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Adenoviral vectors (AdVs) are effective vectors for gene therapy due to their broad tropism, large capacity, and high transduction efficiency, making them widely used as oncolytic vectors and for creating vector-based vaccines. This review also considers the application of adenoviral vectors in oncolytic virotherapy and gene therapy for inherited diseases, analyzing strategies to enhance their efficacy and specificity. However, despite significant progress in this field, the use of adenoviral vectors is limited by their high immunogenicity, low specificity to certain cell types, and limited duration of transgene expression. Various strategies and technologies aimed at improving the characteristics of adenoviral vectors are being developed to overcome these limitations. Significant attention is being paid to the creation of tissue-specific promoters, which allow for the controlled expression of transgenes, as well as capsid modifications that enhance tropism to target cells, which also play a key role in reducing immunogenicity and increasing the efficiency of gene delivery. This review focuses on modern approaches to adenoviral vector modifications made to enhance their effectiveness in gene therapy, analyzing the current achievements, challenges, and prospects for applying these technologies in clinical practice, as well as identifying future research directions necessary for successful clinical implementation. [ABSTRACT FROM AUTHOR]

New prophylactic vaccine platforms are imperative to combat respiratory infections. The efficacy of T and B memory cell-mediated protection, generated through the adenoviral vector, was tested to assess the effectiveness of the new adenoviral-based platforms for infectious diseases. A combination of adenovirus AdV1 (adjuvant), armed with costimulatory ligands (ICOSL and CD40L), and rRBD (antigen: recombinant nonglycosylated spike protein rRBD) was used to promote the differentiation of T and B lymphocytes. Adenovirus AdV2 (adjuvant), without ligands, in combination with rRBD, served as a control. In vitro T-cell responses to the AdV1+rRBD combination revealed that CD8+ platform-specific T-cells increased (37.2±0.7% vs. 23.1±2.1%), and T-cells acted against SARS-CoV-2 via CD8+TEMRA (50.0±1.3% vs. 36.0±3.2%). Memory B cells were induced after treatment with either AdV1+rRBD (84.1±0.8% vs. 82.3±0.4%) or rRBD (94.6±0.3% vs. 82.3±0.4%). Class-switching from IgM and IgD to isotype IgG following induction with rRBD+Ab was observed. RNA-seq profiling identified gene expression patterns related to T helper cell differentiation that protect against pathogens. The analysis determined signaling pathways controlling the induction of protective immunity, including the MAPK cascade, adipocytokine, cAMP, TNF, and Toll-like receptor signaling pathway. The AdV1+rRBD formulation induced IL-6, IL-8, and TNF. RNA-seq of the VERO E6 cell line showed differences in the apoptosis gene expression stimulated with the platforms vs. mock. In conclusion, AdV1+rRBD effectively generates T and B memory cell-mediated protection, presenting promising results in producing CD8+ platform-specific T cells and isotype-switched IgG memory B cells. The platform induces protective immunity by controlling the Th1, Th2, and Th17 cell differentiation gene expression patterns. Further studies are required to confirm its effectiveness.

Simple Summary: The translational gap poses a challenge for therapeutic cancer vaccine development, including HPV-specific therapeutic vaccines. The aim of this study was to evaluate a new human ex vivo PBMC assay that mimics how viral-vectored vaccines induce T-cell responses in vivo. In brief, PBMCs are taken through rounds of stimulation by co-culturing with syngeneic DCs, which have been previously matured and transduced with the viral-vector-based vaccine. We evaluated the assay using a novel HPV16-specific therapeutic vaccine in healthy human donors with pre-existing T-cell responses against HPV16 to simulate a therapeutic setting. The pre-existing T-cell responses were effectively boosted to a larger extent by vaccine-transduced DCs than by peptide-pulsing, and the T cells could specifically recognize and kill HPV16+ human cancer cells. Additionally, this study supports the use of nucleic-acid-based vaccines for optimal cancer recognition and indicates an immunological advantage of targeting the HPV E1 gene. Persistent human papillomavirus (HPV) infection is responsible for practically all cervical and a high proportion of anogenital and oropharyngeal cancers. Therapeutic HPV vaccines in clinical development show great promise in improving outcomes for patients who mount an anti-HPV T-cell response; however, far from all patients elicit a sufficient immunological response. This demonstrates a translational gap between animal models and human patients. Here, we investigated the potential of a new assay consisting of co-culturing vaccine-transduced dendritic cells (DCs) with syngeneic, healthy, human peripheral blood mononuclear cells (PBMCs) to mimic a human in vivo immunization. This new promising human ex vivo PBMC assay was evaluated using an innovative therapeutic adenovirus (Adv)-based HPV vaccine encoding the E1, E2, E6, and E7 HPV16 genes. This new method allowed us to show that vaccine-transduced DCs yielded functional effector T cells and unveiled information on immunohierarchy, showing E1-specific T-cell immunodominance over time. We suggest that this assay can be a valuable translational tool to complement the known animal models, not only for HPV therapeutic vaccines, and supports the use of E1 as an immunotherapeutic target. Nevertheless, the findings reported here need to be validated in a larger number of donors and preferably in patient samples. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis is the main causal agent of pulmonary tuberculosis (TB); the treatment of this disease is long and involves a mix of at least four different antibiotics that frequently lead to abandonment, favoring the surge of drug-resistant mycobacteria (MDR-TB), whose treatment becomes more aggressive, being longer and more toxic. Thus, the search for novel strategies for treatment that improves time or efficiency is of relevance. In this work, we used a murine model of pulmonary TB produced by the MDR-TB strain to test the efficiency of gene therapy with adenoviral vectors codifying TNF (AdTNF), a pro-inflammatory cytokine that has protective functions in TB by inducing apoptosis, granuloma formation and expression of other Th1-like cytokines. When compared to the control group that received an adenoviral vector that codifies for the green fluorescent protein (AdGFP), a single dose of AdTNF at the chronic active stage of the disease produced total survival, decreasing bacterial load and tissue damage (pneumonia), which correlated with an increase in cells expressing IFN-γ, iNOS and TNF in pneumonic areas and larger granulomas that efficiently contain and eliminate mycobacteria. Second-line antibiotic treatment against MDR-TB plus AdTNF gene therapy reduced bacterial load faster within a week of treatment compared to empty vector plus antibiotics or antibiotics alone, suggesting that AdTNF is a new potential type of treatment against MDR-TB that can shorten second-line chemotherapy but which requires further experimentation in other animal models (non-human primates) that develop a more similar disease to human pulmonary TB. [ABSTRACT FROM AUTHOR]

New prophylactic vaccine platforms are imperative to combat respiratory infections. The efficacy of T and B memory cell-mediated protection, generated through the adenoviral vector, was tested to assess the effectiveness of the new adenoviral-based platforms for infectious diseases. A combination of adenovirus AdV1 (adjuvant), armed with costimulatory ligands (ICOSL and CD40L), and rRBD (antigen: recombinant nonglycosylated spike protein rRBD) was used to promote the differentiation of T and B lymphocytes. Adenovirus AdV2 (adjuvant), without ligands, in combination with rRBD, served as a control. In vitro T-cell responses to the AdV1+rRBD combination revealed that CD8+ platform-specific T-cells increased (37.2 ± 0.7% vs. 23.1 ± 2.1%), and T-cells acted against SARS-CoV-2 via CD8+TEMRA (50.0 ± 1.3% vs. 36.0 ± 3.2%). Memory B cells were induced after treatment with either AdV1+rRBD (84.1 ± 0.8% vs. 82.3 ± 0.4%) or rRBD (94.6 ± 0.3% vs. 82.3 ± 0.4%). Class-switching from IgM and IgD to isotype IgG following induction with rRBD+Ab was observed. RNA-seq profiling identified gene expression patterns related to T helper cell differentiation that protect against pathogens. The analysis determined signaling pathways controlling the induction of protective immunity, including the MAPK cascade, adipocytokine, cAMP, TNF, and Toll-like receptor signaling pathway. The AdV1+rRBD formulation induced IL-6, IL-8, and TNF. RNA-seq of the VERO E6 cell line showed differences in the apoptosis gene expression stimulated with the platforms vs. mock. In conclusion, AdV1+rRBD effectively generates T and B memory cell-mediated protection, presenting promising results in producing CD8+ platform-specific T cells and isotype-switched IgG memory B cells. The platform induces protective immunity by controlling the Th1, Th2, and Th17 cell differentiation gene expression patterns. Further studies are required to confirm its effectiveness. [ABSTRACT FROM AUTHOR]

New prophylactic vaccine platforms are imperative to combat respiratory infections. The efficacy of T and B memory cell-mediated protection, generated through the adenoviral vector, was tested to assess the effectiveness of the new adenoviral-based platforms for infectious diseases. A combination of adenovirus AdV1 (adjuvant), armed with costimulatory ligands (ICOSL and CD40L), and rRBD (antigen: recombinant nonglycosylated spike protein rRBD) was used to promote the differentiation of T and B lymphocytes. Adenovirus AdV2 (adjuvant), without ligands, in combination with rRBD, served as a control. In vitro T-cell responses to the AdV1+rRBD combination revealed that CD8+ platform-specific T-cells increased (37.2 ± 0.7% vs. 23.1 ± 2.1%), and T-cells acted against SARS-CoV-2 via CD8+TEMRA (50.0 ± 1.3% vs. 36.0 ± 3.2%). Memory B cells were induced after treatment with either AdV1+rRBD (84.1 ± 0.8% vs. 82.3 ± 0.4%) or rRBD (94.6 ± 0.3% vs. 82.3 ± 0.4%). Class-switching from IgM and IgD to isotype IgG following induction with rRBD+Ab was observed. RNA-seq profiling identified gene expression patterns related to T helper cell differentiation that protect against pathogens. The analysis determined signaling pathways controlling the induction of protective immunity, including the MAPK cascade, adipocytokine, cAMP, TNF, and Toll-like receptor signaling pathway. The AdV1+rRBD formulation induced IL-6, IL-8, and TNF. RNA-seq of the VERO E6 cell line showed differences in the apoptosis gene expression stimulated with the platforms vs. mock. In conclusion, AdV1+rRBD effectively generates T and B memory cell-mediated protection, presenting promising results in producing CD8+ platform-specific T cells and isotype-switched IgG memory B cells. The platform induces protective immunity by controlling the Th1, Th2, and Th17 cell differentiation gene expression patterns. Further studies are required to confirm its effectiveness. [ABSTRACT FROM AUTHOR]

Endogenous retroviruses (ERVs) account for 8% of our genome, and, although they are usually silent in healthy tissues, they become reactivated and expressed in pathological conditions such as cancer. Several studies support a functional role of ERVs in tumour development and progression, specifically through their envelope (Env) protein, which contains a region described as an immunosuppressive domain (ISD). We have previously shown that targeting of the murine ERV (MelARV) Env using virus-like vaccine (VLV) technology, consisting of an adenoviral vector encoding virus-like particles (VLPs), induces protection against small tumours in mice. Here, we investigate the potency and efficacy of a novel MelARV VLV with a mutated ISD (ISDmut) that can modify the properties of the adenoviral vaccine-encoded Env protein. We show that the modification of the vaccine's ISD significantly enhanced T-cell immunogenicity in both prime and prime-boost vaccination regimens. The modified VLV in combination with an α-PD1 checkpoint inhibitor (CPI) exhibited excellent curative efficacy against large established colorectal CT26 tumours in mice. Furthermore, only ISDmut-vaccinated mice that survived CT26 challenge were additionally protected against rechallenge with a triple-negative breast cancer cell line (4T1), showing that our modified VLV provides cross-protection against different tumour types expressing ERV-derived antigens. We envision that translating these findings and technology into human ERVs (HERVs) could provide new treatment opportunities for cancer patients with unmet medical needs. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis is the main causal agent of pulmonary tuberculosis (TB); the treatment of this disease is long and involves a mix of at least four different antibiotics that frequently lead to abandonment, favoring the surge of drug-resistant mycobacteria (MDR-TB), whose treatment becomes more aggressive, being longer and more toxic. Thus, the search for novel strategies for treatment that improves time or efficiency is of relevance. In this work, we used a murine model of pulmonary TB produced by the MDR-TB strain to test the efficiency of gene therapy with adenoviral vectors codifying TNF (AdTNF), a pro-inflammatory cytokine that has protective functions in TB by inducing apoptosis, granuloma formation and expression of other Th1-like cytokines. When compared to the control group that received an adenoviral vector that codifies for the green fluorescent protein (AdGFP), a single dose of AdTNF at the chronic active stage of the disease produced total survival, decreasing bacterial load and tissue damage (pneumonia), which correlated with an increase in cells expressing IFN-γ, iNOS and TNF in pneumonic areas and larger granulomas that efficiently contain and eliminate mycobacteria. Second-line antibiotic treatment against MDR-TB plus AdTNF gene therapy reduced bacterial load faster within a week of treatment compared to empty vector plus antibiotics or antibiotics alone, suggesting that AdTNF is a new potential type of treatment against MDR-TB that can shorten second-line chemotherapy but which requires further experimentation in other animal models (non-human primates) that develop a more similar disease to human pulmonary TB.

The tremendous evolution of genome-editing tools in the last two decades has provided innovative and effective approaches for gene therapy of congenital and acquired diseases. Zinc-finger nucleases (ZFNs), transcription activator- like effector nucleases (TALENs) and CRISPR-Cas9 have been already applied by ex vivo hematopoietic stem cell (HSC) gene therapy in genetic diseases (i.e., Hemoglobinopathies, Fanconi anemia and hereditary Immunodeficiencies) as well as infectious diseases (i.e., HIV), and the recent development of CRISPR-Cas9-based systems using base and prime editors as well as epigenome editors has provided safer tools for gene therapy. The ex vivo approach for gene addition or editing of HSCs, however, is complex, invasive, technically challenging, costly and not free of toxicity. In vivo gene addition or editing promise to transform gene therapy from a highly sophisticated strategy to a "user-friendly' approach to eventually become a broadly available, highly accessible and potentially affordable treatment modality. In the present review article, based on the lessons gained by more than 3 decades of ex vivo HSC gene therapy, we discuss the concept, the tools, the progress made and the challenges to clinical translation of in vivo HSC gene editing. [ABSTRACT FROM AUTHOR]

Adenovirus (Ad) is a non-enveloped linear double-stranded DNA virus with >50 serotypes in humans. Ad vectors have been used as gene delivery vehicles to express transgenes, small interfering RNAs (siRNAs) for gene silencing, or CRISPR/Cas and designer nucleases for genome editing. Although several methods are used to generate Ad vectors, the Ad-making process remains technically challenging and time consuming. Moreover, the Ad-making techniques have not been improved for the past two decades. Gibson DNA Assembly (GDA) technology allows one-step isothermal DNA assembly of multiple overlapping fragments. Here, we developed a one-step construction of Ad (OSCA) system using GDA technology. Specifically, we first engineered several adenoviral recipient vectors that contain the ccdB suicide gene flanked with two 20-bp unique sequences, which serve as universal sites for GDA reactions in the Ad genome ΔE1 region. In two proof-of-principle experiments, we demonstrated that the GDA reactions were highly efficient and that the resulting Ad plasmids could be effectively packaged into Ads. Ad-mediated expression of mouse BMP9 in mesenchymal stem cells was shown to effectively induce osteogenic differentiation both in vitro and in vivo. Collectively, our results demonstrate that the OSCA system drastically streamlines the Ad-making process and should facilitate Ad-based applications in basic, translational, and clinical research.

Adenovirus based vectors are useful tools for vaccine development, gene therapy, and oncolytic virotherapy. Here we describe a novel approach for the genetic engineering of any portion of the adenovirus genome and the reconstruction of either fully replication competent or defective virions. This innovative strategy is rapid, effective and suitable for the manipulation of the entire genome broadening the spectrum of potential applications for the adenovirus system. Our strategy involved insertion of restriction enzyme recognition sequences absent in the native virus into the termini of the adenovirus genome in order to facilitate recovery. These restriction enzyme sites, together with the two inverted terminal repeats and packaging sequences, were synthesized and then subcloned into the pBR322 vector. The remaining internal portion of the adenovirus genome was separated and amplified via PCR into six segments, of which groups of two were joined together by PCR and then subcloned into pBR322 plasmids. During the PCR reaction, an overlap of 30–40 bp was added to the termini of the adjacent fragments, allowing for the subsequent isothermal assembly and correct orientation of all fragments. This approach allows for the genetic modification of each genomic fragment according to the specific research goals, (e.g., deletion, substitution, addition, etc.) To recreate the entire viral genome, the four engineered fragments (each comprised of two adenovirus genomic sections) as well as the pBR322 backbone, were reassembled into a single construct utilizing an isothermal assembly reaction. Finally, the reassembled plasmid containing the entire genome was linearized and transfected into HEK293 cells to recover the complete reconstructed adenoviral vector. Using this approach, we have successfully generated two recombinant reporter adenoviruses, one of human adenovirus serotype 14 and another of serotype 55. The E3 region was replaced by the reporter genes (GFP and Luciferase) to visualize and track the recovery process. Subsequent infection of A549 cells with these reconstructed adenovirus vectors demonstrated that they were replication competent. This method shortens the viral reconstruction process because the one-step isothermal assembly requires less than 4 days, and recombinant adenovirus recovery occurs within 10 days. This new method allows for single or multiple genetic modifications within any portion of the viral genome and can be used to construct or manipulate any adenovirus whose complete genome sequence is known.

Tuberculosis (TB) has been for many years a major public health problem since treatment is long and sometimes ineffective favoring the increase of multidrug-resistant mycobacteria (MDR-TB). Gene therapy is a novel and effective tool to regulate immune responses. In this study we evaluated the therapeutic effect of an adenoviral vector codifying osteopontin (AdOPN), a molecule known for their roles to favor Th1 and Th17 type-cytokine expression which are crucial in TB containment. A single dose of AdOPN administration in BALB/c mice suffering late progressive pulmonary MDR-TB produced significant lower bacterial load and pneumonia, due to higher expression of IFN-γ, IL-12, and IL-17 in coexistence with increase of granulomas in number and size, resulting in higher survival, in contrast with mice treated with the control adenovirus that codify the green fluorescent protein (AdGFP). Combined therapy of AdOPN with a regimen of second line antibiotics produced a better control of bacterial load in lung during the first days of treatment, suggesting that AdOPN can shorten chemotherapy. Taken together, gene therapy with AdOPN leads to higher immune responses against TB infection, resulting in a new potential treatment against pulmonary TB that can co-adjuvant chemotherapy. [ABSTRACT FROM AUTHOR]

The tumour necrosis factor superfamily OX40L and CD70 and their receptors are costimulatory signalling axes critical for adequate T and B cell activation in humans and mice. In this work we inoculated groups of sheep with human recombinant adenovirus type 5 (Ad) expressing Ovis aries (Oa)OX40L or OaCD70 or a control adenoviral vector to determine whether they could improve the immune response to the model antigen OVA. PBMCs and serum samples were obtained for analysis of the adaptive immune response to OVA at days 0, 15, 30 and 90 post-inoculation (pi). Recall responses to OVA were assessed at day 7 and 30 after the second antigen inoculation (pb) at day 90. Administration of these immunomodulatory molecules did not induce unspecific PBMC stimulation. While OaOX40L administration mainly increased TNF-a and IL-4 in PBMC at day 15 pi concomitantly with a slight increase in antibody titer and the number of IFN-g producing cells, we detected greater effects on adaptive immunity after OaCD70 administration. AdOaCD70 inoculation improved antibody titers to OVA at days 30 and 90 pi, and increased anti-OVA-specific IgG-secreting B cell counts when compared to control. Moreover, higher IFN-g production was detected on days 7 pi, 7 pb and 30 pb in PBMCs from this group. Phenotypic analysis of T cell activation showed an increase in effector CD8+ T cells (CD8+ CD62L- CD27-) at day 15 pi in AdOaCD70 group, concurrent with a decrease in early activated cells (CD8+ CD62L-CD27+). Moreover, recall anti-OVA CD8+ T cell responses were increased at 7 pb in the AdOaCD70 group. AdOaCD70 administration could therefore promote CD8+ T cell effector differentiation and long-term activity. In this work we characterized the in vivo adjuvant potential on the humoral and cellular immune response of OaOX40L and OaCD70 delivered by non-replicative adenovirus vectors using the model antigen OVA. We present data highlighting the potency of these molecules as veterinary vaccine adjuvant. [ABSTRACT FROM AUTHOR]

Immunological memory is the basis of protective immunity provided by vaccines and previous infections. Immunological memory can develop from multiple branches of the adaptive immune system, including CD4 T cells, CD8 T cells, B cells, and long‐lasting antibody responses. Extraordinary progress has been made in understanding memory to SARS‐CoV‐2 infection and COVID‐19 vaccines, addressing development; quantitative and qualitative features of different cellular and anatomical compartments; and durability of each cellular component and antibodies. Given the sophistication of the measurements; the size of the human studies; the use of longitudinal samples and cross‐sectional studies; and head‐to‐head comparisons between infection and vaccines or between multiple vaccines, the understanding of immune memory for 1 year to SARS‐CoV‐2 infection and vaccines already supersedes that of any other acute infectious disease. This knowledge may help inform public policies regarding COVID‐19 and COVID‐19 vaccines, as well as the scientific development of future vaccines against SARS‐CoV‐2 and other diseases. [ABSTRACT FROM AUTHOR]

Only two decades after discovering miRNAs, our understanding of the functional effects of deregulated miRNAs in the development of diseases, particularly cancer, has been rapidly evolving. These observations and functional studies provide the basis for developing miRNA-based diagnostic markers or new therapeutic strategies. Adenoviral (Ad) vectors belong to the most frequently used vector types in gene therapy and are suitable for strong short-term transgene expression in a variety of cells. Here, we report the set-up and functionality of an Ad-based miRNA vector platform that can be employed to deliver and express a high level of miRNAs efficiently. This vector platform allows fast and efficient vector production to high titers and the expression of pri-miRNA precursors under the control of a polymerase II promoter. In contrast to non-viral miRNA delivery systems, this Ad-based miRNA vector platform allows accurate dosing of the delivered miRNAs. Using a two-vector model, we showed that Ad-driven miRNA expression was sufficient in down-regulating the expression of an overexpressed and highly stable protein. Additional data corroborated the downregulation of multiple endogenous target RNAs using the system presented here. Additionally, we report some unanticipated synergistic effects on the transduction efficiencies in vitro when cells were consecutively transduced with two different Ad-vectors. This effect might be taken into consideration for protocols using two or more different Ad vectors simultaneously. [ABSTRACT FROM AUTHOR]

Background: Respiratory syncytial virus (RSV) is a significant cause of severe lower respiratory tract disease in children and older adults, but has no approved vaccine. This study assessed the potential of Ad26.RSV.preF to protect against RSV infection and disease in an RSV human challenge model.Methods: In this double-blind, placebo-controlled study, healthy adults aged 18-50 years were randomized 1:1 to receive 1 × 1011 vp Ad26.RSV.preF or placebo intramuscularly. Twenty-eight days postimmunization, volunteers were challenged intranasally with RSV-A (Memphis 37b). Assessments included viral load (VL), RSV infections, clinical symptom score (CSS), safety, and immunogenicity.Results: Postchallenge, VL, RSV infections, and disease severity were lower in Ad26.RSV.preF (n = 27) vs placebo (n = 26) recipients: median VL area under the curve (AUC) quantitative real-time polymerase chain reaction: 0.0 vs 236.0 (P = .012; predefined primary endpoint); median VL-AUC quantitative culture: 0.0 vs 109; RSV infections 11 (40.7%) vs 17 (65.4%); median RSV AUC-CSS 35 vs 167, respectively. From baseline to 28 days postimmunization, geometric mean fold increases in RSV A2 neutralizing antibody titers of 5.8 and 0.9 were observed in Ad26.RSV.preF and placebo, respectively. Ad26.RSV.preF was well tolerated.Conclusions: Ad26.RSV.preF demonstrated protection from RSV infection through immunization in a human challenge model, and therefore could potentially protect against natural RSV infection and disease.Clinical Trials Registration: NCT03334695; CR108398, 2017-003194-33 (EudraCT); VAC18193RSV2002. [ABSTRACT FROM AUTHOR]

The tumour necrosis factor superfamily OX40L and CD70 and their receptors are costimulatory signalling axes critical for adequate T and B cell activation in humans and mice. In this work we inoculated groups of sheep with human recombinant adenovirus type 5 (Ad) expressing Ovis aries (Oa)OX40L or OaCD70 or a control adenoviral vector to determine whether they could improve the immune response to the model antigen OVA. PBMCs and serum samples were obtained for analysis of the adaptive immune response to OVA at days 0, 15, 30 and 90 post-inoculation (pi). Recall responses to OVA were assessed at day 7 and 30 after the second antigen inoculation (pb) at day 90. Administration of these immunomodulatory molecules did not induce unspecific PBMC stimulation. While OaOX40L administration mainly increased TNF-α and IL-4 in PBMC at day 15 pi concomitantly with a slight increase in antibody titer and the number of IFN-γ producing cells, we detected greater effects on adaptive immunity after OaCD70 administration. AdOaCD70 inoculation improved antibody titers to OVA at days 30 and 90 pi, and increased anti-OVA-specific IgG-secreting B cell counts when compared to control. Moreover, higher IFN-γ production was detected on days 7 pi, 7 pb and 30 pb in PBMCs from this group. Phenotypic analysis of T cell activation showed an increase in effector CD8+ T cells (CD8+ CD62L- CD27-) at day 15 pi in AdOaCD70 group, concurrent with a decrease in early activated cells (CD8+ CD62L- CD27+). Moreover, recall anti-OVA CD8+ T cell responses were increased at 7 pb in the AdOaCD70 group. AdOaCD70 administration could therefore promote CD8+ T cell effector differentiation and long-term activity. In this work we characterized the in vivo adjuvant potential on the humoral and cellular immune response of OaOX40L and OaCD70 delivered by non-replicative adenovirus vectors using the model antigen OVA. We present data highlighting the potency of these molecules as veterinary vaccine adjuvant.

Vaccination against the SARS-CoV-2, the virus responsible for the Covid-19 pandemic, represents a major infection control strategy in the absence of effective treatment of the disease to date. Unprecedented mobilization has led to the development of a large number of projects, some of which have already been in test in humans for several months. The first efficacy and safety data are expected in the coming weeks. New vaccine technologies are being evaluated (RNA, replicating or non-replicating viral vectors), further increasing the chances of success. The criteria for evaluating vaccines—despite the exceptional speed of their development—must remain rigorous enough to ensure their acceptance by the population. Beyond their development, mass production and equitable distribution raise many questions. Finally, vaccination can only be successfully implemented if health professionals and the population are convinced of its validity, which implies particular attention to the quality of the information given and the methods of communication.

Chronic liver injury leads to advanced fibrosis, cirrhosis, and hepatocellular carcinoma. Genetical cell treatment related to the use of adenovirus (Ads) has proven to be beneficial and efficient in the recovery of hepatic diseases. Nevertheless, they are highly immunogenic and trigger an immune response where interferons type 1 (IFN-I) play a very important role. Three shRNAs against the Interferon-1 receptor (IFNAR1) were designed and cloned in pENTR/U6 plasmid and amplified in DH5α cells. Huh7 cells were transfected with these plasmids in the presence or absence of 1 × 109 viral particles/ml of adenovirus containing the green fluorescent protein gene used as a reporter. Transfection with the shRNA plasmids partially inhibited the IFNAR1 expression. This inhibition substantially decreased antiviral response, demonstrated by the decrease of IFNAR1, IFN-α, and TNF-α gene expression, and the decrease at protein levels of IFNAR1, Protein kinase RNA-activated (PKR), and phosphorylated STAT1, allowing higher adenoviral transduction and transgene expression. Interestingly it was seen shRNA inhibited macrophage activation. These results suggest that the inhibition of the IFN-I pathway could be a strategy to minimize the immune response against Adenoviral vectors allowing higher Adenovirus transduction extending the transgene expression. [ABSTRACT FROM AUTHOR]

VITT is a rare, life-threatening syndrome characterized by thrombotic symptoms in combination with thrombocytopenia, which may occur in individuals receiving the first administration of adenoviral non replicating vectors (AVV) anti Covid19 vaccines. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is characterized by high levels of serum IgG that bind PF4/polyanion complexes, thus triggering platelet activation. Therefore, identification of the fine pathophysiological mechanism by which vaccine components trigger platelet activation is mandatory. Herein, we propose a multistep mechanism involving both the AVV and the neo-synthetized Spike protein. The former can: i) spread rapidly into blood stream, ii), promote the early production of high levels of IL-6, iii) interact with erythrocytes, platelets, mast cells and endothelia, iv) favor the presence of extracellular DNA at the site of injection, v) activate platelets and mast cells to release PF4 and heparin. Moreover, AVV infection of mast cells may trigger aberrant inflammatory and immune responses in people affected by the mast cell activation syndrome (MCAS). The pre-existence of natural antibodies binding PF4/heparin complexes may amplify platelet activation and thrombotic events. Finally, neosynthesized Covid 19 Spike protein interacting with its ACE2 receptor on endothelia, platelets and leucocyte may trigger further thrombotic events unleashing the WITT syndrome.

VITT is a rare, life-threatening syndrome characterized by thrombotic symptoms in combination with thrombocytopenia, which may occur in individuals receiving the first administration of adenoviral non replicating vectors (AVV) anti Covid19 vaccines. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is characterized by high levels of serum IgG that bind PF4/polyanion complexes, thus triggering platelet activation. Therefore, identification of the fine pathophysiological mechanism by which vaccine components trigger platelet activation is mandatory. Herein, we propose a multistep mechanism involving both the AVV and the neo-synthetized Spike protein. The former can: i) spread rapidly into blood stream, ii), promote the early production of high levels of IL-6, iii) interact with erythrocytes, platelets, mast cells and endothelia, iv) favor the presence of extracellular DNA at the site of injection, v) activate platelets and mast cells to release PF4 and heparin. Moreover, AVV infection of mast cells may trigger aberrant inflammatory and immune responses in people affected by the mast cell activation syndrome (MCAS). The pre-existence of natural antibodies binding PF4/heparin complexes may amplify platelet activation and thrombotic events. Finally, neosynthesized Covid 19 Spike protein interacting with its ACE2 receptor on endothelia, platelets and leucocyte may trigger further thrombotic events unleashing the WITT syndrome. [ABSTRACT FROM AUTHOR]

It is promising to design a vaccine without the vulnerabilities of first-generation vaccines and to develop a cheaper and more attractive vaccine for protection-control in the fight against COVID-19. Keywords: adenoviral vectors; mucosal immunity; next-generation vaccine; SARS-CoV-2; trained innate immunity EN adenoviral vectors mucosal immunity next-generation vaccine SARS-CoV-2 trained innate immunity 2565 2567 3 08/09/22 20220801 NES 220801 Humanity has been facing a major challenge recently from a microbe called SARS-CoV-2. For example, the respiratory mucosal immunity induced by the inhaled aerosol modified vaccinia Ankara Tuberculosis vaccine in humans was not achieved by intradermal injection of the same vaccine.6 Providing long-term protection against current and future variants and sustained vaccine-induced herd immunity is a great challenge. [Extracted from the article]

Abstract Background There is an urgent need for targeted biological therapies for prostate cancer with greater efficacy and less toxicity, particularly for metastatic disease, where current therapies are not curative. Therapeutic adenoviral vectors or oncolytic adenoviruses offer the possibility of a competent, nontoxic therapeutic alternative for prostate cancer. However, free viral particles must be delivered locally, an approach that does not address metastatic disease, and they display poor tumor penetration. To fully exploit the potential of these vectors, we must develop methods that improve intratumoral dissemination and allow for systemic delivery. This study establishes a proof-of-principle rationale for a novel human mesenchymal stem (stromal) cell-based approach to improving vector delivery to tumors. Methods/results We have generated mesenchymal stem cell-derived packaging cells for adenoviruses (E1-modified mesenchymal stem cells) by modifying human mesenchymal stem cells with the adenovirus (type C) E1A/B genes needed for viral replication. Using cell-based assays, we have demonstrated that two adenoviral vectors, replication-defective adenovirus expressing p14 and p53 or conditionally replicating oncolytic adenovirus, packaged by E1A/B-modified mesenchymal stem cells, suppress the growth of prostate cancer cells in culture. Using subcutaneous xenograft models for human prostate cancer in mice, we have shown that E1A/B-modified mesenchymal stem cells display tumor tropism in tumor-bearing nude mice, that E1A/B-modified mesenchymal stem cells disseminate well within tumors, and that replication-defective adenovirus expressing p14 and p53 or conditionally replicating oncolytic adenovirus-loaded E1-modified mesenchymal stem cells suppresses tumor growth in mice. Conclusion The results show that this approach, if optimized, could circumvent the obstacles to efficient gene delivery encountered with current gene delivery approaches and provide an effective, nontoxic therapeutic alternative for metastatic disease.

Abstract Background In non-human primates (NHPs) and humans, partial protection from HIV/SIV infection or suppression of replication is achievable by Env-binding antibodies and Gag-specific CD8+ T-cells targeting protective epitopes. Unfortunately, such T-cell responses are frequently dominated by responses to non-protective, variable epitopes. In this study we attempt to combine three independent approaches, each developed to prevent immunodominance of non-protective epitopes. These approaches were (1) vaccines consisting exclusively of putatively protective p24 Gag highly conserved elements (CEs), (2) vaccines using solely subdominant antigens which were acutely protective in a recent NHP trial, and (3) virus-encoded virus-like particle vaccines (virus-like vaccines/VLVs) using heterologous Env and Gag sequences to enable selection of broadly cross-reactive responses and to avoid immunodominance of non-conserved sequences in prime-boost regimens as previously observed. Methods We vaccinated outbred CD1 mice with HIV-1 clade B Gag/Env encoded in an adenoviral prime and SIVmac239 Gag/Env in an MVA boost. We combined this completely heterologous immunization regimen and the homologous SIVmac239 Gag/Env immunization regimen with an additional prime encoding SIV CEs and accessory antigens Rev, Vif and Vpr (Ad-Ii-SIVCErvv). T-cell responses were analyzed by intracellular cytokine staining of splenocytes and antibody responses by trimer-specific ELISA, avidity and isotype-specific ELISA. Results Env dominance could be avoided successfully in the completely heterologous prime-boost regimen, but Env immunodominance reappeared when Ad-Ii-SIVCErvv was added to the prime. This regimen did however still induce more cross-reactive Gag-specific CD8+ T-cells and Env-specific antibodies. Including Ad-Ii-SIVCErvv in the homologous prime-boost not only elicited accessory antigen-specific CD8+ memory T-cells, but also significantly increased the ratio of Gag- to Env-specific CD8+ T-cells. The CD4+ T-cell response shifted away from structural antigens previously associated with infection-enhancement. Conclusion The homologous Gag/Env prime-boost with Ad-Ii-SIVCErvv prime combined acutely protective CD8+ T-cell responses to subdominant antigens and Env-binding antibodies with chronically protective Gag-specific CD8+ T-cells in outbred mice. This vaccine regimen should be tested in an NHP efficacy trial.

Malignant melanoma is a serious disease in both humans and dogs, and the high metastatic potential results in poor prognosis for many patients. Its similarities with human melanoma make spontaneous canine melanoma an excellent model for comparative studies of novel therapies and tumor biology. Gene therapy using adenoviruses encoding the immunostimulatory gene CD40L (AdCD40L) has shown promise in initial clinical trials enrolling human patients with various malignancies including melanoma. We report a study of local AdCD40L treatment in 32 cases of canine melanoma (23 oral, 5 cutaneous, 3 ungual and 1 conjunctival). Eight patients were World Health Organization (WHO) stage I, 9 were stage II, 12 stage III, and 3 stage IV. One to six intratumoral injections of AdCD40L were given every seven days, combined with cytoreductive surgery in 20 cases and only immunotherapy in 12 cases. Tumor tissue was infiltrated with T and B lymphocytes after treatment, suggesting immune stimulation. The best overall response based on result of immunotherapy included 7 complete responses, 5 partial responses, 5 stable and 2 progressive disease statuses according to the World Health Organization response criteria. Median survival was 285 days (range 20–3435 d). Our results suggest that local AdCD40L therapy is safe and could have beneficial effects in dogs, supporting further treatment development. Clinical translation to human patients is ongoing.

Background: Respiratory syncytial virus (RSV) and influenza cause significant disease burden in older adults. Overlapping RSV and influenza seasonality presents the opportunity to coadminister vaccines for both infections. This study assessed coadministration of the investigational vaccine, Ad26.RSV.preF, an adenovirus serotype 26 (Ad26) vector encoding RSV F protein stabilized in its prefusion conformation (pre-F), with a seasonal influenza vaccine in older adults.Methods: In this phase 2a, double-blind, placebo-controlled study, 180 adults aged ≥60 years received Ad26.RSV.preF plus Fluarix on day 1 and placebo on day 29, or placebo plus Fluarix on day 1 and Ad26.RSV.preF on day 29 (control).Results: The coadministration regimen had an acceptable tolerability profile. Reactogenicity was generally higher after Ad26.RSV.preF versus Fluarix, but symptoms were generally transient and mild or moderate. At 28 days after the first vaccination, the upper confidence intervals of the hemagglutination inhibition antibody geometric mean ratio (control/coadministration) for all influenza strains were <2, demonstrating noninferiority. Robust neutralizing and binding antibody responses to RSV A2 were observed in both groups.Conclusions: Coadministration of Fluarix with Ad26.RSV.preF vaccine had an acceptable safety profile and showed no evidence of interference in immune response. The results are compatible with simultaneous seasonal vaccination with both vaccines.Clinical Trials Registration: NCT03339713. [ABSTRACT FROM AUTHOR]

Inherited retinal diseases encompass a highly heterogenous group of disorders caused by a wide range of genetic variants and with diverse clinical symptoms that converge in the common trait of retinal degeneration. Indeed, mutations in over 270 genes have been associated with some form of retinal degenerative phenotype. Given the immune privileged status of the eye, cell replacement and gene augmentation therapies have been envisioned. While some of these approaches, such as delivery of genes through recombinant adeno-associated viral vectors, have been successfully tested in clinical trials, not all patients will benefit from current advancements due to their underlying genotype or phenotypic traits. Gene editing arises as an alternative therapeutic strategy seeking to correct mutations at the endogenous locus and rescue normal gene expression. Hence, gene editing technologies can in principle be tailored for treating retinal degeneration. Here we provide an overview of the different gene editing strategies that are being developed to overcome the challenges imposed by the post-mitotic nature of retinal cell types. We further discuss their advantages and drawbacks as well as the hurdles for their implementation in treating retinal diseases, which include the broad range of mutations and, in some instances, the size of the affected genes. Although therapeutic gene editing is at an early stage of development, it has the potential of enriching the portfolio of personalized molecular medicines directed at treating genetic diseases.

Inherited retinal diseases encompass a highly heterogenous group of disorders caused by a wide range of genetic variants and with diverse clinical symptoms that converge in the common trait of retinal degeneration. Indeed, mutations in over 270 genes have been associated with some form of retinal degenerative phenotype. Given the immune privileged status of the eye, cell replacement and gene augmentation therapies have been envisioned. While some of these approaches, such as delivery of genes through recombinant adeno-associated viral vectors, have been successfully tested in clinical trials, not all patients will benefit from current advancements due to their underlying genotype or phenotypic traits. Gene editing arises as an alternative therapeutic strategy seeking to correct mutations at the endogenous locus and rescue normal gene expression. Hence, gene editing technologies can in principle be tailored for treating retinal degeneration. Here we provide an overview of the different gene editing strategies that are being developed to overcome the challenges imposed by the post-mitotic nature of retinal cell types. We further discuss their advantages and drawbacks as well as the hurdles for their implementation in treating retinal diseases, which include the broad range of mutations and, in some instances, the size of the affected genes. Although therapeutic gene editing is at an early stage of development, it has the potential of enriching the portfolio of personalized molecular medicines directed at treating genetic diseases. [ABSTRACT FROM AUTHOR]

Purpose: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and ranked top in terms of incidence and mortality in men and women. Recently, improvements in treatment approaches for NSCLC have reported, but still, there is a need to devise innovative treatment strategies, especially to manage the advanced and metastatic stage of NSCLC. Aloperine (ALO), an herbal alkaloid, has exerted anti-cancer effects in many cancers. However, the use of any chemotherapeutic agents is dose limited due to possible adverse effects and drug-resistance issues. Therefore, a combination of chemotherapy with viral-based targeted gene therapy may provide a novel treatment strategy for NSCLC. Methods/results: In this study, the results of the MTT and flow cytometry-based assays showed that Aloperine–Adbic (adenoviral vector expressing p14ARF/p53) combined treatment on NSCLC cells synergistically produced anti-proliferative effects, induced apoptosis, and arrested cell cycle at the G1 phase. Furthermore, the expression analysis suggested that the p53/p21 pathway might contribute to achieving aforesaid cytotoxic effects. The ALO–Adbic combined treatment prolonged the percent survival of NSCLC xenograft models. Conclusion: In conclusion, ALO–Adbic combination can produce synergistic anti-cancer effects at low doses, and may offer a more effective and less toxic new treatment strategy for NSCLC. [ABSTRACT FROM AUTHOR]

With the exception of inactivated vaccines, all SARS-CoV-2 vaccines currently used for clinical application focus on the spike envelope glycoprotein as a virus-specific antigen. Compared to other SARS-CoV-2 genes, mutations in the spike protein gene are more rapidly selected and spread within the population, which carries the risk of impairing the efficacy of spike-based vaccines. It is unclear to what extent the loss of neutralizing antibody epitopes can be compensated by cellular immune responses, and whether the use of other SARS-CoV-2 antigens might cause a more diverse immune response and better long-term protection, particularly in light of the continued evolution towards new SARS-CoV-2 variants. To address this question, we explored immunogenicity and protective effects of adenoviral vectors encoding either the full-length spike protein (S), the nucleocapsid protein (N), the receptor binding domain (RBD) or a hybrid construct of RBD and the membrane protein (M) in a highly susceptible COVID-19 hamster model. All adenoviral vaccines provided life-saving protection against SARS-CoV-2-infection. The most efficient protection was achieved after exposure to full-length spike. However, the nucleocapsid protein, which triggered a robust T-cell response but did not facilitate the formation of neutralizing antibodies, controlled early virus replication efficiently and prevented severe pneumonia. Although the full-length spike protein is an excellent target for vaccines, it does not appear to be the only option for future vaccine design.

The extracellular signal-regulated kinase ERK1/2 is activated in pancreatic acinar cells by cholecystokinin (CCK) and other secretagogues with this activation mediated primarily by protein kinase C (PKC). To identify the responsible PKC isoform, we utilized chemical inhibitors, cell permeant inhibitory peptides and overexpression of individual PKC dominant negative variants by means of adenoviral vectors. While the broad-spectrum PKC inhibitor GF109203X strongly inhibited ERK1/2 activation induced by 100 pM CCK, Go6976 which inhibits the classical PKC isoforms (alpha, beta and gamma), as well as Rottlerin, a specific PKC delta inhibitor, had no inhibitory effect. To test the role of PKC epsilon, we used specific cell permeant peptide inhibitors which block PKC interaction with their intracellular receptors or RACKs. Only PP93 (PKC epsilon peptide inhibitor) inhibited CCK-induced ERK1/2 activation, while PP95, PP101 and PP98, which are PKC alpha, delta and zeta peptide inhibitors respectively, had no effect. We also utilized adenovirus to express dominant negative PKC isoforms in pancreatic acini. Only PKC epsilon dominant negative inhibited CCK-induced ERK1/2 activation. Dominant negative PKC epsilon expression similarly blocked the effect of carbachol and bombesin to activate ERK1/2. Immunoprecipitation results demonstrated that CCK can induce an interaction of c-Raf-1 and PKC epsilon, but not that of other isoforms of Raf or PKC. We conclude that PKC epsilon is the isoform of PKC primarily involved with CCK-induced ERK1/2 activation in pancreatic acinar cells.

Background/Aims: While recombinant adenoviruses are among the most widely-used gene delivery vectors and usually propagated in HEK-293 cells, generating recombinant adenoviruses remains time-consuming and labor-intense. We sought to develop a rapid adenovirus production and amplification (RAPA) line by assessing human Ad5 genes (E1A, E1B19K/55K, pTP, DBP, and DNA Pol) and OCT1 for their contributions to adenovirus production. Methods: Stable transgene expression in 293T cells was accomplished by using piggyBac system. Transgene expression was determined by qPCR. Adenoviral production was assessed with titering, fluorescent markers and/or luciferase activity. Osteogenic activity was assessed by measuring alkaline phosphatase activity. Results: Overexpression of both E1A and pTP led to a significant increase in adenovirus amplification, whereas other transgene combinations did not significantly affect adenovirus amplification. When E1A and pTP were stably expressed in 293T cells, the resultant RAPA line showed high efficiency in adenovirus amplification and production. The produced AdBMP9 infected mesenchymal stem cells with highest efficiency and induced most effective osteogenic differentiation. Furthermore, adenovirus production efficiency in RAPA cells was dependent on the amount of transfected DNA. Under optimal transfection conditions high-titer adenoviruses were obtained within 5 days of transfection. Conclusion: The RAPA cells are highly efficient for adenovirus production and amplification.

In this article, we will review the main vaccination strategies currently being implemented by the health authorities and analyze the main vaccines authorized by the EMA. As practical aspects of vaccination, we must make it clear that until collective immunity is reached, the preventive measures being implemented will have to be kept in place. In the words of the WHO Accelerator Project, "There is no time to waste in the fight against COVID-19. No one is safe until everyone is safe.". [ABSTRACT FROM AUTHOR]

In December 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the outbreak of coronavirus disease 2019 (COVID-19), and the resulting pandemic has caused widespread health problems and social and economic disruption. Thus far in 2021, more than 4 million people worldwide have died from COVID-19, so safe and efficacious vaccines are urgently needed to restore normal economic and social activities. According to the official guidance documents of the World Health Organization (WHO), vaccines based on four major strategies including mRNA, adenoviral vectors, inactivated viruses, and recombinant proteins have entered the stage of emergency use authorization and pre-certification evaluation. The current review summarizes these vaccines and it looks ahead to the development of additional COVID-19 vaccines in the future. [ABSTRACT FROM AUTHOR]

Specifically tailored amino acid–based formulations were previously shown to have a high potential to avoid stress-mediated degradation of complex molecules such as monoclonal antibodies and viral vectors. By using adenovirus 5 (Ad5) as a model, we studied whether such formulations may also efficiently protect viral vectors in thermal stress experiments and during long-term liquid storage. Algorithm-based amino acid preselection using an excipient database and subsequent application of design of experiments (DoE) in combination with a 37°C challenging model enabled the prediction of long-term storage stability of Ad5. By statistical analysis of the Ad5 infectivity, amino acids with significant influence on Ad5 stability were detected after 2 and 3 weeks of liquid storage at 37°C. Ad5 formulations comprising positively selected amino acids did not reveal any loss of infectivity after 24 months in liquid storage at 5°C. By contrast, a 2 log reduction after 3 months and complete loss of infectivity after 18 months was observed with a standard viral vector formulation. By an optimization round, we designed a simple and well-balanced formulation avoiding MgCl 2 , previously considered essential in Ad5 formulations. This work demonstrates the efficacy of an algorithm-based development approach in the formulation development for viral vectors. [ABSTRACT FROM AUTHOR]

Introduction: Despite their appeal as vaccine vectors, adenoviral vectors are yet unable to induce protective immune responses against some weakly immunogenic antigens. Additionally, the maximum doses of adenovirus-based vaccines are limited by vector-induced toxicity, causing vector elimination and diminished immune responses against the target antigen. In order to increase immune responses to the transgene, while maintaining a moderate vector dose, new technologies for improved transgene presentation have been developed for adenoviral vaccine vectors. Areas covered: This review provides an overview of different genetic-fusion adjuvants that aim to improve antigen presentation in the context of adenoviral vector-based vaccines. The influence on both T cell and B cell responses are discussed, with a main focus on two technologies: MHC class II-associated invariant chain and virus-like-vaccines. Expert opinion: Different strategies have been tested to improve adenovirus-based vaccinations with varying degrees of success. The reviewed genetic adjuvants were designed to increase antigen processing and MHC presentation, or promote humoral immune responses with an improved conformational antigen display. While none of the introduced technologies is universally applicable, this review shall give an overview to identify potential improvements for future vaccination approaches. [ABSTRACT FROM AUTHOR]

The calyx of Held, a large glutamatergic presynaptic terminal in the auditory brainstem undergoes developmental changes to support the high action-potential firing rates required for auditory information encoding. In addition, calyx terminals are morphologically diverse, which impacts vesicle release properties and synaptic plasticity. Mitochondria influence synaptic plasticity through calcium buffering and are crucial for providing the energy required for synaptic transmission. Therefore, it has been postulated that mitochondrial levels increase during development and contribute to the morphological-functional diversity in the mature calyx. However, the developmental profile of mitochondrial volumes and subsynaptic distribution at the calyx of Held remains unclear. To provide insight on this, we developed a helper-dependent adenoviral vector that expresses the genetically encoded peroxidase marker for mitochondria, mito-APEX2, at the mouse calyx of Held. We developed protocols to detect labeled mitochondria for use with serial block face scanning electron microscopy to carry out semiautomated segmentation of mitochondria, high-throughput whole-terminal reconstruction, and presynaptic ultrastructure in mice of either sex. Subsequently, we measured mitochondrial volumes and subsynaptic distributions at the immature postnatal day (P)7 and the mature (P21) calyx. We found an increase of mitochondria volumes in terminals and axons from P7 to P21 but did not observe differences between stalk and swelling subcompartments in the mature calyx. Based on these findings, we propose that mitochondrial volumes and synaptic localization developmentally increase to support high firing rates required in the initial stages of auditory information processing. [ABSTRACT FROM AUTHOR]

A resurgence in the development of newer gene therapy systems has led to recent successes in the treatment of B cell cancers, retinal degeneration and neuromuscular atrophy. Gene therapy offers the ability to treat the patient at the root cause of their malady by restoring normal gene function and arresting the pathological progression of their genetic disease. The current standard of care for most genetic diseases is based upon the symptomatic treatment with polypharmacy while minimizing any potential adverse effects attributed to the off-target and drug-drug interactions on the target or other organs. In the kidney, however, the development of gene therapy modifications to specific renal cells has lagged far behind those in other organ systems. Some positive strides in the past few years provide continued enthusiasm to invest the time and effort in the development of new gene therapy vectors for medical intervention to treat kidney diseases. This mini-review will systematically describe the pros and cons of the most commonly tested gene therapy vector systems derived from adenovirus, retrovirus, and adeno-associated virus and provide insight about their potential utility as a therapy for various types of genetic diseases in the kidney. [ABSTRACT FROM AUTHOR]

Adenoviral vectors induce robust epitope‐specific CD8+ T cell responses. Within the repertoire of responses generated both conventional memory evolution and the phenomenon of memory inflation are seen. The rules governing which epitopes inflate are not fully known, but may include a role for both antigen processing and competition. To investigate this, we looked at memory generated from vectors targeting the Gp33‐41 (KAVYNFATC/K9C) epitope from the gp of lymphocytic choriomeningitis virus (LCMV) in mice. This well‐described epitope has both the Gp33‐41 and Gp34‐41 epitopes embedded within it. Vaccination with a full‐length gp or a minigene Ad‐Gp33/K9C vector‐induced conventional memory responses against the immunodominant Gp33/K9C epitope but a strong inflationary response against the Gp34/A8C epitope. These responses showed sustained in vivo function, with complete protection against LCMV infectious challenge. Given the unexpected competition between epitopes seen in the minigene model, we further tested epitope competition using the full‐length Ad‐LacZ (β‐galactosidase) model. Generation of an Ad‐LacZ vector with a single amino acid disruption of the inflationary β‐gal96‐103/D8V epitope transformed the β‐gal497‐504/I8V epitope from conventional to inflationary memory. This work collectively demonstrates the importance of epitope competition within adenoviral vector inserts and is of relevance to future studies using adenoviral vectored immunogens. [ABSTRACT FROM AUTHOR]

Background: There is an urgent need for targeted biological therapies for prostate cancer with greater efficacy and less toxicity, particularly for metastatic disease, where current therapies are not curative. Therapeutic adenoviral vectors or oncolytic adenoviruses offer the possibility of a competent, nontoxic therapeutic alternative for prostate cancer. However, free viral particles must be delivered locally, an approach that does not address metastatic disease, and they display poor tumor penetration. To fully exploit the potential of these vectors, we must develop methods that improve intratumoral dissemination and allow for systemic delivery. This study establishes a proof-of-principle rationale for a novel human mesenchymal stem (stromal) cell-based approach to improving vector delivery to tumors. Methods/results: We have generated mesenchymal stem cell-derived packaging cells for adenoviruses (E1-modified mesenchymal stem cells) by modifying human mesenchymal stem cells with the adenovirus (type C) E1A/B genes needed for viral replication. Using cell-based assays, we have demonstrated that two adenoviral vectors, replication-defective adenovirus expressing p14 and p53 or conditionally replicating oncolytic adenovirus, packaged by E1A/B-modified mesenchymal stem cells, suppress the growth of prostate cancer cells in culture. Using subcutaneous xenograft models for human prostate cancer in mice, we have shown that E1A/B-modified mesenchymal stem cells display tumor tropism in tumor-bearing nude mice, that E1A/B-modified mesenchymal stem cells disseminate well within tumors, and that replication-defective adenovirus expressing p14 and p53 or conditionally replicating oncolytic adenovirus-loaded E1-modified mesenchymal stem cells suppresses tumor growth in mice. Conclusion: The results show that this approach, if optimized, could circumvent the obstacles to efficient gene delivery encountered with current gene delivery approaches and provide an effective, nontoxic therapeutic alternative for metastatic disease. [ABSTRACT FROM AUTHOR]

Gene editing permits changing specific DNA sequences within the vast genomes of human cells. Stem cells are particularly attractive targets for gene editing interventions as their self-renewal and differentiation capabilities consent studying cellular differentiation processes, screening small-molecule drugs, modeling human disorders, and testing regenerative medicines. To integrate gene editing and stem cell technologies, there is a critical need for achieving efficient delivery of the necessary molecular tools in the form of programmable DNA-targeting enzymes and/or exogenous nucleic acid templates. Moreover, the impact that the delivery agents themselves have on the performance and precision of gene editing procedures is yet another critical parameter to consider. Viral vectors consisting of recombinant replication-defective viruses are under intense investigation for bringing about efficient gene-editing tool delivery and precise gene-editing in human cells. In this review, we focus on the growing role that adenoviral vectors are playing in the targeted genetic manipulation of human stem cells, progenitor cells, and their differentiated progenies in the context of in vitro and ex vivo protocols. As preamble, we provide an overview on the main gene editing principles and adenoviral vector platforms and end by discussing the possibilities ahead resulting from leveraging adenoviral vector, gene editing, and stem cell technologies.

Abstract There is a high incidence of adenovirus (AdV) infection in humans due to the presence of more than 60 types of human adenoviruses (HAdVs). The majority of individuals are exposed to one or more HAdV types early in their lives, leading to the development of AdV type-specific neutralizing antibodies. Similarly, immunization or gene therapy with AdV vectors leads to immune responses to the AdV vector. This 'vector immunity' is a concern for AdV vector-based applications for vaccines or gene therapy, especially when the repeated administration of a vector is required. The objective of this investigation was to establish whether AdV neutralizing antibody titers decline sufficiently in a year to permit annual vaccination with the same AdV vector. Naïve or human adenoviral vector group C, type 5 (HAdV-C5)-primed mice were mock-inoculated (with PBS) or inoculated i.m. with 108 PFU of either HAd-GFP [HAdV-C5 vector expressing the green fluorescent protein (GFP)] to mimic the conditions for the first inoculation with an AdV vector-based vaccine. At 1, 3, 6, and 10 months post-HAd-GFP inoculation, naïve- or HAdV-primed animals were vaccinated i.m. with 108 PFU of HAd-H5HA [HAdV-C5 vector expressing hemagglutinin (HA) of H5N1 influenza virus]. There was a significant continual decrease in vector immunity titers with time, thereby leading to significant continual increases in the levels of HA-specific humoral and cell-mediated immune responses. In addition, significant improvement in protection efficacy against challenge with an antigenically heterologous H5N1 virus was observed in HAdV-primed animals at 6 months and onwards. These results indicate that the annual immunization with the same AdV vector may be effective due to a significant decline in vector immunity. [ABSTRACT FROM AUTHOR]

Abstract Cellular immunity established via immunotherapy holds the potential to eliminate solid tumors. Yet, cancer vaccines have failed to induce tumor-reactive T cells of sufficient quality to control disease. The inducible T cell costimulator (ICOS) pathway has been implicated in both the selective induction of immunity over tolerance as well as licensing of IL-17-polarized cellular immunity. Herein, we evaluated the ability of ICOS ligand (ICOSL) to augment the immunogenicity of adenoviral-based vaccination targeting the unglycosylated MUC1 peptide antigen. Vaccination disrupted immunotolerance in a transgenic mouse model recognizing human MUC1 as a self-antigen, inducing robust MUC1-specific immunity. Augmenting vaccination with ICOSL induced a bipolar Th17/Th1 effector profile, marked by increased MUC1-specific IL-17A production and RORγt expression in CD4+ but not CD8+ T cells which predominantly expressed IFNγ/IL-2 and T-bet. The polarization and maintenance of Th17 cells established following ICOSL augmented vaccination was highly durable, with elevated IL-17A and RORγt levels detected in CD4+ T cells up to 10 months after initial immunization. Furthermore, provision of ICOSL significantly enhanced MUC1-specific IgG antibody in response to immunization. ICOSL signaling dramatically influenced CD4+ T cell phenotype, altering gene expression of transcription factors and regulators of effector function following immunization. Interestingly, ICOSL augmentation failed to alter the transcriptional profile of CD8+ T cells following immunization, affecting the magnitude, but not distribution, of gene expression. Collectively, ICOSL supports the induction of durable, antigen-specific Th17/Th1-mediated immunity in vivo , establishing a vaccination platform to enhance CD4+ T cell-mediated antitumor immunity and providing a crucial component of an effective cancer vaccine. [ABSTRACT FROM AUTHOR]

Defining correlates of T cell mediated protection is important in order to accelerate the development of efficient T cell based vaccines conferring long-term immunity. Extensive studies have provided important insight regarding the characteristics and functional properties of the effector and memory CD8 T cells induced by viral vector based vaccines. However, long-term protection has been difficult to achieve with T cell inducing vaccines, and the determinants underlying this loss in protection over time are still not fully defined. In this study we analyzed different parameters of the CD8 T cell response as a function of time after vaccination with a human serotype 5 adenovector expressing the glycoprotein (GP) of LCMV tethered to the MHC class II-associated invariant chain. Using this vector we have previously found that CD8 T cells mediate protection from challenge with GP-expressing Listeria monocytogenes at 60 days post vaccination, but only little protection after further 60 days, and we now confirm this observation. A comparison of vaccine-primed CD8 T cells early and late after vaccination revealed a minor decline in the overall numbers of antigen specific memory CD8 T cells during this interval. More importantly, we also observed phenotypic changes over time with a distinct decline in the frequency and number of KLRG1 + CD8 T cells, and, notably, adoptive transfer studies confirmed that memory CD8 T cells expressing KLRG1 are central to protection from systemic L. monocytogenes infection. Together these findings imply that multiple factors including changes in memory T cell numbers and phenotypic composition over time influence the longevity of CD8 T-cell mediated protection. [ABSTRACT FROM AUTHOR]