Inflammation-induced thrombosis is a common consequence of bacterial infections, such as those caused by Salmonella Typhimurium (STm). The presentation of multi-organ thrombosis post-infection that develops and resolves with organ-specific kinetics raises significant challenges for its therapeutic control. Here, we identify specific inflammatory events driving thrombosis in the spleens and livers of STm-infected mice. IFN-γ or platelet expression of C-type lectin-like receptor CLEC-2, key drivers of thrombosis in liver, are dispensable for thrombosis in the spleen. Platelets, monocytes, and neutrophils are identified as core constituents of thrombi in both organs. Depleting either neutrophils or monocytic cells abrogates thrombus formation. Neutrophils and monocytes secrete TNF and blocking TNF diminishes both thrombosis and inflammation, which correlates with reduced endothelial expression of E-selectin and leukocyte infiltration. Moreover, inhibiting tissue factor and P-selectin glycoprotein ligand-1 pathways impairs thrombosis in both spleen and liver. Therefore, we identify organ-specific, and shared mechanisms driving thrombosis within a single infection. This may inform on tailoring treatments towards infection-induced inflammation, and single- or multi-organ thrombosis, based on the clinical need.

Lipopolysaccharide O-antigen is an immunodominant target of protective antibodies. Variation in O-antigen structures limits antibody-mediated cross-protection between closely-related pathogens including Salmonella Typhimurium (STm) and S . Enteritidis (SEn). Bacterial outer membrane vesicles (OMV) are vaccine platforms presenting surface antigens in their natural conformations. To assess how O-antigen lengths impact antibody responses and control of homologous or heterologous infection, mice were immunized with STm-OMV containing wild-type O-antigen unit repeats (wt-OMV), ≤1 O-antigen unit (wzy-OMV), or no O-antigen units (wbaP-OMV) respectively and challenged with either STm or SEn. Unexpectedly, anti-STm LPS IgG and protection to STm were comparable after immunization with either wt-OMV or wzy-OMV. Anti-porin responses were elevated after immunization with wzy-OMV and wbaP-OMV. A single immunization with any OMV induced minimal cross-protection against SEn, except in blood. In contrast, boosting with O-antigen-expressing OMV enhanced control of SEn infections by >10-fold. These results suggest that i) Antibody to single or variable-length O-antigen units are comparably protective against Salmonella ; ii) Antigens other than immunodominant O-antigens may be targets of cross-reactive antibodies that moderate bacterial burdens; iii) Boosting can enhance the level of cross-protection against related Salmonella serovars and iv) High tissue burdens of Salmonella can be present in the absence of detectable bacteraemia. ### Competing Interest Statement The authors have declared no competing interest.

Introduction Vaccination with Vi capsular polysaccharide (Vi-PS) or protein-Vi typhoid conjugate vaccine (TCV) can protect adults against Salmonella Typhi infections. TCVs offer better protection than Vi-PS in infants and may offer better protection in adults. Potential reasons for why TCV may be superior in adults are not fully understood. Methods and results Here, we immunized wild-type (WT) mice and mice deficient in IgG or IgM with Vi-PS or TCVs (Vi conjugated to tetanus toxoid or CRM197) for up to seven months, with and without subsequent challenge with Vi-expressing Salmonella Typhimurium. Unexpectedly, IgM or IgG alone were similarly able to reduce bacterial burdens in tissues, and this was observed in response to conjugated or unconjugated Vi vaccines and was independent of antibody being of high affinity. Only in the longer-term after immunization (>5 months) were differences observed in tissue bacterial burdens of mice immunized with Vi-PS or TCV. These differences related to the maintenance of antibody responses at higher levels in mice boosted with TCV, with the rate of fall in IgG titres induced to Vi-PS being greater than for TCV. Discussion Therefore, Vi-specific IgM or IgG are independently capable of protecting from infection and any superior protection from vaccination with TCV in adults may relate to responses being able to persist better rather than from differences in the antibody isotypes induced. These findings suggest that enhancing our understanding of how responses to vaccines are maintained may inform on how to maximize protection afforded by conjugate vaccines against encapsulated pathogens such as S . Typhi.

Germinal centers (GCs) are sites where plasma and memory B cells form to generate high-affinity, Ig class-switched antibodies. Specialized stromal cells called follicular dendritic cells (FDCs) are essential for GC formation. During systemic Salmonella Typhimurium (STm) infection GCs are absent, whereas extensive extrafollicular and switched antibody responses are maintained. The mechanisms that underpin the absence of GC formation are incompletely understood. Here, we demonstrate that STm induces a reversible disruption of niches within the splenic microenvironment, including the T and B cell compartments and the marginal zone. Alongside these effects after infection, mature FDC networks are strikingly absent, whereas immature FDC precursors, including marginal sinus pre-FDCs (MadCAM-1+) and perivascular pre-FDCs (PDGFRβ+) are enriched. As normal FDC networks re-establish, extensive GCs become detectable throughout the spleen. Therefore, the reorganization of FDC networks and the loss of GC responses are key, parallel features of systemic STm infections.

Summary Germinal centres (GCs) are sites where plasma and memory B cells form to generate high-affinity, Ig class switched antibodies. Specialised stromal cells called follicular dendritic cells (FDCs) are essential for GC formation. During systemic Salmonella Typhimurium (STm) infection GCs are absent, whereas extensive extrafollicular switched antibody responses are maintained. The mechanisms that underpin the absence of GC formation are incompletely understood. Here, we show that STm-induces a reversible disruption of niches within the splenic microenvironment, including the T and B cell compartments and the marginal zone. Alongside to these effects post-infection, mature FDC networks are strikingly absent, whereas immature FDC precursors, including marginal sinus pre-FDCs (MadCAM-1+) and perivascular Pre-FDCs (PDGFRβ+) are enriched. As normal FDC networks re-establish, extensive GCs become detectable throughout the spleen. Therefore, the reorganisation of FDC networks and the loss of GC responses are key, parallel features of systemic STm infections. Graphical abstract

Mucosal vaccination aims to prevent infection mainly by inducing secretory IgA (sIgA) antibody, which neutralises pathogens and enterotoxins by blocking their attachment to epithelial cells. We previously demonstrated that encapsulated protein antigen CD0873 given orally to hamsters induces neutralising antibodies locally as well as systemically, affording partial protection against Clostridioides difficile infection. The aim of this study was to determine whether displaying CD0873 on liposomes, mimicking native presentation, would drive a stronger antibody response. The recombinant form we previously tested resembles the naturally cleaved lipoprotein commencing with a cysteine but lacking lipid modification. A synthetic lipid (DHPPA-Mal) was designed for conjugation of this protein via its N-terminal cysteine to the maleimide headgroup. DHPPA-Mal was first formulated with liposomes to produce MalLipo; then, CD0873 was conjugated to headgroups protruding from the outer envelope to generate CD0873-MalLipo. The immunogenicity of CD0873-MalLipo was compared to CD0873 in hamsters. Intestinal sIgA and CD0873-specific serum IgG were induced in all vaccinated animals; however, neutralising activity was greatest for the CD0873-MalLipo group. Our data hold great promise for development of a novel oral vaccine platform driving intestinal and systemic immune responses.

Despite high vaccination coverage, Bordetella pertussis the causative agent of whooping cough is still a health concern worldwide. A resurgence of pertussis cases has been reported, particularly in countries using acellular vaccines with waning immunity and pathogen adaptation thought to be responsible. A better understanding of protective immune responses is needed for the development of improved vaccines. In our study, B. pertussis strain B1917 variants presenting a single gene deletion were generated to analyze the role of vaccine components or candidate vaccine antigens as targets for bactericidal antibodies generated after acellular vaccination or natural infection. Our results show that acellular vaccination generates bactericidal antibodies that are only directed against pertactin. Serum bactericidal assay performed with convalescent samples show that disease induces bactericidal antibodies against Prn but against other antigen(s) as well. Four candidate vaccine antigens (CyaA, Vag8, BrkA, and TcfA) have been studied but were not targets for complement-mediated bactericidal antibodies after natural infection. We confirm that Vag8 and BrkA are involved in complement resistance and would be targeted by blocking antibodies. Our study suggests that the emergence and the widespread circulation of Prn-deficient strains is driven by acellular vaccination and the generation of bactericidal antibodies targeting Prn.

Clearance of intracellular infections caused by Salmonella Typhimurium (STm) requires IFN-γ and the Th1-associated transcription factor T-bet. Nevertheless, whereas IFN-γ-/- mice succumb rapidly to STm infections, T-bet-/- mice do not. In this study, we assess the anatomy of immune responses and the relationship with bacterial localization in the spleens and livers of STm-infected IFN-γ-/- and T-bet-/- mice. In IFN-γ-/- mice, there is deficient granuloma formation and inducible NO synthase (iNOS) induction, increased dissemination of bacteria throughout the organs, and rapid death. The provision of a source of IFN-γ reverses this, coincident with subsequent granuloma formation and substantially extends survival when compared with mice deficient in all sources of IFN-γ. T-bet-/- mice induce significant levels of IFN-γ- after challenge. Moreover, T-bet-/- mice have augmented IL-17 and neutrophil numbers, and neutralizing IL-17 reduces the neutrophilia but does not affect numbers of bacteria detected. Surprisingly, T-bet-/- mice exhibit surprisingly wild-type-like immune cell organization postinfection, including extensive iNOS+ granuloma formation. In wild-type mice, most bacteria are within iNOS+ granulomas, but in T-bet-/- mice, most bacteria are outside these sites. Therefore, Th1 cells act to restrict bacteria within IFN-γ-dependent iNOS+ granulomas and prevent dissemination.

Whooping cough is a re-emerging respiratory tract infection. It has become clear that there is a need for better understanding of protective immune responses and variation between Bordetella pertussis strains to aid the development of improved vaccines. In order to survive in the host, B. pertussis has evolved mechanisms to evade complement-mediated killing, including the ability to bind complement-regulatory proteins. Here we evaluate the variation in interactions with the complement system among recently isolated strains. Isolates whose genomes appear highly similar and cluster together on a SNP-based dendrogram were found to vary significantly in resistance to complement-mediated killing and in the deposition of C3b/iC3b, C5b-9 and C1 esterase inhibitor (C1-INH). The key role of Vag8 as a receptor for C1-INH was confirmed and its expression was shown to vary in a panel of isolates. A Vag8 knockout mutant showed increased sensitivity to complement-mediated killing. Antibodies in convalescent sera blocked C1-INH binding to B. pertussis and may play an important role in natural immunity.