Generation of Human Embryonic Stem Cell-Derived Lung Organoids for Modeling Infection and Replication Differences between Human Adenovirus Types 3 and 55 and Evaluating Potential Antiviral Drugs.

Human adenoviruses type 3 (HAdV-3) and type 55 (HAdV-55) are frequently encountered, highly contagious respiratory pathogens with high morbidity rate. In contrast to HAdV-3, one of the most predominant types in children, HAdV-55 is a reemergent pathogen associated with more severe community-acquired pneumonia (CAP) in adults, especially in military camps. However, the infectivity and pathogenicity differences between these viruses remain unknown as in vivo models are not available. Here, we report a novel system utilizing human embryonic stem cells-derived 3-dimensional airway organoids (hAWOs) and alveolar organoids (hALOs) to investigate these two viruses. Firstly, HAdV-55 replicated more robustly than HAdV-3. Secondly, cell tropism analysis in hAWOs and hALOs by immunofluorescence staining revealed that HAdV-55 infected more airway and alveolar stem cells (basal and AT2 cells) than HAdV-3, which may lead to impairment of self-renewal functions post-injury and the loss of cell differentiation in lungs. Additionally, the viral life cycles of HAdV-3 and -55 in organoids were also observed using Transmission Electron Microscopy. This study presents a useful pair of lung organoids for modeling infection and replication differences between respiratory pathogens, illustrating that HAdV-55 has relatively higher replication efficiency and more specific cell tropism in human lung organoids than HAdV-3, which may result in relatively higher pathogenicity and virulence of HAdV-55 in human lungs. The model system is also suitable for evaluating potential antiviral drugs, as demonstrated with cidofovir. IMPORTANCE Human adenovirus (HAdV) infections are a major threat worldwide. HAdV-3 is one of the most predominant respiratory pathogen types found in children. Many clinical studies have reported that HAdV-3 causes less severe disease. In contrast, HAdV-55, a reemergent acute respiratory disease pathogen, is associated with severe community-acquired pneumonia in adults. Currently, no ideal in vivo models are available for studying HAdVs. Therefore, the mechanism of infectivity and pathogenicity differences between human adenoviruses remain unknown. In this study, a useful pair of 3-dimensional (3D) airway organoids (hAWOs) and alveolar organoids (hALOs) were developed to serve as a model. The life cycles of HAdV-3 and HAdV-55 in these human lung organoids were documented for the first time. These 3D organoids harbor different cell types, which are similar to the ones found in humans. This allows for the study of the natural target cells for infection. The finding of differences in replication efficiency and cell tropism between HAdV-55 and -3 may provide insights into the mechanism of clinical pathogenicity differences between these two important HAdV types. Additionally, this study provides a viable and effective in vitro tool for evaluating potential anti-adenoviral treatments.

[Silencing CD46 and DSG2 in host A549 cells inhibits entry of human adenovirus type 3 and type 7 and reduces interleukin-8 release].

OBJECTIVE: To investigate the effect of silencing CD46 and desmoglein 2 (DSG2) in host A549 cells on the entry of human adenovirus type 3 (HAdV-3) and type 7 (HAdV-7) and host cell secretion of inflammatory cytokines. METHODS: RNA interference technique was use to silence the expression of CD46 or DSG2 in human epithelial alveolar A549 cells as the host cells of HAdV-3 or HAdV-7. The binding of the viruses with CD46 and DSG2 were observed with immunofluorescence staining at 0.5 and 1 h after viral infection. The viral load in the host cells was determined with qRT-PCR, and IL-8 secretion level was measured using ELISA. RESULTS: In infected A549 cells, immunofluorescent staining revealed colocalization of HAdV-3 and HAdV-37 with their receptors CD46 and DSG2 at 0.5 h and 2 h after infection, and the copy number of the viruses increased progressively after the infection in a time-dependent manner. In A549 cells with CD46 silencing, the virus titers were significantly lower at 2, 6, 12 and 24 h postinfection in comparison with the cells without gene silencing; the virus titers were also significantly decreased in the cells with DSG2 silencing. The secretion level of IL-8 increased significantly in A549 cells without siRNA transfection following infection with HAdV-3 and HAdV-7 (P < 0.0001), but decreased significantly in cells with CD46 and DSG2 silencing (P < 0.0001). CONCLUSION: HAdV-3 and HAdV-7 enter host cells by binding to their receptors CD46 and DSG2, and virus titer and cytokines release increase with infection time. Silencing CD46 and DSG2 can inhibit virus entry and cytokine IL-8 production in host cells.

Molecular Characteristics of Human Adenovirus Type 3 Circulating in Parts of China During 2014-2018.

Human adenoviruses (HAdVs) are important pathogens causing respiratory infections; 3.5-11% of childhood community-acquired pneumonia is associated with HAdV infection. Human adenovirus type 3 (HAdV-3), leading to severe morbidity and mortality, is one of the most prevalent genotype among adenoviruses responsible for acute respiratory infections (ARIs) in children in China. To identify the genetic variation of HAdV-3 in children with ARIs in China, a molecular epidemiological study was conducted. A total of 54 HAdV-3 isolated strains were obtained from children with ARIs in Beijing, Wenzhou, Shanghai, Shijiazhuang, Hangzhou, Guangzhou, and Changchun from 2014 to 2018. Thirty-two strains of which were selected for whole-genome sequencing, while the hexon, penton base, and fiber genes were sequenced for remaining strains. Bioinformatics analysis was performed on the obtained sequences. The phylogenetic analyses based on whole-genome sequences, major capsid protein genes (hexon, penton base, and fiber), and early genes (E1, E2, E3, and E4) showed that the HAdV-3 strains obtained in this study always clustered together with the reference strains from Chinese mainland, while the HAdV-3 prototype strain formed a cluster independently. Compared with the prototype strain, all strains possessed nine amino acid (AA) substitutions at neutralization antigenic epitopes of hexon. The homology models of the hexon protein of the HAdV-3 prototype and strain BJ20160214 showed that there was no evident structural change at the AA mutation sites. Two AA substitutions were found at the Arg-Gly-Asp (RGD) loop and hypervariable region 1 (HVR1) region of the penton base. A distinct AA insertion (20P) in the highly conserved PPPSY motif of the penton base that had never been reported before was observed. Recombination analysis indicated that partial regions of protein IIIa precursor, penton base, and protein VII precursor genes among all HAdV-3 strains in this study were from HAdV-7. This study showed that the genomes of the HAdV-3 strains in China were highly homologous. Some AA mutations were found at antigenic sites; however, the significance needs further study. Our data demonstrated the molecular characteristics of HAdV-3 circulating in China and was highly beneficial for further epidemiological exploration and the development of vaccines and drugs against HAdV-3.

Construction and Characterization of a Novel Recombinant Attenuated and Replication-Deficient Candidate Human Adenovirus Type 3 Vaccine: "Adenovirus Vaccine Within an Adenovirus Vector".

Human adenoviruses (HAdVs) are highly contagious and result in large number of acute respiratory disease (ARD) cases with severe morbidity and mortality. Human adenovirus type 3 (HAdV-3) is the most common type that causes ARD outbreaks in Asia, Europe, and the Americas. However, there is currently no vaccine approved for its general use. The hexon protein contains the main neutralizing epitopes, provoking strong and lasting immunogenicity. In this study, a novel recombinant and attenuated adenovirus vaccine candidate against HAdV-3 was constructed based on a commercially-available replication-defective HAdV-5 gene therapy and vaccine vector. The entire HAdV-3 hexon gene was integrated into the E1 region of the vector by homologous recombination using a bacterial system. The resultant recombinants expressing the HAdV-3 hexon protein were rescued in AD293 cells, identified and characterized by RT-PCR, Western blots, indirect immunofluorescence, and electron microscopy. This potential vaccine candidate had a similar replicative efficacy as the wild-type HAdV-3 strain. However, and importantly, the vaccine strain had been rendered replication-defective and was incapable of replication in A549 cells after more than twenty-generation passages in AD293 cells. This represents a significant safety feature. The mice immunized both intranasally and intramuscularly by this vaccine candidate raised significant neutralizing antibodies against HAdV-3. Therefore, this recombinant, attenuated, and safe adenovirus vaccine is a promising HAdV-3 vaccine candidate. The strategy of using a clinically approved and replication-defective HAdV-5 vector provides a novel approach to develop universal adenovirus vaccine candidates against all the other types of adenoviruses causing ARDs and perhaps other adenovirus-associated diseases.

Bioinformatics analysis reveals four major hexon variants of human adenovirus type-3 (HAdV-3) as the potential strains for development of vaccine and siRNA-based therapeutics against HAdV-3 respiratory infections.

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.

The possible origin of human adenovirus type 3: Evidence of natural genetic recombination between human and simian adenovirus.

We determined a complete genome sequence of the Korean field strain, KUMC-62, of human adenovirus type 3 (HAdV-3) and performed comparative genome analyses. Interestingly HAdV-3 has a distinct genomic sequence for the fiber CDS region on average 62.46% of nucleotide sequence identity to other types of HAdV-B1, while remaining genomic region of HAdV-3 is very similar (on average 95.71% of nucleotide sequence identity) to other types of HAdV-B1. The blast results showed that the fiber CDS region of HAdV-3 exhibited the highest nucleotide sequence identity with that of simian adenovirus type 32 (SAdV-32), except other strains of HAdV-3. In the Simplot analysis, a potential recombination event was detected between HAdV-7 and SAdV-32, which might have created HAdV-3 in the past. These findings suggest that HAdV-3 highly likely was created by a natural inter-species recombination event between human and non-human primate AdVs.

Genomic diversity of human adenovirus type 3 isolated in Fukui, Japan over a 24-year period.

Recently, human adenovirus type 3 (HAdV-3) has become the most isolated HAdV worldwide. Restriction endonuclease analysis of globally isolated strains of HAdV-3 has uncovered 51 genome types to date. Information on the genome type is important to the epidemiological study of HAdV-3. In this study, analysis of 75 isolates of HAdV- 3 collected over a 24-year period in Fukui revealed: (1) the emergence of three novel genome types (HAdV-3a52, HAdV-3a53 and HAdV-3a54) and two known genome types (HAdV-3a and HAdV-3a54); (2) the spectrum of diseases caused by individual genome types and their major involvement in the paediatric age population; and (3) the co-circulation and replacement of genome types as a usual phenomenon. The rising number of HAdV-3 genome types indicates that the genetic variation of HAdV-3 is more than other HAdVs. Considering the clinical importance of HAdV-3 infection, its genetic diversity underscores the need for its continuous surveillance and genetic characterization.

Mapping the epitope of neutralizing monoclonal antibodies against human adenovirus type 3.

Human adenovirus type 3 (HAdV-3) has produced a global epidemic in recent years causing serious diseases such as pneumonia in both pediatric and adult patients. Development of an effective neutralizing monoclonal antibody (MAb) and identification of its neutralizing epitope is important for the control of HAdV-3 infection. In this study, three neutralizing MAbs were generated, of which MAb 3D7 had a high neutralization titer of 4096 (approximately 0.5 µg/ml) against HAdV-3 infection. In indirect enzyme-linked immunosorbent assays, all three MAbs specifically recognized HAdV-3 virus particles and hexon protein, but did not react with the virus particles or the hexon protein of HAdV-7. Analyses using a series of peptides and chimeric adenovirus particles of epitope mutants revealed that all three MAbs bound to the same exposed region (amino acid positions 244-254 of hexon) in hypervariable region 4 (HVR4), which is highly conserved among global HAdV-3 strains. The amino acids T246 and G250 may be the critical amino acids recognized by these MAbs. MAb 3D7 reduced the recombinant enhanced green fluorescent protein-expressing HAdV-3 (rAd3EGFP) load recovered in the lungs of mice at 3 days post-infection. The generation of MAb 3D7 and the identification of its neutralizing epitope may be useful for therapeutic treatment development, subunit vaccine construction, and virion structural analysis for HAdV-3.

Epitope mapping and characterization of a neutralizing monoclonal antibody against human adenovirus type 3.

Human adenovirus serotype 3 (HAdV-3) has occurred as a global epidemic in recent years causing serious diseases such as pneumonia in pediatric and adult patients. Development of reliable diagnostic reagents and identification of neutralizing epitopes is important for the surveillance and control of infection. In this study, a neutralizing monoclonal antibody (MAb) MAb 1B6 was generated using the HAdV-3 virion. MAb 1B6 specially recognized the HAdV-3 virus particles and the HAdV-3 hexon protein, but not the virus particles or the hexon protein of HAdV-7 and HAdV-4 by western-blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). Analysis using a series of peptides from the hexon protein and chimeric adenovirus (Ad) particles of epitope mutants revealed that MAb 1B6 bound to the exposed region (amino acid positions 414-424 of hexon) in hypervariable region 7 (HVR7). ELISA demonstrated that MAb 1B6 could recognize the corresponding regions of other HAdV-3 genotypes that have some residues substituted. The identification of the neutralizing epitope and the generation of MAb 1B6 may be useful for clinical serotype-specific diagnosis, subunit vaccine construction for HAdV-3 infection, and virion structural analysis.