Search

Adenoviruses are important human pathogens that are widespread and mainly associated with respiratory and gastrointestinal infections. In a previous study on human adenovirus (HAdV) seroprevalence, we observed reduced binding antibody levels against a range of HAdV types in sera collected from students in 2021 compared to sera collected before the SARS-CoV-2 pandemic. In this follow-up study, we wanted to verify this observation in a cohort of regular blood donors for whom serial samples were available. Therefore, HAdV-specific binding antibody levels were analyzed in sera collected over a 5-year period from 2018 to 2022 in a cohort of 60 regular donors to the blood bank of the University Hospital in Greifswald, Germany. Using ELISA-based assays, we quantified the binding antibody responses against 39 HAdV types. On the cohort level, we found largely stable antibody levels over the analyzed time period, with the highest antibody responses against HAdV-C1, -D25, -D26, -E4, -D10, -D27, -C5, -D75, -C2, and -C6. Only minor but significant reductions in comparison to the first serum samples from 2018 were detected for antibody levels in 2021 and 2022 against the low-prevalent types HAdV-A31, -D8, -D20, -D37, -D65, and -D69. On the other hand, we detected fluctuations in antibody levels on the individual level, with strong increases in antibody levels indicative of novel antigen contact. Interestingly, we frequently found simultaneous changes in antibody responses against multiple HAdV types, resulting in strong correlations of antibody responses against distinct clusters of HAdVs suggesting extensive cross-reactivity of HAdV-specific antibodies. To our knowledge, this is the first study of antibodies against a broad range of HAdV types in serum samples collected from a cohort of individuals over a prolonged period, and our data provide important insight into the long-term stability of HAdV-specific antibody levels. In this cohort of regular blood donors, we did not observe any major impact of the SARS-CoV-2 pandemic on HAdV immunity. Correlations of changes in antibody levels against different types indicate cross-reactivity of HAdV-specific antibodies that are important to consider for HAdV vector development. Our data also reveal possible candidates for future development of HAdV-based vectors.

Adenoviruses (Ad) have the potential to induce severe infections in vulnerable patient groups. Therefore, understanding Ad biology and antiviral processes is important to comprehend the signaling cascades during an infection and to initiate appropriate diagnostic and therapeutic interventions. In addition, Ad vector-based vaccines have revealed significant potential in generating robust immune protection and recombinant Ad vectors facilitate efficient gene transfer to treat genetic diseases and are used as oncolytic viruses to treat cancer. Continuous improvements in gene delivery capacity, coupled with advancements in production methods, have enabled widespread application in cancer therapy, vaccine development, and gene therapy on a large scale. This review provides a comprehensive overview of the virus biology, and several aspects of recombinant Ad vectors, as well as the development of Ad vector, are discussed. Moreover, we focus on those Ads that were used in preclinical and clinical applications including regenerative medicine, vaccine development, genome engineering, treatment of genetic diseases, and virotherapy in tumor treatment.

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

More than 100 human adenovirus (Ad) types were identified, of which species D comprises the largest group. Heparan sulfate proteoglycans (HSPGs) were shown to function as cell surface receptors for cell binding and uptake of some Ads, but a systematic analysis of species D Ads is lacking. Previous research focused on Ad5 and blood coagulation factor X (FX) complexes, which revealed that Ad5 can transduce cells with low expression levels of its main coxsackievirus-adenovirus receptor in the presence of high HSPG expression levels in a FX dependent manner. Based on our reporter gene-tagged Ad-library, we explored for the first time a broad spectrum of species D Ads to study the role of HSPG on their cellular uptake. This study was performed on three Chinese Hamster Ovary (CHO) cell lines with different forms of HSPG (only proteoglycan (745), non-sulfated HSPG (606) or sulfated HSPG (K1)). The effect of Ad:FX complexes on Ad uptake was explored in the presence of physiological levels of FX in blood (6–10 µg/mL). We found that sulfation of HSPG plays an important role in cellular uptake and transduction of FX-bound Ad5 but neither HSPG nor FX influenced uptake of all tested species D Ads. Because FX has no influence on transduction efficiencies of species D Ads and therefore may not bind to them, these Ads may not be protected from attack by neutralizing IgM antibodies or the complement pathway, which may have implications for species D Ads used as vaccine and gene therapy vectors.

High pre-existing antibodies against viral vectors reduce their functionality and may lead to adverse complications. To circumvent this problem in future gene therapy approaches, we tested the seroprevalence of a large range of human adenovirus types in patients with neuromuscular disorders (NMDs) to find appropriate viral vector candidates for gene replacement therapy for NMDs. Binding and neutralizing antibodies against 39 human adenovirus types were tested in the sera of 133 patients with NMDs and 76 healthy controls aged 17–92 years. The influence of age, sex, and NMDs on antibody levels was analyzed. The seroprevalence of different adenoviruses in the cohort varied widely. The highest levels of binding antibodies were detected against HAdV-D27, -C1, -D24, -D70, -B14, -C6, -D13, -B34, and -E4, whereas the lowest reactivity was detected against HAdV-F41, -A31, -B11, -D75, -D8, -D65, -D26, -D80, and -D17. The highest neutralizing reactivity was observed against HAdV-B3, -C2, -E4, -C1, -G52, -C5, and -F41, whereas the lowest neutralizing reactivity was observed against HAdV-D74, -B34, -D73, -B37, -D48, -D13, -D75, -D8, -B35, and -B16. We detected no influence of sex and only minor differences between different age groups. Importantly, there were no significant differences between healthy controls and patients with NMDs. Our data show that patients with NMDs have very similar levels of binding and neutralizing antibodies against HAdV compared to healthy individuals, and we identified HAdV-A31, -B16, -B34, -B35, -D8, -D37, -D48, -D73, -D74, -D75, and -D80 as promising candidates for future vector development due to their low binding and neutralizing antibody prevalence.

Rhinoviruses (RV) account for a significant number of asthma exacerbations, and RV species C may be associated with a severe course in vulnerable patient groups. Despite important evidence on the role of RV reported by clinicians and life scientists, there are still unanswered questions regarding their influence on asthma exacerbation in young patients. Thus, we measured the RVspecies-specific IgG titers in our German pediatric exacerbation cohort using a microarray-based technology. For this approach, human sera of patients with exacerbated asthma and wheeze, as well as healthy control subjects (n = 136) were included, and correlation analyses were performed. Concordantly with previously published results, we observed significantly higher cumulative levels of RV species A-specific IgG (p = 0.011) and RV-C-specific IgG (p = 0.051) in exacerbated asthma group compared to age-matched controls. Moreover, atopic wheezers had increased RV-specific IgG levels for species A (p = 0.0011) and species C (p = 0.0009) compared to non-atopic wheezers. Hypothesizing that bacterial infection positively correlates with immune memory against RV, we included nasopharyngeal swab results in our analyses and detected limited correlations. Interestingly, the eosinophil blood titer positively correlated with RV-specific IgG levels. With these observations, we add important observations to the existing data regarding exacerbation in pediatric and adolescent medicine. We propose that scientists and clinicians should pay more attention to the relevance of RV species in susceptible pediatric patients.

Carcinomas are characterized by a widespread upregulation of intercellular junctions that create a barrier to immune response and drug therapy. Desmoglein 2 (DSG2) represents such a junction protein and serves as one adenovirus receptor. Importantly, the interaction between human adenovirus type 3 (Ad3) and DSG2 leads to the shedding of the binding domain followed by a decrease in the junction protein expression and transient tight junction opening. Junction opener 4 (JO-4), a small recombinant protein derived from the Ad3 fiber knob, was previously developed with a higher affinity to DSG2. JO-4 protein has been proven to enhance the effects of antibody therapy and chemotherapy and is now considered for clinical trials. However, the effect of the JO4 mutation in the context of a virus remains insufficiently studied. Therefore, we introduced the JO4 mutation to various adenoviral vectors to explore their infection properties. In the current experimental settings and investigated cell lines, the JO4-containing vectors showed no enhanced transduction compared with their parental vectors in DSG2-high cell lines. Moreover, in DSG2-low cell lines, the JO4 vectors presented a rather weakened effect. Interestingly, DSG2-negative cell line MIA PaCa-2 even showed resistance to JO4 vector infection, possibly due to the negative effect of JO4 mutation on the usage of another Ad3 receptor: CD46. Together, our observations suggest that the JO4 vectors may have an advantage to prevent CD46-mediated sequestration, thereby achieving DSG2-specific transduction.

Bacteria, as well as eukaryotes, principally fungi, of the upper respiratory tract play key roles in the etiopathogenesis of respiratory diseases, whereas the potential role of archaea remains poorly understood. In this review, we discuss the contribution of all three domains of cellular life to human naso- and oropharyngeal microbiomes, i.e., bacterial microbiota, eukaryotes (mostly fungi), as well as the archaeome and their relation to respiratory and atopic disorders in infancy and adolescence. With this review, we aim to summarize state-of-the-art contributions to the field published in the last decade. In particular, we intend to build bridges between basic and clinical science.

To develop adenoviral cell- or tissue-specific gene delivery, understanding of the infection mechanisms of adenoviruses is crucial. Several adenoviral attachment proteins such as CD46, CAR and sialic acid have been identified and studied. However, most receptor studies were performed on non-human cells. Combining our reporter gene-tagged adenovirus library with an in vitro human gene knockout model, we performed a systematic analysis of receptor usage comparing different adenoviruses side-by-side. The CRISPR/Cas9 system was used to knockout CD46 and CAR in the human lung epithelial carcinoma cell line A549. Knockout cells were infected with 22 luciferase-expressing adenoviruses derived from adenovirus species B, C, D and E. HAdV-B16, -B21 and -B50 from species B1 as well as HAdV-B34 and -B35 were found to be CD46-dependent. HAdV-C5 and HAdV-E4 from species E were found to be CAR-dependent. Regarding cell entry of HAdV-B3 and -B14 and all species D viruses, both CAR and CD46 play a role, and here, other receptors or attachment structures may also be important since transductions were reduced but not completely inhibited. The established human knockout cell model enables the identification of the most applicable adenovirus types for gene therapy and to further understand adenovirus infection biology.

The human adenovirus type C5 (HAdV-C5) E1B-55K protein is a multifunctional regulator of HAdV-C5 replication, participating in many processes required for maximal virus production. Its multifunctional properties are primarily regulated by post-translational modifications (PTMs). The most influential E1B-55K PTMs are phosphorylation at highly conserved serine and threonine residues at the C-terminus, and SUMO conjugation to lysines 104 (K104) and 101 (K101) situated in the N-terminal region of the protein, which have been shown to regulate each other. Reversible SUMO conjugation provides a molecular switch that controls key functions of the viral protein, including intracellular trafficking and viral immune evasion. Interestingly, SUMOylation at SUMO conjugation site (SCS) K104 is negatively regulated by another multifunctional HAdV-C5 protein, E4orf6, which is known to form a complex with E1B-55K. To further evaluate the role of E4orf6 in the regulation of SUMO conjugation to E1B-55K, we analyzed different virus mutants expressing E1B-55K proteins with amino acid exchanges in both SCS (K101 and K104) in the presence or absence of E4orf6. We could exclude phosphorylation as factor for E4orf6-mediated reduction of E1B-55K SUMOylation. In fact, we demonstrate that a direct interaction between E1B-55K and E4orf6 is required to reduce E1B-55K SUMOylation. Additionally, we show that an E4orf6-mediated decrease of SUMO conjugation to K101 and K104 result in impaired co-localization of E1B-55K and SUMO in viral replication compartments. These findings indicate that E4orf6 inhibits E1B-55K SUMOylation, which could favor assembly of E4orf6-dependent E3 ubiquitin ligase complexes that are known to degrade a variety of host restriction factors by proteasomal degradation and, thereby, promote viral replication.

Respiratory viruses play an important role in asthma exacerbation, and early exposure can be involved in recurrent bronchitis and the development of asthma. The exact mechanism is not fully clarified, and pathogen-to-host interaction studies are warranted to identify biomarkers of exacerbation in the early phase. Only a limited number of international exacerbation cohorts were studied. Here, we have established a local pediatric exacerbation study in Germany consisting of children with asthma or chronic, recurrent bronchitis and analyzed the viriome within the nasopharyngeal swab specimens derived from the entire cohort (n = 141). Interestingly, 41% of exacerbated children had a positive test result for human rhinovirus (HRV)/human enterovirus (HEV), and 14% were positive for respiratory syncytial virus (RSV). HRV was particularly prevalent in asthmatics (56%), wheezers (50%), and atopic (66%) patients. Lymphocytes were decreased in asthmatics and in HRV-infected subjects, and patients allergic to house dust mites were more susceptible to HRV infection. Our study thus confirms HRV infection as a strong ‘biomarker’ of exacerbated asthma. Further longitudinal studies will show the clinical progress of those children with a history of an RSV or HRV infection. Vaccination strategies and novel treatment guidelines against HRV are urgently needed to protect those high-risk children from a serious course of disease.

Virotherapy research involves the development, exploration, and application of oncolytic viruses that combine direct killing of cancer cells by viral infection, replication, and spread (oncolysis) with indirect killing by induction of anti-tumor immune responses. Oncolytic viruses can also be engineered to genetically deliver therapeutic proteins for direct or indirect cancer cell killing. In this review—as part of the special edition on “State-of-the-Art Viral Vector Gene Therapy in Germany”—the German community of virotherapists provides an overview of their recent research activities that cover endeavors from screening and engineering viruses as oncolytic cancer therapeutics to their clinical translation in investigator-initiated and sponsored multi-center trials. Preclinical research explores multiple viral platforms, including new isolates, serotypes, or fitness mutants, and pursues unique approaches to engineer them towards increased safety, shielded or targeted delivery, selective or enhanced replication, improved immune activation, delivery of therapeutic proteins or RNA, and redirecting antiviral immunity for cancer cell killing. Moreover, several oncolytic virus-based combination therapies are under investigation. Clinical trials in Germany explore the safety and potency of virotherapeutics based on parvo-, vaccinia, herpes, measles, reo-, adeno-, vesicular stomatitis, and coxsackie viruses, including viruses encoding therapeutic proteins or combinations with immune checkpoint inhibitors. These research advances represent exciting vantage points for future endeavors of the German virotherapy community collectively aimed at the implementation of effective virotherapeutics in clinical oncology.

The importance of adenovirus (Ad) research is significantly increasing with respect to virotherapy for vaccine development, tumor, and gene therapy. Due to the different species and subtypes of this virus, the characterization of the biological significance of especially rare Ad is necessary. Previously, rare Ad types 70, 73, and 74 were originally isolated from fecal samples of immunocompromised patients and they represent recombinants of other Ad types. Here we investigated transduction experiments of these reporter gene tagged Ad types in primary cells exemplified by subject-derived primary nasal epithelial cells (NAEPCs). To analyze the transduction rates, we performed flow cytometry, quantitative polymerase chain reaction (PCR), and cytokine analyses 25 h post-infection. We found that, in contrast to Ad type 5 (as a positive control), the transduction rates of NAEPCs with Ad types 70, 73, and 74 were interestingly low. The major Ad receptor (coxsackievirus-adenovirus receptor and CD46) expression levels showed no significant change after infection with Ad types 70, 73 and 74. Moreover, Interleukin 6 (IL-6) was not released after in vitro Ad transduction. Due to the high risk of developing life-threatening complications in immunocompromised patients by these human species D Ads, even more attention needs to be investigated into the development of diagnostic and therapeutic concepts to prevent and treat those opportunistic infections in susceptible patients.

Understanding the response to viral infection in the context of respiratory diseases is of significant importance. Recently, there has been more focus on the role of the nasal epithelium in disease modeling. Here, we provide an overview of different submerged, organotypic 3D and spheroid cell culture models of nasal epithelial cells, which were used to study asthma and allergy with a special focus on virus infection. In detail, this review summarizes the importance, benefits, and disadvantages of patient-derived cell culture models of nasal- and bronchial epithelial cells, including a comparison of these cell culture models and a discussion on why investigators should consider using nasal epithelial cells in their research. Exposure experiments, simple virus transduction analyses as well as genetic studies can be performed in these models, which may provide first insights into the complexity of molecular signatures and may open new doors for drug discovery and biomarker research.

Adenovirus (AdV) infections in the respiratory tract may cause asthma exacerbation and allergic predisposition, and the house dust mite (HDM) may aggravate virus-induced asthma exacerbations. However, the underlying mechanisms of whether and how AdV affects asthmatic patients remains unclear. To address this question, we investigated nasal epithelial cells (NAEPCs) derived from a pediatric exacerbation study cohort for experimental analyses. We analyzed twenty-one different green-fluorescent protein- and luciferase-tagged AdV types in submerged 2D and organotypic 3D cell culture models. Transduction experiments revealed robust transduction of AdV type 5 (AdV5) in NAEPCs, which was associated with an increased uptake of AdV5 in the presence of HDM. In healthy and asthmatic NAEPCs exposed to HDM before infection, we observed a time- and dose-dependent increase of AdV5 uptake associated with upregulation of entry receptors for AdV5. Furthermore, electron microscopic and histologic analyses of 3D cell cultures revealed an impairment of the respiratory cilia after HDM exposition. This ex vivo pilot study shows the impact of AdV infection and HDM exposition in a primary cell culture model for asthma.

The turbulent history of clinical trials in viral gene therapy has taught us important lessons about vector design and safety issues. Much effort was spent on analyzing genotoxicity after somatic integration of therapeutic DNA into the host genome. Based on these findings major improvements in vector design including the development of viral hybrid vectors for somatic integration have been achieved. This review provides a state-of-the-art overview of available hybrid vectors utilizing viruses for high transduction efficiencies in concert with various integration machineries for random and targeted integration patterns. It discusses advantages but also limitations of each vector system.