Browsing by Subject "typhoid fever"

Human genetic variation can be leveraged to understand the subtleties of how common variants with small effect sizes can alter cellular phenotypes and ultimately affect susceptibility to pathogenic disease. By combining GWAS of different phenotypic scales and basic cell biology, we can answer how a particular SNP affects a disease. This body of work elucidates the biological mechanism of how a SNP in VAC14, which encodes a human scaffolding protein involved in phosphoinositide metabolism, alters susceptibility to Typhoid Fever and other pathogens.

Using Hi-HOST (High-throughput Human in vitro Susceptibility Testing), a GWAS platform for cellular host-pathogen traits, we discovered that the ‘A’ allele of rs8060947 was associated with decreased VAC14 protein expression and increased Salmonella Typhi invasion. We experimentally confirmed the phenotype using RNAi to transiently decrease VAC14 protein expression in LCLs and Helas and saw increased Salmonella Typhi invasion. Further studies, using genetic and pharmacological manipulations were able to determine how VAC14 affects Salmonella Typhi invasion. CRISPR knockout VAC14 cells had a robust increase in invasion, and had increased cholesterol accumulation in the cell. Salmonella preferentially docks to cholesterol on the host plasma membrane as one of the first steps involved in invasion. Thus, increasing cholesterol at the plasma membrane increased the number of docked bacteria and ultimately caused higher invasion percentages.

To confirm the relevance of cholesterol and Salmonella Typhi beyond cell culture, we infected the swim bladder of Zebrafish with S. Typhi. Fish were pretreated with Ezetimibe, an FDA approved cholesterol-reducing drug, and then subsequently infected with S. Typhi. Fish treated with Ezetimibe, had decreased cholesterol staining by filipin, and had increased survival from S. Typhi infections. Additionally, because of the optically transparent nature of the zebrafish embryo we were able to image the fish 24hrs after infection and show that ezetimibe treated fish had higher bacterial clearance.

In addition to the fish studies, a collaboration with Dr. Sarah Dunstan (University of Melbourne) was able to retrospectively determine that VAC14 had an effect on human susceptibility to typhoid fever. The ‘A’ allele for SNP rs8060947, which we showed had decreased VAC14 protein expression and increased S. Typhi invasion in cell culture, was found to be more common in people with typhoid fever, suggesting the ‘A’ allele increases human susceptibility to this disease. All together, we have shown that decreased VAC14 expression causes an increase in cellular cholesterol, leading to an increase in docking and invasion of Salmonella and ultimately increasing your chances of acquiring typhoid fever.

The central role of cholesterol in entry of multiple pathogens led us to hypothesize that natural variation or experimental manipulation of VAC14 expression could play a role in pathogens beyond Salmonella. Here we show that its effects extend beyond bacteria to parasites. With cholesterol regulating entry of Plasmodium into hepatocytes, we hypothesized that increasing the amount of cellular cholesterol in hepatocytes will increase Plasmodium entry. These ideas are being tested in collaboration with Maria Toro and Dr. Emily Derbyshire (Duke University). However, unpublished human genetic data already support the idea that VAC14 regulates susceptibility to malaria infection. The same SNP associated with Salmonella invasion (rs8060947) is associated with malaria risk in African populations (Gavin Band and the MalariaGEN Consortium, personal communication).

VAC14 may also affect pathogen entry through its role in regulation of endosomal trafficking. VAC14 forms a complex with the FIG4 phosphatase and PIKfyve kinase to modulate endosomal trafficking through the metabolism of PtdIns(3,5)P2. Recently, FIG4 and PIKfyve were found to be necessary for Ebola entry in a somatic cell genetic screen. Using our VAC14 CRISPR knockout cells we determined that cells mutated for VAC14 had a similar phenotype. Ebola virus-like-particle (VLP) entry decreased dramatically in cells lacking VAC14. While we discovered that VAC14 affects cellular cholesterol, its main reported function is to regulate endosomal trafficking. We hypothesize that lack of VAC14 interferes with proper endosomal maturation and thus prevents the Ebola VLP from reaching its intracellular receptor NPC1 and exiting into the cytoplasm.

The common allele (A) that alters VAC14 expression is associated with decreased protein synthesis, and increased susceptibility to both Salmonella and Malaria infection. On the other hand, decreased VAC14 expression inhibits proper endolysosomal trafficking, inhibiting Ebola infection. These two mechanisms of affecting different infectious diseases may provide opposing forces in an example of balancing selection.

Vaccines save millions of lives every year primarily through the induction of antigen-specific antibodies; however, there is a critical lack of understanding of the mechanistic underpinings of vaccine-elicited, antibody-mediated protection. Vaccinologists have long since recognized neutralization as an important antibody function while comparatively neglecting the role of Fc functions of antibodies. Therefore, the goal of my dissertation was to dissect out the functional roles of different subpopulations of antibodies in preventing infection using a controlled human infection model (CHIM) of a pathogen with a mucosal and systemic phase of infection, Salmonella Typhi. The first objective was to determine which isotypes/subclasses within the polyclonal humoral immune response are protective against S. Typhi infection and through what functional mechanisms. The second objective focused on vaccine-derived monoclonal antibodies to identify biophysical properties that affect the ability to recognize S. Typhi Vi antigen and influence downstream antibody Fc-mediated function. Most immune correlates of vaccine-mediated protection focus on either total IgG responses or bulk polyclonal serum with little-to-no defined understanding of how the quality (avidity) and characteristics (epitope-specificity, functional potency) of specific subpopulations impact protection. In this dissertation, I interrogated the humoral response to vaccination with two Vi polysaccharide vaccine constructs: one plain polysaccharide vaccine (Vi-PS) and one Vi-protein conjugate vaccine (Vi-TT). In chapter 2 of this dissertation, I evaluated the binding and avidity of subclass-specific IgA and IgG antibodies to the Vi polysaccharide. Firstly, I demonstrated that Vi-specific IgA magnitude correlated with protection in the typhoid fever CHIM. In addition, Vi IgG1 and IgA2 avidity were higher amongst protected individuals of both vaccine groups. These findings suggest that a combined IgA, perhaps via IgA2, and IgG1 response may be critical in preventing infection. Although IgA magnitude was associated with protection from S. Typhi infection, I did not identify a threshold concentration of Vi-specific IgA that prevented infection. Therefore, I hypothesized that Fc functions, rather than neutralization, may be critical in the mechanism of IgA-mediated protection. Following the findings of the univariate analysis, a multivariate analysis was conducted as part of a wider scientific collaboration in which binding and avidity data, as well as functional data (including FcR binding and Fc-mediated functions), were included to identify the smallest set of immune measurements that could robustly predict protection. In a composite analysis with both vaccine regimens combined, we identified Vi specific IgA magnitude, IgG2 magnitude, and IgA2 avidity as the top features that were enriched among protected individuals. These responses were also linked to antibody-dependent neutrophil phagocytosis and oxidative burst. However, we found enriched antibody-dependent NK cell activation and complement deposition amongst diagnosed vaccinees. These data suggest that a highly specialized mechanism driven by IgA and IgG mediated neutrophil activation via FcαR and FcγRs impart protection whereas broad innate immune activation can be detrimental. In chapter 3 of this dissertation, I interrogated the humoral response to Vi immunization using recombinantly produced monoclonal antibodies derived from vaccinees. I identified a convergent B cell response amongst vaccinees with most antibodies targeting the immunodominant C3 O-acetyl group of the Vi polysaccharide. However, I also identified four unique subdominant epitopes that varied in their accessibility for antibody binding by performing a de-O-acetylation of the polysaccharide backbone and conducting epitope binning competitive assays. Furthermore, I demonstrated that specific subdominant epitopes on the Vi polysaccharide antigen can be effectively targeted by antibody for phagocytosis and complement deposition. I utilized a kinetics rates-based assay, BioLayer Interferometry, to assess the association rate, dissociation rate, and overall avidity of each monoclonal antibody to the Vi antigen. By conducting correlative analyses of kinetic parameters and functional outcomes, I demonstrated that association rate has no substantial association with Fc function, while dissociation rate is highly associated with Fc function. Taken together, these findings highlight a likely mechanism of protection from infection with S. Typhi through cellular phagocytosis mediated cooperatively by IgA and IgG antibodies. This study also highlights the key epitopes that antibodies elicited by intramuscular Vi vaccination target for functional Fc outcomes which is dependent on epitope exposure and on antibody-antigen binding stability (off-rate). Identifying these specific mechanisms of protective immunity against typhoid fever will facilitate evaluation and licensure of the many Vi conjugate vaccines in development that would otherwise require large scale trials, and also help guide any future development of Vi vaccines. More broadly, the analysis here highlights the importance of evaluating specific subpopulations of antibodies, and their quality, as potential mediators of protection for other vaccine constructs rather than focus only on magnitude of bulk polyclonal responses. This type of analysis deeply enhances our understanding of protective humoral immunity while also informing both the development and the evaluation of new vaccine candidates.