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Viral vector delivery of gene therapy represents a promising approach for the treatment of numerous retinal diseases. Adeno-associated viral vectors (AAV) constitute the primary gene delivery platform; however, their limited cargo capacity restricts the delivery of several clinically relevant retinal genes. In this study, we explore the feasibility of employing high-capacity adenoviral vectors (HC-AdVs) as alternative delivery vehicles, which, with a capacity of up to 36 kb, can potentially accommodate all known retinal gene coding sequences. We utilized HC-AdVs based on the classical adenoviral type 5 (AdV5) and on a fiber-modified AdV5.F50 version, both engineered to deliver a 29.6 kb vector genome encoding a fluorescent reporter construct. The tropism of these HC-AdVs was evaluated in an induced pluripotent stem cell (iPSC)-derived human retinal organoid model. Both vector types demonstrated robust transduction efficiency, with sustained transgene expression observed for up to 110 days post-transduction. Moreover, we found efficient transduction of photoreceptors and Müller glial cells, without evidence of reactive gliosis or loss of photoreceptor cell nuclei. However, an increase in the thickness of the photoreceptor outer nuclear layer was observed at 110 days post-transduction, suggesting potential unfavorable effects on Müller glial or photoreceptor cells associated with HC-AdV transduction and/or long-term reporter overexpression. These findings suggest that while HC-AdVs show promise for large retinal gene delivery, further investigations are required to assess their long-term safety and efficacy. [ABSTRACT FROM AUTHOR]

Background/Objectives: An effective universal influenza vaccine is urgently needed to overcome the limitations of current seasonal influenza vaccines, which are ineffective against mismatched strains and unable to protect against pandemic influenza. Methods: In this study, bovine and human adenoviral vector-based vaccine platforms were utilized to express various combinations of antigens. These included the H5N1 hemagglutinin (HA) stem region or HA2, the extracellular domain of matrix protein 2 of influenza A virus, HA signal peptide (SP), trimerization domain, excretory peptide, and the autophagy-inducing peptide C5 (AIP-C5). The goal was to identify the optimal combination for enhanced immune responses and cross-protection. Mice were immunized using a prime-boost strategy with heterologous adenoviral (Ad) vectors. Results: The heterologous Ad vectors induced robust HA stem-specific humoral and cellular immune responses in the immunized mice. Among the tested combinations, Ad vectors expressing SP + HA stem + AIP-C5 conferred significant protection against group 1 (H1N1 and H5N1) and group 2 (H3N2) influenza A viruses. This protection was demonstrated by lower lung viral titers and reduced morbidity and mortality. Conclusions: The findings support further investigation of heterologous Ad vaccine platforms expressing SP + HA stem + AIP-C5. This combination shows promise as a potential universal influenza vaccine, providing broader protection against influenza A viruses. [ABSTRACT FROM AUTHOR]

Introduction: To analyze the molecular pathogenesis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a small animal model such as mice is needed: human angiotensin converting enzyme 2 (hACE2), the receptor of SARS-CoV-2, needs to be expressed in the respiratory tract of mice. Methods: We conferred SARS-CoV-2 susceptibility in mice by using an adenoviral vector expressing hACE2 driven by an elongation factor 1α (EF1α) promoter with a leftward orientation. Results: In this model, severe pneumonia like human COVID-19 was observed in SARS-CoV-2-infected mice, which was confirmed by dramatic infiltration of inflammatory cells in the lung with efficient viral replication. An early circulating strain of SARS-CoV-2 caused the most severe weight loss when compared to SARS-CoV-2 variants such as Alpha, Beta and Gamma, although histopathological findings, viral replication, and cytokine expression characteristics were comparable Discussion: We found that a distinct proteome of an early circulating strain infected lung characterized by elevated complement activation and blood coagulation, which were mild in other variants, can contribute to disease severity. Unraveling the specificity of early circulating SARS-CoV-2 strains is important in elucidating the origin of the pandemic. [ABSTRACT FROM AUTHOR]

Host–pathogen interactions and the design of vaccines for aquaculture fish viruses are challenging and call for innovative approaches. This study explores the potential of adenoviral (Ad) vectors Ad5 and chimeric Ad5/40 as gene delivery tools for fish brain cells susceptible to neurotropic viruses. For this purpose, European sea bass (Dicentrarchus labrax) DLB-1 and gilthead seabream (Sparus aurata) SaB-1 brain cell lines were infected with Ad5 or Ad5/40 vectors expressing GFP, and we evaluated their capacity for infection by fluorescence microscopy and flow cytometry, as well as their antiviral innate immune response by the transcription of gene markers (irf3 and mx). We found that both vectors are able to infect DLB-1 and SaB-1 brain cell lines to similar levels, as demonstrated by fluorescence microscopy and flow cytometry, though the infection efficiency was low. In addition, infection with Ad vectors regulated the transcription of genes related to the interferon-mediated antiviral immune response. Our results indicate that the Ad5/40 vector achieves better infection and consistent cellular distribution. These findings suggest that these vectors may offer targeted gene delivery and local immune responses. [ABSTRACT FROM AUTHOR]

The use of 3D-printed gene-activated bone grafts represents a highly promising approach in the fields of dentistry and orthopedics. Bioresorbable poly-lactic-co-glycolic acid (PLGA) scaffolds, infused with adenoviral constructs that carry osteoinductive factor genes, may provide an effective alternative to existing bone grafts for the reconstruction of extensive bone defects. This study aims to develop and investigate the properties of 3D scaffolds composed of PLGA and adenoviral constructs carrying the BMP2 gene (Ad-BMP2), both in vitro and in vivo. The elastic modulus of the disk-shaped PLGA scaffolds created using a specialized 3D printer was determined by compressive testing in both axial and radial directions. In vitro cytocompatibility was assessed using adipose-derived stem cells (ADSCs). The ability of Ad-BMP2 to transduce cells was evaluated. The osteoinductive and biocompatible properties of the scaffolds were also assessed in vivo. The Young's modulus of the 3D-printed PLGA scaffolds exhibited comparable values in both axial and radial compression directions, measuring 3.4 ± 0.7 MPa for axial and 3.17 ± 1.4 MPa for radial compression. The scaffolds promoted cell adhesion and had no cytotoxic effect on ADSCs. Ad-BMP2 successfully transduced the cells and induced osteogenic differentiation in vitro. In vivo studies demonstrated that the 3D-printed PLGA scaffolds had osteoinductive properties, promoting bone formation within the scaffold filaments as well as at the center of a critical calvarial bone defect. [ABSTRACT FROM AUTHOR]

Simple Summary: Adenoviral vectors (AdVs) are engineered viruses used to deliver therapeutic genes to specific cells, offering promising solutions for the treatment of genetic diseases. However, their use is limited by issues such as strong immune responses and transient transgene expression. These limitations make them unsuitable for treatments that require long-term gene expression, such as some inherited diseases. This review examines clinical trials using AdVs for gene therapy of cystic fibrosis and ornithine transcarbamylase deficiency, their successes in preclinical testing and failures in practice, and discusses the underlying reasons for the failure of clinical trials. Understanding the reasons may help overcome these barriers to advances in gene therapy for inherited diseases. The review also highlights the achievements in overcoming these barriers. Scientists are modifying the outer structure of these vectors to more precisely target specific cells, attempting to reduce immune responses to AdVs, and improving gene delivery in cystic fibrosis by removing physical barriers such as thick mucus in the lungs. While these vectors are currently most useful for short-term applications such as vaccines and genome editing, ongoing research may open new doors for their use in more complex treatments. These advances have the potential to improve the effectiveness of gene therapy and offer hope to people living with incurable diseases. Adenoviral vectors (AdVs) are effective vectors for gene therapy due to their broad tropism, high capacity, and high transduction efficiency, which makes them actively used as oncolytic vectors and for creating vector vaccines. However, despite their numerous advantages, AdVs have not yet found their place in gene therapy for hereditary diseases. This review provides an overview of AdVs, their features, and clinical trials using them for gene replacement therapy in monogenic diseases and analyzes the reasons for the failures of these studies. Additionally, current research on the modification of AdVs to reduce immune responses and target delivery is discussed. [ABSTRACT FROM AUTHOR]

The outbreak of coronavirus disease 19 (COVID-19) has highlighted the demand for vaccines that are safe and effective in inducing systemic and airway mucosal immunity against the aerosol transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we developed a novel helperdependent adenoviral vector-based COVID-19 mucosal vaccine encoding a full-length SARS-CoV-2 spike protein (HD-Ad-FS). Through intranasal immunization (single-dose and prime-boost regimens), we demonstrated that the HD-Ad-FS was immunogenic and elicited potent systemic and airway mucosal protection in BALB/c mice, transgenic ACE2 (hACE2) mice, and hamsters. We detected high titers of neutralizing antibodies (NAbs) in sera and bronchoalveolar lavages (BALs) in the vaccinated animals. High levels of spikespecific secretory IgA (sIgA) and IgG were induced in the airway of the vaccinated animals. The single-dose HD-Ad-FS elicited a strong immune response and protected animals from SARS-CoV-2 infection. In addition, the prime-boost vaccination induced cross-reactive serum NAbs against variants of concern (VOCs; Beta, Delta, and Omicron). After challenge, VOC infectious viral particles were at undetectable or minimal levels in the lower airway. Our findings highlight the potential of airway delivery of HD-Ad-FS as a safe and effective vaccine platform for generating mucosal protection against SARS-CoV-2 and its VOCs. [ABSTRACT FROM AUTHOR]

The severity of adenovirus infection or the success of adenovirus-vectorized gene therapy largely depends on the efficiency of viral entry into cells. Various drugs can alter viral entry. This study evaluated the effects of dexamethasone, paracetamol, diclofenac, ibuprofen, and ketorolac on adenovirus entry into cells in vitro and in vivo. SiHa cell cultures pretreated with dexamethasone, paracetamol, diclofenac, ibuprofen, ketorolac, or no drug were exposed to the Ad-BGal vector. The percentage of cells showing vector entry was quantified microscopically. In vivo, BALB-C mice pretreated for 7 days with the drugs or no drug were exposed to the Ad-BGal vector intravenously (IV) or via oral (VO). Organs showing vector entry were identified by X-Gal staining and eosin counterstaining. Hepatic areas with adenovirus entry were quantified in µm2. Dexamethasone, paracetamol, and ibuprofen increased adenovirus entry both in vitro and in vivo. Diclofenac increased entry only in vitro. Ketorolac did not affect adenoviral entry. The liver exhibited the most significant changes, with dexamethasone, paracetamol, and ibuprofen increasing adenovirus entry the most. Oral administration of the vector showed that dexamethasone increased its entry into the pharynx. Some physicochemical properties of the drugs (MW (g/mol), LogP, MR [cm3/mol], tPSA, CMR, LogS, and ClogP) were analyzed, and their possible implications on cell membrane properties that could potentially influence adenovirus entry through mechanisms independent of cellular receptors were discussed. Anti-inflammatory drugs could alter adenoviral infections and adenovirus vector-based gene therapies, necessitating further research. [ABSTRACT FROM AUTHOR]

IntroductionTo analyze the molecular pathogenesis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a small animal model such as mice is needed: human angiotensin converting enzyme 2 (hACE2), the receptor of SARS-CoV-2, needs to be expressed in the respiratory tract of mice.MethodsWe conferred SARS-CoV-2 susceptibility in mice by using an adenoviral vector expressing hACE2 driven by an elongation factor 1α (EF1α) promoter with a leftward orientation.ResultsIn this model, severe pneumonia like human COVID-19 was observed in SARS-CoV-2-infected mice, which was confirmed by dramatic infiltration of inflammatory cells in the lung with efficient viral replication. An early circulating strain of SARS-CoV-2 caused the most severe weight loss when compared to SARS-CoV-2 variants such as Alpha, Beta and Gamma, although histopathological findings, viral replication, and cytokine expression characteristics were comparableDiscussionWe found that a distinct proteome of an early circulating strain infected lung characterized by elevated complement activation and blood coagulation, which were mild in other variants, can contribute to disease severity. Unraveling the specificity of early circulating SARS-CoV-2 strains is important in elucidating the origin of the pandemic.

ABSTRACTClassic chimeric hemagglutinin (cHA) was designed to induce immune responses against the conserved stalk domain of HA. However, it is unclear whether combining more than one HA head domain onto one stalk domain is immunogenic and further induce immune responses against influenza viruses. Here, we constructed numerous novel cHAs comprising two or three fuzed head domains from different subtypes grafted onto one stalk domain, designated as cH1-H3, cH1-H7, cH1-H3-H7, and cH1-H7-H3. The three-dimensional structures of these novel cHAs were modelled using bioinformatics simulations. Structural analysis showed that the intact neutralizing epitopes were exposed in cH1-H7 and were predicted to be immunogenic. The immunogenicity of the cHAs constructs was evaluated in mice using a chimpanzee adenoviral vector (AdC68) vaccine platform. The results demonstrated that cH1-H7 expressed by AdC68 (AdC68-cH1-H7) induced the production of high levels of binding antibodies, neutralizing antibodies, and hemagglutinin inhibition antibodies against homologous pandemic H1N1, drifted seasonal H1N1, and H7N9 virus. Moreover, vaccinated mice were fully protected from a lethal challenge with the aforementioned influenza viruses. Hence, cH1-H7 cHAs with potent immunogenicity might be a potential novel vaccine to provide protection against different subtypes of influenza virus.

In this study, the effects of different combinations of the genes Vegf, Ang, and Gdnf injected both using direct virus-mediated injection (adenovirus, Ad5) and umbilical cord blood mononuclear cells (UCBCs) on the processes of stimulation of post-ischemic innervation, angiogenesis, and regeneration in skeletal muscle were investigated in a rat hindlimb chronic ischemia model. It was shown that more pronounced stimulation of angiogenesis and restoration of post-ischemic innervation were achieved both in the early (28 days post-ischemia, dpi) and late (42 dpi) terms of the experiment in the calf muscle when UCBCs delivered the combination of Ad5-Vegf and Ad5-Ang compared to the direct injection of the same vector combination into the area of ischemia. At the same time, the inclusion of Ad5-Gdnf in the combination of Ad5-Vegf and Ad5-Ang directly injected or administered by UCBCs provided a significant increase in the number of centronuclear muscle fibers, indicating stimulation of post-ischemic reparative myogenesis. This study allowed us to determine the most effective gene combinations for angiogenesis and neurogenesis, which, in the future, may serve as the basis for the development of gene and gene cell products for the treatment of chronic lower limb ischemia.

Adenoviral vectors are crucial for gene therapy and vaccine development, offering a platform for gene delivery into host cells. Since the discovery of adenoviruses, first-generation vectors with limited capacity have evolved to third-generation vectors flacking viral coding sequences, balancing safety and gene-carrying capacity. The applications of adenoviral vectors for gene therapy and anti-viral treatments have expanded through the use of in vitro ligation and homologous recombination, along with gene editing advancements such as CRISPR-Cas9. Current research aims to maintain the efficacy and safety of adenoviral vectors by addressing challenges such as pre-existing immunity against adenoviral vectors and developing new adenoviral vectors from rare adenovirus types and non-human species. In summary, adenoviral vectors have great potential in gene therapy and vaccine development. Through continuous research and technological advancements, these vectors are expected to lead to the development of safer and more effective treatments. [ABSTRACT FROM AUTHOR]

Introduction: While TLR ligands derived from microbial flora and pathogens are important activators of the innate immune system, a variety of factors such as intracellular bacteria, viruses, and parasites can induce a state of hyperreactivity, causing a dysregulated and potentially life-threatening cytokine over-response upon TLR ligand exposure. Type I interferon (IFN-αβ) is a central mediator in the induction of hypersensitivity and is strongly expressed in splenic conventional dendritic cells (cDC) and marginal zone macrophages (MZM) when mice are infected with adenovirus. This study investigates the ability of adenoviral infection to influence the activation state of the immune system and underlines the importance of considering this state when planning the treatment of patients. Methods: Infection with adenovirus-based vectors (Ad) or pretreatment with recombinant IFN-β was used as a model to study hypersensitivity to lipopolysaccharide (LPS) in mice, murine macrophages, and human blood samples. The TNF-α, IL-6, IFN-αβ, and IL-10 responses induced by LPS after pretreatment were measured. Mouse knockout models for MARCO, IFN-αβR, CD14, IRF3, and IRF7 were used to probe the mechanisms of the hypersensitive reaction. Results: We show that, similar to TNF-α and IL-6 but not IL-10, the induction of IFN-αβ by LPS increases strongly after Ad infection. This is true both in mice and in human blood samples ex vivo, suggesting that the regulatory mechanisms seen in the mouse are also present in humans. In mice, the scavenger receptor MARCO on IFN-αβ-producing cDC and splenic marginal zone macrophages is important for Ad uptake and subsequent cytokine overproduction by LPS. Interestingly, not all IFN-αβ-pretreated macrophage types exposed to LPS exhibit an enhanced TNF-α and IL-6 response. Pretreated alveolar macrophages and alveolar macrophage-like murine cell lines (MPI cells) show enhanced responses, while bone marrow-derived and peritoneal macrophages show a weaker response. This correlates with the respective absence or presence of the anti-inflammatory IL-10 response in these different macrophage types. In contrast, Ad or IFN-β pretreatment enhances the subsequent induction of IFN-αβ in all macrophage types. IRF3 is dispensable for the LPS-induced IFN-αβ overproduction in infected MPI cells and partly dispensable in infected mice, while IRF7 is required. The expression of the LPS co-receptor CD14 is important but not absolutely required for the elicitation of a TNF-α over-response to LPS in Ad-infected mice. Conclusion: Viral infections or application of virus-based vaccines induces type I interferon and can tip the balance of the innate immune system in the direction of hyperreactivity to a subsequent exposure to TLR ligands. The adenoviral model presented here is one example of how multiple factors, both environmental and genetic, affect the physiological responses to pathogens. Being able to measure the current reactivity state of the immune system would have important benefits for infection-specific therapies and for the prevention of vaccination-elicited adverse effects.

Background/Objectives: An effective universal influenza vaccine is urgently needed to overcome the limitations of current seasonal influenza vaccines, which are ineffective against mismatched strains and unable to protect against pandemic influenza. Methods: In this study, bovine and human adenoviral vector-based vaccine platforms were utilized to express various combinations of antigens. These included the H5N1 hemagglutinin (HA) stem region or HA2, the extracellular domain of matrix protein 2 of influenza A virus, HA signal peptide (SP), trimerization domain, excretory peptide, and the autophagy-inducing peptide C5 (AIP-C5). The goal was to identify the optimal combination for enhanced immune responses and cross-protection. Mice were immunized using a prime-boost strategy with heterologous adenoviral (Ad) vectors. Results: The heterologous Ad vectors induced robust HA stem-specific humoral and cellular immune responses in the immunized mice. Among the tested combinations, Ad vectors expressing SP + HA stem + AIP-C5 conferred significant protection against group 1 (H1N1 and H5N1) and group 2 (H3N2) influenza A viruses. This protection was demonstrated by lower lung viral titers and reduced morbidity and mortality. Conclusions: The findings support further investigation of heterologous Ad vaccine platforms expressing SP + HA stem + AIP-C5. This combination shows promise as a potential universal influenza vaccine, providing broader protection against influenza A viruses.

The variable domain of a heavy-chain antibody (VHH) has the potential to be used to redirect the cell tropism of adenoviral vectors. Here, we attempted to establish platforms to simplify the screening of VHHs for their specific targeting function when being incorporated into the fiber of adenovirus. Both fowl adenovirus 4 (FAdV-4) and simian adenovirus 1 (SAdV-1) have two types of fiber, one of which is dispensable for virus propagation and is a proper site for VHH display. An intermediate plasmid, pMD-FAV4Fs, was constructed as the start plasmid for FAdV-4 fiber2 modification. Foldon from phage T4 fibritin, a trigger for trimerization, was employed to bridge the tail/shaft domain of fiber2 and VHHs against human CD16A, a key membrane marker of natural killer (NK) cells. Through one step of restriction-assembly, the modified fiber2 was transferred to the adenoviral plasmid, which was linearized and transfected to packaging cells. Five FAdV-4 viruses carrying the GFP gene were finally rescued and amplified, with three VHHs being displayed. One recombinant virus, FAdV4FC21-EG, could hardly transduce human 293 or Jurkat cells. In contrast, when it was used at a multiplicity of infection of 1000 viral particles per cell, the transduction efficiency reached 51% or 34% for 293 or Jurkat cells expressing exogenous CD16A. Such a strategy of fiber modification was transplanted to the SAdV-1 vector to construct SAdV1FC28H-EG, which moderately transduced primary human NK cells while the parental virus transduced none. Collectively, we reformed the strategy of integrating VHH to fiber and established novel platforms for screening VHHs to construct adenoviral vectors with a specific tropism. [ABSTRACT FROM AUTHOR]

The outbreak of coronavirus disease 19 (COVID-19) has highlighted the demand for vaccines that are safe and effective in inducing systemic and airway mucosal immunity against the aerosol transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we developed a novel helper-dependent adenoviral vector-based COVID-19 mucosal vaccine encoding a full-length SARS-CoV-2 spike protein (HD-Ad-FS). Through intranasal immunization (single-dose and prime-boost regimens), we demonstrated that the HD-Ad-FS was immunogenic and elicited potent systemic and airway mucosal protection in BALB/c mice, transgenic ACE2 (hACE2) mice, and hamsters. We detected high titers of neutralizing antibodies (NAbs) in sera and bronchoalveolar lavages (BALs) in the vaccinated animals. High levels of spike-specific secretory IgA (sIgA) and IgG were induced in the airway of the vaccinated animals. The single-dose HD-Ad-FS elicited a strong immune response and protected animals from SARS-CoV-2 infection. In addition, the prime-boost vaccination induced cross-reactive serum NAbs against variants of concern (VOCs; Beta, Delta, and Omicron). After challenge, VOC infectious viral particles were at undetectable or minimal levels in the lower airway. Our findings highlight the potential of airway delivery of HD-Ad-FS as a safe and effective vaccine platform for generating mucosal protection against SARS-CoV-2 and its VOCs.

Adenoviruses typically cause mild illnesses, but severe diseases may occur primarily in immunodeficient individuals, particularly children. Recently, adenoviruses have garnered significant interest as a versatile tool in gene therapy, tumor treatment, and vaccine vector development. Over the past two decades, the advent of recombineering, a method based on homologous recombination, has notably enhanced the utility of adenoviral vectors in therapeutic applications. This review summarizes recent advancements in the use of human adenoviral vectors in medicine and discusses the pivotal role of recombineering in the development of these vectors. Additionally, it highlights the current achievements and potential future impact of therapeutic adenoviral vectors.

The nucleoprotein (NP) is a vital target for the heterosubtypic immunity of CD8+ cytotoxic T lymphocytes (CTLs) due to its conservation among influenza virus subtypes. To further enhance the T cell immunity of NP, autophagy-inducing peptide C5 (AIP-C5) from the CFP10 protein of Mycobacterium tuberculosis was used. Mice were immunized intranasally (i.n.) with human adenoviral vectors, HAd-C5-NP(H7N9) or HAd-NP(H7N9), expressing NP of an H7N9 influenza virus with or without the AIP-C5, respectively. Both vaccines developed similar levels of NP-specific systemic and mucosal antibody titers; however, there was a significantly higher number of NP-specific CD8 T cells secreting interferon-gamma (IFN-γ) in the HAd-C5-NP(H7N9) group than in the HAd-NP(H7N9) group. The HAd-C5-NP(H7N9) vaccine provided better protection following the challenge with A/Puerto Rico/8/1934(H1N1), A/Hong Kong/1/68(H3N2), A/chukkar/MN/14951-7/1998(H5N2), A/goose/Nebraska/17097/2011(H7N9), or A/Hong Kong/1073/1999(H9N2) influenza viruses compared to the HAd-NP(H7N9) group. The autophagy transcriptomic gene analysis of the HAd-C5-NP(H7N9) group revealed the upregulation of some genes involved in the positive regulation of the autophagy process. The results support further exploring the use of NP and AIP-C5 for developing a universal influenza vaccine for pandemic preparedness. [ABSTRACT FROM AUTHOR]

Adenoviral vectors based on the human adenovirus species C serotype 5 (HAdV-C5) are commonly used for vector-based gene therapies and vaccines. In the preclinical stages of development, their safety and efficacy are often validated in suitable animal models. However, pre-existing neutralizing antibodies may severely influence study outcomes. Here, we generated a new HAdV-C5-based reporter vector and established a high-throughput screening assay for the multivalent detection of HAdV-C5-neutralizing antibodies in serum. We screened the sera of rhesus macaques at different primate centers, and of rabbits, horses, cats, and dogs, showing that HAdV-C5-neutralizing antibodies can be found in all species, albeit at different frequencies. Our results emphasize the need to prescreen model animals in HAdV-C5-based studies. [ABSTRACT FROM AUTHOR]

The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease. Key messages: Adenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting. An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons. A hybrid promoter allows preferential expression of transgenes in GABAergic neurons. Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model. [ABSTRACT FROM AUTHOR]

Adenoviral vectors, both oncolytic viruses and gene delivery vectors, are among the earliest approved and commercialised vectors for gene therapy. Adenoviruses have high cytotoxicity and immunogenicity. Therefore, lentiviruses or adeno-associated viruses as viral vectors and herpes simplex virus as an oncolytic virus have recently drawn attention. Thus, adenoviral vectors are often considered relatively obsolete. However, their high cargo limit and transduction efficiency are significant advantages over newer viral vectors. This review provides an overview of the new-generation adenoviral vectors. In addition, we describe the modification of the fiber knob region that enhances affinity of adenoviral vectors for cancer cells and the utilisation of cancer-cell-specific promoters to suppress expression of unwanted transgenes in non-malignant tissues.

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes porcine epidemic diarrhea (PED). The emergence of the novel G2b strains of PEDV has increased the need for new vaccines. We generated a recombinant adenovirus expressing the spike (S) protein of G2b PEDV based on human adenovirus serotype 5 (Ad5), named rAd5-PEDV-S. Immunization with rAd5-PEDV-S elicited a significant PEDV-specific humoral immune response in sows, including IgA and IgG in colostrum and serum and circulating neutralizing antibodies. The efficacy of rAd5-PEDV-S was superior to that of the commercial inactivated vaccine, and the intramuscular (IM) route was more effective than the intranasal route. Interestingly, there was no significant difference in immunization effect between the twice and once-immunized IM groups (administered 5 weeks before farrowing). Five-day-old piglets born to sows immunized with the rAd5-PEDV-S through the IM route had less diarrhea and weight loss when challenged with PEDV. Fecal PEDV RNA expression in the PEDV-challenged rAd5-PEDV-S IM group piglets was considerably lower than that in the other groups; all five piglets from the rAd5-PEDV-S IM group survived the infection period. Histopathological examination of small intestinal sections revealed evident shedding of ileum intestinal mucosal epithelial cells in the phosphate-buffered saline (PBS) group. These findings suggest that vaccinating pregnant sows with rAd5-PEDV-S induces immune responses in the pregnant sows and passively protects piglets. Our findings highlight the potential of rAd5-PEDV-S as a candidate vaccine for PED. IMPORTANCE Porcine epidemic diarrhea (PED) is a highly infectious and economically significant gastrointestinal disorder that affects pigs of all ages. Preventing and controlling PED is achieved by immunizing sows with vaccines, enabling passive piglet immunization via colostrum. The prevalence of G2b porcine epidemic diarrhea virus (PEDV) continues in China despite the use of commercial vaccines, raising questions regarding current vaccine efficacy and the need for novel vaccine development. Adenovirus serotype 5 (Ad5) has several advantages, including high transduction efficiency, a wide range of host cells, and the ability to infect cells at various stages. In this study, we expressed the immunogenic proteins of spike (S) using an Ad5 vector and generated a PED vaccine candidate by inducing significant humoral immunity. The rAd5-PEDV-S prevented PED-induced weight loss, diarrhea, and intestinal damage in piglets. This novel vaccine candidate strain possesses the potential for use in the pig breeding industry.

ABSTRACT The failure of COVID-19 vaccines to prevent SARS-CoV-2 infection and transmission, a possibly critical reason was the lack of protective mucosal immunity in the respiratory tract. Here, we evaluated the effects of mucosal and systemic immunity from a novel simian adenovirus-vectored COVID-19 vaccine (Sad23L-nCoV-S) in mice in comparison with Ad5-nCoV-S by intranasal (IN) drip and intramuscular (IM) injection vaccinations. As good as the well-known Ad5-nCoV-S vaccine, a single-dose IN inoculation of 1 × 109 PFU Sad23L-nCoV-S vaccine induced a similar level of IgG S-binding antibody (S-BAb) and neutralizing antibody (NAb) and higher IgA in serum, while IN route raised significantly higher IgG and IgA S-BAb and NAb in bronchoalveolar lavage (BAL), and specific IFN-γ secreting T-cell response in lung compared with IM route, but lower T-cell response in spleen. By prime-boost vaccination regimens with different combinations of IN and IM inoculations of Sad23L-nCoV-S vaccine, the IN-involved vaccination stimulated higher protective mucosal or local immunity in BAL and lung, while the IM-involved immunization induced higher systemic immunity in serum and spleen. A long-term sustained mucosal and systemic NAb and T- cell immunity to SARS-CoV-2 was maintained at high level over 32 weeks by prime-boost vaccination regimens with IN and IM routes. In conclusion, priming or boosting immunization with IN inoculation of Sad23L-nCoV-S vaccine could induce effective mucosal immunity and in combination of IM route could additionally achieve systemic immunity, which provided an important reference for vaccination regimens against respiratory virus infection. IMPORTANCE The essential goal of vaccination is to generate potent and long-term protection against diseases. Several factors including vaccine vector, delivery route, and boosting regimen influence the outcome of prime-boost immunization approaches. The immunization regimens by constructing a novel simian adenovirus-vectored COVID-19 vaccine and employing combination of intranasal and intramuscular inoculations could elicit mucosal neutralizing antibodies against five mutant strains in the respiratory tract and strong systemic immunity. Immune protection could last for more than 32 weeks. Vectored vaccine construction and immunization regimens have positively impacted respiratory disease prevention.

For the development of new adenovirus (AdV)-based vectors, it is important to understand differences in immunogenicity. In a side-by-side in vitro analysis, we evaluated the effect of 40 AdV types covering human AdV (HAdV) species A through G on the expression of 11 activation markers and the secretion of 12 cytokines by AdV-transduced dendritic cells, and the effect on CD8+ T cell proliferation capacity. We found that the expression of activation markers and cytokines differed widely between the different HAdV types, and many types were able to significantly impair the proliferation capacity of CD8+ T cells. Univariate and multivariate regression analyses suggested an important role of type I interferons in mediating this suppression of CD8+ T cells, which we confirmed experimentally in a proliferation assay using a type I interferon receptor blocking antibody. Using Bayesian statistics, we calculated a prediction model that suggests HAdV types HAdV-C1, -D8, -B7, -F41, -D33, -C2, -A31, -B3 and -D65 as the most favorable candidates for vaccine vector development. [ABSTRACT FROM AUTHOR]

The safety profile of the Sputnik V vaccine is generally reassuring. Nevertheless, an enhanced risk of new‐onset of immune‐mediated diseases has been increasingly reported following the adenoviral‐based Covid‐19 vaccine, including inflammatory arthritis, Guillain‐Barré syndrome, optical neuromyelitis, acute disseminated encephalomyelitis, subacute thyroiditis and acute liver injury as well as glomerulopathy. However, no case of autoimmune pancreatitis has been reported yet. Herein, we describe a case of type I autoimmune pancreatitis that may be due to the Sputnik V Covid‐19 vaccine. [ABSTRACT FROM AUTHOR]

Recessive dystrophic epidermolysis bullosa, a devastating skin fragility disease characterized by recurrent skin blistering, scarring, and a high risk of developing squamous cell carcinoma is caused by mutations in COL7A1, the gene encoding type VII collagen, which is the major component of the anchoring fibrils that bind the dermis and epidermis. Ex vivo correction of COL7A1 by gene editing in patients’ cells has been achieved before. However, in vivo editing approaches are necessary to address the direct treatment of the blistering lesions characteristic of this disease. We have now generated adenoviral vectors for CRISPR-Cas9 delivery to remove exon 80 of COL7A1, which contains a highly prevalent frameshift mutation in Spanish patients. For in vivo testing, a humanized skin mouse model was used. Efficient viral transduction of skin was observed after excisional wounds generated with a surgical punch on regenerated patient skin grafts were filled with the adenoviral vectors embedded in a fibrin gel. Type VII collagen deposition in the basement membrane zone of the wounded areas treated with the vectors correlated with restoration of dermal-epidermal adhesion, demonstrating that recessive dystrophic epidermolysis bullosa (RDEB) patient skin lesions can be directly treated by CRISPR-Cas9 delivery in vivo.

Human endogenous retrovirus type W (HERV-W) is expressed in various cancers. We previously developed an adenovirus-vectored cancer vaccine targeting HERV-W by encoding an assembled HERV-W group-specific antigen sequence and the HERV-W envelope sequence Syncytin-1. Syncytin-1 is constitutively fusogenic and forms large multinucleated cell fusions when overexpressed. Consequently, immunising humans with a vaccine encoding Syncytin-1 can lead to the formation of extensive syncytia, which is undesirable and poses a potential safety issue. Here, we show experiments in cell lines that restoring an evolutionary lost cleavage site of the fusion inhibitory R-peptide of Syncytin-1 inhibit cell fusion. Interestingly, this modification of the HERV-W vaccine's fusogenicity increased the expression of the vaccine antigens in vitro. It also enhanced Syncytin-1-specific antibody responses and CD8+-mediated T-cell responses compared to the wildtype vaccine in vaccinated mice, with a notable enhancement in responses to subdominant T-cell epitopes but equal responses to dominant epitopes and similar rates of survival following a tumour challenge. The impairment of cell–cell fusion and the enhanced immunogenicity profile of this HERV-W vaccine strengthens the prospects of obtaining a meaningful immune response against HERV-W in patients with HERV-W-overexpressing cancers. [ABSTRACT FROM AUTHOR]

For the development of new adenovirus (AdV)-based vectors, it is important to understand differences in immunogenicity. In a side-by-side in vitro analysis, we evaluated the effect of 40 AdV types covering human AdV (HAdV) species A through G on the expression of 11 activation markers and the secretion of 12 cytokines by AdV-transduced dendritic cells, and the effect on CD8+ T cell proliferation capacity. We found that the expression of activation markers and cytokines differed widely between the different HAdV types, and many types were able to significantly impair the proliferation capacity of CD8+ T cells. Univariate and multivariate regression analyses suggested an important role of type I interferons in mediating this suppression of CD8+ T cells, which we confirmed experimentally in a proliferation assay using a type I interferon receptor blocking antibody. Using Bayesian statistics, we calculated a prediction model that suggests HAdV types HAdV-C1, -D8, -B7, -F41, -D33, -C2, -A31, -B3 and -D65 as the most favorable candidates for vaccine vector development.

We estimated the risk of death from coronavirus disease 2019 in vaccinated compared with unvaccinated patients. The risk of death was reduced 44% after 1 dose of the Pfizer-BioNTech BNT162b2 vaccine, 55% after 1 dose of the Oxford-Astrazeneca ChAdOx1 vaccine, and 69% after 2 doses of the BNT162b2 vaccine. This is above the protection provided against infection. [ABSTRACT FROM AUTHOR]

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

• Adenoviral vectored vaccine constructs against Q fever were synthesised. • Vaccine constructs elicited robust antigen-specific T cell immunity in mice. • Comparable T cell immunity when co-administering several vaccine constructs. • Formulating constructs with LPS or LPS alone gave protection against challenge. Q fever is a highly infectious zoonosis caused by the Gram-negative bacterium Coxiella burnetii. The worldwide distribution of Q fever suggests a need for vaccines that are more efficacious, affordable, and does not induce severe adverse reactions in vaccine recipients with pre-existing immunity against Q fever. Potential Q fever vaccine antigens include lipopolysaccharide (LPS) and several C. burnetii surface proteins. Antibodies elicited by purified C. burnetii lipopolysaccharide (LPS) correlate with protection against Q fever, while antigens encoded by adenoviral vectored vaccines can induce cellular immune responses which aid clearing of intracellular pathogens. In the present study, the immunogenicity and the protection induced by adenoviral vectored constructs formulated with the addition of LPS were assessed. Multiple vaccine constructs encoding single or fusion antigens from C. burnetii were synthesised. The adenoviral vectored vaccine constructs alone elicited strong cellular immunity, but this response was not correlative with protection in mice. However, vaccination with LPS was significantly associated with lower weight loss post-bacterial challenge independent of co-administration with adenoviral vaccine constructs, supporting further vaccine development based on LPS. [ABSTRACT FROM AUTHOR]

Immune checkpoint blockade based on antibodies has shown great clinical success in patients, but the transitory working manner leads to restricted therapeutic benefits. Herein, a genetically engineered adenovirus is developed as the vector to deliver CRISPR/Cas9 (sgCas9‐AdV) to achieve permanent PD‐L1 gene editing with efficiency up to 78.7% exemplified in Hepa 1‐6 liver cancer cells. Furthermore, the sgCas9‐AdV is loaded into hydrogel made by silk fiber (SgCas9‐AdV/Gel) for in vivo application. The silk‐gel not only promotes local retention of sgCas9‐AdV in tumor tissue, but also masks them from host immune system, thus ensuring effectively gene transduction over 9 days. Bearing these advantages, the sgCas9‐AdV/Gel inhibits Hepa 1‐6 tumor growth with 100% response rate by single‐dose injection, through efficient PD‐L1 disruption to elicit a T cell‐mediated antitumor response. In addition, the sgCas9‐AdV/Gel is also successfully extended into other refractory tumors. In CT26 colon tumor characterized by poor response to anti‐PD‐L1, sgCas9‐AdV/Gel is demonstrated to competent and superior anti‐PD‐L1 antibody to suppress tumor progression. In highly aggressive orthotopic 4T1 mouse breast tumor, such a therapeutic paradigm significantly inhibits primary tumor growth and induces a durable immune response against tumor relapse/metastasis. Thus, this study provides an attractive and universal strategy for immunotherapy. [ABSTRACT FROM AUTHOR]

Tumor-selective viruses are a novel therapeutic approach for treating cancer. Tumor-Specific Immuno Gene Therapy (T-SIGn) vectors are tumor-selective adenoviral vectors designed to express immunomodulatory transgenes. Prolonged activated partial thromboplastin time (aPTT), associated with the presence of antiphospholipid antibodies (aPL), has been observed in patients with viral infections, and following administration of adenovirus-based medicines. aPL may be detected as lupus anticoagulant (LA), anti-cardiolipin (aCL) and/or anti-beta 2 glycoprotein antibodies (aβ2GPI). No subtype alone is definitive for development of clinical sequalae, however, patients who are 'triple positive' have a greater thrombotic risk. Additionally, isolated aCL and aβ2GPI IgM do not appear to add value in thrombotic association to aPL positivity, rather IgG subtypes must also be present to confer an increased risk. Here we report induction of prolonged aPTT and aPL in patients from eight Phase 1 studies who were treated with adenoviral vectors (n = 204). Prolonged aPTT (≥ Grade 2) was observed in 42% of patients, with a peak at 2–3 weeks post-treatment and resolution within ~ 2 months. Among patients with aPTT prolongation, LA, but not aCL IgG nor aβ2GPI IgG, was observed. The transience of the prolongation and discordance between positive LA and negative aCL/aβ2GPI IgG assays is not typical of a prothrombotic state. Among the patients with prolonged aPTT there was no evidence of an increased rate of thrombosis. These findings elucidate the relationship between viral exposure and aPL in the context of clinical trials. They suggest a framework in which hematologic changes can be monitored in patients receiving similar treatments. Clinical trial registration: NCT02028442, NCT02636036, NCT02028117, NCT03852511, NCT04053283, NCT05165433, NCT04830592, NCT05043714. [ABSTRACT FROM AUTHOR]

Oncolytic viruses are a promising technology to attack cancer cells and to recruit immune cells to the tumor site. Since the Lipocalin-2 receptor (LCN2R) is expressed on most cancer cells, we used its ligand LCN2 to target oncolytic adenoviruses (Ads) to cancer cells. Therefore, we fused a Designed Ankyrin Repeat Protein (DARPin) adapter binding the knob of Ad type 5 (knob5) to LCN2 to retarget the virus toward LCN2R with the aim of analyzing the basic characteristics of this novel targeting approach. The adapter was tested in vitro with Chinese Hamster Ovary (CHO) cells stably expressing the LCN2R and on 20 cancer cell lines (CCLs) using an Ad5 vector encoding luciferase and green fluorescent protein. Luciferase assays with the LCN2 adapter (LA) showed 10-fold higher infection compared with blocking adapter (BA) in CHO cells expressing LCN2R and in cells not expressing the LCN2R. Most CCLs showed an increased viral uptake of LA-bound virus compared with BA-bound virus and for five CCLs viral uptake was comparable to unmodified Ad5. Flow cytometry and hexon immunostainings also revealed increased uptake of LA-bound Ads compared with BA-bound Ads in most tested CCLs. Virus spread was studied in 3D cell culture models and nine CCLs showed increased and earlier fluorescence signals for LA-bound virus compared with BA-bound virus. Mechanistically, we show that the LA increases viral uptake only in the absence of its ligand Enterobactin (Ent) and independently of iron. Altogether, we characterized a novel DARPin-based system resulting in enhanced uptake demonstrating potential for future oncolytic virotherapy. [ABSTRACT FROM AUTHOR]

The nucleoprotein (NP) is a vital target for the heterosubtypic immunity of CD8+ cytotoxic T lymphocytes (CTLs) due to its conservation among influenza virus subtypes. To further enhance the T cell immunity of NP, autophagy-inducing peptide C5 (AIP-C5) from the CFP10 protein of Mycobacterium tuberculosis was used. Mice were immunized intranasally (i.n.) with human adenoviral vectors, HAd-C5-NP(H7N9) or HAd-NP(H7N9), expressing NP of an H7N9 influenza virus with or without the AIP-C5, respectively. Both vaccines developed similar levels of NP-specific systemic and mucosal antibody titers; however, there was a significantly higher number of NP-specific CD8 T cells secreting interferon-gamma (IFN-γ) in the HAd-C5-NP(H7N9) group than in the HAd-NP(H7N9) group. The HAd-C5-NP(H7N9) vaccine provided better protection following the challenge with A/Puerto Rico/8/1934(H1N1), A/Hong Kong/1/68(H3N2), A/chukkar/MN/14951-7/1998(H5N2), A/goose/Nebraska/17097/2011(H7N9), or A/Hong Kong/1073/1999(H9N2) influenza viruses compared to the HAd-NP(H7N9) group. The autophagy transcriptomic gene analysis of the HAd-C5-NP(H7N9) group revealed the upregulation of some genes involved in the positive regulation of the autophagy process. The results support further exploring the use of NP and AIP-C5 for developing a universal influenza vaccine for pandemic preparedness.

Adenoviral vectors based on the human adenovirus species C serotype 5 (HAdV-C5) are commonly used for vector-based gene therapies and vaccines. In the preclinical stages of development, their safety and efficacy are often validated in suitable animal models. However, pre-existing neutralizing antibodies may severely influence study outcomes. Here, we generated a new HAdV-C5-based reporter vector and established a high-throughput screening assay for the multivalent detection of HAdV-C5-neutralizing antibodies in serum. We screened the sera of rhesus macaques at different primate centers, and of rabbits, horses, cats, and dogs, showing that HAdV-C5-neutralizing antibodies can be found in all species, albeit at different frequencies. Our results emphasize the need to prescreen model animals in HAdV-C5-based studies.

There are a lot of diseases known today, which are caused by genetic abnormalities. Advances in genetics and biotechnology brought about gene editing technologies that can produce almost any gene, which ultimately led to the emergence of a new class of medicines - gene therapy products (GTPs). The aim of the study was to analyse international experience in development and authorisation of GTPs. The review highlights the challenges in GTP development, related to the search for an optimal approach to therapeutic gene delivery to the target cells. Viral vectors were shown to be a promising gene delivery system, with adenovirus (AV) and adeno-associated virus (AAV) based products demonstrating the highest efficacy and safety. The paper reviews current approaches to gene editing that allow modification of AVs and AAVs to improve GTP efficacy and safety. These modifications are carried out with the aim of, e.g., including a large therapeutic gene into a viral vector, decreasing viral protein expression levels, and decreasing viral vector immunogenicity. The review summarises GTP authorisation procedures in the USA and the European Union, including data on FDA and EMA subcommittees and departments entrusted with advisory functions. The paper mentions that there is one Russian-produced GTP authorised in the Russian Federation, and some other GTPs are in the pipeline. Therefore, the Russian regulatory framework and the Eurasian regulations and recommendations should be updated in order to accommodate for GTP development and authorisation.

The development of an efficient and safe coronavirus disease 2019 (COVID-19) vaccine is a crucial approach for managing the severe acute respiratory disease coronavirus 2 (SARS-CoV-2) pandemic in light of current conditions. In this study, we produced a shortened segment of the optimized SARS-CoV-2 spike gene (2043 bp, termed S1) that was able to encode a truncated S1 protein. The protein was tested to determine if it could elicit efficient immunization in mice against SARS-CoV-2. The presence of the S1 protein was confirmed with immunofluorescence and Western blotting. An adenovirus vaccine bearing the S1 gene fragment (Ad-S1) was administered intramuscularly to mice four times over 4 weeks. SARS-CoV-2 S1 protein humoral immunity was demonstrated in all immunized mice. The serum from immunized mice demonstrated excellent anti-infection activity in vitro. A robust humoral immune response against SARS-CoV-2 was observed in the mice after vaccination with Ad-S1, suggesting that the adenovirus vaccine may aid the development of vaccines against SARS-CoV-2 and other genetically distinct viruses. [ABSTRACT FROM AUTHOR]

The adenovirus vector platform remains one of the most efficient toolboxes for generation of transfer vehicles used in gene therapy and virotherapy to treat tumors, as well as vaccines to protect from infectious diseases. The adenovirus genome and capsids can be modified using highly efficient techniques, and vectors can be produced at high titers, which facilitates their rapid adaptation to current needs and disease applications. Over recent years, the adenovirus vector platform has been in the center of attention for vaccine development against the ongoing coronavirus SARS-CoV-2/COVID-19 pandemic. The worldwide deployment of these vaccines has greatly deepened the knowledge on virus-host interactions and highlighted the need to further improve the effectiveness and safety not only of adenovirus-based vaccines but also of gene therapy and oncolytic virotherapy vectors. Based on the current evidence, we discuss here how adenoviral vectors can be further improved by intelligent molecular design. This review covers the full spectrum of state-of-the-art strategies to avoid vector-induced side effects ranging from the vectorization of non-canonical adenovirus types to novel genome engineering techniques. [ABSTRACT FROM AUTHOR]

Introduction: Approximately 75% of bladder cancer cases are non-muscle invasive at diagnosis. Drug development for non-muscle invasive bladder cancer (NMIBC) has historically lagged behind that of other malignancies. No treatment has demonstrated the ability to overcome drug resistance that ultimately leads to recurrence and progression. Gene therapy is emerging as a promising option for patients with NMIBC., Areas Covered: This review summarizes the clinical application of gene therapy in NMIBC management and discusses recent clinical trials involving the adenoviral vector-based treatment nadofaragene firadenovec, and the oncolytic serotype 5 adenovirus, cretostimogene grenadenorepvec. Nadofaragene received approval by the Food and Drug Administration in December 2022, and cretostimogene has been granted Fast Track Designation and Breakthrough Therapy Designation. Ongoing trials are investigating strategies to augment efficacy and durability of these therapies., Expert Opinion: Gene therapy may overcome resistance mechanisms of other NMIBC treatments, and data suggest a role for combination therapy with additive or synergistic agents. Significant differences in trial design limit comparability of agents across trials, highlighting the need for critical assessment of published findings. While initial investigations were in high-risk patients who recur despite frontline therapy with Bacillus Calmette-Guerin (BCG), there is growing interest in BCG-naïve and intermediate-risk populations.

Vaccination with the adenoviral-vector-based AstraZeneca ChAdOx1 nCov-19 (Vaxzevria) vaccine is efficient and safe. However, in rare cases vaccinated individuals developed life-threatening thrombotic complications, including thrombosis in cerebral sinus and splanchnic veins. Monitoring of the applied vector in vivo represents an important precondition to study the molecular mechanisms underlying vaccine-driven adverse effects now referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). We previously have shown that digital PCR (dPCR) is an excellent tool to quantify transgene copies in vivo. Here, we present a highly sensitive dPCR for in situ quantification of ChAdOx1 nCoV-19 copies. Using this method, we quantified vector copies in human plasma 24, 72, and 168 h post vaccination and in a variety of murine tissues in an experimental vaccination model 30 min post injection. We describe a method for high-sensitivity quantitative detection of ChAdOx1 nCoV-19 with possible implications to elucidate the mechanisms of severe ChAdOx1 nCov-19 vaccine complications.

Hypoxic–ischemic brain damage (HIBD) is a major cause of brain injury in neonates. Bone marrow mesenchymal stem cells (BMSCs) show therapeutic potential for HIBD, and genetic modification may enhance their neuroprotective effects. The goal of this study was to investigate the neuroprotective effects of hepatocyte growth factor (HGF)-overexpressing BMSCs (BMSCs-HGF) against HIBD and their underlying mechanisms.

The objective is to evaluate the effectiveness of placental transfer of maternally derived SARS-CoV-2 IgG antibodies after the vaccination of pregnant women with heterologous CoronaVac–ChAdOx1. Thirty pregnant women were vaccinated with CoronaVac as the first dose, followed by ChAdOx1 3 weeks later. The antibody levels in the maternal blood and in the umbilical cord blood at the time of delivery were determined. The results showed that the vaccination effectively increased antibody levels in both mothers and newborns. The antibody levels in the mothers were strongly correlated with those in the newborns (P

Cancer is one of the leading causes of death in the world, which is the second after heart diseases. Adenoviruses (Ads) have become the promise of new therapeutic strategy for cancer treatment. The objective of this review is to discuss current advances in the applications of adenoviral vectors in cancer therapy. Adenoviral vectors can be engineered in different ways so as to change the tumor microenvironment from cold tumor to hot tumor, including; 1. by modifying Ads to deliver transgenes that codes for tumor suppressor gene (p53) and other proteins whose expression result in cell cycle arrest 2. Ads can also be modified to express tumor specific antigens, cytokines, and other immune-modulatory molecules. The other strategy to use Ads in cancer therapy is to use oncolytic adenoviruses, which directly kills tumor cells. Gendicine and Advexin are replication-defective recombinant human p53 adenoviral vectors that have been shown to be effective against several types of cancer. Gendicine was approved for treatment of squamous cell carcinoma of the head and neck by the Chinese Food and Drug Administration (FDA) agency in 2003 as a first-ever gene therapy product. Oncorine and ONYX-015 are oncolytic adenoviral vectors that have been shown to be effective against some types of cancer. The Chiness FDA agency has also approved Oncorin for the treatment of head and neck cancer. Ads that were engineered to express immune-stimulatory cytokines and other immune-modulatory molecules such as TNF-α, IL-2, BiTE, CD40L, 4-1BBL, GM-CSF, and IFN have shown promising outcome in treatment of cancer. Ads can also improve therapeutic efficacy of immune checkpoint inhibitors and adoptive cell therapy (Chimeric Antigen Receptor T Cells). In addition, different replication-deficient adenoviral vectors (Ad5-CEA, Ad5-PSA, Ad-E6E7, ChAdOx1–MVA and Ad-transduced Dendritic cells) that were tested as anticancer vaccines have been demonstrated to induce strong antitumor immune response. However, the use of adenoviral vectors in gene therapy is limited by several factors such as pre-existing immunity to adenoviral vectors and high immunogenicity of the viruses. Thus, innovative strategies must be continually developed so as to overcome the obstacles of using adenoviral vectors in gene therapy. [ABSTRACT FROM AUTHOR]

Cancer is one of the leading causes of death in the world, which is the second after heart diseases. Adenoviruses (Ads) have become the promise of new therapeutic strategy for cancer treatment. The objective of this review is to discuss current advances in the applications of adenoviral vectors in cancer therapy. Adenoviral vectors can be engineered in different ways so as to change the tumor microenvironment from cold tumor to hot tumor, including; 1. by modifying Ads to deliver transgenes that codes for tumor suppressor gene (p53) and other proteins whose expression result in cell cycle arrest 2. Ads can also be modified to express tumor specific antigens, cytokines, and other immune-modulatory molecules. The other strategy to use Ads in cancer therapy is to use oncolytic adenoviruses, which directly kills tumor cells. Gendicine and Advexin are replication-defective recombinant human p53 adenoviral vectors that have been shown to be effective against several types of cancer. Gendicine was approved for treatment of squamous cell carcinoma of the head and neck by the Chinese Food and Drug Administration (FDA) agency in 2003 as a first-ever gene therapy product. Oncorine and ONYX-015 are oncolytic adenoviral vectors that have been shown to be effective against some types of cancer. The Chiness FDA agency has also approved Oncorin for the treatment of head and neck cancer. Ads that were engineered to express immune-stimulatory cytokines and other immune-modulatory molecules such as TNF-α, IL-2, BiTE, CD40L, 4-1BBL, GM-CSF, and IFN have shown promising outcome in treatment of cancer. Ads can also improve therapeutic efficacy of immune checkpoint inhibitors and adoptive cell therapy (Chimeric Antigen Receptor T Cells). In addition, different replication-deficient adenoviral vectors (Ad5-CEA, Ad5-PSA, Ad-E6E7, ChAdOx1–MVA and Ad-transduced Dendritic cells) that were tested as anticancer vaccines have been demonstrated to induce strong antitumor immune response. However, the use of adenoviral vectors in gene therapy is limited by several factors such as pre-existing immunity to adenoviral vectors and high immunogenicity of the viruses. Thus, innovative strategies must be continually developed so as to overcome the obstacles of using adenoviral vectors in gene therapy.

Proper conformational arrangement of the E2 molecules of bovine viral diarrhoea-mucosal disease virus (BVD-MDV) is crucial to obtain an effective recombinant vaccine candidate against the disease. In this study, we characterised a new molecule composed of two distinct sequences of the E2 glycoprotein of BVD-MDV and the Fc fragment of human immunoglobulin (BVDE2Fc).

Despite emerging contemporary biotechnological methods such as gene- and stem cell-based therapy, there are no clinically established therapeutic strategies for neural regeneration after spinal cord injury. Our previous studies have demonstrated that transplantation of genetically engineered human umbilical cord blood mononuclear cells producing three recombinant therapeutic molecules, including vascular endothelial growth factor (VEGF), glial cell-line derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) can improve morpho-functional recovery of injured spinal cord in rats and mini-pigs. To investigate the efficacy of human umbilical cord blood mononuclear cells-mediated triple-gene therapy combined with epidural electrical stimulation in the treatment of spinal cord injury, in this study, rats with moderate spinal cord contusion injury were intrathecally infused with human umbilical cord blood mononuclear cells expressing recombinant genes VEGF165, GDNF, NCAM1 at 4 hours after spinal cord injury. Three days after injury, epidural stimulations were given simultaneously above the lesion site at C5 (to stimulate the cervical network related to forelimb functions) and below the lesion site at L2 (to activate the central pattern generators) every other day for 4 weeks. Rats subjected to the combined treatment showed a limited functional improvement of the knee joint, high preservation of muscle fiber area in tibialis anterior muscle and increased H/M ratio in gastrocnemius muscle 30 days after spinal cord injury. However, beneficial cellular outcomes such as reduced apoptosis and increased sparing of the gray and white matters, and enhanced expression of heat shock and synaptic proteins were found in rats with spinal cord injury subjected to the combined epidural electrical stimulation with gene therapy. This study presents the first proof of principle study of combination of the multisite epidural electrical stimulation with ex vivo triple gene therapy (VEGF, GDNF and NCAM) for treatment of spinal cord injury in rat models. The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee (approval No. 2.20.02.18) on February 20, 2018.

In response to the SARS-CoV-2 Delta variant, which partially escaped the vaccine-induced immunity provided by two doses of vaccination with CoronaVac (Sinovac), the National Vaccine Committee recommended the heterologous CoronaVac-ChAdOx1 (Oxford–AstraZeneca), a prime–boost vaccine regimen. This pilot study aimed to describe the immunogenicity and adverse events of the heterologous CoronaVac-ChAdOx1 regimen, in comparison with homologous CoronaVac, and homologous ChAdOx1. Between May and August 2021, we recruited a total of 354 participants from four vaccination groups: the CoronaVac-ChAdOx1 vaccinee (n = 155), the homologous CoronaVac vaccinee (n = 32), the homologous ChAdOx1 vaccinee (n = 47), and control group of COVID-19 patients (n = 120). Immunogenicity was evaluated by measuring the level of IgG antibodies against the receptor-binding domain (anti-SRBD) of the SARS-CoV-2 spike protein S1 subunit and the level of neutralizing antibodies (NAbs) against variants of concern (VOCs) using the plaque reduction neutralization test (PRNT) and pseudovirus neutralization test (pVNT). The safety profile was recorded by interviewing at the 1-month visit after vaccination. The anti-SRBD level after the second booster dose of the CoronaVac-ChAdOx1 group at 2 weeks was higher than 4 weeks. At 4 weeks after the second booster dose, the anti-SRBD level in the CoronaVac-ChAdOx1 group was significantly higher than either homologous CoronaVac, the homologous ChAdOx1 group, and Control group (p < 0.001). In the CoronaVac-ChAdOx1 group, the PRNT50 level against the wild-type (434.5 BAU/mL) was the highest; followed by Alpha variant (80.4), Delta variant (67.4), and Beta variant (19.8). The PVNT50 level was also found to be at its highest against the wild-type (432.1); followed by Delta variants (178.3), Alpha variants (163.9), and Beta variant (42.2), respectively. The AEs in the CoronaVac-ChAdOx1 group were well tolerated and generally unremarkable. The CoronaVac-ChAdOx1 heterologous regimen induced higher immunogenicity and a tolerable safety profile. In a situation when only CoronaVac-ChAdOx1 vaccines are available, they should be considered for use in responding to the Delta variant. [ABSTRACT FROM AUTHOR]