Influenza A virus (IAV) infection is a serious respiratory disease associated with significant morbidity and mortality worldwide. Annual vaccination is the most effective way to prevent infection and its potentially severe complications; however, the vaccines currently offered have several drawbacks that limit its availability and protective efficacy. Influenza virus-like particles (VLPs), which lack viral genetic material and are non-infectious, represent a promising vaccine candidate. Previous reports have shown VLPs are more immunogenic than subunit or recombinant proteins, and confer protection upon lethal challenge. A critical component of this protection is mediated by influenza HA-specific neutralizing Abs produced by memory B cells and plasma cells, the cellular products of the germinal center (GC) reaction. While preliminary studies have examined the humoral immune response to VLP vaccination, the current study is the first to characterize the GC response in secondary and tertiary lymphoid tissues. Mice were vaccinated with influenza VLPs using three immunization routes: subcutaneous (s.c.), intramuscular (i.m.), and intranasal (i.n.) and the GC response was assessed over time. Robust GC reactions were induced in the dLNs regardless of vaccination route, though the largest response was generated with VLPs s.c. The pattern of isotype expression was remarkably similar between routes, with IgM+ and IgG2+ B cells representing the majority of the GC B cell population. Mucosal immune responses in the upper (nasal) and lower (lung) airway were measured in mice vaccinated i.n. Marked GC reactions were induced in the nasal-associated lymphoid tissue (NALT), while the pulmonary response was relatively modest and short-lived compared to infection with IAV. Within the GC B cell population, IgM+ and IgG2+ B cells made up the majority, similar to the dLN response. Importantly, the pattern of isotype expression induced by VLPs mimicked the response induced by natural IAV infection, and suggests that VLPs contain the necessary innate immune agonists to induce a TH1 biased response.

Introduction: les follicules lymphoïdes (FL) sont absents du poumon normal mais ont été décrits dans les poumons de patients atteints de mucoviscidose ou de dilatations de bronches non mucoviscidosiques, suggérant un rôle pour l’infection bronchique dans la néogenèse lymphoïde (NL). Nous avons étudié la dynamique de la néogenèse lymphoïde dans l’infection bactérienne. Méthodes: les souris C57BL/6 ont reçu une instillation intratrachéale de billes d’agarose contenant du PAO1 ou du S. aureus (106 CFU/animal) permettant une infection prolongée et ont été comparées à des souris contrôles (billes stériles ou absence de billes). Les souris ont été sacrifiées à J1, J4, J7 et J14. Résultats: l’instillation unique de billes d’agarose contenant du PAO1 ou du S. aureus induit en 14 jours des FL fonctionnels situés sous l’épithélium en regard des zones d’infection. Le marquage pour CXCL12 et CXCL13 est faible chez contrôles, mais présent dans l’épithélium (CXCL13) dès J1 et présent également dans les FL (CXCL12 et CXCL13) à J14 chez les souris infectées. Le traitement des souris par un anticorps anti CXCL12 ou anti CXCL13 n’inhibe pas la formation des FL induite par l’infection à PAO1. Conclusion: nos données suggèrent un rôle pour l’infection bactérienne prolongée et l’épithélium respiratoire dans la NL des bronchopathies chroniques. Notre modèle permet d’évaluer les mécanismes de la formation et de persistance des FL dans le poumon. / Introduction: lymphoid follicles (LF) are absent in normal lungs, but are described in lungs of subjects with cystic fibrosis (CF) or non-CF bronchiectasis, suggesting a role for bacterial infection in lymphoid neogenesis. We aimed to study the dynamic of pulmonary lymphoid neogenesis (LN) during bacterial infection. Methods: C57BL/6 mice were instilled intratracheally with PAO1- or S. aureus-coated (1.106 CFU/mouse) agarose beads (which produced prolonged airway infection) and compared to controls (sterile beads or no instillation). Mice were sacrificed on day (d)1, d4, d7, and d14 after instillation. Results: chronic pulmonary infection with PAO1 or S. aureus induced organised LF in 14 days after a single challenge with PAO1- or S. aureus-coated beads. Bacteria- induced LF were exclusively localized in the subepithelium of infected airways. Staining for CXCL12 and CXCL13 was weak in airway epithelium of controls, but was positive in airway epithelium (CXCL13) at 1 day and in LF (both) of infected mice at 14 days. Treatment with anti CXCL12 or anti CXCL13 Ab did not reduce LN induced by PAO1 infection. Conclusions: chronic bacterial infection and respiratory epithelium could contribute to LN in chronic airway diseases. Our unique model allows to study mechanisms for the formation and maintenance of lung LF.

Influenza is a contagious respiratory disease that has caused at least four pandemics and countless epidemics since the 20th century, impacted millions of people worldwide and the global economy. To date, the predominant influenza species circulating in humans are influenza A and B. Influenza may cause serious illness in all age groups but individuals such as the newborns and senior population whose immune systems are compromised are at higher risk for severe disease. Interestingly, during the outbreak of pandemic 2009 H1N1 (H1N1pdm), it was found that the elderly had the lowest hospitalization rate and an increased proportion of healthy adults developed severe disease. Furthermore, several clinical studies have demonstrated that most H1N1pdm infected children experienced mild to moderate illness and led to the least mortality. The difference of disease outcome in age groups between different influenza infections may be due to several factors, which include differing pathogenicity between the viruses, differential immune status and composition among the age groups, and pre-existing immunity from previous encounter(s) with a similar virus. Since the human clinical data are often complicated by secondary factors such as co-morbidities, I used the ferret model to address these questions. I first compared the clinical and pathological patterns among the pandemic and seasonal influenza strains and found H1N1pdm caused the most severe illness to healthy ferrets. Importantly, the disease severity did not correlate with viral burden but immunopathology. To study the age effect, I found that H1N1pdm infected young ferrets with mild clinical symptoms developed specialized ectopic lymphoid structures and a distinct cytokine expression profile in the lungs, which were absent in adult ferrets with severe illness. I also examined antigenic change in historical H1N1s and anti-H1 responses to explain the pre-existing immunity of H1N1pdm found in the elderly. However, low similarity was found between historical H1N1s and H1N1pdm. Lastly, I conducted a detailed influenza B comparative study. I observed the pathogenic B strain was capable to cause lower respiratory tract infection and pathology like the influenza A viruses. Overall, this thesis provides novel insights for developing therapeutic and prophylactic strategies against influenza infection.

Influenza is a contagious respiratory disease that has caused at least four pandemics and countless epidemics since the 20th century, impacted millions of people worldwide and the global economy. To date, the predominant influenza species circulating in humans are influenza A and B. Influenza may cause serious illness in all age groups but individuals such as the newborns and senior population whose immune systems are compromised are at higher risk for severe disease. Interestingly, during the outbreak of pandemic 2009 H1N1 (H1N1pdm), it was found that the elderly had the lowest hospitalization rate and an increased proportion of healthy adults developed severe disease. Furthermore, several clinical studies have demonstrated that most H1N1pdm infected children experienced mild to moderate illness and led to the least mortality. The difference of disease outcome in age groups between different influenza infections may be due to several factors, which include differing pathogenicity between the viruses, differential immune status and composition among the age groups, and pre-existing immunity from previous encounter(s) with a similar virus. Since the human clinical data are often complicated by secondary factors such as co-morbidities, I used the ferret model to address these questions. I first compared the clinical and pathological patterns among the pandemic and seasonal influenza strains and found H1N1pdm caused the most severe illness to healthy ferrets. Importantly, the disease severity did not correlate with viral burden but immunopathology. To study the age effect, I found that H1N1pdm infected young ferrets with mild clinical symptoms developed specialized ectopic lymphoid structures and a distinct cytokine expression profile in the lungs, which were absent in adult ferrets with severe illness. I also examined antigenic change in historical H1N1s and anti-H1 responses to explain the pre-existing immunity of H1N1pdm found in the elderly. However, low similarity was found between historical H1N1s and H1N1pdm. Lastly, I conducted a detailed influenza B comparative study. I observed the pathogenic B strain was capable to cause lower respiratory tract infection and pathology like the influenza A viruses. Overall, this thesis provides novel insights for developing therapeutic and prophylactic strategies against influenza infection.