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Background: Viral enteric infections are illnesses caused by several types of viruses that primarily affect the gastrointestinal system. Globally, three major viruses associated with gastroenteritis, including norovirus (NoV), rotavirus A (RVA), and human adenovirus (HAdV), have been widely recognized. Accordingly, a rapid and sensitive diagnostic tool, such as immunochromatographic (IC) test, for diarrheal virus detection is required and helpful for rapid diagnosis., Methods: The IP-Triple I IC test kit for simultaneous triple virus detections of NoV, RVA, and HAdV was evaluated for its efficacy by using stool specimens that were known positive for these viruses by real-time RT-PCR or PCR, which was used as the gold standard method., Results: The results revealed that the IP-Triple I IC test kit exhibited a very high level of specificity (100%) for NoV, RVA, and HAdV detections, while the sensitivity was slightly different for these three viruses. The sensitivity of detection for RVA was 86.7%, whereas those for NoV and HAdV were 70.6% and 76.2%, respectively. This IP-Triple I IC kit could detect common antigens of a wide range of NoV, RVA, and HAdV genotypes (NoV GII.2, GII.4, GII.6, GII.7, GII.10, GII.17, RVA G1P[8], G2P[4], G3P[8], G8P[8], G9P[8], HAdV-C1, -C2, -C5, -A12, -F40, and -F41)., Conclusions: Our results revealed that the IP-Triple I IC test kit is highly effective for simultaneous, direct detection of common antigens of several genotypes of NoV, RVA, and HAdV in stool specimens and useful for screening and rapid diagnosis of diarrheal viruses during the seasonal outbreak.

Background: Human adenoviruses (HAdVs) frequently cause common respiratory or gastrointestinal infections among children, adults, individuals with immune deficiencies, and other vulnerable populations with varying degree of symptoms, ranging from mild to server, and in some cases, even fatalities. Despite the significant clinical impact of HAdVs, there is currently no approved vaccine available., Methods: This study explores the potential of the adenovirus type 5 fiber knob (Ad5-FK) to stimulate the production of Ad-specific neutralizing antibodies and T-cell responses in mice. Based on structure predictions, we first expressed Ad5-FK in E. coli and confirmed the assembly of FK into its trimeric form. After testing the binding capability of the trimeric FK to susceptible cells, the immunogenicity of the protein in combination with the c-di-AMP adjuvant was assessed in BALB/c mice., Results: The purified Ad5-FK exhibited self-trimerization and maintained correct conformation akin to the authentic FK structure. This facilitated effective binding to susceptible HEK293 cells. Notably, the protein demonstrated significant inhibition of HEK293 cells infection by rAd5-GFP. Immunization of BALB/c mice with Ad5-FK, or Ad5-FK mixed with c-di-AMP yielded FK-specific antibodies with potent neutralization capacity. Significantly, Ad5-FK was found to elicit a vigorous CD4 + T-cell response in the immunized mice., Conclusion: Our findings underscore the efficacy of FK-based vaccine in eliciting anti-Ad humoral immune response and CD4 T-cell immune reactions essential for protection against viral infections., (© 2024. The Author(s).)

Aim: The human adenovirus (HAdV) is beginning to spread rapidly in children through human, surface and animal vectors. Around 12,000 cases were recognised in 2022 in West Bengal and a shocking number of cases arose throughout India and in other under-developed areas. This is going to be a big threat to public health since no vaccine, awareness or protocol policies were introduced. Early detection, immediate isolation and proper policy developments are the key factors in overcoming the situation. Therefore, we performed this rapid review and discussed probable mitigation strategies, updated research on vaccine development, and treatment strategies to control the outbreaks of mutated HAdV., Design: This is a narrative review of publicly available information., Methods: Here, we extracted updated information and data using the terms HAdV outbreaks, mutations, species, risks and prevention from Google Scholar and PubMed. We considered relevant articles that have discussed prevention strategies, ongoing research, and antiviral drugs for managing HAdV outbreaks., Results: Early detection from throat swabs, isolation and symptomatic treatments are required to minimise viral infections. A massive test needs to be performed to find the affected people. The cases should be immediately isolated. It is recommended to treat high-touch surfaces with heat- or bleach-containing cleaners to prevent the spread of infection. Oxygen support and many broad-spectrum antivirals have been used to treat HAdV. Several studies showed antibody neutralisation and interactions between the natural killer cell receptor KIR3DS1 and HLA-F in infected cells, indicating possible therapeutic options in the future. HAdV-4 and HAdV-7 vaccines have been limitedly approved for administration to military personnel., Conclusion: Isolation, certain safety measures, broad-spectrum antiviral drugs and further research on new vaccines could be useful to prevent this virus from producing a worldwide pandemic. Also, the authorities should ensure the proper therapeutic interventions and nursing care facilities for the infected children., Patient or Public Contribution: Patient or public contribution was not relevant to our work., (© 2024 The Author(s). Nursing Open published by John Wiley & Sons Ltd.)

Human adenovirus (HAdV) pneumonia poses a major health burden for young children, however, factors that contribute to disease severity remain elusive. We analyzed immune cells from bronchoalveolar lavage (BAL) of children with HAdV pneumonia and found that CD19 + CD21 low B cells were significantly enriched in the BAL and were associated with increased autoantibody concentrations and disease severity. Myeloid cells, PD-1 + CD4 + T helper cells and CD21 low B cells formed tertiary lymphoid structures within the respiratory tracts. Myeloid cells promoted autoantibody production by expressing high amounts of B cell activating factor (BAFF). In contrast, PD-1 + CD4 + T helper cells induced production of IgG1 and IgG3 antibodies but suppressed autoreactive IgGs by initiating B cell receptor editing. In summary, this study reveals cellular components involved in protective versus autoreactive immune pathways in the respiratory tract, and these findings provide potential therapeutic targets for severe HAdV lower respiratory tract infections., 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human adenovirus infection (HAdV) may be fatal in patients undergoing allogeneic hematopoietic cell transplantation (HCT). Cidofovir is effective in only a part of the post-HCT HAdV infection. Therefore, posttransplant immune reconstitution is important for HAdV clearance. We describe the detailed immune reconstitution and response of adenovirus-specific T cells in a patient with inborn errors of immunity who had disseminated HAdV infection with hepatitis post-HCT and was treated with cidofovir. Though the patient received cidofovir for only 19 days starting from Day 72 after HCT because of renal dysfunction, we observed T-cell reconstitution, a decrease in HAdV copy number, and amelioration of the symptoms of HAdV infection after Day 90. We initially observed expanded NK and CD8 + CD45RO + memory subsets and later gradual increase of naïve T cells eveloped after cessation of cidofovir treatment. An increase in adenovirus-specific IFN-γ secretion from 2 to 4 months after HCT was confirmed by ELISpot assay. The progression of immune reconstitution and cidofovir treatment are considered to have contributed to survival in this patient. Optimization of transplantation methods, prompt appropriate antiviral medication, and virus-specific T-cell therapy would be necessary as the better strategy for systemic HAdV infection., Competing Interests: Declaration of competing interest We have no conflict of interest to disclose concerning this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Human adenovirus (HAdV) is a prevalent causative agent of acute respiratory disease (ARD) and is frequently responsible for outbreaks, particularly in military environments. Current vaccines do not effectively cover HAdV subtypes commonly found among Korean military personnel, highlighting the need for a new targeted vaccine. This study presents a cost-benefit analysis to evaluate the economic viability of developing and implementing such a vaccine within a military context., Methods: We adopted a societal perspective for this cost-benefit analysis, which included estimating costs associated with vaccine development, production, and distribution over a projected timeline. We assumed a development period of five years, after which vaccine production and administration were initiated in the sixth year. The cost associated with vaccine development, production, and dispensation was considered. The benefits were calculated based on both direct and indirect cost savings from preventing HAdV infections through vaccination. All financial figures were expressed in 2023 US dollars. A sensitivity analysis was conducted to explore the impact of varying factors such as vaccination rate, incidence of infection, vaccine efficacy, and discount rate., Results: For the base case scenario, we assumed a vaccination rate of 100 %, an incidence rate of 0.02, and a vaccine efficacy of 95 %, applying a 3 % discount rate. Initially, in the sixth year, the benefit-cost ratio stood at 0.71, suggesting a cost disadvantage at the onset of vaccination. However, this ratio improved to 1.32 in the following years, indicating a cost benefit from the seventh year onward. The cumulative benefit-cost ratio over a decade reached 2.72. The outcomes from the sensitivity analysis were consistent with these findings., Conclusion: Our cost-benefit analysis demonstrates that the introduction of an HAdV vaccine for the Korean military is economically advantageous, with substantial cost benefits accruing from the seventh year after the commencement of vaccination., 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 © 2024 Elsevier Ltd. All rights reserved.)

Background: Human adenovirus type 55 (hAd55) infection can lead to acute respiratory diseases that often present with severe symptoms. Despite its persistent prevalence in military camps and communities, there are no commercially available vaccines or vaccine candidates undergoing clinical evaluation; therefore, there is an urgent need to address this. In this study, we evaluated the immunogenicity of inactivated hAd55 isolates and investigated the effects of adjuvants and various immunization intervals., Methods and Results: To select a vaccine candidate, four hAd55 strains (6-9, 6-15 (AFMRI 41014), 28-48 (AFMRI 41013), and 12-164 (AFMRI 41012)) were isolated from infected patients in military camps. Sequence analysis revealed no variation in the coding regions of structural proteins, including pentons, hexons, and fibers. Immunization with inactivated hAd55 isolates elicited robust hAd55-specific binding and neutralizing antibody responses in mice, with adjuvants, particularly alum hydroxide (AH), enhancing antibody titers. Co-immunization with AH also induced hAd14-specific neutralizing antibody responses but did not induce hAd11-specific neutralizing antibody responses. Notably, booster immunization administered at a four-week interval resulted in superior immune responses compared with shorter immunization intervals., Conclusions: Prime-boost immunization with the inactivated hAd55 isolate and an AH adjuvant shows promise as a potential approach for preventing hAd55-induced respiratory disease. Further research is needed to evaluate the efficacy and safety of these vaccine candidates in preventing hAd55-associated respiratory illnesses., (© 2024. The Author(s).)

Adenovirus (Ad) vectors based on human adenovirus serotype 5 (Ad5) have attracted significant attention as vaccine vectors for infectious diseases. However, the effectiveness of Ad5 vectors as vaccines is often inhibited by the anti-Ad5 neutralizing antibodies retained by many adults. To overcome this drawback, we focused on human adenovirus serotype 35 (Ad35) vectors with low seroprevalence in adults. Although Ad35 vectors can circumvent anti-Ad5 neutralizing antibodies, vector yields of Ad35 vectors are often inferior to those of Ad5 vectors. In this study, we developed novel Ad35 vectors containing the Ad5 E4 orf 4, 6, and 6/7 or the Ad5 E4 orf 6 and 6/7 for efficient vector production, and compared their properties. These E4-modified Ad35 vectors efficiently propagated to a similar extent at virus titers comparable to those of Ad5 vectors. An Ad35 vector containing the Ad5 E4 orf 4, 6, and 6/7 mediated more efficient transduction than that containing the Ad5 E4 orf 6 and 6/7 in human cultured cells. Furthermore, insertion of an arginine-glycine-aspartate (RGD) peptide in the fiber region of an Ad35 vector containing the Ad5 E4 orf 4, 6, and 6/7 significantly improved the transgene product-specific antibody production following intramuscular administration in mice. The Ad35 vector containing the RGD peptide mediated efficient vaccine effects even in the mice pre-immunized with an Ad5., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hiroyuki Mizuguchi reports financial support was provided by Ministry of Education, Culture, Sports, Sciences, and Technology of Japan. Hiroyuki Mizuguchi reports financial support was provided by Japanese Agency for Medical Research and Development. Hiroyuki Mizuguchi reports financial support was provided by BIKEN Foundation. If there are other authors, they 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 © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Human adenoviruses (HAdVs) are small DNA viruses that generally cause mild disease. Certain strains, particularly those belonging to species B HAdVs, can cause severe pneumonia and have a relatively high mortality rate. Little is known about the molecular aspects of how these highly pathogenic species affect the infected cell and how they suppress innate immunity. The present study provides molecular insights into how species B adenoviruses suppress the interferon signaling pathway. Our study shows that these viruses, unlike HAdV-C2, are resistant to type I interferon. This resistance likely arises due to the highly efficient suppression of interferon-stimulated gene expression. Unlike in HAdV-C2, HAdV-B7 and B14 sequester STAT2 and RNA polymerase II from interferon-stimulated gene promoters in infected cells. This results in suppressed interferon- stimulated gene activation. In addition, we show that RuvBL1 and RuvBL2, cofactors important for RNA polymerase II recruitment to promoters and interferon-stimulated gene activation, are redirected to the cytoplasm forming high molecular weight complexes that, likely, are unable to associate with chromatin. Proteomic analysis also identified key differences in the way these viruses affect the host cell, providing insights into species B-associated high pathogenicity. Curiously, we observed that at the level of protein expression changes to the infected cell, HAdV-C2 and B7 were more similar than those of the same species, B7 and B14. Collectively, our study represents the first such study of innate immune suppression by the highly pathogenic HAdV-B7 and B14, laying an important foundation for future investigations.IMPORTANCEHuman adenoviruses form a large family of double-stranded DNA viruses known for a variety of usually mild diseases. Certain strains of human adenovirus cause severe pneumonia leading to much higher mortality and morbidity than most other strains. The reasons for this enhanced pathogenicity are unknown. Our study provides a molecular investigation of how these highly pathogenic strains might inactivate the interferon signaling pathway, highlighting the lack of sensitivity of these viruses to type I interferon in general while providing a global picture of how viral changes in cellular proteins drive worse disease outcomes., Competing Interests: The authors declare no conflict of interest.

Introduction: The early transcription unit 3 (E3) of human adenoviruses (HAdVs) encodes several immunoevasins, including the E3/49K protein, which is unique for species D of HAdVs. It is expressed as surface transmembrane protein and shed. E3/49K of HAdV-D64 binds to the protein tyrosine phosphatase surface receptor CD45, thereby modulating activation of T and NK cells., Methods: Considering that E3/49K represents the most polymorphic viral protein among species D HAdVs, we demonstrate here that all tested E3/49K orthologs bind to the immunologically important regulator CD45. Thus, this feature is conserved regardless of the pathological associations of the respective HAdV types., Results: It appeared that modulation of CD45 is a unique property restricted to HAdVs of species D. Moreover, E3/49K treatment inhibited B cell receptor (BCR) signaling and impaired BCR signal phenotypes. The latter were highly comparable to B cells having defects in the expression of CD45, suggesting E3/49K as a potential tool to investigate CD45 specific functions., Conclusion: We identified B cells as new direct target of E3/49K-mediated immune modulation, representing a novel viral immunosubversive mechanism., Competing Interests: Author MB was employed by Navita S.r.l. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hildenbrand, Cramer, Bertolotti, Kaiser, Kläsener, Nickel, Reth, Heim, Hengel, Burgert and Ruzsics.)

Human Adenoviruses (HAdV) are nearly ubiquitous pathogens comprising numerous sub-types that infect various tissues and organs. Among many encoded proteins that facilitate viral replication and subversion of host cellular processes, the viral E4orf1 protein has emerged as an intriguing yet under-investigated player in the complex interplay between the virus and its host. E4orf1 has gained attention as a metabolism activator and oncogenic agent, while recent research is showing that E4orf1 may play a more important role in modulating cellular pathways such as PI3K-Akt-mTOR, Ras, the immune response and further HAdV replication stages than previously anticipated. In this review, we aim to explore the structure, molecular mechanisms, and biological functions of E4orf1, shedding light on its potentially multifaceted roles during HAdV infection, including metabolic diseases and oncogenesis. Furthermore, we discuss the role of functional E4orf1 in biotechnological applications such as Adenovirus (AdV) vaccine vectors and oncolytic AdV. By dissecting the intricate relationships between HAdV types and E4orf1 proteins, this review provides valuable insights into viral pathogenesis and points to promising areas of future research., 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 © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Detection of pathogenic viruses for point-of-care applications has attracted great attention since the COVID-19 pandemic. Current virus diagnostic tools are laborious and expensive, while requiring medically trained staff. Although user-friendly and cost-effective biosensors are utilized for virus detection, many of them rely on recognition elements that suffer major drawbacks. Herein, computationally designed epitope-imprinted polymers (eIPs) are conjugated with a portable piezoelectric sensing platform to establish a sensitive and robust biosensor for the human pathogenic adenovirus (HAdV). The template epitope is selected from the knob part of the HAdV capsid, ensuring surface accessibility. Computational simulations are performed to evaluate the conformational stability of the selected epitope. Further, molecular dynamics simulations are executed to investigate the interactions between the epitope and the different functional monomers for the smart design of eIPs. The HAdV epitope is imprinted via the solid-phase synthesis method to produce eIPs using in silico-selected ingredients. The synthetic receptors show a remarkable detection sensitivity (LOD: 10 2 pfu mL -1 ) and affinity (dissociation constant ( K d ): 6.48 × 10 -12 M) for HAdV. Moreover, the computational eIPs lead to around twofold improved binding behavior than the eIPs synthesized with a well-established conventional recipe. The proposed computational strategy holds enormous potential for the intelligent design of ultrasensitive imprinted polymer binders.

Human adenovirus type 7 (HAdV-7) is a significant viral pathogen that causes respiratory infections in children. Currently, there are no specific antiviral drugs or vaccines for children targeting HAdV-7, and the mechanisms of its pathogenesis remain unclear. The NLRP3 inflammasome-driven inflammatory cascade plays a crucial role in the host's antiviral immunity. Our previous study demonstrated that HAdV-7 infection activates the NLRP3 inflammasome. Building upon this finding, our current study has identified the L4 100 kDa protein encoded by HAdV-7 as the primary viral component responsible for NLRP3 inflammasome activation. By utilizing techniques such as co-immunoprecipitation, we have confirmed that the 100 kDa protein interacts with the NLRP3 protein and facilitates the assembly of the NLRP3 inflammasome by binding specifically to the NACHT and LRR domains of NLRP3. These insights offer a deeper understanding of HAdV-7 pathogenesis and contribute to the development of novel antiviral therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Duan, Zhu, Zhang, Wang, Zhang, Tian, Li, Ai, Wang and Xie.)

The exacerbation of pneumonia in children with human adenovirus type 3 (HAdV-3E) is secondary to a Staphylococcus aureus ( S. aureus ) infection. The influence of host-pathogen interactions on disease progression remains unclear. It is important to note that S. aureus infections following an HAdV-3E infection are frequently observed in clinical settings, yet the underlying susceptibility mechanisms are not fully understood. This study utilized an A549 cell model to investigate secondary infection with S. aureus following an HAdV-3E infection. The findings suggest that HAdV-3E exacerbates the S. aureus infection by intensifying lung epithelial cell damage. The results highlight the role of HAdV-3E in enhancing the interferon signaling pathway through RIG-I ( DDX58 ), resulting in the increased expression of interferon-stimulating factors like MX1 , RSAD2 , and USP18 . The increase in interferon-stimulating factors inhibits the NF-κB and MAPK/P38 pro-inflammatory signaling pathways. These findings reveal new mechanisms of action for HAdV-3E and S. aureus in secondary infections, enhancing our comprehension of pathogenesis.

Background: Human adenoviruses (HAdVs) are extensively used as vectors for vaccines development and cancer therapy. People who already have antibodies against HAdVs, on the other hand, would have an impact on the preventative or therapeutic effect. This review focuses primarily on the prevalence of pre-existing antibodies against HAdVs in distinct geographical populations., Summary: After screening, 64 studies from 31 countries between 1962 and 2021 were selected, totaling 39,427 samples. The total prevalence of preexisting antibodies to HAdVs varied by country or location, ranging from 2.00 to 95.70%. Southeast Asia had the highest prevalence (54.57%) while Europe had the lowest (18.17%). The prevalence in practically all developing nations was higher than in developed nations. Adults have a greater frequency than children and newborns in most nations. The primary HAdV antibody types varied by country. Adults in China, the USA, the United Kingdom, and Belgium had the lowest prevalence of preexisting antibodies against HAdV55, HAdV37, HAdV8, and HAdV36, respectively. Children in the USA, China, the United Kingdom, and Japan had the lowest rates of HAdV48, HAdV11, HAdV8, and HAdV40. The frequency of antibodies differed significantly between military and civilian groups., Key Messages: Preexisting antibodies against various types of HAdVs differed greatly throughout worldwide populations. Future development of HAdV-vector vaccines and medicines should focus on preexisting antibodies in target groups rather than a "one-size-fits-all" strategy. It might be advantageous in selecting HAdV vectors for studying the prevalence of preexisting antibodies against HAdVs in different locations and people throughout the world., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Intramuscular delivery of human adenovirus (HAdV)-based vaccines leads to rapid recruitment of neutrophils, which then release antimicrobial peptides/proteins (AMPs). How these AMPs influence vaccine efficacy over the subsequent 24 h is poorly understood. In this study, we asked if human neutrophil protein 1 (HNP-1), an α-defensin that influences direct and indirect innate immune responses to a range of pathogens, impacts the response of human phagocytes to three HAdV species/types (HAdV-C5, -D26, -B35). We show that HNP-1 binds to the capsids and redirects HAdV-C5, -D26, and -B35 to Toll-like receptor 4 (TLR4), which leads to internalization, an NLRP3-mediated inflammasome response, and interleukin 1 beta (IL-1β) release. Surprisingly, IL-1β release was not associated with notable disruption of plasma membrane integrity. These data further our understanding of HAdV vaccine immunogenicity and may provide pathways to extend the efficacy. IMPORTANCE This study examines the interactions between danger-associated molecular patterns and human adenoviruses, and their impact on vaccines. HAdVs and HNP-1 can interact, and these interactions will modify the response of antigen-presenting cells, which will influence vaccine efficacy.

Human adenoviruses (HAdV) cause a variety of infections in human hosts, from self-limited upper respiratory tract infections in otherwise healthy people to fulminant pneumonia and death in immunocompromised patients. Many HAdV enter polarized epithelial cells by using the primary receptor, the Coxsackievirus and adenovirus receptor (CAR). Recently published data demonstrate that a potent neutrophil (PMN) chemoattractant, interleukin-8 (IL-8), stimulates airway epithelial cells to increase expression of the apical isoform of CAR (CAR Ex8 ), which results in increased epithelial HAdV type 5 (HAdV5) infection. However, the mechanism for PMN-enhanced epithelial HAdV5 transduction remains unclear. In this manuscript, the molecular mechanisms behind PMN mediated enhancement of epithelial HAdV5 transduction are characterized using an MDCK cell line that stably expresses human CAR Ex8 under a doxycycline inducible promoter (MDCK-CAR Ex8 cells). Contrary to our hypothesis, PMN exposure does not enhance HAdV5 entry by increasing CAR Ex8 expression nor through activation of non-specific epithelial endocytic pathways. Instead, PMN serine proteases are responsible for PMN-mediated enhancement of HAdV5 transduction in MDCK-CAR Ex8 cells. This is evidenced by reduced transduction upon inhibition of PMN serine proteases and increased transduction upon exposure to exogenous human neutrophil elastase (HNE). Furthermore, HNE exposure activates epithelial autophagic flux, which, even when triggered through other mechanisms, results in a similar enhancement of epithelial HAdV5 transduction. Inhibition of F-actin with cytochalasin D partially attenuates PMN mediated enhancement of HAdV transduction. Taken together, these findings suggest that HAdV5 can leverage innate immune responses to establish infections.

Adenovirus (Ad) is a major causal agent of acute respiratory infections. However, they are a powerful delivery system for gene therapy and vaccines. Some Ad serotypes antagonize the immune system leading to meningitis, conjunctivitis, gastroenteritis, and/or acute hemorrhagic cystitis. Studies have shown that the release of small, membrane-derived extracellular vesicles (EVs) may offer a mechanism by which viruses can enter cells via receptor-independent entry and how they influence disease pathogenesis and/or host protection considering their existence in almost all bodily fluids. We proposed that Ad3 could alter EV biogenesis, composition, and trafficking and may stimulate various immune responses in vitro . In the present study, we evaluated the impact of in vitro infection with Ad3 vector on EV biogenesis and composition in the human adenocarcinoma lung epithelial cell line A549. Cells were infected in an exosome-free media at different multiplicity of infections (MOIs) and time points. The cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorometric calcein-AM. EVs were isolated via ultracentrifugation. Isolated EV proteins were quantified and evaluated via nanoparticle tracking, transmission electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting assays. The cell viability significantly decreased with an increase in MOI and incubation time. A significant increase in particle mean sizes, concentrations, and total EV protein content was detected at higher MOIs when compared to uninfected cells (control group). A549 cell-derived EVs revealed the presence of TSG101, tetraspanins CD9 and CD63, and heat shock proteins 70 and 100 with significantly elevated levels of Rab5, 7, and 35 at higher MOIs (300, 750, and 1500) when compared to the controls. Our findings suggested Ad3 could modulate EV biogenesis, composition, and trafficking which could impact infection pathogenesis and disease progression. This study might suggest EVs could be diagnostic and therapeutic advancement to Ad infections and other related viral infections. However, further investigation is warranted to explore the underlying mechanism(s)., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Ayodeji O. Ipinmoroti et al.)

Human adenovirus types 4 (HAdV4) and 7 (HAdV7) often lead to severe respiratory diseases and occur epidemically in children, adults, immune deficiency patients, and other groups, leading to mild or severe symptoms and even death. However, no licensed adenovirus vaccine has been approved in the market for general use. E3 genes of adenovirus are generally considered nonessential for virulence and replication; however, a few studies have demonstrated that the products of these genes are also functional. In this study, most of the E3 genes were deleted, and two E3-deleted recombinant adenoviruses (ΔE3-rAdVs) were constructed as components of the vaccine. After E3 deletion, the replication efficiencies and cytopathogenicity of ΔE3-rAdVs were reduced, indicating that ΔE3-rAdVs were attenuated after E3 genes deletion. Furthermore, single immunization with live-attenuated bivalent vaccine candidate protects mice against challenge with wild-type human adenovirus types 4 and 7, respectively. Vaccinated mice demonstrated remarkably decreased viral loads in the lungs and less lung pathology compared to the control animals. Taken together, our study confirms the possibility of the two live-attenuated viruses as a vaccine for clinic use and illustrates a novel strategy for the construction of an adenovirus vaccine.

Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination. IMPORTANCE Adenoviral vectors are under investigation for a broad range of therapeutic indications in diverse fields, such as oncology and gene therapy, as well as for vaccination both for human and veterinary use. A wealth of data shows that preexisting immune responses may limit the use of a vector. Particularly in the current climate of global pandemic, there is a need to expand the toolbox with novel adenoviral vectors for vaccine development. Our data demonstrate that we have successfully vectorized a novel adenovirus type candidate with low seroprevalence. The cell transduction data and antigen-specific immune responses induced in vivo demonstrate that this vector is highly promising for the development of gene therapy and vaccine products.

Human adenoviruses (HAdVs) are a major cause for disease in children, in particular after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Currently, effective therapies for HAdV infections in immunocompromised hosts are lacking. To decipher immune recognition of HAdV infection and determine new targets for immune-mediated control, we used an HAdV infection 3D organoid system, based on primary human intestinal epithelial cells. HLA-F, the functional ligand for the activating NK cell receptor KIR3DS1, was strongly up-regulated and enabled enhanced killing of HAdV5-infected cells in organoids by KIR3DS1 + NK cells. In contrast, HLA-A and HLA-B were significantly down-regulated in HAdV5-infected organoids in response to adenoviral E3/glycoprotein19K, consistent with evasion from CD8 + T cells. Immunogenetic analyses in a pediatric allo-HSCT cohort showed a reduced risk to develop severe HAdV disease and faster clearance of HAdV viremia in children receiving KIR3DS1 + / HLA-Bw4 + donor cells compared with children receiving non– KIR3DS1 + / HLA-Bw4 + cells. These findings identify the KIR3DS1/HLA-F axis as a new target for immunotherapeutic strategies against severe HAdV disease.

We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 10 6 TCID 50 ) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge., Competing Interests: Authors EG, MU, AR, JS, PS, LZ, ABez, AS, BM, HA, RW, KD, JB, VP, HG, PL, PS, SR, KN and PS-S were employed by company ImmunityBio, Inc. Author LS was employed by company NantKwest, Inc. Authors ABac, PB and JD were employed by company IosBio. The authors declare that this study received funding from ImmunityBio, Inc. The funder had the following involvement in the study: design and development of the vaccine and all NHP study-related analyses., (Copyright © 2021 Gabitzsch, Safrit, Verma, Rice, Sieling, Zakin, Shin, Morimoto, Adisetiyo, Wong, Bezawada, Dinkins, Balint, Peykov, Garban, Liu, Bacon, Bone, Drew, Sanford, Spilman, Sender, Rabizadeh, Niazi and Soon-Shiong.)

Following repeat exposure to many human adenoviruses (HAdVs), most adults harbour long-lived B- and T-cell responses. Combined, this response typically protects us for years from re-infection by the same HAdV type. In spite of these immune responses, some HAdV types are associated with persistent infections that constitute a life-threatening risk when an individual's T-cell response is compromised. By contrast, patients with B-cell deficiencies do not appear to be at a greater risk of HAdV disease. This dichotomy begs the question of the secondary role of anti-HAdV antibodies during host defence. In this study, we explored IgG-complexed (IC)-HAdV5 and primary human plasmacytoid dendritic cell (pDC) interactions. We found that IC-HAdV5 are efficiently internalized in pDCs, stimulate their activation through TLR9 signalling, and cause apoptosis. These data may help reconcile the enigma of robust immune response to HAdVs, while concurrently allowing persistence.

Reactivation of Human Cytomegalovirus (HCMV) and Human Adenovirus (HAdV) in immunocompromised patients following stem cell transplantation (SCT) or solid organ transplantation (SOT) is associated with high morbidity and mortality. The adoptive transfer of virus-specific CD8 + and CD4 + T cells has been shown to re-establish the antiviral T-cell response and improve clinical outcome. The viral load in immunocompromised patients can efficiently be reduced solely by the infusion of virus-specific CD4 + T cells. The identification of CD4 + T-cell epitopes has mainly focused on a limited number of viral proteins that were characterized as immunodominant. Here, we used in silico prediction to determine promiscuous CD4 + T-cell epitopes from the entire proteomes of HCMV and HAdV. Immunogenicity testing with enzyme-linked immuno spot (ELISpot) assays and intracellular cytokine staining (ICS) revealed numerous novel CD4 + T-cell epitopes derived from a broad spectrum of viral antigens. We identified 17 novel HCMV-derived and seven novel HAdV-derived CD4 + T-cell epitopes that were recognized by > 50% of the assessed peripheral blood mononuclear cell (PBMC) samples. The newly identified epitopes were pooled with previously published, retested epitopes to stimulate virus-specific memory T cells in PBMCs from numerous randomly selected blood donors. Our peptide pools induced strong IFNγ secretion in 46 out of 48 (HCMV) and 31 out of 31 (HAdV) PBMC cultures. In conclusion, we applied an efficient method to screen large viral proteomes for promiscuous CD4 + T-cell epitopes to improve the detection and isolation of virus-specific T cells in a clinical setting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Höttler, März, Lübke, Rammensee and Stevanović.)

Adenovirus vector-based genetic vaccines have emerged as a powerful strategy against the SARS-CoV-2 health crisis. This success is not unexpected because adenoviruses combine many desirable features of a genetic vaccine. They are highly immunogenic and have a low and well characterized pathogenic profile paired with technological approachability. Ongoing efforts to improve adenovirus-vaccine vectors include the use of rare serotypes and non-human adenoviruses. In this review, we focus on the viral capsid and how the choice of genotypes influences the uptake and subsequent subcellular sorting. We describe how understanding capsid properties, such as stability during the entry process, can change the fate of the entering particles and how this translates into differences in immunity outcomes. We discuss in detail how mutating the membrane lytic capsid protein VI affects species C viruses' post-entry sorting and briefly discuss if such approaches could have a wider implication in vaccine and/or vector development.

Human adenoviruses (HAdVs) are a large family of DNA viruses that include more than 100 genotypes divided into seven species (A to G) and induce respiratory tract infections, gastroenteritis, and conjunctivitis. Genetically modified adenoviruses are also used as vaccines, gene therapies, and anticancer treatments. The APOBEC3s are a family of cytidine deaminases that restrict viruses by introducing mutations in their genomes. Viruses developed different strategies to cope with the APOBEC3 selection pressure, but nothing is known on the interplay between the APOBEC3s and the HAdVs. In this study, we focused on three HAdV strains: the B3 and C2 strains, as they are very frequent, and the A12 strain, which is less common but is oncogenic in animal models. We demonstrated that the three HAdV strains induce a similar APOBEC3B upregulation at the transcriptional level. At the protein level, however, APOBEC3B is abundantly expressed during HAdV-A12 and -C2 infection and shows a nuclear distribution. On the contrary, APOBEC3B is barely detectable in HAdV-B3-infected cells. APOBEC3B deaminase activity is detected in total protein extracts upon HAdV-A12 and -C2 infection. Bioinformatic analysis demonstrates that the HAdV-A12 genome bears a stronger APOBEC3 evolutionary footprint than that of the HAdV-C2 and HAdV-B3 genomes. Our results show that HAdV infection triggers the transcriptional upregulation of the antiviral innate effector APOBEC3B. The discrepancies between the APOBEC3B mRNA and protein levels might reflect the ability of some HAdV strains to antagonize the APOBEC3B protein. These findings point toward an involvement of APOBEC3B in HAdV restriction and evolution. IMPORTANCE The APOBEC3 family of cytosine deaminases has important roles in antiviral innate immunity and cancer. Notably, APOBEC3A and APOBEC3B are actively upregulated by several DNA tumor viruses and contribute to transformation by introducing mutations in the cellular genome. Human adenoviruses (HAdVs) are a large family of DNA viruses that cause generally asymptomatic infections in immunocompetent adults. HAdVs encode several oncogenes, and some HAdV strains, like HAdV-A12, induce tumors in hamsters and mice. Here, we show that HAdV infection specifically promotes the expression of the APOBEC3B gene. We report that infection with the A12 strain induces a strong expression of an enzymatically active APOBEC3B protein in bronchial epithelial cells. We provide bioinformatic evidence that HAdVs' genomes and notably the A12 genome are under APOBEC3 selection pressure. Thus, APOBEC3B might contribute to adenoviral restriction, diversification, and oncogenic potential of particular strains.

Despite decades of clinical and preclinical investigations, we still poorly grasp our innate immune response to human adenoviruses (HAdVs) and their vectors. In this study, we explored the impact of lactoferrin on three HAdV types that are being used as vectors for vaccines. Lactoferrin is a secreted globular glycoprotein that influences direct and indirect innate immune response against a range of pathogens following a breach in tissue homeostasis. The mechanism by which lactoferrin complexes increases HAdV uptake and induce maturation of human phagocytes is unknown. We show that lactoferrin redirects HAdV types from species B, C, and D to Toll-like receptor 4 (TLR4) cell surface complexes. TLR4-mediated internalization of the HAdV-lactoferrin complex induced an NLRP3-associated response that consisted of cytokine release and transient disruption of plasma membrane integrity, without causing cell death. These data impact our understanding of HAdV immunogenicity and may provide ways to increase the efficacy of HAdV-based vectors/vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chéneau, Eichholz, Tran, Tran, Paris, Henriquet, Bajramovic, Pugniere and Kremer.)

BACKGROUNDTo understand the features of a replicating vaccine that might drive potent and durable immune responses to transgene-encoded antigens, we tested a replication-competent adenovirus type 4 encoding influenza virus H5 HA (Ad4-H5-Vtn) administered as an oral capsule or via tonsillar swab or nasal spray.METHODSViral shedding from the nose, mouth, and rectum was measured by PCR and culturing. H5-specific IgG and IgA antibodies were measured by bead array binding assays. Serum antibodies were measured by a pseudovirus entry inhibition, microneutralization, and HA inhibition assays.RESULTSAd4-H5-Vtn DNA was shed from most upper respiratory tract-immunized (URT-immunized) volunteers for 2 to 4 weeks, but cultured from only 60% of participants, with a median duration of 1 day. Ad4-H5-Vtn vaccination induced increases in H5-specific CD4+ and CD8+ T cells in the peripheral blood as well as increases in IgG and IgA in nasal, cervical, and rectal secretions. URT immunizations induced high levels of serum neutralizing antibodies (NAbs) against H5 that remained stable out to week 26. The duration of viral shedding correlated with the magnitude of the NAb response at week 26. Adverse events (AEs) were mild, and peak NAb titers were associated with overall AE frequency and duration. Serum NAb titers could be boosted to very high levels 2 to 5 years after Ad4-H5-Vtn vaccination with recombinant H5 or inactivated split H5N1 vaccine.CONCLUSIONReplicating Ad4 delivered to the URT caused prolonged exposure to antigen, drove durable systemic and mucosal immunity, and proved to be a promising platform for the induction of immunity against viral surface glycoprotein targets.TRIAL REGISTRATIONClinicalTrials.gov NCT01443936 and NCT01806909.FUNDINGIntramural and Extramural Research Programs of the NIAID, NIH (U19 AI109946) and the Centers of Excellence for Influenza Research and Surveillance (CEIRS), NIAID, NIH (contract HHSN272201400008C).

The aim of this review is to highlight how, in a syngeneic system, human mononuclear phagocytes respond to environments containing human adenovirus (HAdV) and soluble extracellular proteins that influence their innate immune response. Soluble extracellular proteins, including immunoglobulins, blood clotting factors, proteins of the complement system, and/or antimicrobial peptides (AMPs) can exert direct effects by binding to a virus capsid that modifies interactions with pattern recognition receptors and downstream signaling. In addition, the presence, generation, or secretion of extracellular proteins can indirectly influence the response to HAdVs via the activation and recruitment of cells at the site of infection.

Human adenovirus type 3 (HAdV-3) encompasses 15-87% of all adenoviral respiratory infections. The significant morbidity and mortality, especially among the neonates and immunosuppressed patients, demand the need for a vaccine or a targeted antiviral against this type. However, due to the existence of multiple hexon variants (3Hv-1 to 3Hv-25), the selection of vaccine strains of HAdV-3 is challenging. This study was designed to evaluate HAdV-3 hexon variants for the selection of potential vaccine candidates and the use of hexon gene as a target for designing siRNA that can be used as a therapy. Based on the data of worldwide distribution, duration of circulation, co-circulation and their percentage among all the variants, 3Hv-1 to 3Hv-4 were categorized as the major hexon variants. Phylogenetic analysis and the percentage of homology in the hypervariable regions followed by multi-sequence alignment, zPicture analysis and restriction enzyme analysis were carried out. In the phylogram, the variants were arranged in different clusters. The HVR encoding regions of hexon of 3Hv-1 to 3Hv-4 showed 16 point mutations resulting in 12 amino acids substitutions. The homology in HVRs was 81.81-100%. Therefore, the major hexon variants are substantially different from each other which justifies their inclusion as the potential vaccine candidates. Interestingly, despite the significant differences in the DNA sequence, there were many conserved areas in the HVRs, and we have designed functional siRNAs form those locations. We have also designed immunogenic vaccine peptide epitopes from the hexon protein using bioinformatics prediction tool. We hope that our developed siRNAs and immunogenic vaccine peptide epitopes could be used in the future development of siRNA-based therapy and designing a vaccine against HAdV-3., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Adenovirus (AdV) infections in the respiratory tract may cause asthma exacerbation and allergic predisposition, and the house dust mite (HDM) may aggravate virus-induced asthma exacerbations. However, the underlying mechanisms of whether and how AdV affects asthmatic patients remains unclear. To address this question, we investigated nasal epithelial cells (NAEPCs) derived from a pediatric exacerbation study cohort for experimental analyses. We analyzed twenty-one different green-fluorescent protein- and luciferase-tagged AdV types in submerged 2D and organotypic 3D cell culture models. Transduction experiments revealed robust transduction of AdV type 5 (AdV5) in NAEPCs, which was associated with an increased uptake of AdV5 in the presence of HDM. In healthy and asthmatic NAEPCs exposed to HDM before infection, we observed a time- and dose-dependent increase of AdV5 uptake associated with upregulation of entry receptors for AdV5. Furthermore, electron microscopic and histologic analyses of 3D cell cultures revealed an impairment of the respiratory cilia after HDM exposition. This ex vivo pilot study shows the impact of AdV infection and HDM exposition in a primary cell culture model for asthma.

Purpose of Review: To review the roles of enteric adenovirus types 40 and 41 and nonenteric adenoviruses in the global burden of pediatric diarrhea., Recent Findings: Large studies using highly sensitive, type-specific molecular diagnostics have demonstrated a substantial and previously under-estimated burden of pediatric diarrheal disease because of enteric infections with adenovirus types 40/41. However, the true epidemiology of adenovirus 40/41 remains incompletely understood. Similarly, additional adenovirus types may also be implicated as agents of community-acquired pediatric gastroenteritis but current data are too limited to elucidate their epidemiological role(s), if any., Summary: Efforts at global diarrhea control in low-income and middle-income countries will require combating pediatric gastroenteritis because of enteric adenovirus infections. Future research in these settings using type-specific molecular diagnostics or strain genotyping to fully characterize the epidemiology of adenovirus 40/41 infections, identify non-40/41 adenoviruses significantly associated with gastroenteritis, and develop vaccines effective at preventing adenovirus diarrhea is warranted.

Human Adenovirus (AdV) infection is very common and usually has a significant impact on children. AdV-induced inflammation is believed to be one of the main causes of severe symptoms. However, an inflammatory response profile in the airway in AdV-infected children is still lacking, and the mechanism underlying AdV-induced inflammation in the airway is also poorly understood. In the current study, we determined the expression of a panel of inflammation cytokines in the airway samples from AdV 7 infected children and further investigated the molecular mechanism underlying AdV 7-induced cytokine expression. Our results showed that eight out of 13 tested inflammatory cytokines were significantly increased in nasal washes of AdV 7-infected children comparing to healthy control, with IL-6 showing the highest enhancement. AdV 7 infection of bronchial epithelial cell line and primary airway epithelial cells confirmed that AdV 7 increased IL-6 mRNA and protein expression in an infection dose-dependent manner. Promoter analysis revealed that AdV 7 infection transactivated IL-6 promoter and a NF-κB binding site in IL-6 promoter was involved in the transactivation. Further analysis showed that upon AdV 7 infection, NF-κB p65 was phosphorylated and translocated into nucleus and bound onto IL-6 promoter. Signaling pathway analysis revealed that p38/NF-κB pathway was involved in AdV 7 infection induced IL-6 elevation. Taken together, our study shows that AdV 7 infection triggers the expression of a range of inflammatory cytokines including IL-6 in the airway of infected children, and AdV 7 enhances IL-6 expression by transactivating IL-6 promoter via p38/NF-κB signaling pathway. Findings of our current study have provided more information toward a better understanding of AdV-induced airway inflammation, which might also benefit the development of intervention strategies., (Copyright © 2020 Qi, Wang, Wang and Deng.)

We conducted a seroprevalence study of a large ongoing outbreak of human adenovirus type 55 (HAdV-55) among the military in South Korea. Serum samples were collected between 2018 and 2019 from military-exposed (military group) and non-exposed (non-military group) populations. The plaque reduction neutralization test (PRNT) was used to assess neutralization activity against HAdV-55. A total of 100 sera was collected from the non-military group, of which 18.8% showed HAdV-55 neutralizing antibody activity. Ninety-six sera were tested from the military group, which had significantly higher prevalence of neutralizing antibodies (56.0%, P <0.001). A significantly higher proportion of the military group had PRNT titers ≥1:1,000 than the non-military group (85.7% vs. 50.0%, P = 0.004). Among the military group, 48.9% of active-duty soldiers had PRNT titers ≥1:5,000, while none of the discharged civilians did (P = 0.007). In conclusion, Koreans were exposed to HAdV-55 in their communities, but the exposure risk was higher among people in military service., Competing Interests: The authors have declared that no competing interests exist.

In this review, we discuss the use of oncolytic viruses in cancer immunotherapy treatments in general, with a particular focus on adenoviruses. These serve as a model to elucidate how versatile viruses are, and how they can be used to complement other cancer therapies to gain optimal patient benefits. Historical reports from over a hundred years suggest treatment efficacy and safety with adenovirus and other oncolytic viruses. This is confirmed in more contemporary patient series and multiple clinical trials. Yet, while the first viruses have already been granted approval from several regulatory authorities, room for improvement remains.As good safety and tolerability have been seen, the oncolytic virus field has now moved on to increase efficacy in a wide array of approaches. Adding different immunomodulatory transgenes to the viruses is one strategy gaining momentum. Immunostimulatory molecules can thus be produced at the tumor with reduced systemic side effects. On the other hand, preclinical work suggests additive or synergistic effects with conventional treatments such as radiotherapy and chemotherapy. In addition, the newly introduced checkpoint inhibitors and other immunomodulatory drugs could make perfect companions to oncolytic viruses. Especially tumors that seem not to be recognized by the immune system can be made immunogenic by oncolytic viruses. Logically, the combination with checkpoint inhibitors is being evaluated in ongoing trials. Another promising avenue is modulating the tumor microenvironment with oncolytic viruses to allow T cell therapies to work in solid tumors.Oncolytic viruses could be the next remarkable wave in cancer immunotherapy.

Background: Mycoplasma pneumoniae (M. pneumoniae) is an important pathogen of community-acquired pneumonia (CAP) in children. The coinfection rate of M. pneumoniae pneumonia (MPP) can reach 52% in some areas, but the effects of coinfection with different pathogens have not been clearly recognized., Methods: The cases of MPP hospitalized in Beijing Children's Hospital from 1/1/2014 to 12/31/2016 were screened. MPP patients coinfected with Human adenovirus (HAdV) were categorized into the research group. Patients with single M. pneumoniae infection were categorized into the control group, matching the research group by age and admission time with a ratio of 1:3. Clinical manifestations, laboratory examinations, and disease severity were compared between these two groups., Results: A total of 2540 hospitalized MPP cases were screened in Beijing Children's Hospital, among which thirty cases were enrolled in the research group and ninety cases were enrolled in the control group. The results indicated that patients in the research group had longer hospital stays, longer fever durations and a higher rate of dyspnea, as well as a larger proportion applications of oxygen therapy and noninvasive continuous positive airway pressure (NCPAP). No obvious differences were found in lab examinations within the two groups. Regarding disease severity, the proportions of extremely severe pneumonia and severe disease defined by the clinical score system were higher in the research group than in the control group., Conclusion: Compared with single M. pneumoniae infection, MPP coinfected with HAdV in children was relatively more serious.

Adenovirus (HAdV) infection is a common cause of illness among young children, immunocompromised patients, and transplant recipients. The majority of HAdV infections are self-limited, but recurring infection is frequently encountered in young children and may require hospitalization. In this study, we surveyed the presence of HAdV in tonsillectomy samples and investigated epigenetic conditions that contributed to HAdV reactivation. HAdV DNA was detected from 86.7% donors. The lymphocytes isolated from the samples failed to produce infectious HAdV after incubation, suggesting the viruses remained in a latent status. To determine whether epigenetic factors played a role in HAdV reactivation, isolated lymphocytes were treated with a small compound library. Viral DNA replication and infectious HAdV production were assayed by PCR and by a secondary infection assay. We identified several compounds, mainly pan- and selective histone deacetylase (HDAC) inhibitors, which showed activity to reactivate HAdV from latency. The viruses were isolated and were determined as species C HAdV. Using a model of HAdV lytic infection, we showed that the compounds promoted histone-3 acetylation and association with viral early gene promoters. In addition to demonstrate the palatine tonsils as a reservoir of latent HAdV, this study uncovers a critical role of histone acetylation in HAdV reactivation, linking HAdV latency to recurrent HAdV infection. IMPORTANCE Respiratory tract infection by adenoviruses is among the most common diseases in children, attributing to approximately 20% of hospitalizations of children with acute respiratory infection (ARI). Adenovirus transmits by direct contact, but recurrent infection is common. Ever since its isolation, adenovirus has been known to have the ability to establish persistent or latent infection. We found 87.7% tonsillectomy specimens contained detectable amounts of adenoviral DNA. Isolated lymphocytes did not produce infectious adenoviruses without stimulation. By screening an epigenetic informer compound library, we identified several histone deacetylase inhibitors that promoted adenovirus reactivation that was evidenced by increased viral DNA replication and production of infectious viruses. The human tonsils are covered with bacterial pathogens that may utilize pathogen-associated pattern molecules or metabolites to cause epigenetic activation and proinflammatory gene transcription, which may lead to viral reactivation from latency. The study shows that recurrent adenovirus infection could arise from reactivation of residing virus from previous infections., (Copyright © 2020 American Society for Microbiology.)

Adenoviral vectors are a safe and potently immunogenic vaccine delivery platform. Non-replicating Ad vectors possess several attributes which make them attractive vaccines for infectious disease, including their capacity for high titer growth, ease of manipulation, safety, and immunogenicity in clinical studies, as well as their compatibility with clinical manufacturing and thermo-stabilization procedures. In general, Ad vectors are immunogenic vaccines, which elicit robust transgene antigen-specific cellular (namely CD8 + T cells) and/or humoral immune responses. A large number of adenoviruses isolated from humans and non-human primates, which have low seroprevalence in humans, have been vectorized and tested as vaccines in animal models and humans. However, a distinct hierarchy of immunological potency has been identified between diverse Ad vectors, which unfortunately limits the potential use of many vectors which have otherwise desirable manufacturing characteristics. The precise mechanistic factors which underlie the profound disparities in immunogenicity are not clearly defined and are the subject of ongoing, detailed investigation. It has been suggested that a combination of factors contribute to the potent immunogenicity of particular Ad vectors, including the magnitude and duration of vaccine antigen expression following immunization. Furthermore, the excessive induction of Type I interferons by some Ad vectors has been suggested to impair transgene expression levels, dampening subsequent immune responses. Therefore, the induction of balanced, but not excessive stimulation of innate signaling is optimal. Entry factor binding or receptor usage of distinct Ad vectors can also affect their in vivo tropism following administration by different routes. The abundance and accessibility of innate immune cells and/or antigen-presenting cells at the site of injection contributes to early innate immune responses to Ad vaccination, affecting the outcome of the adaptive immune response. Although a significant amount of information exists regarding the tropism determinants of the common human adenovirus type-5 vector, very little is known about the receptor usage and tropism of rare species or non-human Ad vectors. Increased understanding of how different facets of the host response to Ad vectors contribute to their immunological potency will be essential for the development of optimized and customized Ad vaccine platforms for specific diseases., (Copyright © 2020 Coughlan.)

Human adenovirus serotype 7 (HAdV-7), belonging to species B, has caused severe lower respiratory tract diseases and even deaths recently. However, no adenovirus vaccine or therapeutic is available thus far. In this study, a HAdV-7-specific human monoclonal antibody (HMAb), 3-3E, isolated from single plasma cells obtained from the peripheral blood mononuclear cells of HAdV-7-infected patients showed potent HAdV-7 neutralization activity. The results showed HMAb 3-3E only binds to the hexon protein of intact HAdV-7 or the recombinant hexon protein and it does not bind to other intact virion particles. This could mean the antibody recognizes a conformational epitope of the hexon protein. Further, HMAb 3-3E potently neutralized HAdV-7 in vitro at low concentrations. In vivo studies showed HMAb 3-3E protected from HAdV-7 infection in a murine model. Therefore, HMAb 3-3E is promising as a safe and effective prophylactic and therapeutic treatment for HAdV-7 infection., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Intra-arterial administration of an adenovirus serotype 5 (Ad5) vector in a gene therapy trial caused lethal, systemic inflammation in subject 019 with ornithine transcarbamylase deficiency. This unanticipated inflammatory response was absent in another subject receiving the same vector dose and in 16 subjects receiving lower vector doses. We hypothesized that an immune memory to a previous natural adenovirus infection enhanced the immune response to high-dose systemic Ad5 vector, causing the exaggerated immune response in subject 019. To investigate this, we found that rabbit polyclonal sera to Ad5 and pooled human immunoglobulin (Ig) inhibited Ad5 vector transduction of non-immune cells in vitro, but enhanced transduction and activation of human dendritic cells (DCs). Sera from approximately 7% of normal human subjects and 50% of patients treated topically with Ad5 vectors enhanced Ad5 transduction and activation of DCs, apparently from formation of Ig-Ad5 immune complexes and binding to DCs through FcγR. Subject 019's blood substantially increased Ad5-vector activation of human DC primary cultures at levels exceeding those from normal subjects. Although this study is based on one event in a single subject, the results implicate a pre-existing humoral immune response to Ad5 in the lethal systemic inflammatory response that occurred in subject 019., (Copyright © 2020. Published by Elsevier Inc.)

Background: A number of serious human adenovirus (HAdV) outbreaks have been recently reported: HAdV-B7 (Israel, Singapore, and USA), HAdV-B7d (USA and China), HAdV-D8, -D54, and -C2 (Japan), HAdV-B14p1 (USA, Europe, and China), and HAdV-B55 (China, Singapore, and France)., Methods: To understand the epidemiology of HAdV infections in Singapore, we studied 533 HAdV-positive clinical samples collected from 396 pediatric and 137 adult patients in Singapore from 2012 to 2018. Genome sequencing and phylogenetic analyses were performed to identify HAdV genotypes, clonal clusters, and recombinant or novel HAdVs., Results: The most prevalent genotypes identified were HAdV-B3 (35.6%), HAdV-B7 (15.4%), and HAdV-E4 (15.2%). We detected 4 new HAdV-C strains and detected incursions with HAdV-B7 (odds ratio [OR], 14.6; 95% confidence interval [CI], 4.1-52.0) and HAdV-E4 (OR, 13.6; 95% CI, 3.9-46.7) among pediatric patients over time. In addition, immunocompromised patients (adjusted OR [aOR], 11.4; 95% CI, 3.8-34.8) and patients infected with HAdV-C2 (aOR, 8.5; 95% CI, 1.5-48.0), HAdV-B7 (aOR, 3.7; 95% CI, 1.2-10.9), or HAdV-E4 (aOR, 3.2; 95% CI, 1.1-8.9) were at increased risk for severe disease., Conclusions: Singapore would benefit from more frequent studies of clinical HAdV genotypes to identify patients at risk for severe disease and help guide the use of new antiviral therapies, such as brincidofovir, and potential administration of HAdV 4 and 7 vaccine., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

B1-type human adenoviruses (HAdVs) HAdV-3, HAdV-7, and HAdV-55 have caused epidemics in North America, Asia, and Europe. However, to date, no adenovirus vaccines or antiviral drugs have been approved for general use. In the present work, a scFv-phage immune library was constructed and mouse monoclonal antibody (MMAb) 10G12 was obtained through selection. 10G12 is specific for HAdV-7 and binds the hexon loop1 and loop2 (LP12), resulting in strong neutralization activity against HAdV-7. Additionally, it is stable in serum and buffer at various pH values. The findings provide insight into adenovirus and antibody responses and may facilitate the design and development of adenovirus vaccines and antiviral drugs., (Copyright © 2019 Wang, Lu, Zhou, Chen, Huang, Yu and Yang.)

Human adenovirus commonly causes infections of respiratory, gastrointestinal, genitourinary, and ocular surface mucosae. Although most adenovirus eye infections are mild and self-limited, specific viruses within human adenovirus species D are associated with epidemic keratoconjunctivitis (EKC), a severe and highly contagious ocular surface infection, which can lead to chronic and/or recurrent, visually disabling keratitis. In this review, we discuss the links between adenovirus ontogeny, genomics, immune responses, and corneal pathogenesis, for those viruses that cause EKC., (© 2019 Federation of European Biochemical Societies.)

Preexisting neutralizing antibody (NAb) against human adenovirus serotype 5 (AdHu5) can reduce the immunogenicity of AdHu5 vector-based vaccine, thus inhibiting the host's immune response and utility of other homologous vectors. Common marmoset ( Callithrix jacchus ), a small new world primate, has attracted considerable attention for its potential as a preclinical research model of vaccine development. However, the prevalence of anti-AdHu5 NAb activity in common marmosets bred in China remains unknown. A recombinant adenovirus expressing luciferase and Zs Green reporter genes were constructed to detect NAb against rAdHu5 by flow cytometry (FCM) and chemiluminescence (CL) assay. Five of 25 marmosets (20%) presented AdHu5 NAb detectable by FCM. Four animals had low titer (1/16), while the fifth one reached 1/64. While by CL assay, 7 of 25 (28%) marmosets were anti-AdHu5 NAb positive. Four animals, two of whom were negative by FCM, also had low titer NAb (1/16), suggesting assay discrepancy at low levels. Two marmosets, 1/32 titer by CL, were at 1/16 by FCM. A single animal showed a high titer with both assays (1/128 and 1/64 by CL and FCM, respectively). The CL method was simpler, more sensitive, accurate, and stable. The low prevalence of preexisting anti-AdHu5 NAb in marmosets provides important background information on the feasibility and applicability of using marmosets as a preclinical research model for vaccine development.

Mediastinal fat has been suggested to be associated with cardiovascular diseases such as carotid stiffness, atherosclerosis and coronary artery calcification. We investigated the possible role of Ad-36-induced obesity in the pathogenesis of the coronary artery disease (CAD). Ad-36 DNA was investigated in the anterior mediastinal fat tissue samples of obese adults with CAD. Seventy-five obese adults with left main coronary artery (LMCA) disease, 28 non-obese adults with valvular heart diseases, and 48 healthy individuals without cardiovascular problems were included as the obese patient group (OPG), non-obese patient group (NOG) and healthy control group (HCG), respectively. We also simultaneously investigated Ad-36 antibodies by serum neutralization test (SNA), and measured leptin and adinopectin levels. Ad-36 antibodies were detected only in 10 patients (13.3%) within the 75 OPG. A statistically significant difference was detected between OPG, NOG and HCG in terms of Ad-36 antibody positivity (p>0.05). Ad-36 DNA was not detected in mediastinal tissue samples of OPG and NOP without PCR inhibitors. We suggest that Ad-36 may not have an affinity for mediastinal adipose tissue in obese patients with left main CAD and valvular heart diseases. Ad-36 antibody positivity results are not sufficient to reach a causal relationship.

Human adenovirus (HAdV) causes infections predominantly in early childhood and the tissue tropism of specific HAdV species determines the clinical manifestation, including infections of the gastrointestinal tract, respiratory tract, and keratoconjunctivitis. Why HAdV shows such a tropism has not yet been fully elucidated, but in the intestine different mechanisms for virus entry or resistence to immune modulatory factors have been described. Recently identified antiviral strategies by interferons provide evidence about the repression of E1A and maybe even promote HAdV persistence. The presence of HAdV in a persistent status in the gut is of importance in the setting of pediatric stem cell transplant recipients where HAdV detection in stool usually preceds clinical signs and severe infections are related to mortality. The reactivation of persistent intestinal HAdV infections in these patients needs further investigation also with regard to successful therapy options. In addition, several newly identified recombinant HAdV types have been isolated from stool samples, thus raising the question of possible recombination events in the gut. In this review, intestinal HAdV infections are discussed in relation to the tissue tropism, persistence, recombination, and new in-vitro models to enhance the knowledge about virus-host interactions and support the development of new treatment approaches.

Introduction: Human adenovirus (HAdV) infection can cause substantial morbidity in kidney transplant (KT) recipients. Cell-mediated immunity plays an important role in controlling HAdV infection after KT., Methods: We prospectively (January 2015 to June 2018) investigated the absolute lymphocyte count (ALC) and interferon-γ-producing CD4 + and CD8 + T cells at diagnosis and at viral clearance by an intracellular cytokine assay after stimulating with HAdV whole lysate, hexon, and penton proteins. HAdV infection was defined as the presence of HAdV DNA load in plasma or clinical specimens measured by the polymerase chain reaction assay., Results: Eighteen adult KT recipients were diagnosed with HAdV infection at a median of 16 months (interquartile range [IQR], 2-39) after KT. The majority (94%) had HAdV-associated hemorrhagic cystitis. The median ALC at viral clearance was significantly higher compared with diagnosis (2257 cells/mm 3 [IQR, 1544-3078] vs 1001 cells/mm 3 [IQR, 641-1385]; P < 0.001). Eleven patients underwent measurement of the HAdV-specific T-cell response. The median numbers of CD4 + and CD8 + T cells at viral clearance were significantly higher compared with diagnosis (448 cells/mm 3 [IQR, 248-651] vs 215 cells/mm 3 [IQR, 159-272], P = 0.02; and 623 cells/mm 3 [IQR, 242-772] vs 235 cells/mm 3 [IQR, 129-266], P < 0.01), respectively. The median percentages of penton-specific CD4 + and hexon-specific CD8 + T cells at viral clearance were significantly higher compared with diagnosis (0.012% vs 0%, P = 0.03%; and 0.136% vs 0.016%, P = 0.003, respectively)., Conclusions: Our findings suggest a trend of ALC and HAdV-specific T-cell immune restoration in KT recipients who achieve successful HAdV clearance., (© 2019 Wiley Periodicals, Inc.)

Human adenovirus 5 (HAdV-5) is used as a vector in gene therapy clinical trials, hence its interactions with the host immune system have been widely studied. Previous studies have demonstrated that HAdV-5 binds specifically to murine coagulation factor X (mFX), inhibiting IgM and complement-mediated neutralization. Here, we examined the physical binding of immune components to HAdV-5 by nanoparticle tracking analysis, neutralization assays, mass spectrometry analysis and in vivo experiments. We observed that purified mouse Immunoglobulin M (IgM) antibodies bound to HAdV-5 only in the presence of complement components. Active serum components were demonstrated to bind to HAdV-5 in the presence or absence of mFX, indicating that immune molecules and mFX might bind to different sites. Since binding of mFX to HAdV-5 blocks the neutralization cascade, these findings suggested that not all complement-binding sites may be involved in virion neutralization. Furthermore, the data obtained from serum neutralization experiments suggested that immune molecules other than IgM and IgG may trigger activation of the complement cascade in vitro. In vivo experiments were conducted in immunocompetent C57BL/6 or immuno-deficient Rag2 -/- mice. HAdV-5T* (a mutant HAdV-5 unable to bind to human or mFX) was neutralized to some extent in both mouse models, suggesting that murine immunoglobulins were not required for neutralization of HAdV-5 in vivo. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis of HAdV-5 and HAdV-5T* after exposure to murine sera showed stable binding of C3 and C4b in the absence of mFX. In summary, these results suggest that HAdV-5 neutralization can be mediated by both the classical and alternative pathways and that, in the absence of immunoglobulins, the complement cascade can be activated by direct binding of C3 to the virion.

Detection of human adenoviruses (HAdVs) in nasopharyngeal swab samples by immunofluorescence assay (IFA) will be valuable for diagnosing HAdV infection, which is a leading cause of severe respiratory tract disease, and will help in curbing the spread of HAdV. Monoclonal antibodies employed in IFA for HAdV detection should ideally target highly conserved epitope types. Here, we describe the development of two antigen-binding fragments (Fabs) with specific reactivity to HAdV using phage antibody library technology. When tested with IFA, both Fabs recognized cells infected with several types of HAdV, some of which have been identified in epidemics globally, or associated with outbreaks of severe or fatal acute respiratory diseases. The specificity and cross-reactivity of both Fabs to HAdVs indicated that the generated Fabs could be applied in the development of IFAs to detect HAdVs. Both Fabs bound to the knob proteins, as shown by chemiluminescence enzyme immunoassay and western blot. In addition, epitope mapping showed that both Fabs recognized a conserved linear epitope among several types of HAdV. Two different Fabs recognized the same epitope, suggesting that the epitope triggered the production of at least two kinds of antibodies in the body. The generated Fabs exerted no neutralization against HAdVs. The results demonstrate that both Fabs bind to an epitope that plays no role in neutralization of HAdV., Competing Interests: The authors have declared that no competing interests exist.