did not show any clinical signs, and all survived to 14 day post-infection (dpi), whereas those infected with FAdV-4 showed severe clinical signs with HHS and all died at 4 dpi. Besides, the inoculation of FA4-EGFP in chickens provided efficient protection against lethal challenge with FAdV-4. Compared with an inactivated vaccine, FA4-EGFP induced neutralizing antibodies with higher titers earlier. All these data not only provide a live-attenuated vaccine candidate against the highly pathogenic FAdV-4 but also give a potential insertion site for developing FAdV-4-based vaccine vectors for delivering foreign antigens.

Spotlight on avian pathology: fowl adenovirus (FAdV) in chickens and beyond - an unresolved host-pathogen interplay.

Fowl adenovirus (FAdV) infections in chickens have undergone substantial changes in recent decades, driven by host and pathogen factors. Based on the pathogenesis of inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS), modern broilers are much more inclined to have difficulties keeping the metabolic homeostasis, whereas adenoviral gizzard erosion (AGE) is noticed equally in broilers and egg-layers. Defining the importance of certain serotypes for specific FAdV diseases is a major achievement of recent years but the isolation of viruses from clinically healthy birds remains unexplained, as virulence factors are hardly known and continue to be a "black box". Together with further studies on pathogenesis of FAdV-induced diseases, such knowledge on virulence factors would help to improve protection strategies, which presently mainly concentrate on autogenous vaccines of breeders to prevent vertical transmission.

Oral Vaccination with Replication-Competent Adenovirus in Mice Reveals Dissemination of the Viral Vaccine beyond the Gastrointestinal Tract.

Since the 1970s, replication-competent human adenoviruses 4 and 7 have been used as oral vaccines to protect U.S. soldiers against the severe respiratory diseases caused by these viruses. These vaccines are thought to establish a digestive tract infection conferring protection against respiratory challenge through antibodies. The success of these vaccines makes replication-competent adenoviruses attractive candidates for use as oral vaccine vectors. However, the inability of human adenoviruses to replicate efficiently in laboratory animals has hampered the study of such vectors. Here, we used mouse adenovirus type 1 (MAV-1) in mice to study oral replication-competent adenovirus-based vaccines. We show that MAV-1 oral administration provides protection that recapitulates the protection against homologous respiratory challenge observed with adenovirus 4 and 7 vaccines. Moreover, live oral MAV-1 vaccine better protected against a respiratory challenge than inactivated vaccines. This protection was linked not only with the presence of MAV-1-specific antibodies but also with a better recruitment of effector CD8 T cells. However, unexpectedly, we found that such oral replication-competent vaccine systemically spread all over the body. Our results therefore support the use of MAV-1 to study replication-competent oral adenovirus-based vaccines but also highlight the fact that those vaccines can disseminate widely in the body.IMPORTANCE Replication-competent adenoviruses appear to be promising vectors for the development of oral vaccines in humans. However, the study and development of these vaccines suffer from the lack of any reliable animal model. In this study, mouse adenovirus type 1 was used to develop a small-animal model for oral replication-competent adenovirus vaccines. While this model reproduced in mice what is observed with human adenovirus oral vaccines, it also highlighted that oral immunization with such a replication-competent vaccine is associated with the systemic spread of the virus. This study is therefore of major importance for the future development of such vaccine platforms and their use in large human populations.

Comparative Evaluation of the Vaccine Efficacies of Three Adenovirus-Based Vector Types in the Friend Retrovirus Infection Model.

Adenovirus (AdV)-based vectors are popular experimental vaccine vectors, but despite their ability to induce strong immune responses, their application is impeded by widespread preexisting immunity against many AdV types that can impair or even abrogate the induction of transgene-specific immune responses. Therefore, the development of vectors based on AdV types with a low seroprevalence is important for effective AdV-based immunization in humans. We investigated the immunization efficacy of vectors based on AdV type 48 (Ad48) and Ad50 in the ovalbumin (ova) model as well as the Friend retrovirus (FV) model, which allows testing of the protective effect of vaccine-induced immunity. Using ova-encoding vectors, we found a significantly lower induction of ova-specific CD8+ T cells and antibody responses by Ad48- and Ad50-based vectors than by Ad5-based vectors. Similarly, we found a reduced induction of FV-specific CD8+ T cell responses in Ad48- and Ad50.Leader-Gag-immunized mice compared with that in Ad5-immunized mice; however, some of those mice were able to control the FV infection, and protection correlated with the level of neutralizing antibodies 10 days after FV challenge. Analyses of the AdV-specific antibodies and CD8+ T cells induced by the individual AdV types revealed a high level of cross-reactivity, and the efficacy of Ad48-based immunization was impaired in Ad5-preimmune mice. Our results show that the immunity induced by Ad48- and Ad50-based vectors is reduced compared to that induced by Ad5 and is sufficient to control FV infection in only some of the immunized mice. A high level of cross-reactivity suggests that AdV preimmunity must be considered even when applying rare AdV-based vectors.IMPORTANCE AdV-based vectors are important tools for the development of vaccines against a wide range of pathogens. While AdV vectors are generally considered safe and highly effective, their application can be severely impaired by preexisting immunity due to the widespread seroprevalence of some AdV types. The characterization of different AdV types with regard to immunogenicity and efficacy in challenge models is of great importance for the development of improved AdV-based vectors that allow for efficient immunization despite anti-AdV immunity. We show that the immunity induced by an Ad48-based vector is inferior to that induced by an Ad5-based vector but can still mediate the control of an FV infection in highly FV-susceptible mice. However, the efficacy of Ad48-based immunization was impaired in Ad5-preimmune mice. Importantly, we found cross-reactivity of both the humoral and cellular immune responses raised by the individual AdV types, suggesting that switching to a different AdV type may not be sufficient to circumvent preexisting anti-AdV immunity.

Practice patterns and incidence of adenovirus infection in allogeneic hematopoietic cell transplant recipients: Multicenter survey of transplant centers in the United States.

BACKGROUND: Adenovirus (AdV) is increasingly recognized as a threat to successful outcomes after allogeneic hematopoietic cell transplantation (allo-HCT). Guidelines have been developed to inform AdV screening and treatment practices, but the extent to which they are followed in clinical practice in the United States is still unknown. The incidence of AdV in the United States is also not well documented. The main objectives of the AdVance US study were thus to characterize current AdV screening and treatment practices in the United States and to estimate the incidence of AdV infection in allo-HCT recipients across multiple pediatric and adult transplant centers. METHODS: Fifteen pediatric centers and 6 adult centers completed a practice patterns survey, and 15 pediatric centers and four adult centers completed an incidence survey. RESULTS: The practice patterns survey results confirm that pediatric transplant centers are more likely than adult centers to routinely screen for AdV, and are also more likely to have a preemptive AdV treatment approach compared to adult centers. Perceived risk of AdV infection is a determining factor for whether routine screening and preemptive treatment are implemented. Most pediatric centers screen higher-risk patients for AdV weekly, in blood, and have a preemptive AdV treatment approach. The incidence survey results show that from 2015 to 2017, a total of 1230 patients underwent an allo-HCT at the 15 pediatric transplant centers, and 1815 patients underwent an allo-HCT at the 4 adult transplant centers. The incidences of AdV infection, AdV viremia, and AdV viremia ≥ 1000 copies/mL within 6 months after the first allo-HCT were 23%, 16%, and 9%, respectively, for patients at pediatric centers, and 5%, 3%, and 2%, respectively, for patients at adult centers. CONCLUSIONS: These findings provide a more recent estimate of the incidence of AdV infection in the United States, as well as a multicenter view of practice patterns around AdV infection screening and intervention criteria, in pediatric and adult allo-HCT recipients.

The application of naked DNA plasmid (DrZP3) and recombinant adenovirus (Ad-rZP3) in rat animal model to determine comparative efficacy of ZP3-Immunocontraceptive vaccines.

The common physical and chemical methods for controlling rat pest are less than satisfactory and inhumane. Immunocontraception approach has been considered more humane and it can be accomplished by inducing the relevant host immune response that block further development of reproductive gametes. ZP3 proteins are known to play very important role during sperm-ovum fertilization. It is a self-antigen and only localized in female ovaries. Therefore, an immunization with ZP3 protein elsewhere will induce a generalize host immune response against ZP3 protein. This study employed rat ZP3 (rZP3) gene prepared from its cDNA of Rattus rattus diardii. It was delivered and expressed in vivo by naked plamid DNA (DrZP3) or recombinant ZP3-Adenovirus (Ad-rZP3). Expression studies in vitro with DrZP3 or Ad-ZP3 showed rZP3 proteins were successfully expressed in Vero cells. Hyperimmune serum against rZP3 that were prepared by immunizing several rats with purified rZP3-pichia yeast fusion protein showed it blocked sperms from binding DrZP3-transfected Vero cells. Female Sprague Dawley rats immunized with DrZP3 demonstrated a long-term effect for significant reduction of fertility up to 92.6%. Ovaries from rats immunized with DrZP3 were severely atrophied with disappearance of primordial follicles from ovarian cortex with an increased in the amount of oocyte-free cell clusters. Female rats immunized with Ad-rZP3 demonstrated 27% reduction of fertility. The infertility induced by Ad-rZP3 is comparatively low and ineffective. This could be due to a strong host immune response that suppresses the recombinant virus itself resulted in minimum rZP3 protein presentation to the host immune system. As a result, low antibody titers produced against rZP3 is insufficient to block oocytes from maturity and fertilization. Therefore, immunization with DrZP3 for immunocontraception is more effective than Ad-rZP3 recombinant adenovirus. It is proposed to explore further on the use of adenovirus or other alternative viruses to deliver ZP3 protein and for the development of enhanced expression of rZP3 in target host.

Vaccination with FAdV-8a induces protection against inclusion body hepatitis caused by homologous and heterologous strains.

Fowl aviadenoviruses (FAdV) are important avian pathogens, responsible for several poultry diseases prevalent worldwide, including inclusion body hepatitis (IBH). FAdV intraspecies cross-protection has been clearly demonstrated, but there is little evidence that any interspecies cross-protection exists. The present study aimed to assess the inter- and intraspecies protection between three FAdV field isolates (FAdV-8a, FAdV-8b, FAdV-11) identified in association with severe IBH outbreaks. Inocula prepared using inactivated plaque-purified virus with adjuvant Montanide™ ISA 71VG, were injected intramuscularly into 3-week-old SPF chickens. At 6-weeks of age, the birds were challenged with 106 TCID50 of homologous or heterologous virus intraperitoneally, and full post mortem examination performed at 4 days post-challenge. Various tissues were examined for gross and histological lesions and assessed for the presence of virus by PCR-HRM. All homologous-type vaccine/challenge groups exhibited protection against IBH lesions with no virus detected in the tissues. Unvaccinated groups challenged with virus showed evidence of FAdV-induced lesions; however, FAdV-8a demonstrated lower pathogenicity compared with FAdV-8b and FAdV-11. In the heterologous-type vaccine/challenge groups, FAdV-8a vaccine was shown to protect against challenge with both FAdV-8b and FAdV-11. FAdV-8a and 8b belong to species E and were therefore anticipated to cross-protect. However, FAdV-11 belongs to species D and therefore cross-protection by FAdV-8a was an uncharacteristic and unique finding of this study. Further research is required to disseminate the molecular basis for the interspecies cross-protection between FAdV-8a and FAdV-11. Nonetheless, the FAdV-8a isolate was shown to have substantial potential as a vaccine candidate in countries where FAdV-8a, 8b or 11 are prevalent.

Epidemiological investigation of fowl adenovirus infections in poultry in China during 2015-2018.

BACKGROUND: Fowl adenoviruses (FAdVs) are associated with many diseases, resulting in huge economic losses to the poultry industry worldwide. Since 2015, outbreaks of FAdV infections with high mortality rates have been reported in China. A continued surveillance of FAdVs contributes to understand the epidemiology of the viruses. RESULTS: We isolated 155 FAdV strains from diseased chickens from poultry in China between 2015 and 2018. PCR analysis determined that 123 samples were FAdV species C, 27 were FAdV species E, and five contained two different FAdV strains. The phylogenetic analysis demonstrates that these sequences of hexon regions were clustered into three distinct serotypes: FAdV-4 (79.4%, 123/155), FAdV-8a (13.5%, 21/155) and FAdV-8b (3.9%, 6/155), of which FAdV-4 was the dominant serotype in China. CONCLUSIONS: The characterization of newly prevalent FAdV strains provides valuable information for the development of an effective control strategy for FAdV infections in chickens.

Multicenter phase 1/2 application of adenovirus-specific T cells in high-risk pediatric patients after allogeneic stem cell transplantation.

BACKGROUND: Adenovirus (ADV) reactivation can cause significant morbidity and mortality in children after allogeneic stem cell transplantation. Antiviral drugs can control viremia, but viral clearance requires recovery of cell-mediated immunity. METHOD: This study was an open-label phase 1/2 study to investigate the feasibility of generating donor-derived ADV-specific T cells (Cytovir ADV, Cell Medica) and to assess the safety of pre-emptive administration of ADV-specific T cells in high-risk pediatric patients after allogeneic hematopoietic stem cell transplantation (HSCT) to treat adenoviremia. Primary safety endpoints included graft-versus-host disease (GvHD), and secondary endpoints determined antiviral responses and use of antiviral drugs. RESULTS: Between January 2013 and May 2016, 92 donors were enrolled for the production of ADV T cells at three centers in the United Kingdom (UK), and 83 products were generated from 72 mobilized peripheral blood harvests and 20 steady-state whole blood donations. Eight children received Cytovir ADV T cells after standard therapy and all resolved ADV viremia between 15 and 127 days later. ADV-specific T cells were detectable using enzyme-linked immunospot assay (ELISpot) in the peripheral blood of all patients analyzed. Serious adverse events included Grade II GvHD, Astrovirus encephalitis and pancreatitis. CONCLUSION: The study demonstrates the safety and feasibility of pre-emptively manufacturing peptide pulsed ADV-specific cells for high-risk pediatric patients after transplantation and provides early evidence of clinical efficacy.

A novel monoclonal antibody efficiently blocks the infection of serotype 4 fowl adenovirus by targeting fiber-2.

A recent outbreak of hepatitis-hydropericardium syndrome caused by serotype 4 fowl adenovirus (FAdV-4) has resulted in significant economic losses to the poultry industry worldwide. However, little is known about the molecular pathogenesis of FAdV-4. In this study, a novel monoclonal antibody (mAb) targeting the fiber-2 protein of FAdV-4 was generated, mAb 3C2. Indirect immunofluorescence assay showed that mAb 3C2 neither reacted with serotype 8 fowl adenovirus (FAdV-8) nor reacted with the fiber-1 protein of FAdV-4; it specifically reacted with the fiber-2 protein of FAdV-4. Notably, mAb 3C2 could efficiently immunoprecipitate the fiber-2 protein in chicken liver cells either infected with FAdV-4 or transfected with pcDNA3.1-Fiber2. Moreover, mAb 3C2 demonstrated marked neutralizing activity against FAdV-4 and could efficiently inhibit the infection of FAdV-4 in vitro. Using truncated fiber-2 constructs, the epitope recognized by mAb 3C2 was determined to be located between amino acids 416-448 at the C-terminus of fiber-2. Our data not only provide a foundation for the establishment of a rapid fiber-2 peptide-based diagnostic assay for FAdV-4 but also highlight the critical role of the fiber-2 protein in mediating infection by FAdV-4. Furthermore, the epitope recognized by 3C2 might serve as a novel target for the development of a vaccine targeting FAdV-4.

Newcastle disease virus-attenuated vaccine LaSota played a key role in the pathogenicity of contaminated exogenous virus.

Newcastle disease virus (NDV)-attenuated vaccine has been widely used since the 1950s and made great progress in preventing and controlling Newcastle disease. However, many reports mention exogenous virus contamination in attenuated vaccines, while co-contamination with fowl adenovirus (FAdV) and chicken infectious anaemia virus (CIAV) in the NDV-attenuated vaccine also emerged in China recently, which proved to be an important reason for the outbreaks of inclusion body hepatitis-hydropericardium syndrome in some flocks. It is amazing that exogenous virus contamination at extremely low doses still infected chickens and induced severe disease; thus, we speculated that there must be some interaction between the NDV-attenuated vaccine and the contaminated exogenous viruses within. Accordingly, simulation experiments were launched using FAdV and CIAV isolated from the abovementioned vaccine. The results showed that the pathogenicity of FAdV and CIAV co-infection through the contaminated vaccine was significantly higher than that of direct oral infection, while the synergistic reaction of these viruses and LaSota prompted their multiplication in vivo and disturbed the production of antibodies against each other. This study showed the interactions of FAdV, CIAV and LaSota after using contaminated NDV-attenuated vaccine, helping us to understand how the contaminated exogenous viruses cause infection and induce severe disease at a relatively low dose through the oral route.

Brincidofovir for Asymptomatic Adenovirus Viremia in Pediatric and Adult Allogeneic Hematopoietic Cell Transplant Recipients: A Randomized Placebo-Controlled Phase II Trial.

Adenovirus infection in immunocompromised patients contributes to significant morbidity and mortality, especially after allogeneic hematopoietic cell transplantation (HCT). Brincidofovir (BCV, CMX001) is an orally bioavailable lipid conjugate of cidofovir that has in vitro activity against adenoviruses and other double-stranded DNA viruses. This randomized placebo-controlled phase II trial evaluated pre-emptive treatment with BCV for the prevention of adenovirus disease in pediatric and adult allogeneic HCT recipients with asymptomatic adenovirus viremia. Allogeneic HCT recipients with adenovirus viremia were randomized 1:1:1 to receive oral BCV 100 mg (2 mg/kg if <50 kg) twice weekly (BIW), BCV 200 mg (4 mg/kg if <50 kg) once weekly (QW), or placebo for 6 to 12 weeks, followed by 4 weeks of post-treatment follow-up. For randomization, subjects were stratified by screening absolute lymphocyte count (<300 cells/mm3 versus ≥300 cells/mm3). Assignment to BCV or placebo was double blinded; dose frequency was unblinded. The primary endpoint was the proportion of subjects experiencing treatment failure, defined as either progression to probable or definitive adenovirus disease or confirmed increasing adenovirus viremia (≥1 log10 copies/mL) during randomized therapy. Between June 2011 and December 2012, 48 subjects were randomized to the BCV BIW (n = 14), BCV QW (n = 16), or placebo (n = 18) groups. The proportion of subjects with treatment failure in the BCV BIW group was 21% (odds ratio, .53; 95% confidence interval [CI], .11 to 2.71; P = .45), 38% (odds ratio, 1.23; 95% CI, .30 to 5.05, P = .779) in the BCV QW group, and 33% in the placebo group. All-cause mortality was lower in the BCV BIW (14%) and BCV QW groups (31%) relative to the placebo group (39%), but these differences were not statistically significant. After 1 week of therapy, 8 of 12 subjects (67%) randomized to BCV BIW had undetectable adenovirus viremia (<100 copies/mL), compared with 4 of 14 subjects (29%) randomized to BCV QW and 5 of 15 subjects (33%) randomized to placebo. In a post hoc analysis of subjects with viremia ≥1000 copies/mL at baseline, 6 of 7 BCV BIW subjects (86%) achieved undetectable viremia compared with 2 of 8 placebo subjects (25%; P = .04). Early treatment discontinuation because of adverse events was more common in subjects treated with BCV than with placebo. Diarrhea was the most common event in all groups (57% BCV BIW, 38% BCV QW, 28% placebo), but it led to treatment discontinuation in only 1 subject receiving BCV QW. Events diagnosed as acute graft-versus-host disease, primarily of the gastrointestinal tract, were more frequent in the BCV BIW group (50%) than in the BCV QW (25%) and placebo (17%) groups. There was no evidence of myelotoxicity or nephrotoxicity in BCV-treated subjects. The results of this trial confirm the antiviral activity of BCV against adenoviruses. Further investigation is ongoing to define the optimal treatment strategy for HCT recipients with serious adenovirus infection and disease.

Approach to adenovirus infections in the setting of hematopoietic cell transplantation.

PURPOSE OF REVIEW: To review risk-based pre-emptive treatment for adenovirus (ADV) based on quantitative PCR (qPCR) and to provide an update on clinical trials of brincidofovir (BCV) and ADV-specific cytotoxic T lymphocytes (ADV-CTLs). RECENT FINDINGS: ADV qPCR in various body compartments enables distinction of clinically significant infection and assessment of treatment responses. Plasma ADV qPCR monitoring and aggressive pre-emptive treatment is recommended for high-risk patients. Pre-emptive therapy at low viral load is associated with faster virologic response and improved outcomes. BCV has demonstrated virologic responses against all clinically significant ADV serotypes. Prolonged administration of oral BCV may be limited by gastrointestinal toxicity; an intravenous BCV formulation does not show gastrointestinal toxicity in early studies. ADV-CTLs can be generated from ADV-seropositive individuals using a variety of systems. Banked ADV-CTLs can achieve durable responses in patients for whom no donor-derived cell therapy product is available. SUMMARY: Disseminated ADV disease is associated with substantial mortality in hematopoietic cell transplant recipients. Routine monitoring by plasma ADV qPCR and pre-emptive therapy at low viral load are associated with improved outcomes in high-risk patients. BCV and ADV-CTLs are promising modalities currently undergoing clinical trials.