Human Salmonella Typhi exposure generates differential multifunctional cross‐reactive T‐cell memory responses against Salmonella Paratyphi and invasive nontyphoidal Salmonella

Objective There are no vaccines for most of the major invasive Salmonella strains causing severe infection in humans. We evaluated the specificity of adaptive T memory cell responses generated after Salmonella Typhi exposure in humans against other major invasive Salmonella strains sharing capacity for dissemination. Methods T memory cells from eleven volunteers who underwent controlled oral challenge with wt S. Typhi were characterised by flow cytometry for cross‐reactive cellular cytokine/chemokine effector responses or evidence of degranulation upon stimulation with autologous B‐lymphoblastoid cells infected with either S. Typhi, Salmonella Paratyphi A (PA), S. Paratyphi B (PB) or an invasive nontyphoidal Salmonella strain of the S. Typhimurium serovar (iNTSTy). Results Blood T‐cell effector memory (TEM) responses after exposure to S. Typhi in humans evolve late, peaking weeks after infection in most volunteers. Induced multifunctional CD4+ Th1 and CD8+ TEM cells elicited after S. Typhi challenge were cross‐reactive with PA, PB and iNTSTy. The magnitude of multifunctional CD4+ TEM cell responses to S. Typhi correlated with induction of cross‐reactive multifunctional CD8+ TEM cells against PA, PB and iNTSTy. Highly multifunctional subsets and T central memory and T effector memory cells that re‐express CD45 (TEMRA) demonstrated less heterologous T‐cell cross‐reactivity, and multifunctional Th17 elicited after S. Typhi challenge was not cross‐reactive against other invasive Salmonella. Conclusion Gaps in cross‐reactive immune effector functions in human T‐cell memory compartments were highly dependent on invasive Salmonella strain, underscoring the importance of strain‐dependent vaccination in the design of T‐cell‐based vaccines for invasive Salmonella

Role of circulating T follicular helper subsets following Ty21a immunization and oral challenge with wild type S. Typhi in humans

Despite decades of intense research, our understanding of the correlates of protection against Salmonella Typhi (S. Typhi) infection and disease remains incomplete. T follicular helper cells (TFH), an important link between cellular and humoral immunity, play an important role in the development and production of high affinity antibodies. While traditional TFH cells reside in germinal centers, circulating TFH (cTFH) (a memory subset of TFH) are present in blood. We used specimens from a typhoid controlled human infection model whereby participants were immunized with Ty21a live attenuated S. Typhi vaccine and then challenged with virulent S. Typhi. Some participants developed typhoid disease (TD) and some did not (NoTD), which allowed us to assess the association of cTFH subsets in the development and prevention of typhoid disease. Of note, the frequencies of cTFH were higher in NoTD than in TD participants, particularly 7 days after challenge. Furthermore, the frequencies of cTFH2 and cTFH17, but not cTFH1 subsets were higher in NoTD than TD participants. However, we observed that ex-vivo expression of activation and homing markers were higher in TD than in NoTD participants, particularly after challenge. Moreover, cTFH subsets produced higher levels of S. Typhi-specific responses (cytokines/chemokines) in both the immunization and challenge phases. Interestingly, unsupervised analysis revealed unique clusters with distinct signatures for each cTFH subset that may play a role in either the development or prevention of typhoid disease. Importantly, we observed associations between frequencies of defined cTFH subsets and anti-S. Typhi antibodies. Taken together, our results suggest that circulating TFH2 and TFH17 subsets might play an important role in the development or prevention of typhoid disease. The contribution of these clusters was found to be distinct in the immunization and/or challenge phases. These results have important implications for vaccines aimed at inducing long-lived protective T cell and antibody responses

Association of mixed hematopoietic chimerism with elevated circulating autoantibodies and chronic graft-versus-host disease occurrence.

International audienceBACKGROUND: Use of a reduced-intensity conditioning regimen before an allogeneic hematopoietic cell transplantation is frequently associated with an early state of mixed hematopoietic chimerism. Such a coexistence of both host and donor hematopoietic cells may influence posttransplant alloreactivity and may affect the occurrence and severity of acute and chronic graft-versus-host disease (GVHD) as well as the intensity of the graft-versus-leukemia effect. Here we evaluated the relation between chimerism state after reduced-intensity conditioning transplantation (RICT), autoantibody production, and chronic GVHD (cGVHD)-related pathology. METHODS: Chimerism state, circulating anticardiolipin, and antidouble stranded DNA autoantibody (Ab) titers as well as occurrence of cGVHD-like lesions were investigated in a murine RICT model. RESULTS: We observed a novel association between mixed chimerism state, high levels of pathogenic IgG autoantibodies, and subsequent development of cGVHD-like lesions. Furthermore, we found that the persistence of host B cells, but not dendritic cell origin or subset, was a factor associated with the appearance of cGVHD-like lesions. The implication of host B cells was confirmed by a host origin of autoantibodies. CONCLUSION: Recipient B cell persistence may contribute to the frequency and/or severity of cGVHD after RICT

Salmonella Typhi Bactericidal Antibodies Reduce Disease Severity but Do Not Protect against Typhoid Fever in a Controlled Human Infection Model

Effective vaccines against Salmonella Typhi, a major cause of febrile illness in tropical regions, can have a significant effect as a disease control measure. Earlier work has shown that immunization with either of two Salmonella Typhi vaccines, licensed Ty21a or candidate M01ZH09, did not provide full immunity in a controlled human infection model. Here, we describe the human humoral immune responses to these oral vaccines and their functional role in protection after challenge with S. Typhi. Serum, obtained from healthy volunteers before and after vaccination with Ty21a or M01ZH09 or placebo and before and after oral challenge with wild-type S. Typhi, was assessed for bactericidal activity. Single-dose vaccination with M01ZH09 induced an increase in serum bactericidal antibodies (p = 0.001) while three doses of Ty21a did not. No association between bactericidal activity and protection against typhoid after challenge was seen in either vaccine arm. Bactericidal activity after vaccination correlated significantly with delayed disease onset (p = 0.013), lower bacterial burden (p = 0.006), and decreased disease severity scores (p = 0.021). Depletion of antibodies directed against lipopolysaccharide significantly reduced bactericidal activity (p =  0.009). We conclude that antibodies induced after ingestion of oral live-attenuated typhoid vaccines or after challenge with wild-type S. Typhi exhibit bactericidal activity. This bactericidal activity is mediated by anti-O:LPS antibodies and significantly reduces clinical symptoms but does not provide sterile immunity. This directs future vaccine studies toward other antigens or mechanisms of protection against typhoid

Mucosal-Associated Invariant T cells exhibit distinct functional signatures associated with protection against typhoid fever

We have previously demonstrated that Mucosal-Associated Invariant T (MAIT) cells secrete multiple cytokines after exposure to Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever in humans. However, whether cytokine secreting MAIT cells can enhance or attenuate the clinical severity of bacterial infections remain debatable. This study characterizes human MAIT cell functions in subjects participating in a wild-type S. Typhi human challenge model. Here, we found that MAIT cells exhibit distinct functional signatures associated with protection against typhoid fever. We also observed that the cytokine patterns of MAIT cell responses, rather than the average number of cytokines expressed, are more predictive of typhoid fever outcomes. These results might enable us to objectively, based on functional parameters, identify cytokine patterns that may serve as predictive biomarkers during natural infection and vaccination

Role of circulating T follicular helper subsets following Ty21a immunization and oral challenge with wild type S. Typhi in humans

Despite decades of intense research, our understanding of the correlates of protection against Salmonella Typhi (S. Typhi) infection and disease remains incomplete. T follicular helper cells (TFH), an important link between cellular and humoral immunity, play an important role in the development and production of high affinity antibodies. While traditional TFH cells reside in germinal centers, circulating TFH (cTFH) (a memory subset of TFH) are present in blood. We used specimens from a typhoid controlled human infection model whereby participants were immunized with Ty21a live attenuated S. Typhi vaccine and then challenged with virulent S. Typhi. Some participants developed typhoid disease (TD) and some did not (NoTD), which allowed us to assess the association of cTFH subsets in the development and prevention of typhoid disease. Of note, the frequencies of cTFH were higher in NoTD than in TD participants, particularly 7 days after challenge. Furthermore, the frequencies of cTFH2 and cTFH17, but not cTFH1 subsets were higher in NoTD than TD participants. However, we observed that ex-vivo expression of activation and homing markers were higher in TD than in NoTD participants, particularly after challenge. Moreover, cTFH subsets produced higher levels of S. Typhi-specific responses (cytokines/chemokines) in both the immunization and challenge phases. Interestingly, unsupervised analysis revealed unique clusters with distinct signatures for each cTFH subset that may play a role in either the development or prevention of typhoid disease. Importantly, we observed associations between frequencies of defined cTFH subsets and anti-S. Typhi antibodies. Taken together, our results suggest that circulating TFH2 and TFH17 subsets might play an important role in the development or prevention of typhoid disease. The contribution of these clusters was found to be distinct in the immunization and/or challenge phases. These results have important implications for vaccines aimed at inducing long-lived protective T cell and antibody responses

Oral Wild-Type Salmonella Typhi Challenge Induces Activation of Circulating Monocytes and Dendritic Cells in Individuals Who Develop Typhoid Disease.

A new human oral challenge model with wild-type Salmonella Typhi (S. Typhi) was recently developed. In this model, ingestion of 104 CFU of Salmonella resulted in 65% of subjects developing typhoid fever (referred here as typhoid diagnosis -TD-) 5-10 days post-challenge. TD criteria included meeting clinical (oral temperature ≥38°C for ≥12 h) and/or microbiological (S. Typhi bacteremia) endpoints. One of the first lines of defense against pathogens are the cells of the innate immune system (e.g., monocytes, dendritic cells -DCs-). Various changes in circulating monocytes and DCs have been described in the murine S. Typhimurium model; however, whether similar changes are present in humans remains to be explored. To address these questions, a subset of volunteers (5 TD and 3 who did not develop typhoid despite oral challenge -NoTD-) were evaluated for changes in circulating monocytes and DCs. Expression of CD38 and CD40 were upregulated in monocytes and DCs in TD volunteers during the disease days (TD-0h to TD-96h). Moreover, integrin α4β7, a gut homing molecule, was upregulated on monocytes but not DCs. CD21 upregulation was only identified in DCs. These changes were not observed among NoTD volunteers despite the same oral challenge. Moreover, monocytes and DCs from NoTD volunteers showed increased binding to S. Typhi one day after challenge. These monocytes showed phosphorylation of p38MAPK, NFkB and Erk1/2 upon stimulation with S. Typhi-LPS-QDot micelles. In contrast, monocytes from TD volunteers showed only a moderate increase in S. Typhi binding 48 h and 96 h post-TD, and only Erk1/2 phosphorylation. This is the first study to describe different activation and migration profiles, as well as differential signaling patterns, in monocytes and DCs which relate directly to the clinical outcome following oral challenge with wild type S. Typhi

Importance of Salmonella Typhi-Responsive CD8+ T Cell Immunity in a Human Typhoid Fever Challenge Model

Typhoid fever, caused by the human-restricted organism Salmonella enterica serovar Typhi (S. Typhi), constitutes a major global health problem. The development of improved attenuated vaccines is pressing, but delayed by the lack of appropriate preclinical models. Herein, we report that high levels of S. Typhi-responsive CD8+ T cells at baseline significantly correlate with an increased risk of disease in humans challenged with a high dose (~104 CFU) wild-type S. Typhi. Typhoid fever development was associated with higher multifunctional S. Typhi-responsive CD8+ T effector memory cells at baseline. Early decreases of these cells in circulation following challenge were observed in both S. Typhi-responsive integrin α4β7− and integrin α4β7+ CD8+ T effector memory (TEM) cells, suggesting their potential to home to both mucosal and extra-intestinal sites. Participants with higher baseline levels of S. Typhi-responsive CD8+ T memory cells had a higher risk of acquiring disease, but among those who acquired disease, those with a higher baseline responses took longer to develop disease. In contrast, protection against disease was associated with low or absent S. Typhi-responsive T cells at baseline and no changes in circulation following challenge. These data highlight the importance of pre-existing S. Typhi-responsive immunity in predicting clinical outcome following infection with wild-type S. Typhi and provide novel insights into the complex mechanisms involved in protective immunity to natural infection in a stringent human model with a high challenge dose. They also contribute important information on the immunological responses to be assessed in the appraisal and selection of new generation typhoid vaccines

Salmonella Typhi-specific multifunctional CD8+ T cells play a dominant role in protection from typhoid fever in humans.

BACKGROUND: Typhoid fever, caused by the human-restricted organism Salmonella Typhi (S. Typhi), is a major public health problem worldwide. Development of novel vaccines remains imperative, but is hampered by an incomplete understanding of the immune responses that correlate with protection. METHODS: Recently, a controlled human infection model was re-established in which volunteers received ~10(3) cfu wild-type S. Typhi (Quailes strain) orally. Twenty-one volunteers were evaluated for their cell-mediated immune (CMI) responses. Ex vivo PBMC isolated before and up to 1 year after challenge were exposed to three S. Typhi-infected targets, i.e., autologous B lymphoblastoid cell-lines (B-LCL), autologous blasts and HLA-E restricted AEH B-LCL cells. CMI responses were evaluated using 14-color multiparametric flow cytometry to detect simultaneously five intracellular cytokines/chemokines (i.e., IL-17A, IL-2, IFN-g, TNF-a and MIP-1b) and a marker of degranulation/cytotoxic activity (CD107a). RESULTS: Herein we provide the first evidence that S. Typhi-specific CD8+ responses correlate with clinical outcome in humans challenged with wild-type S. Typhi. Higher multifunctional S. Typhi-specific CD8+ baseline responses were associated with protection against typhoid and delayed disease onset. Moreover, following challenge, development of typhoid fever was accompanied by decreases in circulating S. Typhi-specific CD8+ T effector/memory (TEM) with gut homing potential, suggesting migration to the site(s) of infection. In contrast, protection against disease was associated with low or no changes in circulating S. Typhi-specific TEM. CONCLUSIONS: These studies provide novel insights into the protective immune responses against typhoid disease that will aid in selection and development of new vaccine candidates

A review of operational methods of variational and ensemble-variational data assimilation

Variational and ensemble methods have been developed separately by various research and development groups and each brings its own benefits to data assimilation. In the last decade or so various ways have been developed to combine these methods, especially with the aims of improving the background error covariance matrices and of improving efficiency. The field has become confusing, even to many specialists, and so there is now a need to summarise the methods in order to show how they work, how they are related, what benefits they bring, why they have been developed, how they perform, and what improvements are pending. This paper starts with a reminder of basic variational and ensemble techniques and shows how they can be combined to give the emerging ensemble-variational (EnVar) and hybrid methods. A key part of the paper includes details of how localisation is commonly represented. There has been a particular push to develop four-dimensional methods that are free of linearised forecast models. This paper attempts to provide derivations of the formulations of most popular schemes. These are otherwise scattered throughout the literature or absent. It builds on the nomenclature used to distinguish between methods, and discusses further possible developments to the methods, including the representation of model error