Recombinant Adenovirus Research Articles

GLP1 alleviates oleic acid-propelled lipocalin-2 generation by tumor-infiltrating CD8+ T cells to reduce polymorphonuclear MDSC recruitment and enhances viral immunotherapy in pancreatic cancer.

Recruitment of polymorphonuclear MDSCs (PMN-MDSCs) in the TME suppresses the antitumor activity of tumor-infiltrating CD8+ T cells (CD8+ TILs). Little is known about the role of antitumoral CD8+ TILs in actively initiating an immune-tolerant microenvironment, particularly in the recruitment of PMN-MDSCs. In this study, we found that immunotherapy-activated CD8+ TILs significantly increased PNM-MDSC infiltration in the TME, resulting in antitumor resistance. When CD8+ T cells are activated, lipocalin-2 (LCN2) expression is strongly upregulated, which significantly enhances PMN-MDSC chemotaxis. Mechanistically, immune activation increased fatty acid synthesis in CD8+ T cells, particularly oleic acid (OA), which induced lysosomal membrane permeabilization, releasing cathepsin B and subsequently activating NF-κB to promote LCN2 expression. Moreover, we showed that glucagon-like peptide 1 (GLP1) effectively inhibited OA synthesis in activated CD8+ T cells, reducing LCN2 production. We then developed a recombinant adenovirus encoding GLP1 (AdV-GLP1), which significantly reduced PMN-MDSC infiltration and reinvigorated the antitumor activity of CD8+ TILs. In various pancreatic cancer models, including subcutaneous, orthotopic, and humanized CDX/PDX models, AdV-GLP1 displayed excellent antitumor efficacy. Our work advances the understanding of how immunotherapy-activated CD8+ TILs initiate PMN-MDSC infiltration and provides a clinically relevant strategy to target this interaction and improve cancer immunotherapy.

Treatment Response to Oncolytic Virus in Patient-Derived Breast Cancer and Hypopharyngeal Cancer Organoids: Evaluation via a Microfluidics Organ-on-a-Chip System

In this study, we present an oncolytic virus (OV) evaluation system established using microfluidic organ-on-a-chip (OOC) systems and patient-derived organoids (PDOs), which was used in the development of a novel oncolytic virus, AD4-GHPE. An OV offers advantages such as good targeting ability and minimal side effects, and it has achieved significant breakthroughs when combined with immunotherapy in recent clinical trials. The development of OVs has become an emerging research focus. PDOs can preserve the heterogeneity of in situ tumor tissues, whereas microfluidic OOC systems can automate and standardize various experimental procedures. These systems have been applied in cutting-edge drug screening and cell therapy experiments; however, their use in functionally complex oncolytic viruses remains to be explored. In this study, we constructed a novel recombinant oncolytic adenovirus, AD4-GHPE, and evaluated OOC systems and PDOs through various functional validations in hypopharyngeal and breast cancer organoids. The results confirmed that AD4-GHPE exhibits three antitumor mechanisms, namely, tumor-specific cytotoxicity, a reduction in programmed death ligand 1 (PD-L1) expression in tumor cells to increase CD8+ T-cell activity, and granulocyte–macrophage colony-stimulating factor (GM-CSF) secretion. The evaluation system combining OOC systems and PDOs was efficient and reliable, providing personalized OV treatment recommendations for patients and offering industrialized and standardized research ideas for the development of OVs.

HeterologousPrime-Boost immunizationwithAdenoviral vector and recombinant subunit vaccines strategies against dengue virus type2.

Dengue virus (DENV) remains a significant public health threat in tropical and subtropical regions, with effective antiviral treatments and vaccines still not fully established despite extensive research. A critical aspect of vaccine development for DENV involves selecting proteins from both structural and non-structural regions of the virus to activate humoral and cellular immune responses effectively. In this study, we developed a novel vaccine for dengue virus serotype 2 (DENV2) using a heterologous Prime-Boost strategy that combines an adenoviral vector (Ad) with subunit vaccines. The vaccine design included non-structural protein 1 (NS1), envelope protein domain III (EDIII), and the bc-loop of envelope domain II (EDII) as conserved epitopes. These antigens were fused into a single construct P1 and inserted into the pAdTrack-CMV vector to produce a recombinant adenovirus (rAd5-P1) via homologous recombination in E. coli. The examination of the immune response indicated that strong humoral and cellular immunity was generated in various groups of mice. Additionally, the group receiving a heterologous regimen of recombinant adenovirus and protein showed a superior balance of humoral and cellular immunity in terms of IgG2a/IgG1 and INF-γ /IL-4 ratios. These findings validate the vaccine design's ability to utilize both structural and non-structural proteins to generate strong immune responses on two platforms. The promising results from the heterologous regimen highlight its potential as an effective DENV2 vaccine candidate. This research offers significant insights into developing safe and effective DEN vaccines, contributing to efforts to control DENV infections.

Detection of recombinant type of human adenovirus C composed of type 89 and type 5 in Aichi Prefecture, Japan, 2016-2019

Recombinant human adenoviruses (HAdVs) have been reported from many countries, including Japan. We report a recombinant HAdV-C type detected in feces with gastroenteritis infection or throat swabs with upper respiratory tract inflammation of six children in Aichi Prefecture. This type may have been prevalent in the area. Between April 2014 and March 2019, we attempted to detect HAdVs in 9,483 specimens (fecal, throat swab, urine, and conjunctival swab) collected from patients with suspected viral infection. A total of 220 HAdV-C isolates were obtained and serotyped by neutralization test (NT). Of these, 62 HAdV-C strains (HAdV-C1: 15, -C2: 19, -C5: 22, -C6: 6) were genotyped by sequencing for the three regions, penton base, hexon, and fiber regions, respectively. All 62 strains were grouped into the same genotype as serotype for the hexon and fiber region. In contrast, phylogenetic analysis showed 6 of the 22 strains with serotype HAdV-C5 were grouped into HAdV-C89 for the penton base region. This type was detected as a recombinant HAdV causing SARI in China between 2017 and 2021, was found to have been persistently detected in transnational Aichi Prefecture over the same period.

Vaccination against SARS-CoV-2 provides low-level cross-protection against common cold coronaviruses in mouse and non-human primate animal models.

The common cold coronaviruses are a source of ongoing morbidity and mortality particularly among elderly and immunocompromised individuals. While cross-reactive immune responses against multiple coronaviruses have been described following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination, it remains unclear if these confer any degree of cross-protection against the common cold coronaviruses. A recombinant fowl adenovirus vaccine expressing the SARS-CoV-2 spike protein (FAdV-9-S19) was generated, and protection from SARS-CoV-2 challenge was shown in K18-hACE2 mice. Vaccinated mice were also challenged with the common cold coronaviruses human coronavirus (HCoV)-OC43 and HCoV-NL63 by the intranasal route, and viral shedding and lung burden were reduced in these groups compared to unvaccinated animals. Histopathological analysis of lung tissues revealed significantly less inflammation and lower pathology scores in mice that received FAdV-9-S19 . Because no mouse model for the coronavirus HCoV-229E exists, we vaccinated and challenged cynomolgus macaques to evaluate cross-protection against HCoV-229E. Animals were monitored for clinical signs of disease and viral shedding. Infectious virus was detected in both groups throughout the course of infection; however, vaccinated animals showed reduced viral shedding at multiple time points after infection. Histopathological analysis of lung tissues following challenge also indicated a more moderate disease in the vaccinated animals. Therefore, vaccination with FAdV-9-S19 also provided a moderate cross-protection against HCoV-229E disease in the cynomolgus macaques infection model. Our study demonstrates that vaccination with a recombinant fowl adenovirus expressing SARS-CoV-2 spike protein can provide a low-level cross-protection against beta- and alphacoronaviruses. These findings are important for the design of future pan-coronavirus vaccines.IMPORTANCEThe common cold coronaviruses are a source of ongoing morbidity and mortality particularly among elderly and immunocompromised individuals, and no vaccine is currently available. Cross-reactive immune responses have been described following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination; however, it remains unclear what degree of cross-protection they confer against the common cold coronaviruses. We demonstrate that both humoral and cell-mediated immune responses provide a low-level cross-protection, resulting in reduced viral load and pathology for the common cold coronaviruses OC43 and NL63 in mouse models. Additionally, we present a novel non-human primate (NHP) model of infection with the common cold coronavirus 229E, demonstrating that it mimics the disease observed in humans and can serve as a model for future vaccine studies, as cross-protection was also observed. This is significant as it suggests that current vaccines could provide a low-level protection against other coronaviruses and could serve as part of vaccination strategy against future novel coronaviruses.

Exploring the impact of climatic variations on infectious gastroenteritis and a review of current antiviral vaccines

Weather and seasonal changes are recognized contributors to the prevalence of infectious diseases, including gastroenteritis, particularly in developing countries. Infectious gastroenteritis, characterized by symptoms such as diarrhea, vomiting, and abdominal pain, is predominantly caused by pathogens like rotavirus and norovirus. Environmental changes, such as abrupt temperature drops and heavy rainfall, facilitate the proliferation and transmission of these pathogens. Studies reveal a significant correlation between low temperatures, increased rainfall, and higher hospitalization rates for rotavirus and norovirus infections. Developing countries face compounded challenges due to financial constraints, inadequate healthcare infrastructure, poor sanitation, and malnutrition, which exacerbate disease burden. Preventive measures, particularly vaccinations, have proven effective in mitigating viral gastroenteritis. Rotavirus vaccines, such as RotaTeq™ and Rotarix™, have demonstrated efficacy in reducing disease severity and hospitalization rates. However, norovirus vaccine development remains complex due to genetic diversity, rapid mutation rates, and the absence of standardized models for testing efficacy. Promising candidates like HIL-214, a bivalent VLP vaccine, and novel approaches such as plant-based vaccines and recombinant adenovirus platforms have shown potential in preclinical and early clinical trials. Notable advancements include immunization strategies employing double-stranded RNA adjuvants and the use of gnotobiotic pig models to study vaccine efficacy. This study underscores the urgent need for enhanced research into the interplay between weather patterns and gastroenteritis incidence. Addressing vaccine development challenges is crucial to mitigating the global health burden of infectious gastroenteritis in the face of climate change.

Construction and immunological evaluation of recombinant adenovirus vaccines of new novel NADC34-PRRSV strains in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive and respiratory diseases in sow herds and piglets. The emergence of ORF5 RFLP 1-7-4-like (NADC34-like) PRRSV strain in China has brought a new round of challenges to PRRSV prevention. In addition, recombinant adenovirus vaccine candidates against the newly emerged NADC34-like strain were constructed in the study; the immunogenicity of the vaccine was investigated in piglets. After inoculation with PRRSV recombinant adenovirus, specific antibodies, neutralizing antibodies, and levels of IFN-γ and IL-4 cytokines were detected in serum. Thirty-five days after immunization, the levels of IFN-γ and IL-4 cytokines in the pac-Ad5-34-GP3, pac-Ad5-34-GP5, and pac-Ad5-34-GP35 experimental groups were significantly higher (p < 0.05) than those of the PBS and the adenovirus group. All vaccines can cause corresponding Th1 and Th2 immune responses based on animal experimental results. After the challenge, no obvious clinical symptoms were observed in the immune groups compared with the control group, vaccinated animals could reduce the occurrence of viremia, and the occurrence of viremia was alleviated, with no obvious pathological changes in the lungs, indicating that recombinant adenovirus vaccine could provide a good protective immunity and produce a good humoral and cellular immune response at the same time. It shows that the recombinant adenovirus vaccine group has better protection against the virus. Provide vaccine reserve and theoretical support for the emergence of new PRRSV subtypes in China.

Immune Response Elicited by Recombinant Adenovirus-Delivered Glycoprotein B and Nucleocapsid Protein UL18 and UL25 of HSV-1 in Mice.

Due to the complex pathogenic and immune escape mechanisms of herpes simplex virus type 1 (HSV-1), especially the failure of induced immune responses to block the initial cell-to-cell transmission of the virus from skin cells to neurons, the body struggles to establish effective prevention and control methods, resulting in the failure of currently developed vaccines. Previous studies have highlighted the crucial roles of surface glycoproteins and nucleocapsid proteins in activating the body's immune defense system against HSV-1 infection. In this study, recombinant adenoviruses were used as vectors to generate adenoviruses carrying the nucleocapsid protein genes UL18 and UL25, as well as the surface glycoprotein gene gB. This approach aimed to mimic the protein expression process that occurs following viral infection of the host and to investigate the immune response characteristics induced by UL18, UL25, and gB proteins. The findings revealed that UL18, UL25, and gB proteins could all trigger the expression of genes associated with innate immune responses; however, the specific genes induced varied in type and level. Furthermore, all three proteins were capable of promoting the proliferation of CD8+ T cells in the lymph nodes. Notably, only UL18 and gB could elicit a Th1 cell immune response. Interestingly, among these proteins, only UL18 could also induce a relatively higher IL-4 level, indicating a Th2 cell immune response. In addition to cellular immunity, all three proteins stimulated the production of specific IgG antibodies. Notably, UL18 induced higher and more sustained levels of specific IgG antibodies in mice. By contrast, only glycoprotein gB induced lower levels of neutralizing antibodies in mice. Moreover, when these mice were challenged with HSV-1, the co-immunization with UL18 and gB provided better protection than gB alone. In conclusion, HSV-1 surface glycoproteins and nucleocapsid proteins exhibit differences in their ability to induce innate and adaptive immunity in the body, suggesting potential avenues for vaccine design by leveraging their complementary advantages.

In situ blockade of TNF-TNFR2 axis via oncolytic adenovirus improves antitumor efficacy in solid tumors.

Tumor necrosis factor (TNF) has been recognized as an immune activation factor in tumor immunotherapy. Our study demonstrated that TNF blockade markedly enhanced the antitumor efficacy of oncolytic adenovirus (AdV) therapy. To minimize systemic side effects, we engineered a recombinant oncolytic AdV encoding a TNF inhibitor (AdV-TNFi) to confine TNF blockade within the tumor microenvironment (TME). AdV-TNFi significantly improved therapeutic outcomes across various solid tumor models, including four murine and two golden hamster cancers. Immune cell profiling identified CD8+ T cells as the primary mediators of AdV-TNFi-induced antitumor effects, rather than CD4+ T or NK cells. Additionally, AdV-TNFi significantly decreased the infiltration of suppressive myeloid-derived immune cells within the TME and promoted long-term antitumor immune surveillance. Further investigation indicated that TNFR2, more than TNFR1, is pertinent to the immunosuppressive TME, with a recombinant AdV encoding anti-TNFR2 demonstrating comparable antitumor efficacy to AdV-TNFi. Moreover, AdV-TNFi enhanced the antitumor efficacy of gemcitabine and immune checkpoint blockades (ICBs), such as anti-PD-L1 and anti-TIGIT antibodies, in pancreatic carcinoma, and the anti-EGFR antibody in colon carcinoma. In conclusion, intratumoral blockade of the TNF/TNFR2 axis using AdV augments cancer immunotherapy efficacy while mitigating the risks associated with systemic TNF or TNFR2 suppression, warranting further clinical investigation.

Transglutaminase 2 regulates endothelial cell calcification via IL-6-mediated autophagy.

Endothelial cell (EC) calcification is an important marker of atherosclerotic calcification. ECs play a critical role not only in atherogenesis but also in intimal calcification, as they have been postulated to serve as a source of osteoprogenitor cells that initiate this process. While the role of transglutaminase 2 (TG2) in cellular differentiation, survival, apoptosis, autophagy, and cell adhesion is well established, the mechanism underlying the TG2-mediated regulation of EC calcification is yet to be fully elucidated. The TG2 gene was overexpressed or silenced by using siRNA and recombinant adenovirus. RT-PCR and WB were used to analyze the relative expression of target genes and proteins. 5-BP method analyzed TG2 activity. mCherry-eGFP-LC3 adenovirus and transmission electron microscopy analyzed EC autophagy level. Calcium concentrations were measured by using a calcium colorimetric assay kit. Alizarin red S staining assay analyzed EC calcification level. Elisa analyzed IL-6 level. Establishing EC calcification model by using a calcification medium (CM). Our findings demonstrated that CM increased TG2 activity and expression, which activated the NF-κB signaling pathway, and induced IL-6 autocrine signaling in ECs. Furthermore, IL-6 activated the JAK2/STAT3 signaling pathway to suppress cell autophagy and promoted ECs calcification. ECs are not only critical for atherogenesis but also believed to be a source of osteoprogenitor cells that initiate intimal calcification. Previous research has shown that TG2 plays an important role in the development of VC, but the mechanism by which it exerts this effect is not yet fully understood. Our results demonstrated that TG2 forms complexes with NF-κB components inhibition of autophagy promoted endothelial cell calcification through EndMT. Therefore, our research investigated the molecular mechanism of EC calcification, which can provide new insights into the pathogenesis of atherosclerosis.

&lt;i&gt;In vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt; tropism and biodistribution of recombinant simian adenovirus type 25

Introduction. Recombinant adenoviruses are widely used in the development of vaccines for a variety of infectious diseases. Despite numerous clinical studies, only a few types of human (types 5 and 26) and simian (isolate Y25) adenoviruses are currently used to produce vaccine formulations. Different types of adenoviruses vary in their cellular tropism, which plays a key role in their ability to elicit an immune response. The aim of this study was to investigate the cellular tropism of the simian adenovirus type 25 in vitro and its biodistribution in vivo in comparison with human adenoviruses types 5 and 26. Materials and methods. The efficiency of in vitro transduction was evaluated on 15 different cell lines using recombinant adenovirus vectors expressing the enhanced green fluorescent protein (EGFP) reporter gene. In vivo biodistribution and bioluminescence imaging were evaluated in BALB/c mice after administration of recombinant adenoviral vectors encoding the luciferase reporter gene. The acute toxicity of a recombinant simian adenovirus type 25 vector was assessed in mice and rats following intramuscular or intravenous administration. Results. Recombinant simian adenovirus effectively transduces a wide range of cells. At the same time, a higher tropism to human glioblastoma cells (GL-6) was found compared to the other two studied adenoviruses. In vivo experiments have shown that recombinant adenoviruses are mainly localized at the injection site, and transgene expression persists for 21 days. Acute toxicity studies demonstrated that simian adenovirus type 25 vector was well-tolerated, with no animal deaths or detectable toxic effects. Conclusion. The new platform based on the recombinant simian adenovirus type 25 is not inferior to the existing and well-established delivery systems based on human adenovirus types 5 and 26. Due to its high level of gene transfer and favorable safety profile, the use of the simian adenovirus type 25 in medicine has the potential to offer many benefits for the development of vaccines against future infectious diseases.

An engineered Japanese encephalitis virus mRNA-lipid nanoparticle immunization induces protective immunity in mice.

Japanese encephalitis virus (JEV) and Zika virus (ZIKV) pose a severe threat to human health. Our previous research results, as well as those of other research groups, indicated that antibodies (Abs) induced by JEV infection or JEV vaccine vaccination could enhance ZIKV infection in vitro and exacerbate the mortality of ZIKV-infected mice, vice versa, which is known as antibody-dependent enhancement (ADE). Although studies on other flaviviruses revealed that altering the amino acid residues located in the fusion loop (FL) of envelope (E) protein can reduce the level of flavivirus-cross-reactive Abs, thereby abating the ADE of heterologous flavivirus infection, it is unclear whether this strategy is equally applicable to JEV. In this study, we constructed recombinant adenoviruses and nucleotide-modified mRNA-lipid nanoparticle (LNP) encoding JEV wild-type E protein or E protein mutant (designated as Ad5-JEV-EWT and Ad5-JEV-Emut; JEV-EWT mRNA-LNP, and JEV-Emut mRNA-LNP). We evaluated the immunogenicity of these vaccine candidates in mice and the capacity of vaccine-immune mouse sera to neutralize JEV infection or mediate ADE of ZIKV infection in vitro and in vivo. Ad5-JEV-Emut or JEV-Emut mRNA-LNP immunization induced ZIKV-cross-reactive Ab response which is dramatically lower than that induced by Ad5-JEV-EWT and JEV-EWT mRNA-LNP, respectively. The levels of JEV-neutralizing Abs induced by Ad5-JEV-Emut or JEV-Emut mRNA-LNP are comparable to that induced by Ad5-JEV-EWT and JEV-EWT mRNA-LNP, respectively. The ability of Abs induced by Ad5-JEV-Emut to enhance ZIKV infection in vitro is attenuated as compared with that induced by Ad5-JEV-EWT. Moreover, JEV-Emut mRNA-LNP immunization elicited potent T cell response similar to JEV-EWT mRNA-LNP in mice. Mice immunized with each mRNA-LNP exhibited lower level of serum viral load than the mock-immunized mice post JEV challenge. Mice receiving JEV-EWT mRNA-LNP-immune mouse sera exhibited ADE post ZIKV challenge whereas passively transferred JEV-Emut mRNA-LNP-immune mouse sera did not lead to obvious ADE of ZIKV infection in recipient mice. Most importantly, maternally acquired Abs did not enhance the mortality of 1-day-old neonates born to JEV-Emut mRNA-LNP-immunized mice post ZIKV challenge. These results suggest that optimizing the FL sequence of JEV could significantly reduce the level of JEV/ZIKV-cross-reactive Abs and abrogate the ADE of ZIKV infection, providing a promising strategy to develop effective and safety JEV vaccine.

Dissecting immunological mechanisms underlying influenza viral nucleoprotein-induced mucosal immunity against diverse viral strains

ABSTRACT The nucleoprotein (NP) of type A influenza virus (IAV) is highly conserved across all virus strains, making it an attractive candidate antigen for universal vaccines. While various studies have explored NP-induced mucosal immunity, here we interrogated the mechanistic differences between intramuscular (IM) and intranasal (IN) delivery of a recombinant adenovirus carrying NP fused with a bifunctional CD40 ligand. Despite being less effective than IM delivery in inducing systemic cellular immune responses and antibody-dependent cellular cytotoxicity (ADCC), IN immunization elicited superior antigen-specific recall humoral and cellular response in the nasal associated lymphoid tissue (NALT) of the upper respiratory tract, the initial site of immune recognition and elimination of inhaled pathogens. IN vaccination also induced significantly stronger pulmonary T cell responses in the lower respiratory tract than IM vaccination, in particular the CD8 T cells. Moreover, blocking lymphocyte circulation abrogated IM but not IN immunization induced protection, illustrating the critical role of local memory immune response upon viral infection. Notably, the CD40-targeted nasal delivery not only improved the magnitude but also the breadth of protection, including against lethal challenge with a newly isolated highly pathogenic avian H5N1 strain. These findings are informative for the design of universal mucosal vaccines, where the predominant mode of protection is independent of neutralizing antibodies.

Apolipoprotein A1-encoding recombinant adenovirus remodels cholesterol metabolism in tumors and the tumor microenvironment to inhibit hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a prevalent malignant tumor requiring effective treatments. Oncolytic viruses induce anti-tumor responses but have limited efficacy. Apolipoprotein A1 (ApoA1) inhibits inflammation, modulates immunity, and promotes anti-oxidation. This study aims to construct an oncolytic adenovirus (Ad5)-ApoA1 for superior anti-tumor effects. We analyzed ApoA1 expression in tumors and its prognostic significance using public databases. Subsequently, we engineered a recombinant oncolytic adenovirus Ad5-ApoA1 and assessed its replication and oncolytic efficacy in vitro and in nude mice. The impact of Ad5-ApoA1 on the tumor microenvironment of HCC was evaluated through flow cytometry, transcriptome sequencing, single-cell sequencing, and other methodologies. Additionally, mechanisms of immune microenvironment modulation by Ad5-ApoA1 were explored. ApoA1 expression was down-regulated with HCC progression and significantly positively correlated with the prognosis of HCC patients. Ad5-ApoA1 exhibited robust oncolytic activity but showed no therapeutic effect on nude mice. However, it significantly inhibited HCC growth and prolonged the survival period of both healthy-immune and humanized immune-reconstituted NCG mice. Furthermore, Ad5-ApoA1 significantly promoted the expression of IFN-γ and GzmB in CD8+ T cells while inhibiting the expression of PD-1 and LAG-3. Notably, the cholesterol content in the CD8+ T cells studied was significantly correlated with the expression of PD-1 and LAG-3, with ApoA1 promoting cholesterol efflux and reducing cholesterol levels. Ad5-ApoA1 activates CD8+ T cells by promoting large-scale viral replication. High levels of ApoA1 protein expression promote cholesterol efflux, inhibit CD8+ T cell depletion, and reduce inflammatory factors, ultimately leading to superior therapeutic effects on hepatocellular carcinoma.

Protective efficacy of a recombinant adenovirus expressing novel dual F and HN proteins of bovine parainfluenza virus type 3

Bovine parainfluenza virus type 3 (BPIV3) is a viral respiratory pathogen that infects cattle and causes significant economic losses. We generated a recombinant adenovirus called rHAd5-F + HN by expressing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein of BPIV3 using the human adenovirus serotype 5 (rHAd5). We evaluated its effects on humoral and cellular immune responses in mice (n = 45) and calves (n = 9). Serum antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI), and neutralising antibodies (NAb). After boosting immunity with rHAd5-F + HN, mice produced significantly higher levels of antibodies against the BPIV3 genotype A and genotype C strains. The production of antibodies exceeded those produced by adenoviruses rHAd5-F and rHAd5-HN, which express the F and HN glycoprotein, respectively. The percentages of splenic CD3+/CD8+T lymphocytes and IL-4+ cytokines in rHAd5-F + HN mice were considerably higher than those in the control group. Mice immunised with rHAd5-F + HN exhibited much lower viral loads in the lungs and tracheas compared to the control group. Additionally, the lungs of mice vaccinated with rHAd5-F + HN showed no notable histopathological changes. On the other hand, rHAd5-F + HN produced a humoral immune response in calves. Following the booster intramuscular injection with the rHAd5-F + HN, the serum antibody levels against BPIV3 genotype C strain were 1:20 452, 1:1024, and 1:426 in calves, as detected by ELISA, HI, and NAb, respectively. The HI and NAb levels against the BPIV3 genotype A strain were 1:213 and 1:85 in calves, respectively. These results indicate that rHAd5-F + HN effectively induced immunity against BPIV3 infection.

Enhanced Humoral and Cellular Immune Responses Elicited by Adenoviral Delivery of SARS-CoV-2 Receptor-Binding Motif Fused to Human Fc.

Background/Objectives: The receptor binding motif (RBM) of the SARS-CoV-2 spike protein is critical for viral entry into host cells. Development of a vaccine targeting this region is a promising strategy for COVID-19 prevention. To enhance the immunogenicity of SARS-CoV-2 vaccines, we developed an adenoviral vector expressing the RBM from the SARS-CoV-2 spike protein that fused to the human Fc (hFc) domain. Methods: The recombinant RBM_hFc fusion protein was successfully cloned into the pacAd5CMV-N-pA (pAd5) vector and expressed in HEK293 cells as a ~40 kDa protein. A recombinant adenovirus encoding RBM_hFc was subsequently generated and confirmed by cytopathic effect assay. Results: Western blot analysis verified the expression of RBM_hFc in the adenovirus (AdV). ELISA assays, validated for IgG detection, demonstrated a twofold increase in IgG antibody levels (M-1.090 at 450 nm; SD-±0.326; and 95% CI-0.250 [0.839 to 1.340]) in sera from BALB/c mice immunized with Ad/RBM_hFc, compared to the negative control group. Result suggests a robust humoral immune response induced by the Ad/RBM_hFc vaccine. Moreover, ELISpot assays demonstrated a tenfold increase in IFN-γ -producing cells (M-440 spot-forming cells; SD-±124.976; and 95% CI-75.522 [364.478 to 515.522]) in mice immunized with AdV/RBM_hFc compared to the negative control group. Result proved that AdV/RBM_hFc-stimulated a robust cellular immune response in animal model. Conclusions: Our findings indicate that the RBM_hFc fusion protein enhances both humoral and cellular immune responses. These results suggest the potential of adenoviral vectors carrying RBM_hFc as vaccine candidates. However, comprehensive evaluation of the protective efficacy of these adenoviral vectors will necessitate rigorous experimental studies.

Effects of chemokine-binding protein in visceral ovine aphthae on immune regulation response

ORFV of the family poxvirus，which produces a pustular dermatitis both in humans and animals.,Previous studies have found an fatal case caused by the infection of ORFV in the viscera. However, the mechanisms of ORFV how to infect the viscera remain unknown. Our sequencing results revealed that the CBP of the visceral infection strain lacked a 24-base pair segment at position 217 comparison to the oral infection strain. Subsequently, we successfully packaged the recombinant adenoviruses pAd-CBP-K and pAd-CBP-N in HEK-293A cells and carried it to infect lymphocytes. RT-PCR analysis showed that the CBP protein was expressed in lymphocytes, and pAd-CBP-N group exhibited a significantly higher CBP expression level compared to the pAd-CBP-K group. At 4, 8, and 12 hours post-infection, both pAd-CBP-K and pAd-CBP-N were found to downregulate the expression of MIP-1 and CCL-5 in the supernatant of lymphocytes. However, the expression of IL-2, IL-6, IL-8, IL-12, INF-γ, and TNF-α showed a significant up-regulation. Furthermore, the inflammatory factors relative expression levels of IL-2, IL-6, IL-8, IL-8, IL-12, IFN-γ and TNF-α were significantly up-regulated in the both group. Interestingly, a significant increase in the expression of IL-6, IL-8 and TNF-α were detected in the pAd-CBP-N group at both 8 and 12 hours compared to pAd-CBP-N. Taken together, these findings showed that CBP can regulate the expression of free chemokines and activate the expression of inflammatory factors, and provide a basis for follow-up research.

Novel combination therapy using recombinant oncolytic adenovirus silk hydrogel and PD-L1 inhibitor for bladder cancer treatment

Recombinant oncolytic adenovirus offers a novel and promising cancer treatment approach, but its standalone efficacy remains limited. This study investigates a combination treatment strategy by co-administering recombinant oncolytic Adv-loaded silk hydrogel with a PD-L1 inhibitor for patients with bladder cancer to enhance treatment outcomes. Bladder cancer tissues from mice were collected and subjected to single-cell sequencing, identifying CRB3 as a key gene in malignant cells. Differential expression and functional enrichment analyses were performed, validating CRB3’s inhibitory role through in vitro experiments showing suppression of bladder cancer cell proliferation, migration, and invasion. Recombinant oncolytic adenoviruses encoding CRB3 and GM-CSF were constructed and encapsulated in silk hydrogel to enhance drug loading and release efficiency. In vivo experiments demonstrated that the nano-composite hydrogel significantly inhibited tumor growth and increased immune infiltration in tumor tissues. Co-administration of adenovirus silk hydrogel (Adv-CRB3@gel) with a PD-L1 inhibitor significantly enhanced T-cell infiltration and tumor killing. The combination of recombinant oncolytic Adv-loaded nano-composite hydrogel encoding CRB3 and GM-CSF with a PD-L1 inhibitor improves bladder cancer treatment outcomes by effectively recruiting T cells, providing a novel therapeutic strategy.

H Antigen expression modulates epidermal Keratinocyte Integrity and differentiation

BackgroundABO blood group antigens (ABH antigens) are carbohydrate chains glycosylated on epithelial and red blood cells. Recent findings suggest reduced ABH expression in psoriasis and atopic dermatitis, a chronic inflammatory skin disease with retained scale. H antigen, a precursor for A and B antigens, is synthesized by fucosyltransferase 1 (FUT1). Desmosomes, critical for skin integrity, are known to require N-glycosylation for stability. We investigate the impact of H antigens, a specific type of glycosylation, on desmosomes in keratinocytes.MethodPrimary human keratinocytes were transfected with FUT1 siRNA or recombinant adenovirus for FUT1 overexpression. Cell adhesion and desmosome characteristics and their underlying mechanisms were analyzed.ResultThe knockdown of FUT1, responsible for H2 antigen expression in the skin, increased cell-cell adhesive strength and desmosome size in primary cultured keratinocytes without altering the overall desmosome structure. Desmosomal proteins, including desmogleins or plakophilin, were upregulated, suggesting enhanced desmosome assembly. Reduced H2 antigen expression via FUT1 knockdown led to increased keratinocyte differentiation, evidenced by elevated expression of differentiation markers. Epidermal growth factor receptor (EGFR) has been described to be associated with FUT1 and promotes cell migration and differentiation. The effects of FUT1 knockdown were recapitulated by an EGFR inhibitor concerning desmosomal proteins and cellular differentiation. Further investigation demonstrated that the FUT1 knockdown reduced EGFR signaling by lowering the levels of EGF ligands rather than directly regulating EGFR activity. Moreover, FUT1 overexpression reversed the effects observed in FUT1 knockdown, resulting in the downregulation of desmosomal proteins and differentiation markers while increasing both mRNA and protein levels of EGFR ligands.ConclusionThe expression level of FUT1 in the epidermis appears to influence cell-cell adhesion and keratinocyte differentiation status, at least partly through regulation of H2 antigen and EGFR ligand expression. These observations imply that the fucosylation of the H2 antigen by FUT1 could play a significant role in maintaining the molecular composition and regulation of desmosomes and suggest a possible involvement of the altered H2 antigen expression in skin diseases, such as psoriasis and atopic dermatitis.

Outbreak of severe acute respiratory infections caused by recombinant human adenovirus type B 7/3 in hospitalized infants from a nursery in Dakar, April 2024

ObjectivesAcute respiratory infections are among the leading cause of mortality in children under 5 years of age worldwide, with most of these deaths due to bronchiolitis and pneumonia. We investigated and analyzed a pediatric outbreak of acute respiratory infections that resulted in the hospitalization of four infants in a nursery in Dakar in late April 2024. MethodsNasopharyngeal specimens were collected from infants and tested for a panel of respiratory pathogens by multiplex real-time reverse transcription–polymerase chain reaction. Subsequently, the positive samples underwent next-generation sequencing for molecular analysis. ResultsHuman adenovirus (HAdV) was the principal etiologic agent detected in hospitalized infants with pneumonia and nearly half (46.7%; seven of 15) of the suspected cases identified during the investigation at the nursery. All the HAdV isolates were classified as being of subgroup B1. Molecular characterization revealed that infants attending the nursery were infected with a recombinant HAdV strain containing an adenovirus serotype 7 penton and serotype 3 hexon and fiber proteins. ConclusionsOur findings reinforce previous evidence that recombination leads to the emergence of new adenovirus strains with epidemic and lethal potential. These results emphasize the need to strengthen surveillance in inpatient settings across the country.