Comparison of the Antibodies in Lymphocyte Supernatant and Antibody-Secreting Cell Assays for Measuring Intestinal Mucosal Immune Response to a Novel Oral Typhoid Vaccine (M01ZH09)

ABSTRACTEnteric fever caused bySalmonella entericaserovar Paratyphi A infection has emerged as an important public health problem. Recognizing that in randomized controlled field trials oral immunization with attenuatedS. entericaserovar Typhi live vaccine Ty21a conferred significant cross-protection againstS. Paratyphi B but notS. Paratyphi A disease, we undertook a clinical study to ascertain whether humoral immune responses could explain the field trial results. Ty21a immunization of adult residents of Maryland elicited predominantly IgA antibody-secreting cells (ASC) that recognizeS. Typhi lipopolysaccharide (LPS). Cross-reactivity toS. Paratyphi A LPS was significantly lower than that toS. Paratyphi B LPS. ASC producing IgG and IgA that bind LPS from each of theseSalmonellaserovars expressed CD27 and integrin α4β7 (gut homing), with a significant proportion coexpressing CD62L (secondary lymphoid tissue homing). No significant differences were observed in serum antibody against LPS of the different serovars. Levels of IgA B memory (BM) cells toS. Typhi LPS were significantly higher than those againstS. Paratyphi A or B LPS, with no differences observed betweenS. Paratyphi A and B. The response of IgA BMto outer membrane proteins (OMP) fromS. Typhi was significantly stronger than that to OMP ofS. Paratyphi A but similar to that to OMP ofS. Paratyphi B. The percentages of IgG or IgA BMresponders to LPS or OMP from theseSalmonellastrains were similar. Whereas cross-reactive humoral immune responses toS. Paratyphi A or B antigens are demonstrable following Ty21a immunization, they cannot explain the efficacy data gleaned from controlled field trials.

Section: Discussionsupporting

confidence: 74%

Live Oral Typhoid Vaccine Ty21a Induces Cross-Reactive Humoral Immune Responses against Salmonella enterica Serovar Paratyphi A andS. Paratyphi B in Humans

Wahid

Simon

Zafar

et al. 2012

“…These results suggest that measurement of the MP IgA responses warrants further development as a diagnostic test for individuals with suspected typhoid. The MP includes (15), and ASC and ALS responses that measure transient presence of mucosal lymphocytes in peripheral circulation after intestinal activation have been reported in vaccinees receiving oral attenuated strains of serovar Typhi (21,22,24) but have not been previously described in individuals with wild-type infection. The ALS specimen that we have used to measure the responses are those derived from cells which have mucosal priming in the gut.…”

Section: Discussionmentioning

confidence: 99%

“…We resuspended isolated PBMC to a concentration of 10 7 cells/ml in RPMI complete medium RPMI 1640 (Gibco, Gaithersburg, MD) with 10% heat-inactivated fetal bovine serum (HyClone, Ogden, UT), 100 U of penicillin/ml, 100 g of streptomycin/ml, 100 mM pyruvate, and 200 mM Lglutamine (Gibco) (31). We incubated cells for 48 h at 37°C with 5% CO 2 , collected supernatants, and added a protease inhibitor solution as previously described (21,31). Plasma and ALS specimens were tested for typhoid-specific antibodies by enzyme-linked immunosorbent assay (ELISA).…”

Section: Methodsmentioning

confidence: 99%

“…This migration peaks 1 to 2 weeks after intestinal infection and may be measured by using peripheral blood mononuclear cells (PBMC) in an antibody-secreting cell (ASC) assay (19,26) or in supernatants recovered from harvested PBMC (the "antibody in lymphocyte supernatant" [ALS] assay) (7,31). Although ALS and ASC responses have previously been measured after immunization with oral live attenuated typhoid vaccines, detailed analyses of ALS or ASC responses in individuals with wild-type typhoid fever are lacking (21,24). In order to gain further insight into mucosal immune responses during wild-type serovar Typhi infection, we undertook a study to characterize the serum and ALS responses to serovar Typhi among individuals with suspected typhoid fever in Bangladesh.…”

mentioning

confidence: 99%

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Salmonella enterica Serovar Typhi-Specific Immunoglobulin A Antibody Responses in Plasma and Antibody in Lymphocyte Supernatant Specimens in Bangladeshi Patients with Suspected Typhoid Fever

Sheikh

Bhuiyan

Khanam

et al. 2009

Many currently available diagnostic tests for typhoid fever lack sensitivity and/or specificity, especially in areas of the world where the disease is endemic. In order to identify a diagnostic test that better correlates with typhoid fever, we evaluated immune responses to Salmonella enterica serovar Typhi (serovar Typhi) in individuals with suspected typhoid fever in Dhaka, Bangladesh. We enrolled 112 individuals with suspected typhoid fever, cultured day 0 blood for serovar Typhi organisms, and performed Widal assays on days 0, 5, and 20. We harvested peripheral blood lymphocytes and analyzed antibody levels in supernatants collected on days 0, 5, and 20 (using an antibody-in-lymphocyte-supernatant [ALS] assay), as well as in plasma on these days. We measured ALS reactivity to a serovar Typhi membrane preparation (MP), a formalin-inactivated whole-cell preparation, and serovar Typhi lipopolysaccharide. We measured responses in healthy Bangladeshi, as well as in Bangladeshi febrile patients with confirmed dengue fever or leptospirosis. We categorized suspected typhoid fever individuals into different groups (groups I to V) based on blood culture results, Widal titer, and clinical features. Responses to MP antigen in the immunoglobulin A isotype were detectable at the time of presentation in the plasma of 81% of patients. The ALS assay, however, tested positive in all patients with documented or highly suspicious typhoid, suggesting that such a response could be the basis of improved diagnostic pointof-care-assay for serovar Typhi infection. It can be important for use in epidemiological studies, as well as in difficult cases involving fevers of unknown origin.Salmonella enterica serovar Typhi (serovar Typhi) is the cause of typhoid fever, an illness that affects over 20,000,000 individuals worldwide each year, killing over 200, 000 (5, 8, 16). The largest burden of typhoid fever is borne by impoverished individuals in resource-poor areas of the world. Serovar Typhi is a human-restricted invasive enteric pathogen which, after ingestion, crosses the intestinal mucosa, is taken up by gutassociated lymphoreticular tissues, and enters the systemic circulation. Both mucosal and systemic host immune responses are stimulated after infection. Serovar Typhi is an intracellular pathogen, and antibody and cell-mediated immune responses occur after infection or immunization with live oral attenuated typhoid vaccines (10,25,34).Diagnostic tests for typhoid fever often lack sensitivity and/or specificity, especially in areas of the world that are endemic for typhoid fever, where clinically distinguishing typhoid fever from other febrile illnesses is difficult (5, 17, 39). Microbiologic culturing of blood is approximately 30 to 70% sensitive, with the highest sensitivity being associated with an absence of prior use of antibiotics and the culturing of larger volumes of blood, features that complicate this mode of diagnosis in young children (5,6,8,36). Microbiologic culturing of bone marrow aspirates is more sensitive than bl...

“…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).…”

mentioning

confidence: 99%

Characterization of Anti- Salmonella enterica Serotype Typhi Antibody Responses in Bacteremic Bangladeshi Patients by an Immunoaffinity Proteomics-Based Technology

Charles

Sheikh

Krastins

et al. 2010

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 mucosa...