Celia Alpuche-Aranda

Gallstones play a significant role in Salmonella spp. gallbladder colonization and carriage

Salmonella enterica serovar Typhi can colonize the gallbladder and persist in an asymptomatic carrier state that is frequently associated with the presence of gallstones. We have shown that salmonellae form bile-mediated biofilms on human gallstones and cholesterol-coated surfaces in vitro. Here, we test the hypothesis that biofilms on cholesterol gallbladder stones facilitate typhoid carriage in mice and men. Naturally resistant (Nramp1+/+) mice fed a lithogenic diet developed cholesterol gallstones that supported biofilm formation during persistent serovar Typhimurium infection and, as a result, demonstrated enhanced fecal shedding and enhanced colonization of gallbladder tissue and bile. In typhoid endemic Mexico City, 5% of enrolled cholelithiasis patients carried serovar Typhi, and bacterial biofilms could be visualized on gallstones from these carriers whereas significant biofilms were not detected on gallstones from Escherichia coli infected gallbladders. These findings offer direct evidence that gallstone biofilms occur in humans and mice, which facilitate gallbladder colonization and shedding.

The PhoP virulence regulon and live oral Salmonella vaccines

The PhoP virulence regulon is essential to Salmonella typhimurium mouse typhoid fever pathogenesis and survival within macrophages. This virulence regulon is composed of the PhoP (transcriptional regulator) and PhoQ (environmental sensor) proteins and the genetic loci they positively (pags for PhoP activated genes) and negatively (prgs for PhoP repressed genes) regulate. Three regulated loci pagC, pagD, and prgH, when singly mutated, affect the virulence of S. typhimurium for mice. Strains with phoP locus mutations are effective as live vaccines in mice, and strains with a constitutive regulatory mutation, a point mutation in PhoQ, can protect mice against typhoid fever when only very few organisms are administered. The addition of various PhoP regulon mutations further attenuates aroA mutants of S. typhimurium, suggesting that these mutations would be useful in further attenuating vaccine strains with metabolic pathway mutations. The phoP, phoQ, pagC, and pagD genes are highly conserved between S. typhimurium and S. typhi and may be valuable as mutations in live vaccines for human typhoid fever. A plasmid suicide vector that allows deletion of the pagC gene and stable insertion of heterologous antigen genes within the deleted pagC locus has been constructed and used successfully in S. typhimurium and S. typhi.

Salmonella Downregulates Nod-like Receptor Family CARD Domain Containing Protein 4 Expression To Promote Its Survival in B Cells by Preventing Inflammasome Activation and Cell Death

Salmonella infects and survives within B cells, but the mechanism used by the bacterium to promote its survival in these cells is unknown. In macrophages, flagellin secreted by Salmonella activates the Nod-like receptor (NLR) family CARD domain containing protein 4 (NLRC4) inflammasome, leading to the production of IL-1β and pyroptosis of infected cells. In this study, we demonstrated that the NLRC4 inflammasome is functional in B cells; however, in Salmonella-infected B cells, IL-1β secretion is prevented through the downregulation of NLRC4 expression. A functional Salmonella pathogenicity island 1 type III secretion system appears to be required for this process. Furthermore, infection induces Yap phosphorylation and promotes the interaction of Yap with Hck, thus preventing the transcriptional activation of NLRC4. The ability of Salmonella to inhibit IL-1β production also prevents B cell death; thus, B cells represent an ideal niche in which Salmonella resides, thereby promoting its persistence and dissemination.

Survival of Salmonella enterica serovar Typhimurium within late endosomal-lysosomal compartments of B lymphocytes is associated with the inability to use the vacuolar alternative major histocompatibility complex class I antigen-processing pathway.

ABSTRACT Gamma interferon (IFN-γ)-activated macrophages use an alternative processing mechanism to present Salmonella antigens to CD8+ T lymphocytes. This pathway involves processing of antigen in a vacuolar compartment followed by secretion and loading of antigenic peptides to major histocompatibility complex class I (MHC-I) molecules on macrophage cell surface and bystander cells. In this study, we have shown that B lymphocytes are not able to process Salmonella antigens using this alternative pathway. This is due to differences in Salmonella enterica serovar Typhimurium-containing vacuoles (SCV) when comparing late endosomal-lysosomal processing compartments in B lymphocytes to those in macrophages. The IFN-γ-activated IC21 macrophage cell line and A-20 B-cell line were infected with live or dead Salmonella enterica serovar Typhimurium. The SCV in B cells were in a late endosomal-lysosomal compartment, whereas SCV in macrophages were remodeled to a noncharacteristic late endosomal-lysosomal compartment over time. Despite the difference in SCV within macrophages and B lymphocytes, S. enterica serovar Typhimurium survives more efficiently within the IFN-γ-activated B cells than in activated macrophage cell lines. Similar results were found during in vivo acute infection. We determined that a lack of remodeling of late endosomal-lysosomal compartments by live Salmonella infection in B lymphocytes is associated with the inability to use the alternative MHC-I antigen-processing pathway, providing a survival advantage to the bacterium. Our data also suggest that the B lymphocyte late endosome-lysosome environment allows the expression of Salmonella virulence mechanisms favoring B lymphocytes in addition to macrophages and dendritic cells as a reservoir during in vivo infection.

Antibody Persistence in Adults Two Years after Vaccination with an H1N1 2009 Pandemic Influenza Virus-Like Particle Vaccine.

The influenza virus is a human pathogen that causes epidemics every year, as well as potential pandemic outbreaks, as occurred in 2009. Vaccination has proven to be sufficient in the prevention and containment of viral spreading. In addition to the current egg-based vaccines, new and promising vaccine platforms, such as cell culture-derived vaccines that include virus-like particles (VLPs), have been developed. VLPs have been shown to be both safe and immunogenic against influenza infections. Although antibody persistence has been studied in traditional egg-based influenza vaccines, studies on antibody response durations induced by VLP influenza vaccines in humans are scarce. Here, we show that subjects vaccinated with an insect cell-derived VLP vaccine, in the midst of the 2009 H1N1 influenza pandemic outbreak in Mexico City, showed antibody persistence up to 24 months post-vaccination. Additionally, we found that subjects that reported being revaccinated with a subsequent inactivated influenza virus vaccine showed higher antibody titres to the pandemic influenza virus than those who were not revaccinated. These findings provide insights into the duration of the antibody responses elicited by an insect cell-derived pandemic influenza VLP vaccine and the possible effects of subsequent influenza vaccination on antibody persistence induced by this VLP vaccine in humans.

Dengue vaccine: local decisions, global consequences

Abstract As new vaccines against diseases that are prevalent in low- and middle-income countries gradually become available, national health authorities are presented with new regulatory and policy challenges. The use of CYD-TDV – a chimeric tetravalent, live-attenuated dengue vaccine – was recently approved in five countries. Although promising for public health, this vaccine has only partial and heterogeneous efficacy and may have substantial adverse effects. In trials, children who were aged 2–5 years when first given CYD-TDV were seven times more likely to be hospitalized for dengue, in the third year post-vaccination, than their counterparts in the control group. As it has not been clarified whether this adverse effect is only a function of age or is determined by dengue serostatus, doubts have been cast over the long-term safety of this vaccine in seronegative individuals of any age. Any deployment of the vaccine, which should be very cautious and only considered after a rigorous evaluation of the vaccine’s risk–benefit ratio in explicit national and subnational scenarios, needs to be followed by a long-term assessment of the vaccine’s effects. Furthermore, any implementation of dengue vaccines must not weaken the political and financial support of preventive measures that can simultaneously limit the impacts of dengue and several other mosquito-borne pathogens.

Subversion of innate and adaptive immune activation induced by structurally modified lipopolysaccharide from Salmonella typhimurium

Salmonella are successful pathogens that infect millions of people every year. During infection, Salmonella typhimurium changes the structure of its lipopolysaccharide (LPS) in response to the host environment, rendering bacteria resistant to cationic peptide lysis in vitro. However, the role of these structural changes in LPS as in vivo virulence factors and their effects on immune responses and the generation of immunity are largely unknown. We report that modified LPS are less efficient than wild‐type LPS at inducing pro‐inflammatory responses. The impact of this LPS‐mediated subversion of innate immune responses was demonstrated by increased mortality in mice infected with a non‐lethal dose of an attenuated S. typhimurium strain mixed with the modified LPS moieties. Up‐regulation of co‐stimulatory molecules on antigen‐presenting cells and CD4+ T‐cell activation were affected by these modified LPS. Strains of S. typhimurium carrying structurally modified LPS are markedly less efficient at inducing specific antibody responses. Immunization with modified LPS moiety preparations combined with experimental antigens, induced an impaired Toll‐like receptor 4‐mediated adjuvant effect. Strains of S. typhimurium carrying structurally modified LPS are markedly less efficient at inducing immunity against challenge with virulent S. typhimurium. Hence, changes in S. typhimurium LPS structure impact not only on innate immune responses but also on both humoral and cellular adaptive immune responses.

Salmonella Modulates B Cell Biology to Evade CD8+ T Cell-Mediated Immune Responses

Although B cells and antibodies are the central effectors of humoral immunity, B cells can also produce and secrete cytokines and present antigen to helper T cells. The uptake of antigen is mainly mediated by endocytosis; thus, antigens are often presented by MHC-II molecules. However, it is unclear if B cells can present these same antigens via MHC-I molecules. Recently, Salmonella bacteria were found to infect B cells, allowing possible antigen cross-processing that could generate bacterial peptides for antigen presentation via MHC-I molecules. Here, we will discuss available knowledge regarding Salmonella antigen presentation by infected B cell MHC-I molecules and subsequent inhibitory effects on CD8+ T cells for bacterial evasion of cell-mediated immunity.

Salmonella impairs CD8 T cell response through PD-1: PD-L axis

We have shown that Salmonella remains for a long period of time within B cells, plasma cells, and bone marrow B cell precursors, which might allow persistence and dissemination of infection. Nonetheless, how infected cells evade CD8 T cell response has not been characterized. Evidence indicates that some pathogens exploit the PD-1: PD-L (PD-L1 and PD-L2) interaction to inhibit CD8 T cells response to contribute the chronicity of the infection. To determine whether the PD-1: PD-L axis plays a role during Salmonella infection; we evaluated PD-1 expression in antigen-specific CD8 T cells and PD-1 ligands in Salmonella-infected cells. Our results show that infected B cells and macrophages express continuously co-stimulatory (CD40, CD80, and CD86) and inhibitory molecules (PD-L1 and PD-L2) in early and late stages of chronic Salmonella infection, while antigen-specific CD8 T cells express in a sustained manner PD-1 in the late stages of infection. Blocking this axis restores the ability of the CD8 T cells to proliferate and eliminate primary infected APCs. Therefore, a continuous PD-1: PDL interaction might be a mechanism employed by Salmonella to negatively regulate Salmonella-specific CD8 T cell cytotoxic response in order to remain within the host for a long period of time.

Salmonella induces PD-L1 expression in B cells.

Salmonella persists for a long time in B cells; however, the mechanism(s) through which infected B cells avoid effector CD8 T cell responses has not been characterized. In this study, we show that Salmonella infects and survives within all B1 and B2 cell subpopulations. B cells are infected with a Salmonella typhimurium strain expressing an ovalbumin (OVA) peptide (SIINFEKL) to evaluate whether B cells process and present Salmonella antigens in the context of MHC-I molecules. Our data showed that OVA peptides are presented by MHC class I K(b)-restricted molecules and the presented antigen is generated through proteasomal degradation and vacuolar processing. In addition, Salmonella-infected B cells express co-stimulatory molecules such as CD40, CD80, and CD86 as well as inhibitory molecules such as PD-L1. Thus, the cross-presentation of Salmonella antigens and the expression of activation molecules suggest that infected B cells are able to prime and activate specific CD8(+) T cells. However, the Salmonella infection-stimulated expression of PD-L1 suggests that the PD-1/PD-L1 pathway may be involved in turning off the cytotoxic effector response during Salmonella persistent infection, thereby allowing B cells to become a reservoir for the bacteria.