Date Published: May 1, 2019
Publisher: Public Library of Science
Author(s): Hyesuk Seo, Rahul M. Nandre, Jerome Nietfeld, Zhenhai Chen, Qiangde Duan, Weiping Zhang, Nicholas J Mantis.
Antibodies that block the adherence of enterotoxigenic Escherichia coli (ETEC) to host intestinal epithelial cells are protective. Multiepitope-fusion-antigens (MEFAs) carrying epitopes of ETEC adhesin major subunits or tip minor subunits induced antibodies against ETEC adherence. Adherence inhibition effectiveness of antibodies induced by major subunit epitopes versus minor tip subunit epitopes, however, has not been comparatively characterized. In this study, we immunized mice with a major subunit MEFA or a tip MEFA, evaluated MEFA anti-adhesin immunogenicity, and examined induced-antibodies against bacteria in vitro adherence or in vivo colonization in mice. Mice subcutaneously immunized with major subunit MEFA CFA/I/II/IV or tip MEFA showed no adverse effects and developed strong antigen-specific antibody responses. Data showed that antibodies derived from two MEFAs were equally effective against adherence of the bacteria expressing CS1, CS2, CS3, CS4/CS6, CS5/CS6, or CS6 adhesin in vitro. Subsequently, we immunized mice with CFA/I fimbriae, major subunit CfaB, or minor tip adhesin subunit CfaE. We found that antibodies induced by CFA/I, CfaB and CfaE equally inhibited in vitro adherence of ETEC strain H10407. Furthermore, we immunized mice with CFA/I fimbriae, CfaB, or CfaE, and then challenged the mice with H10407. Data showed that although not significantly, fewer H10407 bacteria colonized the immunized mice. These results suggest that ETEC adhesin major subunit and minor tip subunit should be equally effective in inducing neutralizing anti-adhesin antibodies, and that major subunit CFA/I/II/IV MEFA or tip MEFA, perhaps combined with toxoid fusion 3xSTaN12S-mnLTR192G/L211A, can be used for development of broadly protective vaccines against ETEC diarrhea.
Diarrheal disease remains a major global health problem as it continues to be a leading cause of death in children younger than 5 years of age in the developing countries [1, 2]. Enterotoxigenic Escherichia coli (ETEC) bacteria are among the leading causes of children’s diarrhea [1–3]. ETEC associated diarrhea contributes to a significant portion of the children annual death rate [2, 4]. ETEC bacteria are also the most common cause of diarrhea in children and adults travelling from developed countries to ETEC endemic regions, civil and military personnel deployed at developing countries, and perhaps immunocompromised patients [5–7]. Unfortunately, there is no licensed vaccine against ETEC diarrhea [8, 9].
Data from the current study indicated that antibodies derived from ETEC major structural subunits and minor tip subunits, or epitopes of a major subunit and a minor tip subunit, appear to be equivalently effective against bacteria adherence in vitro. Since ETEC tip subunits or adhesive subunits play an essential role in bacterial inter-reaction with host cell receptors, anti-tip subunit antibodies which directly block attachment of ETEC fimbriae to host cell receptors can effectively protect against ETEC bacterial colonization and thus diarrheal disease. Antibodies against a fimbrial major structural subunit, unless this major subunit is also the adhesive subunit which is directly involved in bacterial adherence, do not serve as a direct blockage to prevent the recognition between a fimbria and host receptors. A recent study, however, demonstrated that anti-fimbria antibodies, particularly antibodies to ETEC major structural subunits can physically damage fimbria adherence function . Anti-fimbria or anti-major subunit antibodies recognize adjacent major subunits and lock extension and contraction movement of coil-structured fimbriae. That makes fimbria “stiff and tangled”, resulting in reduction or prevention of ETEC fimbria adherence to host cells . Since the molecular sizes of IgG and more particularly IgA antibodies to CFA major subunits are much larger compared to a typical ETEC tip subunit (15 to 50 kDa), anti-major subunit antibodies recognized the major structural subunits adjacent to the tip subunit can physically prevent a tip from adhering to host receptors (S2 Fig). The high copy number of a major subunit in an ETEC fimbria also intensify the interaction between anti-major-subunit antibodies and fimbriae. Thus, anti-major subunit antibodies can be equivalently effective as anti-tip antibodies to inhibit fimbrial adherence to host cells.