Adenovirus Serotype Research Articles

A fowl adenovirus serotype 4 (FAdV-4) Fiber2 subunit vaccine candidate provides complete protection against challenge with virulent FAdV-4 strain in chickens

Hypervirulent fowl adenovirus serotype 4 (FAdV-4)-induced hepatitis-hydropericardium syndrome (HHS) with high mortality causes huge economic losses to the poultry industry worldwide. However, commercially available vaccines against FAdV-4 infection remain scarce. Here, we prepared a subunit vaccine candidate derived from the bacterially expressed recombinant Fiber2 protein (termed as rFiber2 subunit vaccine) of FAdV-4 GZ-QL strain (a hypervirulent strain isolated in Guizhou province) and a recombinant plasmid pVAX1-Fiber2 as DNA vaccine candidate (termed as Fiber2 DNA vaccine). The immune effects of different dosages (50, 100, and 150 μg) of these were evaluated through immunization and challenge studies in chickens. Three injections of the rFiber2 subunit vaccine or the Fiber2 DNA vaccine induced robust humoral and cellular immune responses in chickens, which was assessed based on the secretion of high-level neutralizing antibodies, Th1- (IL-2, IFN-γ) and Th2-type cytokines (IL-4, IL-6). Importantly, the efficacy of the rFiber2 subunit vaccine was significantly higher (80 %–100 %) compared with the Fiber2 DNA vaccine (50 %–60 %) and a commercial inactivated vaccine (80 %). Collectively, these results suggest that the rFiber2 subunit and Fiber2 DNA vaccine candidate induced remarkable humoral and cellular immune responses, while the rFiber2 subunit vaccine candidate possesses better potential in the fight against FAdV-4 infection, laying foundations for the effective control of HHS in chickens.

Chimeric Helper-Dependent Adenoviruses Transduce Retinal Ganglion Cells and Müller Cells in Human Retinal Explants.

Purpose: Despite numerous recent advances in retinal gene therapy using adeno-associated viruses (AAVs) as delivery vectors, there remains a crucial need to identify viral vectors with the ability to transduce specific retinal cell types and that have a larger carrying capacity than AAV. In this study, we evaluate the retinal tropism of 2 chimeric helper-dependent adenoviruses (HDAds), helper-dependent adenovirus serotype 5 (HDAd5)/3 and HDAd5/35, both ex vivo using human retinal explants and in vivo using rats. Methods: We transduced cultured human retinal explants with HDAd5/3 and HDAd5/35 carrying an eGFP vector and evaluated tropism and transduction efficiency using immunohistochemistry. To assess in vivo transduction efficiency, subretinal injections were performed in wild-type Sprague-Dawley rats. For both explants and subretinal injections, we delivered 10 μL (1 × 106 vector genomes/mL) and assessed tropism at 7- and 14-days post-transduction, respectively. Results: HDAd5/3 and HDAd5/35 both transduced human retinal ganglion cells (RGCs) and Müller cells, but not photoreceptors, in human retinal explants. However, subretinal injections in albino rats resulted in transduction of the retinal pigmented epithelium only, highlighting species-specific differences in retinal tropism and the value of a human explant model when testing vectors for eventual human gene therapy. Conclusions: Chimeric HDAds are promising candidates for the delivery of large genes, multiple genes, or neuroprotective factors to Müller cells and RGCs. These vectors may have utility for targeted therapy of neurodegenerative diseases primarily involving retinal ganglion or Müller cell types, such as glaucoma or macular telangiectasia type 2.

The Revolving Door of Adenovirus Cell Entry: Not All Pathways Are Equal.

Adenoviruses represent exceptional candidates for wide-ranging therapeutic applications, from vectors for gene therapy to oncolytics for cancer treatments. The first ever commercial gene therapy medicine was based on a recombinant adenovirus vector, while most recently, adenoviral vectors have proven critical as vaccine platforms in effectively controlling the global coronavirus pandemic. Here, we discuss factors involved in adenovirus cell binding, entry, and trafficking; how they influence efficiency of adenovirus-based vectors; and how they can be manipulated to enhance efficacy of genetically modified adenoviral variants. We focus particularly on endocytosis and how different adenovirus serotypes employ different endocytic pathways to gain cell entry, and thus, have different intracellular trafficking pathways that subsequently trigger different host antiviral responses. In the context of gene therapy, the final goal of the adenovirus vector is to efficiently deliver therapeutic transgenes into the target cell nucleus, thus allowing its functional expression. Aberrant or inefficient endocytosis can impede this goal, therefore, it should be considered when designing and constructing adenovirus-based vectors.

Isolation and Molecular Characterization of Fowl Adenoviruses Associated with Inclusion Body Hepatitis-hydropericardium Syndrome in Broiler Chickens in Egypt

Avian adenoviruses are an extremely diversified group of pathogens that recently triggering a variety of problems for poultry production. In particular, Inclusion Body Hepatitis-Hydropericardium Syndrome (IBH-HPS), which has been observed in broiler birds from 3 to 6 weeks of age and is associated with anemia, hemorrhagic disorders, hydropericardium, and high mortality. The disease has been reported worldwide, and recently it was reported in many Egyptian farms, causing severe economic losses. Therefore, the current study aimed to isolate, and genetically type the most common Adenovirus serotypes associated with this syndrome in Egyptian farms. A total of 50 broiler chicken farms (3-6 weeks old) located in different Egyptian governorates were examined. Macroscopically, the diseased flock revealed hydropericardium, enlarged friable livers with ecchymotic hemorrhages, and varying mortality rates (1 to 7.5%). Histopathologically, severe diffuse necrotizing enteritis, hepatitis, pericarditis, and diffuse lymphoid depletion of the spleen were the most prominent lesions. Liver tissues and cloacal swabs were collected from all examined flocks for FAdVs detection by conventional polymerase chain reaction (PCR) targeting the L1 loop in the hexon gene. The PCR products were sequenced for typing of the detected viruses. It was found that 10 out of 50 flocks examined were PCR positive for FAdVs (20%). Phylogenetic analysis of the sequenced genes revealed that the obtained viruses clustered with reference strains belonging to FAdV type D and E serotype 2, 11, and 8a respectively. The isolation of both FAdV type D and FAdV type E were carried out on a primary cell culture chicken embryo liver cell (CEL) and the presence of these viruses was confirmed by PCR after the appearance of cytopathic effect (CPE). From this study, it could be concluded that both FAdVs types D and E are the most common adenoviruses circulating in poultry farms suffering from hydropericardium and inclusion body hepatitis.

Dual-Antigen COVID-19 Vaccine Subcutaneous Prime Delivery With Oral Boosts Protects NHP Against SARS-CoV-2 Challenge.

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 106 TCID50) 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.

Protective Efficacy of Rhesus Adenovirus COVID-19 Vaccines against Mouse-Adapted SARS-CoV-2.

ABSTRACTThe global COVID-19 pandemic has sparked intense interest in the rapid development of vaccines as well as animal models to evaluate vaccine candidates and to define immune correlates of protection. We recently reported a mouse-adapted SARS-CoV-2 virus strain (MA10) with the potential to infect wild-type laboratory mice, driving high levels of viral replication in respiratory tract tissues as well as severe clinical and respiratory symptoms, aspects of COVID-19 disease in humans that are important to capture in model systems. We evaluated the immunogenicity and protective efficacy of novel rhesus adenovirus serotype 52 (RhAd52) vaccines against MA10 challenge in mice. Baseline seroprevalence is lower for rhesus adenovirus vectors than for human or chimpanzee adenovirus vectors, making these vectors attractive candidates for vaccine development. We observed that RhAd52 vaccines elicited robust binding and neutralizing antibody titers, which inversely correlated with viral replication after challenge. These data support the development of RhAd52 vaccines and the use of the MA10 challenge virus to screen novel vaccine candidates and to study the immunologic mechanisms that underscore protection from SARS-CoV-2 challenge in wild-type mice.IMPORTANCE We have developed a series of SARS-CoV-2 vaccines using rhesus adenovirus serotype 52 (RhAd52) vectors, which exhibit a lower seroprevalence than human and chimpanzee vectors, supporting their development as novel vaccine vectors or as an alternative adenovirus (Ad) vector for boosting. We sought to test these vaccines using a recently reported mouse-adapted SARS-CoV-2 (MA10) virus to (i) evaluate the protective efficacy of RhAd52 vaccines and (ii) further characterize this mouse-adapted challenge model and probe immune correlates of protection. We demonstrate that RhAd52 vaccines elicit robust SARS-CoV-2-specific antibody responses and protect against clinical disease and viral replication in the lungs. Further, binding and neutralizing antibody titers correlated with protective efficacy. These data validate the MA10 mouse model as a useful tool to screen and study novel vaccine candidates, as well as the development of RhAd52 vaccines for COVID-19.

Human Adenovirus Serotypes Efficiently Transducing HEK293 Cells: An In Vitro Propagation of HAdv

Generally, a gene which is inserted directly into a cell does not operate independently. Instead, the transmission of the gene is genetically modified by a biological messenger called a vector, consists of a transgene and a large DNA sequence as a backbone. Since they can deliver the new gene by infecting the cell, such viruses are also used as vectors. The adenovirus is a non-enveloped virus that can be tailored to transfer DNA to target cells, and it has sparked a lot of interest in the field, particularly in clinical trial therapy techniques. For the new age production of COVID-19 vaccine, development of different mammalian cell lines like HEK293 (most reliable growth and prosperity for transfection) and recombinant adenoviral vectors have become the first priority for biopharmaceutical giants and globally approved vaccine manufacturers to scale up their vaccine production. Adenoviruses have an icosahedral shape, with a protein coat encasing the viral double-stranded DNA genome. Because the adenovirus genome is relatively small, it's a good candidate for insertion of foreign DNA. The adenovirus E1A gene is deleted, and the virus loses its capacity to replicate. This ability can be restored during cell culture propagation by employing cells that produce the E1A protein, for example. Hence, in this mini research, I have shared an overview of the propagation of adenoviral vectors, i.e. recombinant adenovirus SARS CoV-2 vector in HEK-293 cell suspension culture.

Development of a subunit vaccine based on fiber2 and hexon against fowl adenovirus serotype 4

Hepatitis-hydropericardium syndrome (HHS) is widespread in China and causes high chicken mortality that results in great economic losses. A safe and effective vaccine is needed, and a subunit vaccine has potential for development. In this study, a truncated region of the FAdV-4 fiber 2 fused with coding sequence of one epitope of hexon was expressed in a prokaryotic expression system, and the immune protective effects of different doses of recombinant fiber 2 subunit vaccine on SPF chickens were compared. The recombinant fiber2 (Gly275- Pro479 aa)-hexon (Met21-Val51 aa) protein (rFH) obtained in Escherichia coli showed good solubility. The chicken survival rate at the lowest dose (2.5 μg/bird) was 75% (6/8), and at higher doses (≥5 μg/bird) was 100% (8/8) in challenge experiment. Two chickens in the 2.5 μg/bird treatment showed severe lesions, while birds in the higher dose treatments showed no obvious tissue damage as determined by histopathologic analysis of liver and spleen. Absolute quantitative real-time PCR showed no viral load in the ≥5 μg/bird treatments, but two chickens in the 2.5 μg/bird treatment had high viral loads. The challenge experience demonstrated that the rFH vaccine provided 100% protection at ≥5 μg/bird. These results suggested that rFH protein as an effective vaccine to protect against FAdV-4 and provided a new idea for the development of vaccine against HHS.

Transcriptome Analysis Reveals the Potential Role of Long Noncoding RNAs in Regulating Fowl Adenovirus Serotype 4-Induced Apoptosis in Leghorn Male Hepatocellular Cells.

Hepatitis-hydropericardium syndrome (HHS) is caused by fowl adenovirus serotype 4 (FAdV-4) and has resulted in considerable economic losses to the poultry industry globally. FAdV-4 elicits apoptosis in host cells. Long noncoding RNAs (lncRNAs) have emerged as important regulatory RNAs with profound effects on various biological processes, including apoptosis. However, it remains unknown whether lncRNAs participate in FAdV-4-induced apoptosis. In this study, RNA sequencing was applied to determine the transcription of cellular lncRNA in leghorn male hepatocellular (LMH) cells infected with FAdV-4. Cellular RNA transcription analysis demonstrated that FAdV-4 infection elicited 1798 significantly differentially expressed (DE) lncRNAs in infected LMH cells at 24 h post-infection (hpi) compared to mock control infection. In addition, 2873 DE mRNAs were also found. Target prediction and analyses revealed that 775 DE lncRNAs whose 671 target mRNAs were among the DE mRNAs were involved in several signaling pathways, including the AMPK signaling pathway, p53 signaling pathway and insulin signaling pathway. From these 775 DE lncRNAs, we identified 71 DE lncRNAs related to apoptosis based on their target gene functions. Subsequently, lncRNA 54128 was selected from the 71 identified DE lncRNAs, and its role in FAdV-4-induced apoptosis was verified. LncRNA 54128 interference significantly suppressed the rate of apoptosis, which was accompanied by reduced BMP4 transcription levels. To the best of our knowledge, this is the first study to analyze host lncRNA transcription during FAdV-4 infection. Our findings provide a better understanding of host responses to FAdV-4 infection and provide new directions for understanding the potential association between lncRNAs and FAdV-4 pathogenesis.

Quadruple-editing of the MAPK and PI3K pathways effectively blocks the progression of KRAS-mutated colorectal cancer cells.

Mutated KRAS promotes the activation of the MAPK pathway and the progression of colorectal cancer (CRC) cells. Aberrant activation of the PI3K pathway strongly attenuates the efficacy of MAPK suppression in KRAS‐mutated CRC. The development of a novel strategy targeting a dual pathway is therefore highly essential for the therapy of KRAS‐mutated CRC. In this study, a quadruple‐depleting system for the KRAS, MEK1, PIK3CA, and MTOR genes based on CRISPR/SaCas9 was developed. Adenovirus serotype 5 (ADV5) was integrated with two engineered proteins, an adaptor and a protector, to form ADV‐protein complex (APC) for systemic delivery of the CRISPR system. Quadruple‐editing could significantly inhibit the MAPK and PI3K pathways in CRC cells with oncogenic mutations of KRAS and PIK3CA or with KRAS mutation and compensated PI3K activation. Compared with MEK and PI3K/MTOR inhibitors, quadruple‐editing induced more significant survival inhibition on primary CRC cells with oncogenic mutations of KRAS and PIK3CA. The adaptor specifically targeting EpCAM and the hexon‐shielding protector could dramatically enhance ADV5 infection efficiency to CRC cells and significantly reduce off‐targeting tropisms to many organs except the colon. Moreover, quadruple‐editing intravenously delivered by APC significantly blocked the dual pathway and tumor growth of KRAS‐mutated CRC cells, without influencing normal tissues in cell‐ and patient‐derived xenograft models. Therefore, APC‐delivered quadruple‐editing of the MAPK and PI3K pathways shows a promising therapeutic potential for KRAS‐mutated CRC.

Drug resistance via radixin-mediated increase of P-glycoprotein membrane expression during SNAI1-induced epithelial-mesenchymal transition in HepG2 cells.

Epithelial-mesenchymal transition (EMT) plays a role in cancer metastasis as well as in drug resistance through various mechanisms, including increased drug efflux mediated by P-glycoprotein (P-gp). In this study, we investigated the activation mechanism of P-gp, including its regulatory factors, during EMT in hepatoblastoma-derived HepG2 cells. HepG2 cells were transfected with SNAI1 using human adenovirus serotype 5 vector. We quantified mRNA and protein expression levels using qRT-PCR and western blot analysis, respectively. P-gp activity was evaluated by uptake assay, and cell viability was assessed by an MTT assay. P-gp protein expression on plasma membrane was higher in SNAI1-transfected cells than in Mock cells, although there was no difference in P-gp protein level in whole cells. Among the scaffold proteins such as ezrin, radixin and moesin (ERM), only radixin was increased in SNAI1-transfected cells. Uptake of both Rho123 and paclitaxel was decreased in SNAI1-transfected cells, and this decrease was blocked by verapamil, a P-gp inhibitor. The reduced susceptibility of SNAI1-transfected cells to paclitaxel was reversed by elacridar, another P-gp inhibitor. Increased expression of radixin during SNAI1-induced EMT leads to increased P-gp membrane expression in HepG2 cells, enhancing P-gp function and thereby increasing drug resistance.

Molecular typing and pathogenicity assessment of fowl adenovirus associated with inclusion body hepatitis in chicken from India

Recently, inclusion body hepatitis (IBH) outbreaks have been increasingly reported in different regions of India, particularly in broiler flocks. The present study was undertaken to characterize fowl adenovirus associated with IBH in chicken and assessment of its pathogenicity. Liver samples were collected from fowl adenovirus (FAdV) suspected 100 commercial broiler and six broiler breeder flocks from eleven different States of India from 2016 to 2019. All the samples were subjected to 897-bp FAdV hexon gene-specific PCR for confirmation and primary chicken liver cells were used to isolate the field FAdVs. Sequencing and phylogenetic analysis of 897-bp FAdV hexon gene revealed that all the isolates have showed close evolutionary relationship with fowl adenovirus serotype 11 of species D. For pathogenicity assessment, 0.5ml of 106.5 TCID50/ml of field FAdV serotype 11 isolate was orally inoculated in 1-day-old SPF chicks and observed for 21days. This experimental study revealed that there was no mortality in infected chicks and showed clinical signs of dullness, depression and diarrhoea between third and fifth day of oral inoculation. The FAdV was reisolated and confirmed by PCR from experimentally infected chicken. Based on this study, among all serotypes, FAdV serotype 11 is involved in pathogenesis of inclusion body hepatitis in broiler-type chickens in India.

The fowl adenovirus serotype 4 (FAdV-4) induce cellular pathway in chickens to produce interferon and antigen-presented molecules (MHCI/II)

FAdV-4 is the major strain of adenovirus that responsible for hydro-pericardial syndrome (HPS) in poultry. In this study, the virus's specific gene fragments were isolated from clinically suspected cases and amplified by PCR. Finally, after a viral infection to investigate the immune response of the host, the gene expression of MHC (major histo-compatible) molecules (MHCIα, MHCIIβ), Ii (Invariant Chain) gene, inflammatory cytokines (IFN-β, IFN-γ, and IL-1β), and transcription factors (MDA5, STING, IRF7, and NF-kB) were detected by real-time PCR (fluorescence technology). The results of sequence comparison showed that the clinically isolated virus was 100% homologous to a virulent strain of avian adenovirus group C serotype 4 (FAdV-4), which were named AH-FAdV-4. The TCID50 and pathogenicity of the virus were determined that was 106.52/0.1 mL with a mortality rate of 100% in chickens and 0% in ducks. Furthermore, results showed that the expression level of MHCIα, MHCIIβ, and Ii genes in chicken embryo kidney cells significantly (P < 0.01) upregulated (increased) after infection, which was 43, 5.2, and 2.5 times higher than the control group. With the addition of PDTC, an inhibitor of NF-kB, then the expression level of MHCIα, MHCIIβ, and Ii was decreased significantly (P < 0.01) than the control group. The transcription levels of these genes were decreased 0.64, 0.27, and 0.26 respectively. Simultaneously, the expression levels of IFN-β, IFN-γ, and IL-1β were also significantly (P < 0.01) up-regulated (increased) 7.8, 22.7, and 5 times higher than the control group. It was found that up-regulation of STING and NF-κB pathways are directly involved in the regulation of inflammatory cytokines (IFN-β, IFN-γ, and IL-1β), MHC molecules (MHCIα, MHCIIβ), and Ii gene. The results also showed that the gene regulation pathways consecutively increased the expression levels of MDA5, STING, IRF7, and NF-kB. It is conducted that the expression levels of cytokines, MHC molecules, and li gene were increased by STING and NF-kB pathways.

Intranasal plus subcutaneous prime vaccination with a dual antigen COVID-19 vaccine elicits T-cell and antibody responses in mice

We have developed a COVID-19 vaccine, hAd5 S-Fusion + N-ETSD, that expresses SARS-CoV-2 spike (S) and nucleocapsid (N) proteins with modifications to increase immune responses delivered using a human adenovirus serotype 5 (hAd5) platform. Here, we demonstrate subcutaneous (SC) prime and SC boost vaccination of CD-1 mice with this dual-antigen vaccine elicits T-helper cell 1 (Th1) biased T-cell and humoral responses to both S and N that are greater than those seen with hAd5 S wild type delivering only unmodified S. We then compared SC to intranasal (IN) prime vaccination with SC or IN boosts and show that an IN prime with an IN boost is as effective at generating Th1 biased humoral responses as the other combinations tested, but an SC prime with an IN or SC boost elicits greater T cell responses. Finally, we used a combined SC plus IN (SC + IN) prime with or without a boost and found the SC + IN prime alone to be as effective in generating humoral and T-cell responses as the SC + IN prime with a boost. The finding that SC + IN prime-only delivery has the potential to provide broad immunity—including mucosal immunity—against SARS-CoV-2 supports further testing of this vaccine and delivery approach in animal models of viral challenge.

Safety and Immunogenicity of ChAd63/MVA Pfs25-IMX313 in a Phase I First-in-Human Trial

BackgroundTransmission blocking vaccines targeting the sexual-stages of the malaria parasite could play a major role to achieve elimination and eradication of malaria. The Plasmodium falciparum Pfs25 protein (Pfs25) is the most clinically advanced candidate sexual-stage antigen. IMX313, a complement inhibitor C4b-binding protein that forms heptamers with the antigen fused to it, improve antibody responses. This is the first time that viral vectors have been used to induce antibodies in humans against an antigen that is expressed only in the mosquito vector.MethodsClinical trial looking at safety and immunogenicity of two recombinant viral vectored vaccines encoding Pfs25-IMX313 in healthy malaria-naive adults. Replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding Pfs25-IMX313, were delivered by the intramuscular route in a heterologous prime-boost regimen using an 8-week interval. Safety data and samples for immunogenicity assays were taken at various time-points.ResultsThe reactogenicity of the vaccines was similar to that seen in previous trials using the same viral vectors encoding other antigens. The vaccines were immunogenic and induced both antibody and T cell responses against Pfs25, but significant transmission reducing activity (TRA) was not observed in most volunteers by standard membrane feeding assay.ConclusionBoth vaccines were well tolerated and demonstrated a favorable safety profile in malaria-naive adults. However, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine formulation.Trial Registration Clinicaltrials.gov NCT02532049.

Development of colloidal gold-based test strip for rapid detection of serotype 4 fowl adenovirus

The outbreaks of hepatitis-hydropericardium syndrome (HPS) caused by serotype 4 fowl adenovirus (FAdV-4) have spread from broilers to laying hens, breeders, geese and Cherry Valley duck, resulting in high economic losses to the poultry industry globally. In this study, a rapid colloidal gold test strip for detection of FAdV-4 was developed by using two monoclonal antibodies (mAbs) against the Fiber-2 of FAdV-4. Specificity analysis revealed that the test strip only reacted with FAdV-4, but not with other pathogens including different serotypes of fowl adenovirus and other avian pathogens tested. The limit of the detection (LOD) of the strip could reach as low as 0.1 μg/0.1 mL of GST-Fiber-2 protein and 1 × 105 TCID50/0.1 mL of FAdV-4, respectively. Moreover, the test strip could be efficiently applied in detecting tissue samples from diseased chickens with HPS. Comparison analysis further revealed that the test strip showed good compatibility with PCR assay for detection of virus isolates and clinical samples. In conclusion, our test strip provides an efficient on-site diagnostic method in a quick and convenient manner for detection of FAdV-4, especially in resource-limited areas.

Investigation of Adenovirus and Rotavirus Frequencies and Seasonal Distribution in Patients with Gastroenteritis

Purpose: The aim of the study was to determine the incidence and seasonal distribution of rotavirus group A and enteric adenovirus serotypes 40 and 41 in all age groups admitted to our hospital with acute gastroenteritis. Materials and Methods: Results of the patients from all ages with the pre-diagnosis of acute gastroenteritis between January 2016 and December 2019 were retrospectively evaluated. A total of 10,049 patient results in which rotavirus antigen was investigated in stool samples and 11.482 patient results in which adenovirus antigen was investigated in stool samples were included in the study. Rotavirus and adenovirus positivities were investigated by using immunochromatographic method. Results: In the distribution of rotavirus and adenovirus antigen positivity according to years, the highest positivity for both viral agents was recorded in 2016 and in winter months. According to this result, rotavirus positivity rate was 18.4% and adenovirus positivity rate was 3.5% in 2016. While the frequency of rotavirus antigen positivity was the highest in the age group between 0-2 years the frequency of adenovirus antigen positivity was the highest in the age group between 3-5 years. Conclusion: The epidemiology of the disease must absolutely be followed up with surveillance studies within the scope of fight against viral gastroenteritis. Our study in which there are data of four years will contribute to establishing the country data.

Abstract 1912: Evasion of neutralizing antibodies and enhanced anti-tumor effects of hexon-modified Delta-24-RGD oncolytic adenovirus

Abstract Oncolytic viruses are a new form of cancer immunotherapy that harnesses the combined abilities of the virus to directly lyse cancer cells and to induce immunogenic environments to activate anti-tumor immunity. Our oncolytic adenovirus Delta-24-RGD has demonstrated significant clinical benefits for a subset of patients with recurrent malignant gliomas in a recent phase 1 clinical trial. However, the backbone of Delta-24-RGD is derived from adenovirus serotype 5 to which 40 percent of the human population has prior exposure and harbor neutralizing antibodies that can block virus infection and replication. We hypothesized that replacing the major targets of antibodies in the oncolytic virus will protect it from neutralizing antibodies, resulting in enhanced efficacy of the cancer virotherapy. Because hexon hypervariable regions are the dominant targets of antibodies, we replaced these regions with those from a lesser prevalent serotype 43 to create a hybrid virus called Delta-24-RGD-H43m. Transmission electron microscopy imaging of the viruses revealed the assembly of viable virions after infection with either Delta-24-RGD or Delta-24-RGD-H43m in human and mouse cancer cell lines, signifying successful virus replication, that was corroborated by quantification of viral replication using hexon-staining dilution method. Accordingly, the inherent anti-tumor effect of the virus was not perturbed after the genetic modification, as Delta-24-RGD and Delta-24-RGD-H43m showed comparable efficacies both in vitro (P= 0.568) and in vivo using mice without prior immunity to adenovirus (P= 0.228). Importantly, the oncolytic ability of Delta-24-RGD-H43m was not compromised by neutralizing antibodies generated against adenovirus serotype 5 that efficiently neutralized Delta-24-RGD (P&amp;lt; 0.0001) as assessed by measuring cell viability after virus infection. In immunized mice with prior adenovirus exposure, Delta-24-RGD-H43m treatment yielded higher occurrences of complete tumor remission compared to Delta-24-RGD (89% versus 67%). Despite our growing understanding of viruses, the immune system, and cancer, the specific interaction between oncolytic viruses and the host immune system is still very limited. Strategies to protect the therapeutic viruses from virus-specific immune responses should prolong their persistence in tumors and result in better therapeutic outcomes. Further research in this field will aid the development of enhanced cancer virotherapies. Citation Format: Dong Ho Shin, Teresa T. Nguyen, Hong Jiang, Xuejun Fan, Yanhua Yi, Sagar S. Sohoni, Sumit Gupta, Ashley Ossimetha, Candelaria Gomez-Manzano, Juan Fueyo. Evasion of neutralizing antibodies and enhanced anti-tumor effects of hexon-modified Delta-24-RGD oncolytic adenovirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1912.

Abstract 1689: Exosomes derived from tumor cells treated with immunostimulatory oncolytic virotherapy exert immune activation instead of immunosuppression

Abstract Immunostimulatory gene therapy using oncolytic viruses - OV - for combined gene transfer and tumor-specific oncolysis is a promising immunotherapy for cancer. Due to neutralizing antibodies in the blood, OVs are commonly administered by intratumoral delivery. The immune activation is both local and systemic as activated dendritic cells in the injected lesion travel to lymph nodes to activate tumor-reactive T cells that can in turn act on both injected and non-injected lesions. In the present project, we demonstrate that OV-infected tumor cells released exosomes that cargo mRNA and protein coded by the human immunostimulatory genes expressed by the OV. Tumor cell-derived exosomes are commonly immunosuppressive because they cargo immunosuppressive molecules expressed by the tumor. We hypothesized that exosomes derived from OV-infected tumor cells may instead be immunostimulatory. To investigate the role of exosomes derived from OV-treated tumors, human melanoma Mel526 cells were infected by delolimogene mupadenorepvec -LOAd703 - which is an adenoviral serotype 5/35 OV armed with costimulatory molecules CD40L and 4-1BBL. LOAd703 could induce expression of CD40L and 4-1BBL on the melanoma cell surface and kill the cells by replication-induced oncolysis after 48-72 hours culture as evaluated by MTS. Exosomes were purified prior oncolysis and investigated for the presence of CD40L and 4-1BBL mRNA and protein, and for their capacity to stimulate immune responses. The results demonstrated that the exosomes can indeed cargo CD40L and 4-1BBL both as mRNA as shown by PCR, and protein as shown by ELISA, western blot, flow cytometry and electron microscopy post LOAd703 tumor cell infection. Furthermore, the exosomes from CD40L and 4-1BBL-expressing tumor cells could stimulate robust dendritic cell activation with enhanced levels of MHC class I and II as well as costimulatory molecules CD80, CD86 and CD83. In contrast, MHC class I and the costimulatory molecules were reduced on dendritic cells stimulated with exosomes derived from non-infected tumor cells, which is in line with previous knowledge of exosomes as immunosuppressants in the tumor. However, PD-L1 was increased on dendritic cells stimulated with the exosomes from CD40L and 4-1BBL-expressing tumor cells. Hence, LOAd703 therapy may benefit from combination with PD-L1 or PD-1 checkpoint blockade. In conclusion, tumor-derived exosomes released by OV-infected tumor cells can cargo the immunostimulatory transgenes expressed by the OVs and are then converted from immunosuppressive to immune activating exosomes as shown by their capacity to activate immune cells such as dendritic cells. Exosomes that cargo immunostimulatory transgenes from OV-infected tumor cells may support the systemic immunity gained by OVs after local intratumoral delivery. Citation Format: Alireza Labani-Motlagh, Sedigheh Naseri, Jessica Wenthe, Emma Eriksson, Angelica Loskog. Exosomes derived from tumor cells treated with immunostimulatory oncolytic virotherapy exert immune activation instead of immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1689.

Single-Dose Immunization With a Chimpanzee Adenovirus-Based Vaccine Induces Sustained and Protective Immunity Against SARS-CoV-2 Infection.

The development of a safe and effective vaccine against SARS-CoV-2, the causative agent of pandemic coronavirus disease-2019 (COVID-19), is a global priority. Here, we aim to develop novel SARS-CoV-2 vaccines based on a derivative of less commonly used rare adenovirus serotype AdC68 vector. Three vaccine candidates were constructed expressing either the full-length spike (AdC68-19S) or receptor-binding domain (RBD) with two different signal sequences (AdC68-19RBD and AdC68-19RBDs). Single-dose intramuscular immunization induced robust and sustained binding and neutralizing antibody responses in BALB/c mice up to 40 weeks after immunization, with AdC68-19S being superior to AdC68-19RBD and AdC68-19RBDs. Importantly, immunization with AdC68-19S induced protective immunity against high-dose challenge with live SARS-CoV-2 in a golden Syrian hamster model of SARS-CoV-2 infection. Vaccinated animals demonstrated dramatic decreases in viral RNA copies and infectious virus in the lungs, as well as reduced lung pathology compared to the control animals. Similar protective effects were also found in rhesus macaques. Taken together, these results confirm that AdC68-19S can induce protective immune responses in experimental animals, meriting further development toward a human vaccine against SARS-CoV-2.