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Enterotoxigenic Escherichia coli (ETEC), which produces heat-labile toxin (LT), is a common cause of travelers' diarrhea (TD). The B subunit of ETEC LT is immunologically related to the B subunit of Vibrio cholerae toxin (CT). In this pilot study we evaluated the whole-blood gamma interferon response to CT B in 17 U.S. adults traveling to Mexico. Only one of nine subjects who demonstrated a cellular immune response as determined by whole-blood gamma interferon production to CT B on arrival to Mexico developed diarrhea, whereas five of eight without a cellular response developed diarrhea. Markers of the cellular immune response to ETEC LT could help in identifying individuals immune to ETEC LT, and these markers deserve additional study.
Enterotoxigenic Escherichia coli (ETEC) is the most common cause of travelers' diarrhea (TD) in U.S. visitors to Mexico (1). ETEC isolates can produce heat-labile toxin (LT), heat-stable toxin (ST), or both toxins simultaneously (8). The LT of ETEC is highly homologous to the Vibrio cholerae toxin (CT) (4) and is composed of five beta-subunits that bind to the intestinal epithelial cell and a single alpha-unit that activates intracellular adenyl cyclase by virtue of its ADP-ribosylating activity. In addition to being an enterotoxin, LT is a potent mucosal adjuvant (5).
In travelers, natural exposure to the ETEC LT is associated with a humoral immune response to LT; however, serum LT antibody titers resulting from naturally acquired ETEC LT do not correlate with long-lasting protection (11, 12). This suggests that other components of the immune system are important in developing protective immunity to ETEC LT.
Little is known about the protective effect that cellular immunity provides against ETEC LT and V. cholerae. In mice, immunization with the B subunit of the ETEC LT induces early CD4+ Th1-type and late CD4+ Th2-type activation in Peyer's patches (10). In humans, individuals suffering from acute cholera show increased CD4+ and CD8+ T-cell proliferative responses against V. cholerae during the acute and convalescent stages of infection. Gut-homing CD4+ T cells (β7+), gut-homing CD8+ T cells (β7+), and gut-homing B cells (CD19+ β7+) also increase during the late convalescent stage of cholera compared to the acute stage of disease (2). This suggests that cellular immunity is important in the response to V. cholerae infection.
We conducted a pilot prospective study to evaluate the cellular immune response to ETEC LT as evidenced by the whole-blood gamma interferon response to the B subunit of CT in U.S. adults traveling to a region where ETEC is endemic. The study was approved by the Committee for the Protection of Human Subjects of the University of Texas Health Science Center at Houston.
(This paper was presented in part at the 2008 Joint 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy and 47th Infectious Diseases Society of America Annual Meeting in Washington, DC [abstract no. P-1615].)
Seventeen white non-Hispanic U.S. adults traveling to Cuernavaca, Mexico, for 3 weeks during July and August 2007 were enrolled in the study. Thirteen were women (76.5%), and the mean age at arrival was 28.6 years (standard deviation [SD], 11.6). TD was defined as the passage of three or more unformed stools within a 24-hour period plus one or more abdominal symptom of enteric infection. Six (35.3%) participants developed TD. In this pilot study, gender, age, and previous travel history were not significantly associated with the occurrence of TD (Table (Table1).1). The mean onset for TD was 12.3 days (SD, 5.6), and there was a mean of 19.5 unformed stools (SD, 4.0) per stay.
We evaluated the whole-blood gamma interferon response (WBGIR) against the B subunit of CT as a surrogate of the ETEC LT immune response on arrival to Mexico and prior to departure. Briefly, 10 ml of venous blood was collected in a heparinized tube from each participant and 1 ml of whole blood was cultured per well of a 24-well ultra-low attachment polystyrene tissue culture plate (Corning, NY) within 2 h after collection. The whole blood was then stimulated with either 100 μl sterile phosphate-buffered saline (PBS; negative control), 100 μl of 1 μg/ml phytohemagglutinin-L (positive control), or 100 μl of 5 μg/ml CT subunit B. The tissue culture plates were incubated for 24 h at 37°C with 5% CO2 and 100% humidity. Plasma was then harvested and the gamma interferon concentration was determined by using a commercially available enzyme immunoassay according to the manufacturer's instructions (Quantiferon, Valencia, CA). A positive gamma interferon response was defined as >0.5 IU/ml after 24 h of antigen stimulation.
Nine (52.3%) travelers had positive WBGIR on arrival to Mexico. Six participants had a previous travel history to Mexico. Only one recalled suffering from TD during a prior trip to Mexico. Previous travel history to Mexico within the prior year was not associated with a positive WBGIR (3/9 versus 3/8; P = not significant [NS]).
Individuals developing TD had lower mean WBGIR at arrival than those developing no symptoms (0.41 ± 0.60 versus 1.14 ± 1.27; P = 0.04). Of interest, individuals with negative WBGIR were more likely to experience TD (P = 0.05). Five out of the eight (62.5%) travelers with negative WBGIR at arrival developed TD. In contrast, only one out of nine (11.1%) travelers with positive WBGIR at arrival developed TD (Table (Table2).2). Furthermore, individuals with positive WBGIR passed a lower number of unformed stools (8.8 versus 17.0) per stay. WBGIR conversion at departure occurred in four (50%) of the eight travelers with negative response at arrival, two of whom suffered from TD while in Mexico (Table (Table22).
In the present study, we found that individuals possessing a Th-1 cellular immune response against ETEC LT as evidenced by a WBGIR to the B subunit of CT were less likely to develop diarrhea when traveling to an area where ETEC LT is endemic than individuals without such a response. This report is the first prospective study describing an association between the cellular immune response to the CT B subunit and TD during natural exposure. The small sample size did not allow us to determine if the WBGIR to CT B was protective of ETEC LT diarrhea, but it appeared to correlate with protection against TD overall. This protective effect of immunity to LT against TD is consistent with previous observations of vaccines with an LT component that provided protection against other enteropathogens (3, 7), presumably by preventing the LT-mediated conditioning of the intestinal epithelium that renders it more susceptible to infection (9).
Our previous studies in travelers have shown that subjects with polymorphisms in the promoter region of the interleukin-10 (IL-10) gene that are associated with high IL-10 production are more likely to develop ETEC LT-associated diarrhea (6). How this observation relates to the whole-blood gamma interferon response to LT is unknown; it is plausible that the increased susceptibility to ETEC LT is mediated by IL-10-induced blocking of gamma interferon production by T cells. Whether CD4+ or NKT or other T cells are responsible for the WBGIR seen in this study and the relationships to IL-10 single-nucleotide polymorphisms remain to be characterized.
This small study will require validation in a larger prospective study that can examine the contribution of the humoral immune response to LT and ETEC colonization factors, host genetics, and cytokine responses during the course of the disease. We plan to examine the utility of the WBGIR CT B assay for evaluation of susceptibility to LT and to refine host correlates of protection against ETEC LT.
We are indebted to Judy Guillen, Gabriela Meza, Lily Carlin, and the administration and staff of Universidad Internacional in Cuernavaca, Morelos, Mexico, for their assistance with this project.
This work was supported by the following sources: NIH R01 AI54948-01 to P.C.O. and NIH DK56338, which funds the Texas Gulf Coast Digestive Diseases Center.
We report that there are no conflicts of interest related to this work.
Published ahead of print on 10 March 2010.