Viral-vectored boosting of OmcB or CPAF-specific T cell responses fail to enhance protection from Chlamydia muridarum in infection immune mice and elicits a non-protective CD8-dominant response in naïve mice.

A vaccine is needed to combat the Chlamydia epidemic. Replication-deficient viral vectors are safe and induce antigen-specific T cell memory. We tested the ability of intramuscular immunization with modified vaccinia virus Ankara (MVA) or Chimpanzee Adenovirus (ChAd) expressing chlamydial outer membrane protein, OmcB, or the secreted protein, CPAF, to enhance T cell immunity and protection in mice previously infected with plasmid-deficient Chlamydia muridarum CM972, and to elicit protection in naïve mice. MVA.OmcB or MVA.CPAF increased antigen-specific T cells in CM972-immune mice ∼150 and 50-fold respectively but failed to improve bacterial clearance. ChAd.OmcB/MVA.OmcB prime-boost immunization of naïve mice elicited a CD8-dominant T cell response that failed to protect. ChAd.CPAF/ChAd.CPAF prime-boost also induced a CD8-dominant response with a marginal reduction in burden. Challenge of ChAd.CPAF-immunized mice genetically deficient in CD4 or CD8 T cells showed that protection was entirely CD4-dependent. CD4-deficient mice had prolonged infection, while CD8-deficient mice had higher frequencies of CPAF-specific CD4 T cells, earlier clearance, and reduced burden compared to wild-type controls. These data reinforce the essential nature of the CD4 T cell response in protection from chlamydial genital infection in mice and the need for vaccine platforms that drive CD4-dominant responses.

Toxic Epidermal Necrolysis After COVID-19 Vaccination: A Case Report and Review of Literature.

Following vaccination for COVID-19, various cutaneous adverse reactions (CARs) are reported. Here is an Asian male in late 50's who developed necrotic skin with mucosal involvement 10 days following booster dose of ChAdOx1 nCov-19 vaccination. Based on disease course and morphology, toxic epidermal necrolysis (TEN) was suspected. The patient developed respiratory distress and was intubated, intravenous immunoglobulin (IVIG) administered at 2 g/kg body weight following which skin lesions healed in fourth week, the patient was discharged after 50 days of intensive care unit (ICU) stay. Severe CARs are rare following vaccination, of two components in ChAdOx1nCoV-19 adenoviral vector vaccine, virotopes cause T-cell mediated granulysin and granzyme B release leading to epidermal detachment and mucosal involvement of conducting airways causing respiratory failure. CARs can also occur in whom first and second dose was uneventful. Supportive therapy and prevention of sepsis are mainstay of management. Though the use of IVIG has shown conflicting results, our case was successfully managed with IVIG.

Evaluation of two inoculation routes of an adenovirus-mediated viral protein inhibitor in a Crimean-Congo hemorrhagic fever mouse model.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne nairovirus with a wide geographic spread that can cause severe and lethal disease. No specific medical countermeasures are approved to combat this illness. The CCHFV L protein contains an ovarian tumor (OTU) domain with a cysteine protease thought to modulate cellular immune responses by removing ubiquitin and ISG15 post-translational modifications from host and viral proteins. Viral deubiquitinases like CCHFV OTU are attractive drug targets, as blocking their activity may enhance cellular immune responses to infection, and potentially inhibit viral replication itself. We previously demonstrated that the engineered ubiquitin variant CC4 is a potent inhibitor of CCHFV replication in vitro. A major challenge of the therapeutic use of small protein inhibitors such as CC4 is their requirement for intracellular delivery, e.g., by viral vectors. In this study, we examined the feasibility of in vivo CC4 delivery by a replication-deficient recombinant adenovirus (Ad-CC4) in a lethal CCHFV mouse model. Since the liver is a primary target of CCHFV infection, we aimed to optimize delivery to this organ by comparing intravenous (tail vein) and intraperitoneal injection of Ad-CC4. While tail vein injection is a traditional route for adenovirus delivery, in our hands intraperitoneal injection resulted in higher and more widespread levels of adenovirus genome in tissues, including, as intended, the liver. However, despite promising in vitro results, neither route of in vivo CC4 treatment resulted in protection from a lethal CCHFV infection.

Development of adenoviral vectors that transduce Purkinje cells and other cerebellar cell-types in the cerebellum of a humanized mouse model.

Viral vector gene therapy has immense promise for treating central nervous system (CNS) disorders. Although adeno-associated virus vectors (AAVs) have had success, their small packaging capacity limits their utility to treat the root cause of many CNS disorders. Adenoviral vectors (Ad) have tremendous potential for CNS gene therapy approaches. Currently, the most common vectors utilize the Group C Ad5 serotype capsid proteins, which rely on the Coxsackievirus-Adenovirus receptor (CAR) to infect cells. However, these Ad5 vectors are unable to transduce many neuronal cell types that are dysfunctional in many CNS disorders. The human CD46 (hCD46) receptor is widely expressed throughout the human CNS and is the primary attachment receptor for many Ad serotypes. Therefore, to overcome the current limitations of Ad vectors to treat CNS disorders, we created chimeric first generation Ad vectors that utilize the hCD46 receptor. Using a "humanized" hCD46 mouse model, we demonstrate these Ad vectors transduce cerebellar cell types, including Purkinje cells, that are refractory to Ad5 transduction. Since Ad vector transduction properties are dependent on their capsid proteins, these chimeric first generation Ad vectors open new avenues for high-capacity helper-dependent adenovirus (HdAd) gene therapy approaches for cerebellar disorders and multiple neurological disorders.

Redirect Tropism of Fowl Adenovirus 4 Vector by Modifying Fiber2 with Variable Domain of Heavy-Chain Antibody.

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.

Type I Interferon, Induced by Adenovirus or Adenoviral Vector Infection, Regulates the Cytokine Response to Lipopolysaccharide in a Macrophage Type-Specific Manner.

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-αß) 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-ß 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-αß, and IL-10 responses induced by LPS after pretreatment were measured. Mouse knockout models for MARCO, IFN-αßR, 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-αß 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-αß-producing cDC and splenic marginal zone macrophages is important for Ad uptake and subsequent cytokine overproduction by LPS. Interestingly, not all IFN-αß-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-ß pretreatment enhances the subsequent induction of IFN-αß in all macrophage types. IRF3 is dispensable for the LPS-induced IFN-αß 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.

High-Throughput Screening for the Prevalence of Neutralizing Antibodies against Human Adenovirus Serotype 5.

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.

Development of NP-Based Universal Vaccine for Influenza A Viruses.

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.

Vaccine-Induced Immune Thrombotic Thrombocytopenia: Clinicopathologic Features and New Perspectives on Anti-PF4 Antibody-Mediated Disorders.

INTRODUCTION: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet severe adverse complication first identified during the global vaccination effort against SARS-CoV-2 infection, predominantly observed following administration of the ChAdOx1-S (Oxford-AstraZeneca) and Ad26.CoV2.S (Johnson & Johnson/Janssen) adenoviral vector-based vaccines. Unlike other anti-platelet factor 4 (PF4) antibody-mediated disorders, such as heparin-induced thrombocytopenia (HIT), VITT arises with the development of platelet-activating anti-PF4 antibodies 4-42 days post-vaccination, typically featuring thrombocytopenia and thrombosis at unusual sites. AIM: To explore the unique properties, pathogenic mechanisms, and long-term persistence of VITT antibodies in patients, in comparison with other anti-PF4 antibody-mediated disorders. DISCUSSION: This review highlights the complexity of VITT as it differs in antibody behavior and clinical presentation from other anti-PF4-mediated disorders, including the high incidence rate of cerebral venous sinus thrombosis (CVST) and the persistence of anti-PF4 antibodies, necessitating a re-evaluation of long-term patient care strategies. The nature of VITT antibodies and the underlying mechanisms triggering their production remain largely unknown. CONCLUSION: The rise in awareness and subsequent prompt recognition of VITT is paramount in reducing mortality. As vaccination campaigns continue, understanding the role of adenoviral vector-based vaccines in VITT antibody production is crucial, not only for its immediate clinical implications, but also for developing safer vaccines in the future.

Exploring the DNA2-PNA heterotriplex formation in targeting the Bcl-2 gene promoter: A structural insight by physico-chemical and microsecond-scale MD investigation.

Peptide Nucleic Acids (PNAs) represent a promising tool for gene modulation in anticancer treatment. The uncharged peptidyl backbone and the resistance to chemical and enzymatic degradation make PNAs highly advantageous to form stable hybrid complexes with complementary DNA and RNA strands, providing higher stability than the corresponding natural analogues. Our and other groups' research has successfully shown that tailored PNA sequences can effectively downregulate the expression of human oncogenes using antigene, antisense, or anti-miRNA approaches. Specifically, we identified a seven bases-long PNA sequence, complementary to the longer loop of the main G-quadruplex structure formed by the bcl2midG4 promoter sequence, capable of downregulating the expression of the antiapoptotic Bcl-2 protein and enhancing the anticancer activity of an oncolytic adenovirus. Here, we extended the length of the PNA probe with the aim of including the double-stranded Bcl-2 promoter among the targets of the PNA probe. Our investigation primarily focused on the structural aspects of the resulting DNA2-PNA heterotriplex that were determined by employing conventional and accelerated microsecond-scale molecular dynamics simulations and chemical-physical analysis. Additionally, we conducted preliminary biological experiments using cytotoxicity assays on human A549 and MDA-MB-436 adenocarcinoma cell lines, employing the oncolytic adenovirus delivery strategy.

Reported COVID-19 vaccines side effects among Algerian athletes: a comparison between inactivated virus, adenoviral vector, and mRNA COVID-19 vaccines.

OBJECTIVES: Many types of COVID19 vaccines are administered globally, yet there is not much evidence regarding their side effects among athletes. This study evaluated the selfreported postvaccination side effects of inactivated virus, adenoviral vector, and mRNA COVID19 vaccines among Algerian athletes. METHODS: A cross-sectional survey-based study was carried out in Algeria between March 01 and 4 April 2022. The study used a validated questionnaire with twenty-five multiple-choice items covering the participants' anamnestic characteristics, post-vaccination side effects (their onset and duration), post-vaccination medical care, and risk factors. RESULTS: A total of 273 athletes completed the survey. Overall, (54.6%) of the athletes reported at least one local side effect, while (46.9%) reported at least one systemic side effect. These side effects were more prevalent among the adenoviral vector group compared to the inactivated virus and mRNA groups. The most common local side effect was injection site pain (29.9%), while Fever (30.8%) was the most prevalent systemic side effect. The age group of 31-40 years, allergy, previous infection with COVID-19, and the first dose of vaccines were associated with an increased risk of side effects for all groups of COVID-19 vaccines. Logistic regression analysis further revealed that compared to males, the incidence of reported side effects was significantly higher in females (odd ratio (OR) = 1.16; P = 0.015*) only for the adenoviral vector vaccine group. In addition, a significantly higher percentage of athletes group of high dynamic/moderate static or high dynamic /high static components suffered from post-vaccination side effects compared to the group of athletes with high dynamic/low static components (OR = 14.68 and 14.71; P < 0.001, respectively). CONCLUSIONS: The adenoviral vector vaccines have the highest rate of side effects, followed by the inactivated virus and mRNA COVID-19 vaccines. COVID­19 vaccines were well-tolerated among Algerian athletes and there were no reports of serious side effects. Nevertheless, further long-term follow-up study with a larger sample size of athletes (from different types and sports categories) is warranted to establish the long-term safety of the COVID-19 vaccine.

One HA stalk topping multiple heads as a novel influenza vaccine.

Classic 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.

Immunogenicity and protective efficacy of recombinant adenovirus expressing a novel genotype G2b PEDV spike protein in protecting newborn piglets against PEDV.

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.

Comparative Biodistribution Study of Baculoviral and Adenoviral Vector Vaccines against SARS-CoV-2.

Various types of vaccines have been developed against COVID-19, including vector vaccines. Among the COVID-19 vaccines, AstraZeneca's chimpanzee adenoviral vaccine was the first to be commercialized. For viral vector vaccines, biodistribution studies are critical to vaccine safety, gene delivery, and efficacy. This study compared the biodistribution of the baculoviral vector vaccine (AcHERV-COVID19) and the adenoviral vector vaccine (Ad-COVID19). Both vaccines were administered intramuscularly to mice, and the distribution of the SARS-CoV-2 S gene in each tissue was evaluated for up to 30 days. After vaccination, serum and various tissue samples were collected from the mice at each time point, and IgG levels and DNA copy numbers were measured using an enzyme-linked immunosorbent assay and a quantitative real-time polymerase chain reaction. AcHERV-COVID19 and Ad-COVID19 distribution showed that the SARS-CoV-2 spike gene remained predominantly at the injection site in the mouse muscle. In kidney, liver, and spleen tissues, the AcHERV-COVID19 group showed about 2-4 times higher persistence of the SARS-CoV-2 spike gene than the Ad-COVID19 group. The distribution patterns of AcHERV-COVID19 and Ad-COVID19 within various organs highlight their contrasting biodistribution profiles, with AcHERV-COVID19 exhibiting a broader and prolonged presence in the body compared to Ad-COVID19. Understanding the biodistribution profile of AcHERV-COVID19 and Ad-COVID19 could help select viral vectors for future vaccine development.

Enhancement of mucosal innate and adaptive immunity following intranasal immunization of mice with a bovine adenoviral vector.

Introduction: Nonhuman adenoviral (AdV) gene delivery platforms have significant value due to their ability to elude preexisting AdV vector immunity in most individuals. Previously, we have demonstrated that intranasal (IN) immunization of mice with BAd-H5HA, a bovine AdV type 3 (BAdV3) vector expressing H5N1 influenza virus hemagglutinin (HA), resulted in enhanced humoral and cell-mediated immune responses. The BAd-H5HA IN immunization resulted in complete protection following the challenge with an antigenically distinct H5N1 virus compared to the mouse group similarly immunized with HAd-H5HA, a human AdV type 5 (HAdV5) vector expressing HA. Methods: Here, we attempted to determine the activation of innate immune responses in the lungs of mice inoculated intranasally with BAd-H5HA compared to the HAd-H5HA-inoculated group. Results: RNA-Seq analyses of the lung tissues revealed differential expression (DE) of genes involved in innate and adaptive immunity in animals immunized with BAd-H5HA. The top ten enhanced genes were verified by RT-PCR. Consistently, there were transient increases in the levels of cytokines (IL-1α, IL-1ß, IL-5, TNF- α, LIF, IL-17, G-CSF, MIP-1ß, MCP-1, MIP-2, and GM-CSF) and toll-like receptors in the lungs of the group inoculated with BAdV vectors compared to that of the HAdV vector group. Conclusion: These results demonstrate that the BAdV vectors induce enhanced innate and adaptive immunity-related factors compared to HAdV vectors in mice. Thus, the BAdV vector platform could be an excellent gene delivery system for recombinant vaccines and cancer immunotherapy.

mRNA and Adenoviral Vector Vaccine Platforms Utilized in COVID-19 Vaccines: Technologies, Ecosystem, and Future Directions.

New technological platforms, such as mRNA and adenoviral vector vaccines, have been utilized to develop coronavirus disease 2019 (COVID-19) vaccines. These new modalities enable rapid and flexible vaccine design and cost-effective and swift manufacturing, effectively combating pandemics caused by mutating viruses. Innovation ecosystems, including universities, startups, investors, and governments are crucial for developing these cutting-edge technologies. This review summarizes the research and development trajectory of these vaccine technologies, their investments, and the support surrounding them, in addition to the technological details of each technology. In addition, this study examines the importance of an innovation ecosystem in developing novel technologies, comparing it with the case of Japan, which has lagged behind in COVID-19 vaccine development. It also explores the direction of vaccine development in the post-COVID-19 era.

Comparative analysis of the impact of 40 adenovirus types on dendritic cell activation and CD8+ T cell proliferation capacity for the identification of favorable immunization vector candidates.

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.

Comparison of Three Different Methods of Transfection for the Production of Recombinant Adenovirus Expressing Human Carcinoembryonic Antigen Gene.

Adenoviral vectors (AdVs) are widely used as a gene delivery vehicle and vaccine design due to their genetic stability, transfer capacity of large genes, production at high titers, and remarkable efficacy of transduction. One of the most important applications of AdVs is in cancer immunotherapy. Tumor-associated antigens are overexpressed in cancer cells; however, they cannot induce immune responses sufficiently. Therefore, the immune system must be stimulated against these antigens to kill the cancer cells. This study described the construction steps of a recombinant AdV expressing human carcinoembryonic antigen (CEA) gene. Furthermore, in order to achieve a high titer of the virus, an efficient transfection was required. Three various transfection reagents were compared to achieve the best method of transfection. Carcinoembryonic antigen was cloned into the pAdV and transfected into the A293 cells using three different reagents, including polyethylenimine (PEI), calcium phosphate, and DMRIE-C. The PEI had the highest transfection efficiency, which was selected for the transfection of the recombinant plasmid. It has low toxicity for cells and is suitable for large-scale transfection. The virus produced in this study can be applied as a vaccine in cancer immunotherapy for stimulating the immune system against CEA-expressing tumors.

A novel simian adenovirus-vectored COVID-19 vaccine elicits effective mucosal and systemic immunity in mice by intranasal and intramuscular vaccination regimens.

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.

Preferential expression of SCN1A in GABAergic neurons improves survival and epileptic phenotype in a mouse model of Dravet syndrome.

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.