Regulation of pulmonary plasma cell responses during secondary infection with influenza virus

During secondary infection with influenza virus, plasma cells (PCs) develop within the lung, providing a local source of antibodies. However, the site and mechanisms that regulate this process are poorly defined. Here, we show that while circulating memory B cells entered the lung during rechallenge and were activated within inducible bronchus-associated lymphoid tissues (iBALTs), resident memory B (BRM) cells responded earlier, and their activation occurred in a different niche: directly near infected alveoli. This process required NK cells but was largely independent of CD4 and CD8 T cells. Innate stimuli induced by virus-like particles containing ssRNA triggered BRM cell differentiation in the absence of cognate antigen, suggesting a low threshold of activation. In contrast, expansion of PCs in iBALTs took longer to develop and was critically dependent on CD4 T cells. Our work demonstrates that spatially distinct mechanisms evolved to support pulmonary secondary PC responses, and it reveals a specialized function for BRM cells as guardians of the alveoli

Influenza A Virus: From Infection to Prevention. Long-term Effects of an Early Life IAV Infection in vivo and Optimization of the Live Attenuated Influenza A Vaccine Backbone Using Mouse Models

Influenza A virus (IAV) is one of the major causative agents of acute respiratory infections in humans. Influenza disease causes up to 650,000 deaths every year with its burden highest among high-risk populations. A major complication during IAV infections is the increased susceptibility to bacterial secondary infections, however the impact on the commensal bacterial community is still poorly understood. This is particularly important during childhood, when IAV infections are frequent and the microbiota community is still developing. Vaccination is the best way to prevent influenza disease. The live attenuated influenza vaccine (LAIV) provides good protection and induces mucosal responses important in blocking transmission. However, due to safety concerns, key target populations that could benefit from the advantages of this vaccine, are left out. The aim of this thesis was two-fold. To optimize the current LAIV backbone and increase its safety profile and to explore the effects of a childhood IAV infection on the development of the host microbiota and their impact in adult life using a mouse model. To address the first aim, we performed targeted mutagenesis on the backbone of the LAIV and rescued an optimized LAIV (optiLAIV) with a higher attenuation degree. We characterized optiLAIV in two mouse models representing infants under 2-years old and highly susceptible populations to viral infections. In neonatal mice, we showed that optiLAIV is cleared faster from the upper respiratory tract (URT) while still maintaining its protective ability against two challenge models. Additionally, we showed that in adult mice lacking a key player of the interferon signaling response, optiLAIV presented reduced replication in the lower respiratory tract (LRT) and caused no signs of morbidity compared to a 50% mortality rate observed in animals vaccinated with LAIV. OptiLAIV induced UPR-related genes in a human nasal epithelial tissue model suggesting this pathway as a potential mechanism of attenuation. Our results present an optimized LAIV candidate that could be explored as a safer alternative to the licensed LAIV in high-risk patient groups. To address the second aim, we used a neonatal mouse model, in which we report that a single subclinical IAV infection leads to a significant decrease in the bacterial abundance of the small intestine in adulthood. This observation was accompanied by an enrichment of Enterobacteriales and a reduction of Clostridiales. Furthermore, IAV-imprinted male animals had an increased body weight and lower energy expenditure compared to mock imprinted animals. Cohousing experiments abrogated the body weight differences observed while a highfat-high-sucrose diet enhanced the phenotype, suggesting a microbiota-dependent effect. Our results show that an acute respiratory infection during childhood induces long-term dysbiosis with possible consequences on host metabolic processes. Together, our findings highlight the importance of preventing IAV infections during childhood and propose an alternative strategy to do so.</p

Visualization of respiratory commensal bacteria in context of their natural host environment

Commensal microbes are an integral component of mammalian physiology. 16S rRNA gene-specific next generation sequencing from DNA of total organs, swabs or lavages has revolutionized the characterization of bacterial communities in virtually every ecological niche of the body. Culturomics, next allowed the isolation and characterization of commensal bacteria in the lab and the establishment of artificial communities of bacteria, which were eventually reintroduced in model organisms. Spatial organization of microbiota within a given host environment is critical to the physiological or pathological phenotypes provoked by commensal microbiota. In situ hybridization (ISH) is a complementary technique to sequencing and culturing to visualize the presence of individual bacterial operational taxonomic unit (OTUs) in context of the colonized organ. We recently applied highly sensitive in situ RNA hybridization to detection of commensal bacteria in low abundance respiratory tract samples of mice housed under specific pathogen free conditions. This technique allows species-specific detection of living bacteria using RNAScopeTM technology, while preserving the natural environment of the organ. We here provide a detailed step-by-step protocol describing the detection of commensal lung bacteria in respiratory tissue

A single respiratory tract infection early in life reroutes healthy microbiome development and affects adult metabolism in a preclinical animal model

In adult animals, acute viral infections only temporarily alter the composition of both respiratory and intestinal commensal microbiota, potentially due to the intrinsic stability of this microbial ecosystem. In stark contrast, commensal bacterial communities are rather vulnerable to perturbation in infancy. Animal models proved that disruption of a balanced microbiota development e.g., by antibiotics treatment early in life, increases the probability for metabolic disorders in adults. Importantly, infancy is also a phase in life with high incidence of acute infections. We postulated that acute viral infections in early life might pose a similarly severe perturbation and permanently shape microbiota composition with long-term physiological consequences for the adult host. As a proof of concept, we infected infant mice with a sub-lethal dose of influenza A virus. We determined microbiota composition up to early adulthood (63 days) from small intestine by 16S rRNA gene-specific next-generation sequencing. Infected mice underwent long-lasting changes in microbiota composition, associated with increase in fat mass. High-fat-high-glucose diet promoted this effect while co-housing with mock-treated animals overwrote the weight gain. Our data suggest that in the critical phase of infancy even a single silent viral infection could cast a long shadow and cause long-term microbiota perturbations, affecting adult host physiology

Optimizing the live attenuated influenza A vaccine backbone for high-risk patient groups

Together with inactivated influenza vaccines (IIV), live attenuated influenza vaccines (LAIV) are an important tool to prevent influenza A virus (IAV) illnesses in patients. LAIVs present the advantages to have a needle-free administration and to trigger a mucosal immune response. LAIV is approved for healthy 2- to 49-year old individuals. However, due to its replicative nature and higher rate of adverse events at-risk populations are excluded from the benefits of this vaccine. Using targeted mutagenesis, we modified the nonstructural protein 1 of the currently licensed LAIV in order to impair its ability to bind the host cellular protein CPSF30 and thus its ability to inhibit host mRNA poly-adenylation. We characterized our optimized LAIV (optiLAIV) in three different mouse models mimicking healthy and high-risk patients. Using a neonatal mouse model, we show faster clearance of our optimized vaccine compared to the licensed LAIV. Despite lower replication, optiLAIV equally protected mice against homosubtypic and hetesubtypic influenza strain challenges. We confirmed the safer profile of optiLAIV in Stat1-/-mice (highly susceptible to viral infections) by showing no signs of morbidity compared to a 50% mortality rate observed following LAIV inoculation. Using a human nasal 3D tissue model, we showed an increased induction of ER stress-related genes following immunization with optiLAIV. Induction of ER stress was previously shown to improve antigen-specific immune responses and is proposed as the mechanism of action of the licensed adjuvant AS03. This study characterizes a safer LAIV candidate in two mouse models mimicking infants and severely immunocompromised patients and proposes a simple attenuation strategy that could broaden LAIV application and reduce influenza burden in high-risk populations.IMPORTANCELive attenuated influenza vaccine (LAIV) is a needle-free, mucosal vaccine approved for healthy 2- to 49-year old individuals. Its replicative nature and higher rate of adverse events excludes at-risk populations. We propose a strategy to improve LAIV safety and explore the possibility to expand its applications in children under 2-year old and immunocompromised patients. Using a neonatal mouse model, we show faster clearance of our optimized vaccine (optiLAIV) compared to the licensed LAIV. Despite lower replication, optiLAIV equally protected mice against influenza virus challenges. We confirmed the safer profile of optiLAIV in Stat1-/-mice (highly susceptible to viral infections) by showing no signs of morbidity compared to a 50% mortality rate from LAIV. OptiLAIV could expand the applications of the current LAIV and help mitigate the burden of IAV in susceptible populations

Silent neonatal influenza A virus infection primes systemic antimicrobial immunity

Infections with influenza A viruses (IAV) cause seasonal epidemics and global pandemics. The majority of these infections remain asymptomatic, especially among children below five years of age. Importantly, this is a time, when immunological imprinting takes place. Whether early-life infections with IAV affect the development of antimicrobial immunity is unknown. Using a preclinical mouse model, we demonstrate here that silent neonatal influenza infections have a remote beneficial impact on the later control of systemic juvenile-onset and adult-onset infections with an unrelated pathogen, Staphylococcus aureus , due to improved pathogen clearance and clinical resolution. Strategic vaccination with a live attenuated IAV vaccine elicited a similar protection phenotype. Mechanistically, the IAV priming effect primarily targets antimicrobial functions of the developing innate immune system including increased antimicrobial plasma activity and enhanced phagocyte functions and antigen-presenting properties at mucosal sites. Our results suggest a long-term benefit from an exposure to IAV during the neonatal phase, which might be exploited by strategic vaccination against influenza early in life to enforce the host’s resistance to later bacterial infections. </p

Influenza A viruses limit NLRP3-NEK7-complex formation and pyroptosis in human macrophages

Pyroptosis is a fulminant form of macrophage cell death, contributing to release of pro-inflammatory cytokines. In humans, it depends on caspase 1/4-activation of gasdermin D and is characterized by the release of cytoplasmic content. Pathogens apply strategies to avoid or antagonize this host response. We demonstrate here that a small accessory protein (PB1-F2) of contemporary H5N1 and H3N2 influenza A viruses (IAV) curtails fulminant cell death of infected human macrophages. Infection of macrophages with a PB1-F2-deficient mutant of a contemporary IAV resulted in higher levels of caspase-1 activation, cleavage of gasdermin D, and release of LDH and IL-1β. Mechanistically, PB1-F2 limits transition of NLRP3 from its auto-repressed and closed confirmation into its active state. Consequently, interaction of a recently identified licensing kinase NEK7 with NLRP3 is diminished, which is required to initiate inflammasome assembly