Replication-defective Adenovirus Research Articles

Establishment of a novel cell line for producing replication-competent adenovirus-free adenoviruses

Adenoviruses are commonly utilized as viral vectors for gene therapy, genetic vaccines, and recombinant protein expression. To generate replication-defective adenoviruses, E1-complementing cell lines such as HEK293A are utilized; however, limitations remain. Repeated passage of E1-deleted virus in HEK293A cells increases the occurrence of replication-competent adenoviruses (RCAs). In the present study, we developed a novel cell line originating from human primary cells. L132 cells were transduced two times with E1-encoded retrovirus and three times with E1A-encoded retrovirus. Finally, we selected the most productive L132 cell line for generation of RCA-free adenovirus, GT541. GT541 can serve as an alternative cell line to HEK293A and other adenovirus-producing cells.

Overall survival after treatment with CAN-2409 plus valacyclovir in combination with continued ICI in patients with stage III/IV NSCLC with an inadequate response to ICI.

8634 Background: Patients with unresectable non-small cell lung cancer (NSCLC) with an inadequate response to immune checkpoint inhibitors (ICI) have limited therapeutic options and expected median overall survival (mOS) of 11.6 to 14.5m (PMID: 35658002 ). The immunosuppressive tumor microenvironment and the lack of a specific T cell response underpin inadequate clinical response. CAN-2409 is a replication-defective adenovirus encoding the HSV-tk gene. Transduced cells activate orally delivered valacyclovir, resulting in immunogenic cell death, and in situ immunization against the patient’s tumor antigens, eliciting a specific immune response against the injected tumor and uninjected metastases. Methods: NCT04495153 is an open-label, phase 2 clinical trial of CAN-2409 + valacyclovir in combination with continued ICI in patients with non-resectable, stage III/IV NSCLC, refractory or resistant to anti-PD-(L)1. Patients were enrolled in 2 cohorts (C) depending on disease status at enrolment: C1, stable disease or C2, progressive disease. Two doses of CAN-2409 (5x1011vp) were given 5-7w apart via bronchoscopic or percutaneous injection into lung tumor, disease-positive lymph node, or peripheral metastasis, each followed by oral prodrug. Patients were assessed for safety, immunologic biomarkers, and OS. Results: As of 16Jan2024, 73 patients were treated (safety population: ≥1 dose of CAN-2409) and 44 were evaluable per protocol (received 2 doses of CAN-2409+ valacyclovir and 12-week CT scan). Baseline treated pt median age was 67 [43-88] yrs, 44% female, 68% on aPD(L)1 alone and 32% aPD(L)1 + pemetrexed regimen. We observed no dose-limiting toxicities or ≥grade 4 treatment-related AEs. mOS of the evaluable population was estimated as 22.0m (95% CI: 14.3m, NA; median follow up (mFU) time of 18.2m). In C2 (n = 38) mOS was 20.6 (mFU 19.2.m) and in C1 (n = 6) was N.A. (mFU 15.2m). mOS in the safety population (n = 73) was 14.3.m (mFU16.8m). 64% of evaluable patients had systemic clinical response with shrinkage of both injected and uninjected lesions. We found a significant correlation between increases in T cell populations after the second injection of CAN-2409 (circulating memory and effector memory CD4+ and CD8+T cells, CD4+ Ki67+IFNg+ and CD4+granzymeB+ T cells, CD8+granzymeB+Ki67+ T cells, T regulatory cells) and subsequent OS (R2 between 0.181 and 0.354 and p = 0.043 and 0.004). Conclusions: Experimental treatment with CAN-2409+valacyclovir in NSCLC pts following an inadequate response to ICI is well tolerated and results in the induction of a cytotoxic and memory circulating T cell response associated with shrinkage of injected and uninjected lesions and a raised tail of the survival curve. mOS of the evaluable population was estimated as 22.0m, markedly longer than the expected mOS of 11.6-14.5m. Clinical trial information: NCT04495153 .

Prophylactic treatment with PEGylated bovine IFNλ3 effectively bridges the gap in vaccine-induced immunity against FMD in cattle.

Foot-and-mouth disease (FMD) is a vesicular disease of cloven-hoofed animals with devastating economic implications. The current FMD vaccine, routinely used in enzootic countries, requires at least 7 days to induce protection. However, FMD vaccination is typically not recommended for use in non-enzootic areas, underscoring the need to develop new fast-acting therapies for FMD control during outbreaks. Interferons (IFNs) are among the immune system's first line of defense against viral infections. Bovine type III IFN delivered by a replication defective adenovirus (Ad) vector has effectively blocked FMD in cattle. However, the limited duration of protection-usually only 1-3 days post-treatment (dpt)-diminishes its utility as a field therapeutic. Here, we test whether polyethylene glycosylation (PEGylation) of recombinant bovine IFNλ3 (PEGboIFNλ3) can extend the duration of IFN-induced prevention of FMDV infection in both vaccinated and unvaccinated cattle. We treated groups of heifers with PEGboIFNλ3 alone or in combination with an adenovirus-based FMD O1Manisa vaccine (Adt-O1M) at either 3 or 5 days prior to challenge with homologous wild type FMDV. We found that pre-treatment with PEGboIFNλ3 was highly effective at preventing clinical FMD when administered at either time point, with or without co-administration of Adt-O1M vaccine. PEGboIFNλ3 protein was detectable systemically for >10 days and antiviral activity for 4 days following administration. Furthermore, in combination with Adt-O1M vaccine, we observed a strong induction of FMDV-specific IFNγ+ T cell response, demonstrating its adjuvanticity when co-administered with a vaccine. Our results demonstrate the promise of this modified IFN as a pre-exposure prophylactic therapy for use in emergency outbreak scenarios.

The Biodistribution of Replication-Defective Simian Adenovirus 1 Vector in a Mouse Model.

The administration route affects the biodistribution of a gene transfer vector and the expression of a transgene. A simian adenovirus 1 vector carrying firefly luciferase and GFP reporter genes (SAdV1-GFluc) were constructed, and its biodistribution was investigated in a mouse model by bioluminescence imaging and virus DNA tracking with real-time PCR. Luciferase activity and virus DNA were mainly found in the liver and spleen after the intravenous administration of SAdV1-GFluc. The results of flow cytometry illustrated that macrophages in the liver and spleen as well as hepatocytes were the target cells. Repeated inoculation was noneffective because of the stimulated serum neutralizing antibodies (NAbs) against SAdV-1. A transient, local expression of low-level luciferase was detected after intragastric administration, and the administration could be repeated without compromising the expression of the reporter gene. Intranasal administration led to a moderate, constant expression of a transgene in the whole respiratory tract and could be repeated one more time without a significant increase in the NAb titer. An immunohistochemistry assay showed that respiratory epithelial cells and macrophages in the lungs were transduced. High luciferase activity was restricted at the injection site and sustained for a week after intramuscular administration. A compromised transgene expression was observed after a repeated injection. When these mice were intramuscularly injected for a third time with the human adenovirus 5 (HAdV-5) vector carrying a luciferase gene, the luciferase activity recovered and reached the initial level, suggesting that the sequential use of SAdV-1 and HAdV-5 vectors was practicable. In short, the intranasal inoculation or intramuscular injection may be the preferred administration routes for the novel SAdV-1 vector in vaccine development.

Bioinformatic, Biochemical, and Immunological Mining of MHC Class I Restricted T Cell Epitopes for a Marburg Nucleoprotein Microparticle Vaccine.

The Marburg virus (MARV), the virus responsible for Marburg hemorrhagic fever (MHF), is considered a top-priority pathogen for vaccine development. Recent outbreaks in Equatorial Africa have highlighted the urgency of MARV because of its high fatality rate and historical concerns about potential weaponization. Currently, there are no licensed vaccines for MARV. Existing vaccine candidates rely on attenuated recombinant vesicular stomatitis virus carrying MARV glycoprotein (VSVΔG) or the chimpanzee replication-defective adenovirus 3 vector ChAd3-MARV. Although these platforms provide significant protection in animal models, they face challenges because of their limited thermal stability and the need for cold storage during deployment in resource-poor areas. An alternative approach involves using adjuvanted poly (lactic-co-glycolic acid) (PLGA) microparticles loaded with synthetic peptides representing MHC class I-restricted T cell epitopes. This vaccine platform has demonstrated effectiveness in protecting against SARS-CoV-2 and EBoV disease in animal models and has the advantage of not requiring cold storage and remaining stable at room temperature for over six months. This report outlines the design, manufacturing, and in vivo immunogenicity testing of PLGA microparticle human vaccines designed to prevent Marburg hemorrhagic fever.

Regulatory T cell-like response to SARS-CoV-2 in Jamaican fruit bats (Artibeus jamaicensis) transduced with human ACE2.

Insectivorous Old World horseshoe bats (Rhinolophus spp.) are the likely source of the ancestral SARS-CoV-2 prior to its spillover into humans and causing the COVID-19 pandemic. Natural coronavirus infections of bats appear to be principally confined to the intestines, suggesting fecal-oral transmission; however, little is known about the biology of SARS-related coronaviruses in bats. Previous experimental challenges of Egyptian fruit bats (Rousettus aegyptiacus) resulted in limited infection restricted to the respiratory tract, whereas insectivorous North American big brown bats (Eptesicus fuscus) showed no evidence of infection. In the present study, we challenged Jamaican fruit bats (Artibeus jamaicensis) with SARS-CoV-2 to determine their susceptibility. Infection was confined to the intestine for only a few days with prominent viral nucleocapsid antigen in epithelial cells, and mononuclear cells of the lamina propria and Peyer's patches, but with no evidence of infection of other tissues; none of the bats showed visible signs of disease or seroconverted. Expression levels of ACE2 were low in the lungs, which may account for the lack of pulmonary infection. Bats were then intranasally inoculated with a replication-defective adenovirus encoding human ACE2 and 5 days later challenged with SARS-CoV-2. Viral antigen was prominent in lungs for up to 14 days, with loss of pulmonary cellularity during this time; however, the bats did not exhibit weight loss or visible signs of disease. From day 7, bats had low to moderate IgG antibody titers to spike protein by ELISA, and one bat on day 10 had low-titer neutralizing antibodies. CD4+ helper T cells became activated upon ex vivo recall stimulation with SARS-CoV-2 nucleocapsid peptide library and exhibited elevated mRNA expression of the regulatory T cell cytokines interleukin-10 and transforming growth factor-β, which may have limited inflammatory pathology. Collectively, these data show that Jamaican fruit bats are poorly susceptible to SARS-CoV-2 but that expression of human ACE2 in their lungs leads to robust infection and an adaptive immune response with low-titer antibodies and a regulatory T cell-like response that may explain the lack of prominent inflammation in the lungs. This model will allow for insight of how SARS-CoV-2 infects bats and how bat innate and adaptive immune responses engage the virus without overt clinical disease.

The co-delivery of adenovirus-based immune checkpoint vaccine elicits a potent anti-tumor effect in renal carcinoma

Immune-based checkpoint therapy has made significant progress in cancer treatment, but its therapeutic effect is limited. A replication-defective adenovirus (Ad) vaccine encoding tumor antigen carbonic anhydrase IX (CAIX) combined with Ad-encoding immune checkpoint PD-L1 was developed to treat renal carcinoma. Three tumor models, subcutaneous, lung metastasis and orthotopic tumor were established, and Ad vaccines were used to immunize them and evaluate the vaccine’s therapeutic effect. Compared to the single Ad vaccine group, the subcutaneous tumor growth was significantly reduced in Ad-CAIX/Ad-PD-L1 combination group. Co-immunization of Ad-CAIX/Ad-PD-L1 enhanced the induction and maturation of CD11c+ or CD8+CD11c+ DCs in the spleen and tumor and promoted the strong tumor-specific CD8+ T cell immune responses. In vivo CD8 T cell deletion assay showed that the anti-tumor effect of the Ad-CAIX/Ad-PD-L1 vaccine was mainly dependent on functional CD8+ T cell immune responses. Furthermore, the Ad-CAIX/Ad-PD-L1 vaccine effectively inhibited tumor growth and lung metastasis in metastatic or orthotopic models. These results indicate that the combination strategy of the immune checkpoint vaccine shows promising potential as an approach for malignant tumor therapy.

Adenovirus vector-mediated single chain variable fragments target the nucleocapsid protein of porcine epidemic diarrhea virus and protect against viral infection in piglets.

Porcine epidemic diarrhea virus (PEDV) mainly infects the intestinal epithelial cells of pigs, causing porcine epidemic diarrhea (PED). In particular, the virus causes severe diarrhea, dehydration, and death in neonatal piglets. Maternal immunity effectively protects neonatal piglets from PEDV infection; however, maternal antibodies can only prevent PEDV attachment and entry into target cells, but have no effects on intracellular viruses. Intracellular antibodies targeting virus-encoded proteins are effective in preventing viral infection. We previously identified four single chain variable fragments (scFvs), ZW1-16, ZW3-21, ZW1-41, and ZW4-16, which specifically targeted the PEDV N protein and significantly inhibited PEDV replication and up-regulated interferon-λ1 (IFN-λ1) expression in host cells. In our current study, the four scFvs were subcloned into replication-defective adenovirus vectors to generate recombinant adenoviruses rAdV-ZW1-16, rAdV-ZW3-21, rAdV-ZW1-41, and rAdV-ZW4-16. ScFvs were successfully expressed in Human Embryonic Kidney 293 (HEK293) cells and intestinal porcine epithelial cell line J2 (IPEC-J2) and were biosafe for piglets as indicated by body temperature and weight, scFv excretion in feces, IFN-γ and interleukin-4 (IL-4) expression in jejunum, and pathological changes in porcine tissue after oral administration. Western blotting, immunofluorescence, and immunohistochemical analyses showed that scFvs were expressed in porcine jejunum. The prophylactic effects of rAdV-ZW, a cocktail of the four rAdV-scFvs, on piglet diarrhea caused by PEDV was investigated. Clinical symptoms in piglets orally challenged with PEDV, following a two-time treatment with rAdV-ZW, were significantly reduced when compared with PEDV-infected piglets treated with phosphate buffered saline (PBS) or rAdV-wild-type. Also, no death and jejunal lesions were observed. ScFv co-localization with the PEDV N protein in vivo was also observed. Next, the expression of pro-inflammatory serum cytokines such as tumor necrosis factor-α (TNF-α), IL-6, IL-8, IL-12, and IFN-λ was assessed by enzyme-linked immunosorbent assay (ELISA), which showed that scFvs significantly suppressed PEDV-induced pro-inflammatory cytokine expression and restored PEDV-inhibited IFN-λ expression. Therefore, our study supported a promising role for intracellular scFvs targeting the PEDV N protein to prevent and treat diarrhea in PEDV-infected piglets.

Heterologous chimpanzee adenovirus vector immunizations for SARS-CoV-2 spike and nucleocapsid protect hamsters against COVID-19

Available COVID-19 vaccine only provide protection for a limited time due in part to the rapid emergence of viral variants with spike protein mutations, necessitating the generation of new vaccines to combat SARS-CoV-2. Two serologically distinct replication-defective chimpanzee-origin adenovirus (Ad) vectors (AdC) called AdC6 and AdC7 expressing early SARS-CoV-2 isolate spike (S) or nucleocapsid (N) proteins, the latter expressed as a fusion protein within herpes simplex virus glycoprotein D (gD), were tested individually or as a mixture in a hamster COVID-19 SARS-CoV-2 challenge model. The S protein expressing AdC (AdC-S) vectors induced antibodies including those with neutralizing activity that in part cross-reacted with viral variants. Hamsters vaccinated with the AdC-S vectors were protected against serious disease and showed accelerated recovery upon SARS-CoV-2 challenge. Protection was enhanced if AdC-S vectors were given together with the AdC vaccines that expressed the gD N fusion protein (AdC-gDN). In contrast hamsters that just received the AdC-gDN vaccines showed only marginal lessening of symptoms compared to control animals. These results indicate that immune response to the N protein that is less variable than the S protein may potentiate and prolong protection achieved by the currently used S protein based genetic COVID-19 vaccines.

Preclinical evaluation of safety and immunogenicity of a primary series intranasal COVID-19 vaccine candidate (BBV154) and humoral immunogenicity evaluation of a heterologous prime-boost strategy with COVAXIN (BBV152).

Most if not all vaccine candidates developed to combat COVID-19 due to SARS-CoV-2 infection are administered parenterally. As SARS-CoV-2 is transmitted through infectious respiratory fluids, vaccine-induced mucosal immunity could provide an important contribution to control this pandemic. ChAd-SARS-CoV-2-S (BBV154), a replication-defective chimpanzee adenovirus (ChAd)-vectored intranasal (IN) COVID-19 vaccine candidate, encodes a prefusion-stabilized version of the SARS-CoV-2 spike protein containing two proline substitutions in the S2 subunit. We performed preclinical evaluations of BBV154 in mice, rats, hamsters and rabbits. Repeated dose toxicity studies presented excellent safety profiles in terms of pathology and biochemical analysis. IN administration of BBV154 elicited robust mucosal and systemic humoral immune responses coupled with Th1 cell-mediated immune responses. BBV154 IN vaccination also elicited potent variant (omicron) cross neutralization antibodies. Assessment of anti-vector (ChAd36) neutralizing antibodies following repeated doses of BBV154 IN administration showed insignificant titers of ChAd36 neutralizing antibodies. However, the immune sera derived from the same animals displayed significantly higher levels of SARS-CoV-2 virus neutralization (p<0.003). We also evaluated the safety and immunogenicity of heterologous prime-boost vaccination with intramuscular (IM) COVAXIN-prime followed by BBV154 IN administration. COVAXIN priming followed by BBV154 IN-booster showed an acceptable reactogenicity profile comparable to the homologous COVAXIN/COVAXIN or BBV154/BBV154 vaccination model. Heterologous vaccination of COVAXIN-prime and BBV154 booster also elicited superior (p<0.005) and cross variant (omicron) protective immune responses (p<0.013) compared with the homologous COVAXIN/COVAXIN schedule. BBV154 has successfully completed both homologous and heterologous combination schedules of human phase 3 clinical trials and received the restricted emergency use approval (in those aged above 18 years) from the Drugs Controller General of India (DCGI).

Development of a recombinant adenovirus-vectored vaccine against both infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss)

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout Oncorhynchus mykiss, and the concurrent infection of the two viruses is very common among modern trout hatcheries, which has caused huge economic losses to the rainbow trout farming industry. To prevent and control the spread of IHNV and IPNV in juvenile trout simultaneously, in this study a bivalent recombinant adenovirus vaccine with IHNV Glycoprotein (G) and IPNV VP2 genes was developed. After immunizing juvenile trout with this bivalent vaccine via the immersion route, the expression levels of IHNV G and IPNV VP2 and the representative immune genes in vaccinated and control rainbow trout were tested to evaluate the correlation of immune responses with the expression of viral genes. The neutralizing antibody level induced by this bivalent vaccine as well as the protection efficacy of the vaccine against IHNV and IPNV was also evaluated. The results showed that IHNV G and IPNV VP2 were successfully expressed in juvenile trout, and all the innate and adaptive immune genes were up-regulated. This indicated that the level of the innate and adaptive immune responses were significantly increased, which might be induced by the high expression of the two viral proteins. Compared with the controls, high levels of neutralizing antibodies against IHNV and IPNV were induced in the vaccinated trout. Besides, the bivalent recombinant adenovirus vaccine showed high protection rate against IHNV, with the relative percent survival (RPS) of 81.25%, as well as against IPNV, with the RPS of 78.95%. Taken together, our findings clearly demonstrated that replication-defective adenovirus can be developed as a qualified vector for fish vaccines and IHNV G and IPNV VP2 were two suitable antigenic genes that could induce effective immune protection against these two pathogens. This study provided new insights into developing bivalent vectored vaccines and controlling the spread of IHNV and IPNV simultaneously in juvenile trout.

Single-Shot ChAd3-MARV Vaccine in Modified Formulation Buffer Shows 100% Protection of NHPs.

Marburg virus (MARV) is a virus of high human consequence with a case fatality rate of 24-88%. The global health and national security risks posed by Marburg virus disease (MVD) underscore the compelling need for a prophylactic vaccine, but no candidate has yet reached regulatory approval. Here, we evaluate a replication-defective chimpanzee adenovirus type 3 (ChAd3)-vectored MARV Angola glycoprotein (GP)-expressing vaccine against lethal MARV challenge in macaques. The ChAd3 platform has previously been reported to protect against the MARV-related viruses, Ebola virus (EBOV) and Sudan virus (SUDV), and MARV itself in macaques, with immunogenicity demonstrated in macaques and humans. In this study, we present data showing 100% protection against MARV Angola challenge (versus 0% control survival) and associated production of GP-specific IgGs generated by the ChAd3-MARV vaccine following a single dose of 1 × 1011 virus particles prepared in a new clinical formulation buffer designed to enhance product stability. These results are consistent with previously described data using the same vaccine in a different formulation and laboratory, demonstrating the reproducible and robust protective efficacy elicited by this promising vaccine for the prevention of MVD. Additionally, a qualified anti-GP MARV IgG ELISA was developed as a critical pre-requisite for clinical advancement and regulatory approval.

Comparison of replicating and nonreplicating vaccines against SARS-CoV-2.

Most gene-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are nonreplicating vectors. They deliver the gene or messenger RNA to the cell to express the spike protein but do not replicate to amplify antigen production. This study tested the utility of replication in a vaccine by comparing replication-defective adenovirus (RD-Ad) and replicating single-cycle adenovirus (SC-Ad) vaccines that express the SARS-CoV-2 spike protein. SC-Ad produced 100 times more spike protein than RD-Ad and generated significantly higher antibodies against the spike protein than RD-Ad after single immunization of Ad-permissive hamsters. SC-Ad–generated antibodies climbed over 14 weeks after single immunization and persisted for more than 10 months. When the hamsters were challenged 10.5 months after single immunization, a single intranasal or intramuscular immunization with SC-Ad-Spike reduced SARS-CoV-2 viral loads and damage in the lungs and preserved body weight better than vaccination with RD-Ad-Spike. This demonstrates the utility of harnessing replication in vaccines to amplify protection against infectious diseases.

Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

IntroductionThe on-target off-tumor toxicity of chimeric antigen receptor-engineered T cells (CAR-T) might lead to fatal side effects in cancer patients, which remains as a major obstacle to the clinical application of CAR-T immunotherapy. The off-tumor on-target normal tissue toxicity of CAR-T cells needs to be evaluated in preclinical studies using rational animal models. ObjectivesWe aim to develop a rational animal model for assessing the off-tumor on-target normal tissue toxicity of various CAR-T cell designs quickly. MethodsWe used a recombinant adenovirus type 5 carrying human HER2/ERBB2 (Ad5-HER2) or CD47 gene (Ad5-CD47) to rapidly generate a mouse model with tunable human antigen expression on normal liver tissue to determine immunotoxicity of traditional CAR-T and hypoxia-response CAR-T cells in vivo. ResultsThe obvious liver damage and lymphocyte infiltration were not observed in mice with human antigen-high livers 8 days post-infection. Interestingly, the lethal liver damage, systemic cytokine release and CAR-T cells infiltration in liver were only observed in mice that received traditional CAR-T cells, but not in hypoxia-response CAR-T cells. ConclusionAdenovirus-based expression of target antigen in normal mouse tissue may be a useful method for assessing on-target CAR-T cell toxicity in normal tissues, especially various CAR-T cell designs that have the potency of conditional regulation in tumor microenvironment (TME).

Boosting of SARS-CoV-2 immunity in nonhuman primates using an oral rhabdoviral vaccine

An orally active vaccine capable of boosting SARS-CoV-2 immune responses in previously infected or vaccinated individuals would help efforts to achieve and sustain herd immunity. Unlike mRNA-loaded lipid nanoparticles and recombinant replication-defective adenoviruses, replicating vesicular stomatitis viruses with SARS-CoV-2 spike glycoproteins (VSV-SARS2) were poorly immunogenic after intramuscular administration in clinical trials. Here, by G protein trans-complementation, we generated VSV-SARS2(+G) virions with expanded target cell tropism. Compared to parental VSV-SARS2, G-supplemented viruses were orally active in virus-naive and vaccine-primed cynomolgus macaques, powerfully boosting SARS-CoV-2 neutralizing antibody titers. Clinical testing of this oral VSV-SARS2(+G) vaccine is planned.

Tissue distribution and transcriptional regulation of CCN5 in the heart after myocardial infarction

CCN5 is a divergent member of the cellular communication network factor (CCN) family in that it lacks the carboxyl terminal cystine knot domain common to the other CCN family members. CCN5 has been reported to antagonize the profibrotic actions of CCN2 and to inhibit myocardial collagen deposition and fibrosis in chronic pressure overload of the heart. However, what mechanisms that regulate CCN5 activity in the heart remain unknown. Recombinant, replication defective adenovirus encoding firefly luciferase under control of the human CCN5 promoter was prepared and used to investigate what mechanisms regulate CCN5 transcription in relevant cells. Tissue distribution of CCN5 in hearts from healthy mice and from mice subjected to myocardial infarction was investigated. Contrary to the profibrotic immediate early gene CCN2, we find that CCN5 is induced in the late proliferation and maturation phases of scar healing. CCN5 was identified principally in endothelial cells, fibroblasts, smooth muscle cells, and macrophages. Our data show that CCN5 gene transcription and protein levels are induced by catecholamines via β2-adrenergic receptors. Myocardial induction of CCN5 was further confirmed in isoproterenol-infused mice. We also find that CCN5 transcription is repressed by TNF-α, an inflammatory mediator highly elevated in early phases of wound healing following myocardial infarction. In conclusion, CCN5 predominates in endothelial cells, fibroblasts, and macrophages of the differentiating scar tissue and its transcription is conversely regulated by β2-adrenergic agonists and TNF-α.

SARI inhibits growth and reduces survival of oral squamous cell carcinomas (OSCC) by inducing endoplasmic reticulum stress

AimsSARI (suppressor of activator protein (AP)-1, regulated by interferon (IFN) was identified as a novel tumor suppressor by applying subtraction hybridization to terminally differentiating human melanoma cells. The anti-tumor activity of SARI and the correlation between expression and cancer aggression and metastasis has been examined in multiple cancers, but its potential role in oral squamous cell carcinomas (OSCC) has not been explored. MethodsSARI expression was monitored in tumor tissues of OSCC patients by performing immunohistochemistry. Ectopic expression of SARI was achieved using a replication defective adenovirus expressing SARI (Ad.SARI). A nude mouse xenograft model was used to evaluate the in vivo efficacy of SARI. Endoplasmic reticulum (ER) stress was monitored in SARI infected OSCC cells by confocal microscopy. Key findingIn this study, we demonstrate that SARI expression is significantly lower in OSCC tumor tissue as compared to normal adjacent tissue. Ectopic expression of SARI induces cancer-specific cell death in human OSCC cell lines and in a paclitaxel plus cisplatin non-responder OSCC patient-derived (PDC1) cell line. Mechanistically, SARI inhibits zinc finger protein GLI1 expression through induction of endoplasmic reticulum (ER) stress. Using a nude mouse xenograft model, we show that intratumoral injections of Ad.SARI significantly reduce PDC1 tumor burden, whereas treatment with an ER stress inhibitor efficiently rescues tumors from growth inhibition. SignificanceOverall, our data provides a link between induction of ER stress and inhibition of the GLI1/Hedgehog signaling pathway and the tumor suppressive activity of SARI in the context of OSCC.

GRAd-COV2, a gorilla adenovirus-based candidate vaccine against COVID-19, is safe and immunogenic in younger and older adults.

Safe and effective vaccines against coronavirus disease 2019 (COVID-19) are essential for ending the ongoing pandemic. Although impressive progress has been made with several COVID-19 vaccines already approved, it is clear that those developed so far cannot meet the global vaccine demand alone. We describe a COVID-19 vaccine based on a replication-defective gorilla adenovirus expressing the stabilized prefusion severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein named GRAd-COV2. We assessed the safety and immunogenicity of a single-dose regimen of this vaccine in healthy younger and older adults to select the appropriate dose for each age group. For this purpose, a phase 1, dose-escalation, open-labeled trial was conducted including 90 healthy participants (45 aged 18 to 55 years old and 45 aged 65 to 85 years old) who received a single intramuscular administration of GRAd-COV2 at three escalating doses. Local and systemic adverse reactions were mostly mild or moderate and of short duration, and no serious adverse events were reported. Four weeks after vaccination, seroconversion to spike protein and receptor binding domain was achieved in 43 of 44 young volunteers and in 45 of 45 older participants. Consistently, neutralizing antibodies were detected in 42 of 44 younger-age and 45 of 45 older-age volunteers. In addition, GRAd-COV2 induced a robust and T helper 1 cell (TH1)–skewed T cell response against the spike protein in 89 of 90 participants from both age groups. Overall, the safety and immunogenicity data from the phase 1 trial support the further development of this vaccine.

Adenovirus transduction to express human ACE2 causes obesity-specific morbidity in mice, impeding studies on the effect of host nutritional status on SARS-CoV-2 pathogenesis

The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6 N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology or viral replication was observed between AdV transduced lean and obese groups, infected with SARS-CoV-2, but certain cytokines were expressed more significantly in infected obese mice compared to the lean ones. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection, as described here and elsewhere, may be inappropriate for nutrition studies.

A recombinant bovine adenoviral mucosal vaccine expressing mycobacterial antigen-85B generates robust protection against tuberculosis in mice

SummaryAlthough the BCG vaccine offers partial protection, tuberculosis remains a leading cause of infectious disease death, killing ∼1.5 million people annually. We developed mucosal vaccines expressing the autophagy-inducing peptide C5 and mycobacterial Ag85B-p25 epitope using replication-defective human adenovirus (HAdv85C5) and bovine adenovirus (BAdv85C5) vectors. BAdv85C5-infected dendritic cells (DCs) expressed a robust transcriptome of genes regulating antigen processing compared to HAdv85C5-infected DCs. BAdv85C5-infected DCs showed enhanced galectin-3/8 and autophagy-dependent in vitro Ag85B-p25 epitope presentation to CD4 T cells. BCG-vaccinated mice were intranasally boosted using HAdv85C5 or BAdv85C5 followed by infection using aerosolized Mycobacterium tuberculosis (Mtb). BAdv85C5 protected mice against tuberculosis both as a booster after BCG vaccine (>1.4-log10 reduction in Mtb lung burden) and as a single intranasal dose (>0.5-log10 reduction). Protection was associated with robust CD4 and CD8 effector (TEM), central memory (TCM), and CD103+/CD69+ lung-resident memory (TRM) T cell expansion, revealing BAdv85C5 as a promising mucosal vaccine for tuberculosis.