Environmental Impact Assessment for the Use of an Orally Aerosolized Adenovirus Type-5 Vector-Based COVID-19 Vaccine in Randomized Clinical Trials.

BACKGROUND: An orally aerosolized adenovirus type-5 vector-based coronavirus disease 2019 (COVID-19) vaccine (Ad5-nCoV) has recently been authorized for boosting immunization in China. Our study aims to assess the environmental impact of the use of aerosolized Ad5-nCoV. METHODS: We collected air samples from rooms, swabs from the desks on which the vaccine nebulizer was set, mask samples from participants, and blood samples of nurses who administered the inoculation in the clinical trials. The viral load of adenovirus type-5 vector in the samples and the antibody levels against the wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain in serum were detected. RESULTS: Only one (4.00%) air sample collected before initiation of vaccination was positive and most air samples collected during and after vaccination were positive (97.96%, 100%, respectively). All nurses in trial A showed at least 4-fold increase of the neutralizing antibody against SARS-CoV-2 after initiation of the study. In trial B, the proportion of positive mask samples was 72.97% at 30 minutes after vaccination, 8.11% at day 1, and 0% at days 3, 5, and 7. CONCLUSIONS: Vaccination with the orally aerosolized Ad5-nCoV could result in some spillage of the vaccine vector viral particles in the environment and cause human exposure. Clinical Trials Registration. NCT04840992 and NCT05303584.

In-silico evaluation of adenoviral COVID-19 vaccination protocols: Assessment of immunological memory up to 6 months after the third dose.

Background: The immune response to adenoviral COVID-19 vaccines is affected by the interval between doses. The optimal interval is unknown. Aim: We aim to explore in-silico the effect of the interval between vaccine administrations on immunogenicity and to analyze the contribution of pre-existing levels of antibodies, plasma cells, and memory B and T lymphocytes. Methods: We used a stochastic agent-based immune simulation platform to simulate two-dose and three-dose vaccination protocols with an adenoviral vaccine. We identified the model's parameters fitting anti-Spike antibody levels from individuals immunized with the COVID-19 vaccine AstraZeneca (ChAdOx1-S, Vaxzevria). We used several statistical methods, such as principal component analysis and binary classification, to analyze the correlation between pre-existing levels of antibodies, plasma cells, and memory B and T cells to the magnitude of the antibody response following a booster dose. Results and conclusions: We find that the magnitude of the antibody response to a booster depends on the number of pre-existing memory B cells, which, in turn, is highly correlated to the number of T helper cells and plasma cells, and the antibody titers. Pre-existing memory T cytotoxic cells and antibodies directly influence antigen availability hence limiting the magnitude of the immune response. The optimal immunogenicity of the third dose is achieved over a large time window, spanning from 6 to 16 months after the second dose. Interestingly, after any vaccine dose, individuals can be classified into two groups, sustainers and decayers, that differ in the kinetics of decline of their antibody titers due to differences in long-lived plasma cells. This suggests that the decayers may benefit from a tailored boosting schedule with a shorter interval to avoid the temporary loss of serological immunity.

Efficacy of hydrogen peroxide wipes for decontamination of AZD1222 adenovirus COVID-19 vaccine strain on pharmaceutical industry materials.

This study aimed to evaluate the performance of accelerated hydrogen peroxide® wipes (HPW) for decontamination of the chimpanzee adenovirus AZD1222 vaccine strain used in the production of recombinant COVID-19 vaccine in a pharmaceutical industry. Two matrices were tested on stainless-steel (SS) and low-density-polyethylene (LDP) surfaces: formulated recombinant COVID-19 vaccine (FCV) and active pharmaceutical ingredient (API). The samples were spiked, dried and the initial inoculum, possible residue effect (RE) and titre reduction after disinfection with HPW were determined. No RE was observed. The disinfection procedure with HPW resulted in complete decontamination the of AZD1222 adenovirus strain in FCV (≥7·46 and ≥7·49 log10 infectious unit [IFU] ml-1 for SS and LDP carriers respectively) and API (≥8·79 and ≥8·78 log10 IFU ml-1 for SS and LDP carriers respectively). In conclusion, virucidal activity of HPW was satisfactory against the AZD1222 adenovirus strain and can be a good option for disinfection processes of SS and LPD surfaces in pharmaceutical industry facilities during recombinant COVID-19 vaccine production. This procedure is simple and can be also applied on safety unit cabins and sampling bags made of LDP as well.

Functional Assessment of Missense Variants in the ABCC6 Gene Implicated in Pseudoxanthoma Elasticum, a Heritable Ectopic Mineralization Disorder.

Pseudoxanthoma elasticum, a heritable multisystem ectopic mineralization disorder, is caused by inactivating mutations in the ABCC6 gene. The encoded protein, ABCC6, a transmembrane transporter, has a specialized efflux function in hepatocytes by contributing to plasma levels of inorganic pyrophosphate, a potent inhibitor of mineralization in soft connective tissues. Reduced plasma inorganic pyrophosphate levels underlie the ectopic mineralization in pseudoxanthoma elasticum. In this study, we characterized the pathogenicity of three human ABCC6 missense variants using an adenovirus-mediated liver-specific ABCC6 transgene expression system in an Abcc6-/- mouse model of pseudoxanthoma elasticum. Variants p.L420V and p.R1064W were found benign because they had abundance and plasma membrane localization in hepatocytes similar to the wild-type human ABCC6 transgene, normalized plasma inorganic pyrophosphate levels, and prevented mineralization in the dermal sheath of vibrissae in muzzle skin, a phenotypic hallmark in the Abcc6-/- mice. In contrast, p.S400F was shown to be pathogenic because it failed to normalize plasma inorganic pyrophosphate levels and had no effect on ectopic mineralization despite its normal expression and proper localization in hepatocytes. These results showed that adenovirus-mediated hepatic ABCC6 expression in Abcc6-/- mice can provide a model system to effectively elucidate the multifaceted functional consequences of human ABCC6 missense variants identified in patients with pseudoxanthoma elasticum.

Nonclinical safety assessment of repeated administration and biodistribution of ChAd3-EBO-Z Ebola candidate vaccine.

ChAd3-EBO-Z is an investigational adenovirus-based vaccine for the prevention of Ebola virus disease. Two nonclinical studies were performed to evaluate the biodistribution, local tolerance and potential local and systemic toxic effects of this vaccine. In the biodistribution study, rats received a single intramuscular injection of either ChAd3-EBO-Z or saline. Enlargement of the draining lymph nodes, starting on day 2, was noticed in ChAd3-EBO-Z-treated rats, indicating that an immune response had taken place. Viral DNA was mainly found at the injection sites and in the draining lymph nodes, from where it progressively disappeared during the observation period, while it was found only transiently and occasionally in other organs. In the repeated-dose toxicity study, either ChAd3-EBO-Z or saline was administered intramuscularly to rabbits on two occasions with a 2-week interval. General health status, rectal temperature, local tolerance, ophthalmology, hematology, coagulation and blood chemistry parameters were monitored. Macroscopic and microscopic evaluations were performed. Treatment-related changes included a transient increase in neutrophil count, C-reactive protein and fibrinogen levels, and a transient decrease in platelet count. As expected, microscopic observations 3 days after the second injection were related to the elicited inflammatory reaction, and these inflammatory responses had almost completely disappeared 29 days after the second immunization. In conclusion, the vaccine was locally and systemically well-tolerated and the viral vector was partially or totally cleared from the organs where it disseminated, supporting the clinical development of the vaccine.

Mating strategy is determinant of adenovirus prevalence in European bats.

Adenoviruses are double-strained DNA viruses found in a great number of vertebrates, including humans. In order to understand their transmission dynamics, it is crucial, even from a human health perspective, to investigate how host traits influence their prevalence. Bats are important reservoirs for adenoviruses, and here we use the results of recent screenings in Western Europe to evaluate the association between characteristic traits of bat species and their probability of hosting adenoviruses, taking into account their phylogenetic relationships. Across species, we found an important phylogenetic component in the presence of adenoviruses and mating strategy as the most determinant factor conditioning the prevalence of adenoviruses across bat species. Contrary to other more stable mating strategies (e.g. harems), swarming could hinder transmission of adenoviruses since this strategy implies that contacts between individuals are too short. Alternatively, bat species with more promiscuous behavior may develop a stronger immune system. Outstandingly high prevalence of adenoviruses was reported for the Iberian species Pipistrellus pygmaeus, P. kuhlii and Nyctalus lasiopterus and we found that in the latter, males were more likely to be infected by adenoviruses than females, due to the immunosuppressing consequence of testosterone during the mating season. As a general trend across species, we found that the number of adenoviruses positive individuals was different across localities and that the difference in prevalence between populations was correlated with their geographic distances for two of the three studied bat species (P. pygmaeus and P.kuhlii). These results increase our knowledge about the transmission mechanisms of adenoviruses.

Assessment of viral and bacterial contamination of fresh and ripened semi-hard cheeses.

This study aimed to evaluate viral and bacterial contamination from typical Brazilian cheeses, such as Minas (fresh) and Prato (ripened), commercially obtained in the Greater Metropolitan Region of the State of Rio de Janeiro, Brazil. Minas [30], Prato [30] and sliced Prato [30] cheese samples were investigated for norovirus genogroup I and II (NoV GI-II) and human adenovirus (HAdV) by direct nucleic acid extraction using TRIzol and amplification by TaqMan based quantitative polymerase chain reaction. Listeria monocytogenes, Salmonella spp., coagulase-positive staphylococci (CPS) and fecal coliforms were also assessed by using standard counting methods. NoV GI and GII were detected in one sample (1.1%) each and HAdV in nine samples (10.0%) while bacteriological analysis revealed five samples (5.5%) contaminated with L. monocytogenes, 27 (30.0%) with fecal coliforms and 10 (11.1%) with CPS. Salmonella spp. was not detected in any sample. Viruses were detected in 11 samples (12.2%), of which 9 met the microbiological criteria used to evaluate the microbiological quality of the cheeses, stressing the importance of considering virological parameters for monitoring this food matrix.

Gene Therapy for Retinal Degeneration.

Biallelic mutations in the RPE65 gene are associated with inherited retinal degenerations/dystrophies (IRD) and disrupt the visual cycle, leading to loss of vision. A new adenoviral vector-based gene therapy surgically delivered to retinal cells provides normal human RPE65 protein that can restore the visual cycle and some vision. To view this Bench to Bedside, open or download the PDF.

Assessment of diversity of adenovirus DNA polymerase gene in recreational waters facilitated by ultracentrifugal concentration.

Adenoviruses (AdV) are related to respiratory and gastrointestinal diseases in animals and human beings. Their wide genetic diversity in water bodies and their resistance to environmental conditions allow the use of AdV as a reliable marker for detection of fecal contamination. In this work, the diversity of AdV along Belo Stream - in the city of Caxias do Sul, Rio Grande do Sul, Brazil - was evaluated. Samples were compared in both concentrated and unconcentrated forms. The identification of different AdV species was performed by amplifying a partial sequence of the DNA polymerase gene. AdV was detected in 24 out of 55 concentrated samples (43.6%) and the following species were identified: human adenovirus (HAdV) species C (4/55; 7.2%), D (6/55; 10.9%), E (2/55; 3.6%), and F (9/55; 16.3%). AdV related to other mammalian hosts, such as bovine adenovirus (1/55, 1.8%) and murine adenovirus (2/55, 3.6%), have also been identified; 23.6% (13/55) of the unconcentrated samples were positive, and identified as HAdV species C (6/55, 10.9%), D (1/55, 1.8%), and F (6/55, 10.9%). Results obtained evidenced the presence and the great diversity of AdV, mainly of human origin, circulating in Belo Stream. As expected, the concentration step performed helped to detect AdV in more samples.

Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells.

BACKGROUND: Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. METHODS: Pediatric pAECs derived from children with CF (pAECCF) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. RESULTS: Data showed that pAECCF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAECCF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. SIGNIFICANCE: The current study demonstrates that the halide assay can be adapted for pediatric pAECCF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations.

Social Control of Hypothalamus-Mediated Male Aggression.

How environmental and physiological signals interact to influence neural circuits underlying developmentally programmed social interactions such as male territorial aggression is poorly understood. We have tested the influence of sensory cues, social context, and sex hormones on progesterone receptor (PR)-expressing neurons in the ventromedial hypothalamus (VMH) that are critical for male territorial aggression. We find that these neurons can drive aggressive displays in solitary males independent of pheromonal input, gonadal hormones, opponents, or social context. By contrast, these neurons cannot elicit aggression in socially housed males that intrude in another male's territory unless their pheromone-sensing is disabled. This modulation of aggression cannot be accounted for by linear integration of environmental and physiological signals. Together, our studies suggest that fundamentally non-linear computations enable social context to exert a dominant influence on developmentally hard-wired hypothalamus-mediated male territorial aggression.

Assessment of Specificity of an Adenovirus Targeted to HER3/4.

Gene therapy with viral vectors, such as adenovirus (Ad), targeted to the human epidermal growth factor receptors 3 and 4 (HER3/4) are potentially useful for cancer therapy. Testing the expression of a reporter gene from these viruses in target cells is essential to determine functionality of the targeted virus. A competition assay with a relevant ligand (heregulin, HRG) can provide convincing evidence that blocking binding to the HER3/4 receptor results in decreased reporter gene expression. Labeling individual viruses with a fluorescent molecule allows examination of the targeted virus in specific steps in the infection. Virus internalization into cell lines can be determined using antibody-labeled receptors, and the virus colocalization with receptors can also be visualized. Characterization of a targeted virus in this fashion is important to demonstrate that the targeting of the virus functions in an expected manner, and provides support for larger-scale testing of the virus. Information acquired in these experiments may also be useful to inform and improve on the design of future targeted viruses.

Future Prospects for the Development of Cost-Effective Adenovirus Vaccines.

Vaccination is one of the most efficient tools for disease prevention, and a continuously growing field of research. However, despite progress, we still need more efficient and cost-effective vaccines that would improve access to those in need. In this review, we will describe the status of virus-vectored vaccine technology with a focus on adenoviral-based vaccines. Adenovirus (Ad) vaccines have proven to be efficient in military vaccinations against Ad4 and Ad7 and as highly efficient vectored vaccines against rabies. The question of how other adenovirus-based vaccines can become as efficient as the rabies vaccine is the underlying theme in this review. Here, we will first give an overview of the basic properties of vectored vaccines, followed by an introduction to the characteristics of adenoviral vectors and previously tested modifications of the vector backbone and expression cassettes, with a focus on how they can contribute to increased vaccine cost-effectiveness. Finally, we will highlight a few successful examples of research that have attempted to improve the use of adenoviral-based vaccines by improving the transgene immunogenicity.

Low-Cost Charged-Coupled Device (CCD) Based Detectors for Shiga Toxins Activity Analysis.

To improve food safety there is a need to develop simple, low-cost sensitive devices for detection of food-borne pathogens and their toxins. We describe a simple, low-cost webcam-based detector which can be used for various optical detection modalities, including fluorescence, chemiluminescence, densitometry, and colorimetric assays. The portable battery-operated CCD-based detection system consists of four modules: (1) a webcam to measure and record light emission, (2) a sample plate to perform assays, (3) a light emitting diode (LED) for illumination, and (4) a portable computer to acquire and analyze images. To demonstrate the technology, we used a cell based assay for fluorescence detection of the activity of the food borne Shiga toxin type 2 (Stx2), differentiating between biologically active toxin and inactive toxin which is not a risk. The assay is based on Shiga toxin inhibition of cell protein synthesis measured through inhibition of the green fluorescent protein (GFP). In this assay, GFP emits light at 509 nm when excited with a blue LED equipped with a filter at 486 nm. The emitted light is then detected with a green filter at 535 nm. Toxin activity is measured through a reduction in the 509 nm emission. In this system the level of detection (LOD) for Stx2 was 0.1 pg/ml, similar to the LOD of commercial fluorometers. These results demonstrate the utility and potential of low cost detectors for toxin activity. This approach could be readily adapted to the detection of other food-borne toxins.

An Adenoviral Vector Encoding Full-Length Dectin-1 Promotes Aspergillus-Induced Innate Immune Response in Macrophages.

INTRODUCTION: The incidence of invasive pulmonary aspergillosis (IPA) has increased significantly over the last two decades. Alveolar macrophages (AMs) represent the first line of pulmonary host response to Aspergillus conidia. Recognition of conidia by AMs involves Dectin-1 (CLEC7A), which is a conserved structure to combine ß-glucans. The deficiency of Dectin-1 results in impaired fungal killing and uncontrolled growth of Aspergillus fumigatus. Thus, we hypothesized that high expression of Dectin-1 would enhance the host recognition and fungal killing. METHODS: We set out to develop an adenoviral vector encoding full-length Dectin-1 (Ad-Dectin-1-EGFP) and then transfect it to MH-S cells. Transfect cell model was verified by using real-time RT-PCR, Western blot, flow cytometric, and confocal microscopic assays. And also, the function of Dectin-1 was explored by measuring cytokine release and killing ability during the course of A. fumigatus infection. RESULTS: We constructed a recombinant adenovirus which could upregulate the expression of Dectin-1 and verified that Dectin-1 was expressed on cell membrane. The function of Dectin-1 was also demonstrated by its ability in promoting the production of cytokines and increasing the killing ability during the course of A. fumigatus infection. CONCLUSIONS: An adenoviral vector was successfully applied to the production of a recombinant adenovirus encoding full-length Dectin-1, and also, its function in Aspergillus-induced innate immune response was demonstrated.

Assessment of transfection of AdCMV-EGFP to rat submandibular gland cells.

We evaluated the efficiency of transfecting adenoviral vectors encoding enhanced green fluorescent protein (AdCMV-EGFP) into rat submandibular gland cells and the effects of gene transfer on cell proliferation and secretory function. Isolated submandibular gland cells were transfected with different titers (or multiplicity of infection, MOI) of AdCMV-EGFP. The transfection efficiency was evaluated by quantifying EGFP-positive cells by inverted fluorescence microscopy, cell proliferation by MTT assay, and cell secretory activity by measuring α-amylase in culture medium. A transfection efficiency of up to 70.8% was achieved in submandibular gland cells. MTT assay showed that increased viral titers resulted in significant inhibition of cell proliferation, which occurs on day 5 post-transfection. Simultaneously, the amylase levels started to reduce with a significant decrease on day 7 after transfection. The results show that AdCMV-EGFP transfection of submandibular gland cells at higher MOI results in cytotoxicity, decreased cell proliferation, and secretory function. However, the lower adenoviral titers (e.g., 200 particles/cell) could be an efficient and safe labeling tool for gene transfer to submandibular gland cells.