Current state and future perspectives on de facto population markers for normalization in wastewater-based epidemiology: A systematic literature review.

Wastewater-based epidemiology (WBE) and wastewater surveillance have become a valuable complementary data source to collect information on community-wide exposure through the measurement of human biomarkers in influent wastewater (IWW). In WBE, normalization of data with the de facto population that corresponds to a wastewater sample is crucial for a correct interpretation of spatio-temporal trends in exposure and consumption patterns. However, knowledge gaps remain in identifying and validating suitable de facto population biomarkers (PBs) for refinement of WBE back-estimations. WBE studies that apply de facto PBs (including hydrochemical parameters, utility consumption data sources, endo- and exogenous chemicals, biological biomarkers and signalling records) for relative trend analysis and absolute population size estimation were systematically reviewed from three databases (PubMed, Web of Science, SCOPUS) according to the PRISMA guidelines. We included in this review 81 publications that accounted for daily variations in population sizes by applying de facto population normalization. To date, a wide range of PBs have been proposed for de facto population normalization, complicating the comparability of normalized measurements across WBE studies. Additionally, the validation of potential PBs is complicated by the absence of an ideal external validator, magnifying the overall uncertainty for population normalization in WBE. Therefore, this review proposes a conceptual tier-based cross-validation approach for identifying and validating de facto PBs to guide their integration for i) relative trend analysis, and ii) absolute population size estimation. Furthermore, this review also provides a detailed evaluation of the uncertainty observed when comparing different de jure and de facto population estimation approaches. This study shows that their percentual differences can range up to ±200 %, with some exceptions showing even larger variations. This review underscores the need for collaboration among WBE researchers to further streamline the application of de facto population normalization and to evaluate the robustness of different PBs in different socio-demographic communities.

The natural history of CVB3 myocarditis in C57BL/6J mice: an extended in-depth characterization.

BACKGROUND AND AIMS: Viral infections are the leading cause of myocarditis. Besides acute cardiac complications, late-stage sequelae such as myocardial fibrosis may develop, importantly impacting the prognosis. Coxsackievirus B3 (CVB)-induced myocarditis in mice is the most commonly used translational model to study viral myocarditis and has provided the majority of our current understanding of the disease pathophysiology. Nevertheless, the late stages of disease, encompassing fibrogenesis and arrhythmogenesis, have been underappreciated in viral myocarditis research to date. The present study investigated the natural history of CVB-induced myocarditis in C57BL/6J mice, expanding the focus beyond the acute phase of disease. In addition, we studied the impact of sex and inoculation dose on the disease course. METHODS AND RESULTS: C57BL/6J mice (12 weeks old; n=154) received a single intraperitoneal injection with CVB to induce viral myocarditis, or vehicle (PBS) as control. Male mice (n=92) were injected with 5 × 105 (regular dose)(RD) or 5 × 106 (high dose)(HD) plaque-forming units of CVB, whereas female mice received the RD only. Animals were sacrificed 1, 2, 4, 8 and 11 weeks after CVB or PBS injection. Virally inoculated mice developed viral disease with a temporary decline in general condition and weight loss, which was less pronounced in female animals (P<0.001). In male CVB mice, premature mortality occurred between days 8-23 after inoculation (RD: 21%, HD: 20%), whereas all female animals survived. Over the course of disease, cardiac inflammation progressively subsided, with faster resolution in female mice. There were no substantial group differences in the composition of the inflammatory cell infiltrates: predominance of cytotoxic T cells at day 7 and 14, and a switch from arginase1-reactive macrophages to iNOS-reactive macrophages from day 7 to 14 were the main findings. There was concomitant development and maturation of different patterns of myocardial fibrosis, with enhanced fibrogenesis in male mice. Virus was almost completely cleared from the heart by day 14. Serum biomarkers of cardiac damage and cardiac expression of remodeling genes were temporarily elevated during the acute phase of disease. Cardiac CTGF gene upregulation was less prolonged in female CVB animals. In vivo electrophysiology studies at weeks 8 and 11 demonstrated that under baseline conditions (i.e. in the absence of proarrhythmogenic drugs), ventricular arrhythmias could only be induced in CVB animals. The cumulative arrhythmia burden throughout the entire stimulation protocol was not significantly different between CVB and control groups. CONCLUSION: CVB inoculation in C57BL/6J mice represents a model of acute self-limiting viral myocarditis, with progression to different patterns of myocardial fibrosis. Sex, but not inoculation dose, seems to modulate the course of disease.

HPV-specific antibodies in female genital tract secretions captured via first-void urine retain their neutralizing capacity.

Human papillomavirus (HPV) vaccines, primarily relying on neutralizing antibodies, have proven highly effective. Recently, HPV-specific antibodies have been detected in the female genital tract secretions captured by first-void urine (FVU), offering a minimally invasive diagnostic approach. In this study, we investigated whether HPV16-specific antibodies present in FVU samples retain their neutralizing capacity by using pseudovirion-based neutralization assays. Paired FVU and serum samples (vaccinated n = 25, unvaccinated n = 25, aged 18-25) were analyzed using two orthogonal pseudovirion-based neutralization assays, one using fluorescence microscopy and the other using luminescence-based spectrophotometry. Results were compared with HPV16-specific IgG concentrations and correlations between neutralizing antibodies in FVU and serum were explored. The study demonstrated the presence of neutralizing antibodies in FVU using both pseudovirion-based neutralization assays, with the luminescence-based assay showing higher sensitivity for FVU samples, while the fluorescence microscopy-based assay exhibited better specificity for serum and overall higher reproducibility. High Spearman correlation values were calculated between HPV16-IgG and HPV16-neutralizing antibodies for both protocols (rs: 0.54-0.94, p < .001). Significant Spearman correlations between FVU and serum concentrations were also established for all assays (rs: 0.44-0.91, p < .01). This study demonstrates the continued neutralizing ability of antibodies captured with FVU, supporting the hypothesis that HPV vaccination may reduce autoinoculation and transmission risk to the sexual partner. Although further protocol optimizations are warranted, these findings provide a foundation for future research and larger cohort studies that could have implications for the optimal design, evaluation, and implementation of HPV vaccination programs.

Lack of functional TCR-epitope interaction is associated with herpes zoster through reduced downstream T cell activation.

The role of T cell receptor (TCR) diversity in infectious disease susceptibility is not well understood. We use a systems immunology approach on three cohorts of herpes zoster (HZ) patients and controls to investigate whether TCR diversity against varicella-zoster virus (VZV) influences the risk of HZ. We show that CD4+ T cell TCR diversity against VZV glycoprotein E (gE) and immediate early 63 protein (IE63) after 1-week culture is more restricted in HZ patients. Single-cell RNA and TCR sequencing of VZV-specific T cells shows that T cell activation pathways are significantly decreased after stimulation with VZV peptides in convalescent HZ patients. TCR clustering indicates that TCRs from HZ patients co-cluster more often together than TCRs from controls. Collectively, our results suggest that not only lower VZV-specific TCR diversity but also reduced functional TCR affinity for VZV-specific proteins in HZ patients leads to lower T cell activation and consequently affects the susceptibility for viral reactivation.

Achieving High-Precision, Low-Cost Microfluidic Chip Fabrication with Flexible PCB Technology.

Soft lithography has long remained the state of the art to generate the necessary micropatterning for molded microfluidic (MF) chips. Previous attempts to use printed circuit boards (PCBs) as a cheap and accessible alternative to expensive lithographed molds for the production of PDMS MF chip prototypes have shown their limitations. A more in-depth exploration of using PCBs as a mold substrate and a novel methodology of using flexible PCBs to produce highly accurate MF chips is reported here for the first time. Cross sections highlight the improved accuracy of this method, and peel testing is performed to demonstrate suitable adhesion between the glass substrate and PDMS cast. Positive cell growth viability showcases this novel method as a high-accuracy, high-accessibility, low-cost prototyping method for microfluidic chips while still maintaining all favorable properties provided by the PDMS material.

In vitro modelling of bacterial pneumonia: a comparative analysis of widely applied complex cell culture models.

Bacterial pneumonia greatly contributes to the disease burden and mortality of lower respiratory tract infections among all age groups and risk profiles. Therefore, laboratory modelling of bacterial pneumonia remains important for elucidating the complex host-pathogen interactions and to determine drug efficacy and toxicity. In vitro cell culture enables for the creation of high-throughput, specific disease models in a tightly controlled environment. Advanced human cell culture models specifically, can bridge the research gap between the classical two-dimensional cell models and animal models. This review provides an overview of the current status of the development of complex cellular in vitro models to study bacterial pneumonia infections, with a focus on air-liquid interface models, spheroid, organoid, and lung-on-a-chip models. For the wide scale, comparative literature search, we selected six clinically highly relevant bacteria (Pseudomonas aeruginosa, Mycoplasma pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Streptococcus pneumoniae, and Staphylococcus aureus). We reviewed the cell lines that are commonly used, as well as trends and discrepancies in the methodology, ranging from cell infection parameters to assay read-outs. We also highlighted the importance of model validation and data transparency in guiding the research field towards more complex infection models.

Unravelling the immune signature of herpes zoster: Insights into pathophysiology and the HLA risk profile.

The varicella-zoster virus (VZV) infects over 95% of the population. VZV reactivation causes herpes zoster (HZ), known as shingles, primarily affecting the elderly and immunocompromised individuals. However, HZ can also occur in otherwise healthy individuals. We analyzed the immune signature and risk profile in HZ patients using a genome-wide association study across different UK Biobank HZ cohorts. Additionally, we conducted one of the largest HZ HLA association studies to date, coupled with transcriptomic analysis of pathways underlying HZ susceptibility. Our findings highlight the significance of the MHC locus for HZ development, identifying five protective and four risk HLA alleles. This demonstrates that HZ susceptibility is largely governed by variations in the MHC. Furthermore, functional analyses revealed the upregulation of type I interferon and adaptive immune responses. These findings provide fresh molecular insights into the pathophysiology and the activation of innate and adaptive immune responses triggered by symptomatic VZV reactivation.

All Eyes on the Prefusion-Stabilized F Construct, but Are We Missing the Potential of Alternative Targets for Respiratory Syncytial Virus Vaccine Design?

Respiratory Syncytial Virus (RSV) poses a significant global health concern as a major cause of lower respiratory tract infections (LRTIs). Over the last few years, substantial efforts have been directed towards developing vaccines and therapeutics to combat RSV, leading to a diverse landscape of vaccine candidates. Notably, two vaccines targeting the elderly and the first maternal vaccine have recently been approved. The majority of the vaccines and vaccine candidates rely solely on a prefusion-stabilized conformation known for its highly neutralizing epitopes. Although, so far, this antigen design appears to be successful for the elderly, our current understanding remains incomplete, requiring further improvement and refinement in this field. Pediatric vaccines still have a long journey ahead, and we must ensure that vaccines currently entering the market do not lose efficacy due to the emergence of mutations in RSV's circulating strains. This review will provide an overview of the current status of vaccine designs and what to focus on in the future. Further research into antigen design is essential, including the exploration of the potential of alternative RSV proteins to address these challenges and pave the way for the development of novel and effective vaccines, especially in the pediatric population.

Diagnosing arthropod-borne flaviviruses: non-structural protein 1 (NS1) as a biomarker.

In recent decades, the presence of flaviviruses of concern for human health in Europe has drastically increased,exacerbated by the effects of climate change - which has allowed the vectors of these viruses to expand into new territories. Co-circulation of West Nile virus (WNV), Usutu virus (USUV), and tick-borne encephalitis virus (TBEV) represents a threat to the European continent, and this is further complicated by the difficulty of obtaining an early and discriminating diagnosis of infection. Moreover, the possibility of introducing non-endemic pathogens, such as Japanese encephalitis virus (JEV), further complicates accurate diagnosis. Current flavivirus diagnosis is based mainly on RT-PCR and detection of virus-specific antibodies. Yet, both techniques suffer from limitations, and the development of new assays that can provide an early, rapid, low-cost, and discriminating diagnosis of viral infection is warranted. In the pursuit of ideal diagnostic assays, flavivirus non-structural protein 1 (NS1) serves as an excellent target for developing diagnostic assays based on both the antigen itself and the antibodies produced against it. This review describes the potential of such NS1-based diagnostic methods, focusing on the application of flaviviruses that co-circulate in Europe.

Subtractive Immunization as a Method to Develop Respiratory Syncytial Virus (RSV)-Specific Monoclonal Antibodies.

Respiratory Syncytial Virus (RSV) is a significant cause of lower respiratory tract infections in the young, the elderly, and in immunodeficient patients. As such, the virus represents an important cause of morbidity and mortality worldwide. Development of monoclonal antibodies against RSV has resulted in a commercial prophylaxis, palivizumab (Synagis®), and different antibodies that have improved our understanding of the structure of the viral proteins. In this study, a different immunization technique, subtractive immunization, was evaluated for its applicability to develop RSV-specific antibodies. One hybridoma which produced antibodies with the strongest staining of RSV infected cells, ATAC-0025, was selected for further characterization. This antibody belongs to the IgG1 class, has neutralizing capacity and recognizes the envelope F-protein. The antibody has a broad reactivity against a range of RSV reference strains and clinical isolates.

In-depth biological characterization of two black soldier fly anti-Pseudomonas peptides reveals LPS-binding and immunomodulating effects.

As effector molecules of the innate immune system, antimicrobial peptides (AMPs) have gathered substantial interest as a potential future generation of antibiotics. Here, we demonstrate the anti-Pseudomonas activity and lipopolysaccharide (LPS)-binding ability of HC1 and HC10, two cecropin peptides from the black soldier fly (Hermetia Illucens). Both peptides are active against a wide range of Pseudomonas aeruginosa strains, including drug-resistant clinical isolates. Moreover, HC1 and HC10 can bind to lipid A, the toxic center of LPS and reduce the LPS-induced nitric oxide and cytokine production in murine macrophage cells. This suggests that the peptide-LPS binding can also lower the strong inflammatory response associated with P. aeruginosa infections. As the activity of AMPs is often influenced by the presence of salts, we studied the LPS-binding activity of HC1 and HC10 in physiological salt concentrations, revealing a strong decrease in activity. Our research confirmed the early potential of HC1 and HC10 as starting points for anti-Pseudomonas drugs, as well as the need for structural or formulation optimization before further preclinical development can be considered. IMPORTANCE The high mortality and morbidity associated with Pseudomonas aeruginosa infections remain an ongoing challenge in clinical practice that requires urgent action. P. aeruginosa mostly infects immunocompromised individuals, and its prevalence is especially high in urgent care hospital settings. Lipopolysaccharides (LPSs) are outer membrane structures that are responsible for inducing the innate immune cascade upon infection. P. aeruginosa LPS can cause local excessive inflammation, or spread systemically throughout the body, leading to multi-organ failure and septic shock. As antimicrobial resistance rates in P. aeruginosa infections are rising, the research and development of new antimicrobial agents remain indispensable. Especially, antimicrobials that can both kill the bacteria themselves and neutralize their toxins are of great interest in P. aeruginosa research to develop as the next generation of drugs.

The RSVPreF3-AS01 vaccine elicits broad neutralization of contemporary and antigenically distant respiratory syncytial virus strains.

The RSVPreF3-AS01 vaccine, containing the respiratory syncytial virus (RSV) prefusion F protein and the AS01 adjuvant, was previously shown to boost neutralization responses against historical RSV strains and to be efficacious in preventing RSV-associated lower respiratory tract diseases in older adults. Although RSV F is highly conserved, variation does exist between strains. Here, we characterized variations in the major viral antigenic sites among contemporary RSV sequences when compared with RSVPreF3 and showed that, in older adults, RSVPreF3-AS01 broadly boosts neutralization responses against currently dominant and antigenically distant RSV strains. RSV-neutralizing responses are thought to play a central role in preventing RSV infection. Therefore, the breadth of RSVPreF3-AS01-elicited neutralization responses may contribute to vaccine efficacy against contemporary RSV strains and those that may emerge in the future.

Time series modelling for wastewater-based epidemiology of COVID-19: A nationwide study in 40 wastewater treatment plants of Belgium, February 2021 to June 2022.

BACKGROUND: Wastewater-based epidemiology (WBE) has been implemented to monitor surges of COVID-19. Yet, multiple factors impede the usefulness of WBE and quantitative adjustment may be required. AIM: We aimed to model the relationship between WBE data and incident COVID-19 cases, while adjusting for confounders and autocorrelation. METHODS: This nationwide WBE study includes data from 40 wastewater treatment plants (WWTPs) in Belgium (02/2021-06/2022). We applied ARIMA-based modelling to assess the effect of daily flow rate, pepper mild mottle virus (PMMoV) concentration, a measure of human faeces in wastewater, and variants (alpha, delta, and omicron strains) on SARS-CoV-2 RNA levels in wastewater. Secondly, adjusted WBE metrics at different lag times were used to predict incident COVID-19 cases. Model selection was based on AICc minimization. RESULTS: In 33/40 WWTPs, RNA levels were best explained by incident cases, flow rate, and PMMoV. Flow rate and PMMoV were associated with -13.0 % (95 % prediction interval: -26.1 to +0.2 %) and +13.0 % (95 % prediction interval: +5.1 to +21.0 %) change in RNA levels per SD increase, respectively. In 38/40 WWTPs, variants did not explain variability in RNA levels independent of cases. Furthermore, our study shows that RNA levels can lead incident cases by at least one week in 15/40 WWTPs. The median population size of leading WWTPs was 85.1 % larger than that of non­leading WWTPs. In 17/40 WWTPs, however, RNA levels did not lead or explain incident cases in addition to autocorrelation. CONCLUSION: This study provides quantitative insights into key determinants of WBE, including the effects of wastewater flow rate, PMMoV, and variants. Substantial inter-WWTP variability was observed in terms of explaining incident cases. These findings are of practical importance to WBE practitioners and show that the early-warning potential of WBE is WWTP-specific and needs validation.

Lack of strong innate immune reactivity renders macrophages alone unable to control productive Varicella-Zoster Virus infection in an isogenic human iPSC-derived neuronal co-culture model.

With Varicella-Zoster Virus (VZV) being an exclusive human pathogen, human induced pluripotent stem cell (hiPSC)-derived neural cell culture models are an emerging tool to investigate VZV neuro-immune interactions. Using a compartmentalized hiPSC-derived neuronal model allowing axonal VZV infection, we previously demonstrated that paracrine interferon (IFN)-α2 signalling is required to activate a broad spectrum of interferon-stimulated genes able to counteract a productive VZV infection in hiPSC-neurons. In this new study, we now investigated whether innate immune signalling by VZV-challenged macrophages was able to orchestrate an antiviral immune response in VZV-infected hiPSC-neurons. In order to establish an isogenic hiPSC-neuron/hiPSC-macrophage co-culture model, hiPSC-macrophages were generated and characterised for phenotype, gene expression, cytokine production and phagocytic capacity. Even though immunological competence of hiPSC-macrophages was shown following stimulation with the poly(dA:dT) or treatment with IFN-α2, hiPSC-macrophages in co-culture with VZV-infected hiPSC-neurons were unable to mount an antiviral immune response capable of suppressing a productive neuronal VZV infection. Subsequently, a comprehensive RNA-Seq analysis confirmed the lack of strong immune responsiveness by hiPSC-neurons and hiPSC-macrophages upon, respectively, VZV infection or challenge. This may suggest the need of other cell types, like T-cells or other innate immune cells, to (co-)orchestrate an efficient antiviral immune response against VZV-infected neurons.

Risk Factors Associated with Severe RSV Infection in Infants: What Is the Role of Viral Co-Infections?

The respiratory syncytial virus (RSV) represents the leading cause of viral lower respiratory tract infections (LRTI) in children worldwide and is associated with significant morbidity and mortality rates. The clinical picture of an RSV infection differs substantially between patients, and the role of viral co-infections is poorly investigated. During two consecutive winter seasons from October 2018 until February 2020, we prospectively included children up to 2 years old presenting with an acute LRTI, both ambulatory and hospitalized. We collected clinical data and tested nasopharyngeal secretions for a panel of 16 different respiratory viruses with multiplex RT-qPCR. Disease severity was assessed with traditional clinical parameters and scoring systems. A total of 120 patients were included, of which 91.7% were RSV positive; 42.5% of RSV-positive patients had a co-infection with at least one other respiratory virus. We found that patients suffering from a single RSV infection had higher pediatric intensive care unit (PICU) admission rates (OR = 5.9, 95% CI = 1.53 to 22.74), longer duration of hospitalization (IRR = 1.25, 95% CI = 1.03 to 1.52), and a higher Bronchiolitis Risk of Admission Score (BRAS) (IRR = 1.31, 95% CI = 1.02 to 1.70) compared to patients with RSV co-infections. No significant difference was found in saturation on admission, O2 need, or ReSViNET-score. In our cohort, patients with a single RSV infection had increased disease severity compared to patients with RSV co-infections. This suggests that the presence of viral co-infections might influence the course of RSV bronchiolitis, but heterogeneity and small sample size in our study prevents us from drawing strong conclusions. IMPORTANCE RSV is worldwide the leading cause of serious airway infections. Up to 90% of children will be infected by the age of 2. RSV symptoms are mostly mild and typically mimic a common cold in older children and adolescents, but younger children can develop severe lower respiratory tract disease, and currently it is unclear why certain children develop severe disease while others do not. In this study, we found that children with a single RSV infection had a higher disease severity compared to patients with viral co-infections, suggesting that the presence of a viral co-infection could influence the course of an RSV bronchiolitis. As preventive and therapeutic options for RSV-associated disease are currently limited, this finding could potentially guide physicians to decide which patients might benefit from current or future treatment options early in the course of disease, and therefore, warrants further investigation.

Inactivation of SARS-CoV-2 and Other Enveloped and Non-Enveloped Viruses with Non-Thermal Plasma for Hospital Disinfection.

As recently highlighted by the SARS-CoV-2 pandemic, viruses have become an increasing burden for health, global economy, and environment. The control of transmission by contact with contaminated materials represents a major challenge, particularly in hospital environments. However, the current disinfection methods in hospital settings suffer from numerous drawbacks. As a result, several medical supplies that cannot be properly disinfected are not reused, leading to severe shortages and increasing amounts of waste, thus prompting the search for alternative solutions. In this work, we report that non-thermal plasma (NTP) can effectively inactivate SARS-CoV-2 from non-porous and porous materials commonly found in healthcare facilities. We demonstrated that 5 min treatment with a dielectric barrier discharge NTP can inactivate 100% of SARS-CoV-2 (Wuhan and Omicron strains) from plastic material. Using porcine respiratory coronavirus (surrogate for SARS-CoV-2) and coxsackievirus B3 (highly resistant non-enveloped virus), we tested the NTP virucidal activity on hospital materials and obtained complete inactivation after 5 and 10 min, respectively. We hypothesize that the produced reactive species and local acidification contribute to the overall virucidal effect of NTP. Our results demonstrate the potential of dielectric barrier discharge NTPs for the rapid, efficient, and low-cost disinfection of healthcare materials.

Simultaneous carriage of multiple serotypes of Group B Streptococcus: Systematic review and meta-analysis.

BACKGROUND: Epidemiological studies evaluating the distribution of Group B Streptococcus (GBS) serotypes are crucial for serotype-specific vaccine development and post-licensure surveillance. However, there is a paucity of data about the prevalence of simultaneous carriage of multiple serotypes. METHODS: We conducted a systematic review of three databases (Medline, Embase, PubMed) to identify studies reporting GBS serotype co-carriage at the same anatomical site (multiple serotypes in one sample) or different anatomical sites (paired samples from one individual with different serotypes). We conducted a random-effects meta-analysis to evaluate the prevalence of co-carriage. RESULTS: 18 articles met the inclusion criteria, representing at least 12,968 samples from 14 countries. In a random-effects meta-analysis, we identified that 10 % (95 % CI: 4-19) of the positive samples taken from one anatomical site have more than one serotype, and 11 % (95 % CI: 5-20) of positive participants with samples taken from two anatomical sites carried different serotypes. When reported, the number of serotypes simultaneously carried ranged from 1 to 4. The serotypes most often associated with co-carriage are III (20.3 %), V (20.3 %) and Ia (19.5 %). CONCLUSION: This systematic review demonstrates that co-carriage is a minor but definite phenomenon, but the data are too limited to give a precise picture of the current epidemiology. Co-colonisation detection needs to be taken into consideration in the design and methods of future GBS carriage surveillance studies to estimate and evaluate the potential for serotype replacement once vaccines are introduced.

Development of a live biotherapeutic throat spray with lactobacilli targeting respiratory viral infections.

Respiratory viruses such as influenza viruses, respiratory syncytial virus (RSV), and coronaviruses initiate infection at the mucosal surfaces of the upper respiratory tract (URT), where the resident respiratory microbiome has an important gatekeeper function. In contrast to gut-targeting administration of beneficial bacteria against respiratory viral disease, topical URT administration of probiotics is currently underexplored, especially for the prevention and/or treatment of viral infections. Here, we report the formulation of a throat spray with live lactobacilli exhibiting several in vitro mechanisms of action against respiratory viral infections, including induction of interferon regulatory pathways and direct inhibition of respiratory viruses. Rational selection of Lactobacillaceae strains was based on previously documented beneficial properties, up-scaling and industrial production characteristics, clinical safety parameters, and potential antiviral and immunostimulatory efficacy in the URT demonstrated in this study. Using a three-step selection strategy, three strains were selected and further tested in vitro antiviral assays and in formulations: Lacticaseibacillus casei AMBR2 as a promising endogenous candidate URT probiotic with previously reported barrier-enhancing and anti-pathogenic properties and the two well-studied model strains Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum WCFS1 that display immunomodulatory capacities. The three strains and their combination significantly reduced the cytopathogenic effects of RSV, influenza A/H1N1 and B viruses, and HCoV-229E coronavirus in co-culture models with bacteria, virus, and host cells. Subsequently, these strains were formulated in a throat spray and human monocytes were employed to confirm the formulation process did not reduce the interferon regulatory pathway-inducing capacity. Administration of the throat spray in healthy volunteers revealed that the lactobacilli were capable of temporary colonization of the throat in a metabolically active form. Thus, the developed spray with live lactobacilli will be further explored in the clinic as a potential broad-acting live biotherapeutic strategy against respiratory viral diseases.

T cell immunity in HSV-1- and VZV-infected neural ganglia.

Herpesviruses hijack the MHC class I (MHC I) and class II (MHC II) antigen-presentation pathways to manipulate immune recognition by T cells. First, we illustrate herpes simplex virus-1 (HSV-1) and varicella-zoster virus (VZV) MHC immune evasion strategies. Next, we describe MHC-T cell interactions in HSV-1- and VZV- infected neural ganglia. Although studies on the topic are scarce, and use different models, most reports indicate that neuronal HSV-1 infection is mainly controlled by CD8+ T cells through noncytolytic mechanisms, whereas VZV seems to be largely controlled through CD4+ T cell-specific immune responses. Autologous human stem-cell-derived in vitro models could substantially aid in elucidating these neuroimmune interactions and are fit for studies on both herpesviruses.

Activation of Interferon-Stimulated Genes following Varicella-Zoster Virus Infection in a Human iPSC-Derived Neuronal In Vitro Model Depends on Exogenous Interferon-α.

Varicella-zoster virus (VZV) infection of neuronal cells and the activation of cell-intrinsic antiviral responses upon infection are still poorly understood mainly due to the scarcity of suitable human in vitro models that are available to study VZV. We developed a compartmentalized human-induced pluripotent stem cell (hiPSC)-derived neuronal culture model that allows axonal VZV infection of the neurons, thereby mimicking the natural route of infection. Using this model, we showed that hiPSC-neurons do not mount an effective interferon-mediated antiviral response following VZV infection. Indeed, in contrast to infection with Sendai virus, VZV infection of the hiPSC-neurons does not result in the upregulation of interferon-stimulated genes (ISGs) that have direct antiviral functions. Furthermore, the hiPSC-neurons do not produce interferon-α (IFNα), a major cytokine that is involved in the innate antiviral response, even upon its stimulation with strong synthetic inducers. In contrast, we showed that exogenous IFNα effectively limits VZV spread in the neuronal cell body compartment and demonstrated that ISGs are efficiently upregulated in these VZV-infected neuronal cultures that are treated with IFNα. Thus, whereas the cultured hiPSC neurons seem to be poor IFNα producers, they are good IFNα responders. This could suggest an important role for other cells such as satellite glial cells or macrophages to produce IFNα for VZV infection control.