Pseudo-affinity purification and formulation of a cell-culture derived whole influenza virus vaccine using magnetic sulfated cellulose particles

The production of viral vaccines usually employs different unit operations where formulation and filling are the final steps of downstream processing (DSP). However, complex DSP is often hard to realize in research laboratories focusing on novel vaccine candidates. Moreover, there are no real ready-to-use tools for high-throughput DSP of whole virus particles that can speed up development. Because of these needs we developed a new platform for easy and straightforward whole virus particle purification and formulation based on magnetic sulfated cellulose particles (MSCP)1,2. Proof of concept was carried out with an influenza A/Puerto Rico/8/34 (H1N1) whole virus vaccine for the immunization of mice. The virus particles were produced in suspension MDCK cells, clarified, inactivated, and concentrated using a standard protocol. After diafiltration to low salt buffer, the virus particles were bound to the MSCP and the virus loaded MSCP were washed and resuspended in formulation buffer. Please click Additional Files below to see the full abstract

In vivo Neutralization of Pro-inflammatory Cytokines During Secondary Streptococcus pneumoniae Infection Post Influenza A Virus Infection

An overt pro-inflammatory immune response is a key factor contributing to lethal pneumococcal infection in an influenza pre-infected host and represents a potential target for therapeutic intervention. However, there is a paucity of knowledge about the level of contribution of individual cytokines. Based on the predictions of our previous mathematical modeling approach, the potential benefit of IFN-γ- and/or IL-6-specific antibody-mediated cytokine neutralization was explored in C57BL/6 mice infected with the influenza A/PR/8/34 strain, which were subsequently infected with the Streptococcus pneumoniae strain TIGR4 on day 7 post influenza. While single IL-6 neutralization had no effect on respiratory bacterial clearance, single IFN-γ neutralization enhanced local bacterial clearance in the lungs. Concomitant neutralization of IFN-γ and IL-6 significantly reduced the degree of pneumonia as well as bacteremia compared to the control group, indicating a positive effect for the host during secondary bacterial infection. The results of our model-driven experimental study reveal that the predicted therapeutic value of IFN-γ and IL-6 neutralization in secondary pneumococcal infection following influenza infection is tightly dependent on the experimental protocol while at the same time paving the way toward the development of effective immune therapies

Mitochondria:at the crossroads of regulating lung epithelial cell function in chronic obstructive pulmonary disease

Disturbances in mitochondrial structure and function in lung epithelial cells have been implicated in the pathogenesis of various lung diseases, including chronic obstructive pulmonary disease (COPD). Such disturbances affect not only cellular energy metabolism but also alter a range of indispensable cellular homeostatic functions in which mitochondria are known to be involved. These range from cellular differentiation, cell death pathways, and cellular remodeling to physical barrier function and innate immunity. all of which are known to be impacted by exposure to cigarette smoke and have been linked to COPD pathogenesis. Next to their well-established role as the first physical frontline against external insults, lung epithelial cells are immunologically active. Malfunctioning epithelial cells with defective mitochondria are unable to maintain homeostasis and respond adequately to further stress or injury, which may ultimately shape the phenotype of lung diseases. In this review, we provide a comprehensive overview of the impact of cigarette smoke on the development of mitochondrial dysfunction in the lung epithelium and highlight the consequences for cell function, innate immune responses, epithelial remodeling, and epithelial barrier function in COPD. We also discuss the applicability and potential therapeutic value of recently proposed strategies for the restoration of mitochondrial function in the treatment of COPD

Characterization of Maladaptive Processes in Acute, Chronic and Remission Phases of Experimental Colitis in C57BL/6 Mice

Inflammatory bowel disease (IBD) is a chronic recurrent inflammatory disease with unknown etiology. Dextran sulfate sodium (DSS) induced colitis is a widely used mouse model in IBD research. DSS colitis involves activation of the submucosal immune system and can be used to study IBD-like disease characteristics in acute, chronic, remission and transition phases. Insight into colon inflammatory parameters is needed to understand potentially irreversible adaptations to the chronification of colitis, determining the baseline and impact of further inflammatory episodes. We performed analyses of non-invasive and invasive colitis parameters in acute, chronic and remission phases of the DSS colitis in C57BL/6 mice. Non-invasive colitis parameters poorly reflected inflammatory aspects of colitis in chronic remission phase. We found invasive inflammatory parameters, positively linked to repeated DSS-episodes, such as specific colon weight, inflamed colon area, spleen weight, absolute cell numbers of CD4+ and CD8+ T cells as well as B cells, blood IFN-γ level, colonic chemokines BLC and MDC as well as the prevalence of Turicibacter species in feces. Moreover, microbial Lactobacillus species decreased with chronification of disease. Our data point out indicative parameters of recurrent gut inflammation in context of DSS colitis

Clostridioides difficile Activates Human Mucosal-Associated Invariant T Cells

Clostridioides difficile infection (CDI) causes severe inflammatory responses at the intestinal mucosa but the immunological mechanisms underlying CDI-related immunopathology are still incompletely characterized. Here we identified for the first time that both, non-toxigenic strains as well as the hypervirulent ribotypes RT027 and RT023 of Clostridioides difficile (formerly Clostridium difficile), induced an effector phenotype in mucosal-associated invariant T (MAIT) cells. MAIT cells can directly respond to bacterial infections by recognizing MR1-presented metabolites derived from the riboflavin synthesis pathway constituting a novel class of antigens. We confirmed functional riboflavin synthesis of C. difficile and found fixed bacteria capable of activating primary human MAIT cells in a dose-dependent manner. C. difficile-activated MAIT cells showed an increased and MR1-dependent expression of CD69, proinflammatory IFNγ, and the lytic granule components granzyme B and perforin. Effector protein expression was accompanied by the release of lytic granules, which, in contrast to other effector functions, was mainly induced by IL-12 and IL-18. Notably, this study revealed hypervirulent C. difficile strains to be most competent in provoking MAIT cell responses suggesting MAIT cell activation to be instrumental for the immunopathology observed in C. difficile-associated colitis. In conclusion, we provide first evidence for a link between C. difficile metabolism and innate T cell-mediated immunity in humans

Presence of Infected Gr-1intCD11bhiCD11cint Monocytic Myeloid Derived Suppressor Cells Subverts T Cell Response and Is Associated With Impaired Dendritic Cell Function in Mycobacterium avium-Infected Mice

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunomodulatory function. To study the mechanism by which MDSC affect antimicrobial immunity, we infected mice with two M. avium strains of differential virulence, highly virulent Mycobacterium avium subsp. avium strain 25291 (MAA) and low virulent Mycobacterium avium subsp. hominissuis strain 104 (MAH). Intraperitoneal infection with MAA, but not MAH, caused severe disease and massive splenic infiltration of monocytic MDSC (M-MDSC; Gr-1intCD11bhiCD11cint) expressing inducible NO synthase (Nos2) and bearing high numbers of mycobacteria. Depletion experiments demonstrated that M-MDSC were essential for disease progression. NO production by M-MDSC influenced antigen-uptake and processing by dendritic cells and proliferation of CD4+ T cells. M-MDSC were also induced in MAA-infected mice lacking Nos2. In these mice CD4+ T cell expansion and control of infection were restored. However, T cell inhibition was only partially relieved and arginase (Arg) 1-expressing M-MDSC were accumulated. Likewise, inhibition of Arg1 also partially rescued T cell proliferation. Thus, mycobacterial virulence results in the induction of M-MDSC that block the T cell response in a Nos2- and Arg1-dependent manner

CD47 restricts antiviral function of alveolar macrophages during influenza virus infection

CD47 is an ubiquitously expressed surface molecule with significant impact on immune responses. However, its role for antiviral immunity is not fully understood. Here, we revealed that the expression of CD47 on immune cells seemed to disturb the antiviral immune response as CD47-deficient mice (CD47−/−) showed an augmented clearance of influenza A virus (IAV). Specifically, we have shown that enhanced viral clearance is mediated by alveolar macrophages (aMФ). Although aMФ displayed upregulation of CD47 expression during IAV infection in wildtype mice, depletion of aMФ in CD47−/− mice during IAV infection reversed the augmented viral clearance. We have also demonstrated that CD47 restricts hemoglobin (HB) expression in aMФ after IAV and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, with HB showing antiviral properties by enhancing the IFN-β response. Our study showed a negative role for CD47 during antiviral immune responses in the lung by confining HB expression in aMФ