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Evaluating the A-Subunit of the Heat-Labile Toxin (LT) As an Immunogen and a Protective Antigen Against Enterotoxigenic Escherichia coli (ETEC).

Norton EB, Branco LM, Clements JD - PLoS ONE (2015)

Bottom Line: In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits.In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A).Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.

ABSTRACT
Diarrheal illness contributes to malnutrition, stunted growth, impaired cognitive development, and high morbidity rates in children worldwide. Enterotoxigenic Escherichia coli (ETEC) is a major contributor to this diarrheal disease burden. ETEC cause disease in the small intestine by means of colonization factors and by production of a heat-labile enterotoxin (LT) and/or a small non-immunogenic heat-stable enterotoxin (ST). Overall, the majority of ETEC produce both ST and LT. LT induces secretion via an enzymatically active A-subunit (LT-A) and a pentameric, cell-binding B-subunit (LT-B). The importance of anti-LT antibodies has been demonstrated in multiple clinical and epidemiological studies, and a number of potential ETEC vaccine candidates have included LT-B as an important immunogen. However, there is limited information about the potential contribution of LT-A to development of protective immunity. In the current study, we evaluate the immune response against the A-subunit of LT as well as the A-subunit's potential as a protective antigen when administered alone or in combination with the B-subunit of LT. We evaluated human sera from individuals challenged with a prototypic wild-type ETEC strain as well as sera from individuals living in an ETEC endemic area for the presence of anti-LT, anti-LT-A and anti-LT-B antibodies. In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits. In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A). Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined. Taken together, these data support the inclusion of both LT-A and LT-B in prospective vaccines against ETEC.

No MeSH data available.


Related in: MedlinePlus

ETEC-challenged human serum pool contains antibodies to both A- and B-subunits of LT.(A) ETEC challenge serum (pooled 10 days after oral H10407 challenge) anti-LT, anti-A, and anti-B antibody responses detected by ELISA (gray line, circles) compared to commercially purchased control sera (black lines, open circles) using dilutions of each sample. (B) ETEC-challenge serum (1) or control serum (2) immunoblot testing for anti-LT antibodies using unboiled LT-loaded lanes or boiled LT-loaded lanes. In unboiled SDS-PAGE gels, LT runs as an 84 kD polymeric protein, pentameric B-subunit (56 kD), and LT-A (28 kD). When boiled and subjected to SDS-PAGE, LT separates into LT-A (28 kD) and monomeric LT-B (11.5 kD). (C) ETEC-challenge serum or control serum anti-LT, anti-A, or anti-B responses detected with a modified Immunoblot using a slot blot apparatus to load 0.1 μg protein (LT, A, A1, or B) with raw images (top) and quantified band density of these images for unboiled, loaded proteins graphed (bottom).
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pone.0136302.g001: ETEC-challenged human serum pool contains antibodies to both A- and B-subunits of LT.(A) ETEC challenge serum (pooled 10 days after oral H10407 challenge) anti-LT, anti-A, and anti-B antibody responses detected by ELISA (gray line, circles) compared to commercially purchased control sera (black lines, open circles) using dilutions of each sample. (B) ETEC-challenge serum (1) or control serum (2) immunoblot testing for anti-LT antibodies using unboiled LT-loaded lanes or boiled LT-loaded lanes. In unboiled SDS-PAGE gels, LT runs as an 84 kD polymeric protein, pentameric B-subunit (56 kD), and LT-A (28 kD). When boiled and subjected to SDS-PAGE, LT separates into LT-A (28 kD) and monomeric LT-B (11.5 kD). (C) ETEC-challenge serum or control serum anti-LT, anti-A, or anti-B responses detected with a modified Immunoblot using a slot blot apparatus to load 0.1 μg protein (LT, A, A1, or B) with raw images (top) and quantified band density of these images for unboiled, loaded proteins graphed (bottom).

Mentions: We first analyzed human immune serum for antibody response to LT holotoxin or subunits using a pool of sera from individuals challenged 10-days previously with ETEC isolate H10407 (O78:H11:K80 LT+ ST+), a prototypical strain of enterotoxigenic E. coli which reproducibly elicits diarrhea in human volunteer studies [42]. Commercially purchased human serum was included as a negative control. As seen in Fig 1, this ETEC-challenge serum tested positive for anti-LT, anti-A, and anti-B antibodies by ELISA (Fig 1A) in plates coated with native LT, LT-A or LT-B. The ELISA results indicated that antibodies to both the A-subunit and B-subunit were present in the sera of individuals infected with an LT-producing strain. The ELISA primarily detects antibodies against conformational epitopes and the use of A-subunit contaminated with traces of holotoxin or B-subunit could give a misleading impression of the presence of A-subunit antibodies. These studies used recombinantly produced LT-A and LT-B to reduce that possibility. In addition, we used immunoblots with boiled and unboiled preparations of LT to distinguish between the presence of antibodies against conformational and linear epitopes of LT in the H10407 challenge serum pool. In unboiled SDS-PAGE gels, LT runs as an 84 kD polymeric protein, pentameric B-subunit (56 kD), and LT-A (28 kD). When boiled and subjected to SDS-PAGE, LT separates into LT-A (28 kD) and monomeric LT-B (11.5 kD). When immunoblots with samples of unboiled LT were probed with H10407 challenge serum (Fig 1B), antibodies to LT, pentameric B (B5) and LT-A were observed, while predominantly anti-A antibodies were detected in the boiled LT preparation. This suggests that the anti-B response is against conformational epitopes, while the anti-A response is likely directed against both linear and conformational determinants. We confirmed this finding by probing immunoblots loaded with boiled or unboiled purified LT, LT-A, LT-A1, or LT-B with H10407 challenge serum (Fig 1C). Taken together, our results show that individuals infected with a wild-type ETEC strain developed antibodies to LT, LT-A, LT-A1 and LT-B.


Evaluating the A-Subunit of the Heat-Labile Toxin (LT) As an Immunogen and a Protective Antigen Against Enterotoxigenic Escherichia coli (ETEC).

Norton EB, Branco LM, Clements JD - PLoS ONE (2015)

ETEC-challenged human serum pool contains antibodies to both A- and B-subunits of LT.(A) ETEC challenge serum (pooled 10 days after oral H10407 challenge) anti-LT, anti-A, and anti-B antibody responses detected by ELISA (gray line, circles) compared to commercially purchased control sera (black lines, open circles) using dilutions of each sample. (B) ETEC-challenge serum (1) or control serum (2) immunoblot testing for anti-LT antibodies using unboiled LT-loaded lanes or boiled LT-loaded lanes. In unboiled SDS-PAGE gels, LT runs as an 84 kD polymeric protein, pentameric B-subunit (56 kD), and LT-A (28 kD). When boiled and subjected to SDS-PAGE, LT separates into LT-A (28 kD) and monomeric LT-B (11.5 kD). (C) ETEC-challenge serum or control serum anti-LT, anti-A, or anti-B responses detected with a modified Immunoblot using a slot blot apparatus to load 0.1 μg protein (LT, A, A1, or B) with raw images (top) and quantified band density of these images for unboiled, loaded proteins graphed (bottom).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549283&req=5

pone.0136302.g001: ETEC-challenged human serum pool contains antibodies to both A- and B-subunits of LT.(A) ETEC challenge serum (pooled 10 days after oral H10407 challenge) anti-LT, anti-A, and anti-B antibody responses detected by ELISA (gray line, circles) compared to commercially purchased control sera (black lines, open circles) using dilutions of each sample. (B) ETEC-challenge serum (1) or control serum (2) immunoblot testing for anti-LT antibodies using unboiled LT-loaded lanes or boiled LT-loaded lanes. In unboiled SDS-PAGE gels, LT runs as an 84 kD polymeric protein, pentameric B-subunit (56 kD), and LT-A (28 kD). When boiled and subjected to SDS-PAGE, LT separates into LT-A (28 kD) and monomeric LT-B (11.5 kD). (C) ETEC-challenge serum or control serum anti-LT, anti-A, or anti-B responses detected with a modified Immunoblot using a slot blot apparatus to load 0.1 μg protein (LT, A, A1, or B) with raw images (top) and quantified band density of these images for unboiled, loaded proteins graphed (bottom).
Mentions: We first analyzed human immune serum for antibody response to LT holotoxin or subunits using a pool of sera from individuals challenged 10-days previously with ETEC isolate H10407 (O78:H11:K80 LT+ ST+), a prototypical strain of enterotoxigenic E. coli which reproducibly elicits diarrhea in human volunteer studies [42]. Commercially purchased human serum was included as a negative control. As seen in Fig 1, this ETEC-challenge serum tested positive for anti-LT, anti-A, and anti-B antibodies by ELISA (Fig 1A) in plates coated with native LT, LT-A or LT-B. The ELISA results indicated that antibodies to both the A-subunit and B-subunit were present in the sera of individuals infected with an LT-producing strain. The ELISA primarily detects antibodies against conformational epitopes and the use of A-subunit contaminated with traces of holotoxin or B-subunit could give a misleading impression of the presence of A-subunit antibodies. These studies used recombinantly produced LT-A and LT-B to reduce that possibility. In addition, we used immunoblots with boiled and unboiled preparations of LT to distinguish between the presence of antibodies against conformational and linear epitopes of LT in the H10407 challenge serum pool. In unboiled SDS-PAGE gels, LT runs as an 84 kD polymeric protein, pentameric B-subunit (56 kD), and LT-A (28 kD). When boiled and subjected to SDS-PAGE, LT separates into LT-A (28 kD) and monomeric LT-B (11.5 kD). When immunoblots with samples of unboiled LT were probed with H10407 challenge serum (Fig 1B), antibodies to LT, pentameric B (B5) and LT-A were observed, while predominantly anti-A antibodies were detected in the boiled LT preparation. This suggests that the anti-B response is against conformational epitopes, while the anti-A response is likely directed against both linear and conformational determinants. We confirmed this finding by probing immunoblots loaded with boiled or unboiled purified LT, LT-A, LT-A1, or LT-B with H10407 challenge serum (Fig 1C). Taken together, our results show that individuals infected with a wild-type ETEC strain developed antibodies to LT, LT-A, LT-A1 and LT-B.

Bottom Line: In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits.In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A).Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.

ABSTRACT
Diarrheal illness contributes to malnutrition, stunted growth, impaired cognitive development, and high morbidity rates in children worldwide. Enterotoxigenic Escherichia coli (ETEC) is a major contributor to this diarrheal disease burden. ETEC cause disease in the small intestine by means of colonization factors and by production of a heat-labile enterotoxin (LT) and/or a small non-immunogenic heat-stable enterotoxin (ST). Overall, the majority of ETEC produce both ST and LT. LT induces secretion via an enzymatically active A-subunit (LT-A) and a pentameric, cell-binding B-subunit (LT-B). The importance of anti-LT antibodies has been demonstrated in multiple clinical and epidemiological studies, and a number of potential ETEC vaccine candidates have included LT-B as an important immunogen. However, there is limited information about the potential contribution of LT-A to development of protective immunity. In the current study, we evaluate the immune response against the A-subunit of LT as well as the A-subunit's potential as a protective antigen when administered alone or in combination with the B-subunit of LT. We evaluated human sera from individuals challenged with a prototypic wild-type ETEC strain as well as sera from individuals living in an ETEC endemic area for the presence of anti-LT, anti-LT-A and anti-LT-B antibodies. In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits. In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A). Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined. Taken together, these data support the inclusion of both LT-A and LT-B in prospective vaccines against ETEC.

No MeSH data available.


Related in: MedlinePlus