| Abstract: | Salmonella enterica serotype Typhi is the cause of typhoid fever and a human-restricted pathogen. Currently available typhoid vaccines provide 50 to 90% protection for 2 to 5 years, and available practical diagnostic assays to identify individuals with typhoid fever lack sensitivity and/or specificity. Identifying immunogenic S. Typhi antigens expressed during human infection could lead to improved diagnostic assays and vaccines. Here we describe a platform immunoaffinity proteomics-based technology (IPT) that involves the use of columns charged with IgG, IgM, or IgA antibody fractions recovered from humans bacteremic with S. Typhi to capture S. Typhi proteins that were subsequently identified by mass spectrometry. This screening tool identifies immunogenic proteins recognized by antibodies from infected hosts. Using this technology and the plasma of patients with S. Typhi bacteremia in Bangladesh, we identified 57 proteins of S. Typhi, including proteins known to be immunogenic (PagC, HlyE, OmpA, and GroEL) and a number of proteins present in the human-restricted serotypes S. Typhi and S. Paratyphi A but rarely found in broader-host-range Salmonella spp. (HlyE, CdtB, PltA, and STY1364). We categorized identified proteins into a number of major groupings, including those involved in energy metabolism, protein synthesis, iron homeostasis, and biosynthetic and metabolic functions and those predicted to localize to the outer membrane. We assessed systemic and mucosal anti-HlyE responses in S. Typhi-infected patients and detected anti-HlyE responses at the time of clinical presentation in patients but not in controls. These findings could assist in the development of improved diagnostic assays.Salmonella enterica serotype Typhi is a human-restricted pathogen that is the primary cause of enteric fever. It is estimated that S. Typhi infects over 20 million individuals and kills approximately 200,000 people globally each year (4). Currently, commercially available typhoid vaccines provide approximately 50 to 75% protection for 2 to 5 years (21), although an anti-typhoid Vi conjugate vaccine demonstrated 90% protection in 2- to 5-year-old children in a large field trial (23). Available and practical diagnostic tests for typhoid fever lack sensitivity and/or specificity (28). Identifying immunogenic S. Typhi antigens expressed during human infection could lead to improved diagnostic assays and vaccines.Infection with S. Typhi begins with the ingestion of contaminated water or food. The bacteria invade the gastrointestinal mucosa, translocate to the lymphoid follicles, where they survive and replicate within macrophages, and then disseminate via the bloodstream to the liver, spleen, and intestinal lymph nodes (14). The incubation period is typically 8 to 14 days (22), and symptoms include fever, abdominal pain, anorexia, weakness, potential complications of intestinal perforation, encephalopathy, and gastrointestinal bleeding (14, 34). Clinical studies demonstrate that S. Typhi infection stimulates both an intestinal mucosal and systemic humoral and cellular immune response (14, 34). S. Typhi is a facultative intracellular pathogen of macrophages, and both cellular and antibody-mediated immune responses are known to play roles in controlling and clearing S. Typhi infection (37). Despite this, there are limited data on antigen-specific cellular responses during wild-type S. Typhi infection in humans. Analyses of cellular immune responses during S. Typhi infection have largely used whole-cell preparations or flagellar antigens and have focused predominately on measuring immune responses in recipients of oral live attenuated typhoid vaccines, not in individuals with wild-type disease (24, 25, 40-42, 49).Antibody responses during wild-type infection have been better studied but have focused largely on a relatively small number of antigens, including O antigen (lipopolysaccharide LPS]), H antigen (flagellar component), polysaccharide capsular antigen (Vi antigen), heat shock proteins such as GroEL, and outer membrane proteins such as OmpC and -F (13, 34). In addition, gut-derived IgA antibody-secreting cells that recognize LPS, a membrane preparation, or whole-killed S. Typhi organisms can be detected in the peripheral blood following natural S. Typhi infection or oral typhoid vaccination (16, 43, 50, 54). These cells eventually return home to the gastrointestinal mucosa, where they secrete secretory IgA antibody (36, 43).A number of immunoaffinity-based techniques that screen protein libraries of pathogens to identify immunogenic antigens have been developed (12, 17, 38), and we have previously reported using one such approach, in vivo-induced-antigen technology (IVIAT), to identify immunogenic S. Typhi antigens expressed during human infection (12). Another previously described technique, proteomics-based expression library screening (PELS), involves using antibody-charged columns to capture antigens produced by an Escherichia coli-based expression system containing an inducible library of a pathogen of interest, with subsequent elution and identification of bound proteins using mass spectrometric analysis (17). Here we describe using a modification of this approach that we have termed immunoaffinity proteomics-based technology (IPT). IPT involves directly screening the pathogen of interest using columns charged with IgG, IgM, or IgA antibody fractions recovered from the blood of infected humans. We applied IPT to S. Typhi to gain further insights into immunogenic antigens expressed in patients bacteremic with S. Typhi in Bangladesh. |
