MecVax supplemented with CFA MEFA-II induces functional antibodies against 12 adhesins (CFA/I, CS1-CS7, CS12, CS14, CS17, and CS21) and 2 toxins (STa, LT) of enterotoxigenic Escherichia coli (ETEC)

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains that produce various adhesins and one or two enterotoxins are the leading causes of children's diarrhea and travelers' diarrhea. MecVax, a multivalent ETEC vaccine candidate, consists of two proteins, an adhesin multiepitope fusion antigen (MEFA) that stimulates antibodies to the seven most important ETEC adhesins (CFA/I and CS1-CS6) and a toxoid fusion antigen which stimulates antibodies against ETEC enterotoxins (heat-labile toxin and heat-stable toxin). CFA MEFA-II, another polyvalent MEFA protein, has been demonstrated to stimulate antibodies to another five important ETEC adhesins (CS7, CS12, CS14, CS17, and CS21). We hypothesize that MecVax coverage and efficacy can be expanded if MecVax could stimulate antibodies to all 12 adhesins. In this study, we supplemented MecVax with CFA MEFA-II, examined broad immunity to the 12 targeted ETEC adhesins and 2 ETEC toxins (STa, LT) in mice, and assessed mouse antibody functions for inhibiting the adherence of the 12 adhesins and neutralizing the enterotoxicity of 2 toxins, thus assessing the potential application of a broadly protective pan-ETEC vaccine. Mice intramuscularly immunized with MecVax and CFA MEFA-II developed robust antibody responses to the 12 ETEC adhesins and 2 toxins; furthermore, mouse serum antibodies showed functional activities against the adherence from each of the targeted adhesins and the enterotoxicity of either toxin. Data also indicated that CFA MEFA-II was antigenically compatible with MecVax. These results demonstrated that the inclusion of CFA MEFA-II further expands MecVax broad immunogenicity and protection coverage, suggesting the feasibility of developing a vaccine against all important diarrheal ETEC strains.IMPORTANCEThere are no vaccines licensed for Enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea and the most common cause of travelers' diarrhea. Since ETEC strains produce over 25 adhesins and 2 distinctive enterotoxins, heterogeneity is a key obstacle to vaccine development. MecVax, a multivalent ETEC vaccine candidate, induces protective antibodies against the seven most important adhesins (CFA/I and CS1-CS6) associated with two-thirds of ETEC clinical cases. However, ETEC prevalence shifts chronically and geographically, and other adhesins are also associated with clinical cases. MecVax would become a pan-ETEC vaccine if it also protects against the remaining important adhesins. This study demonstrated that MecVax supplemented with adhesin protein CFA MEFA-II induces functional antibodies against 12 important ETEC adhesins (CFA/I, CS1-CS7, CS12, CS14, CS17, and CS21), enabling the development of a more broadly protective ETEC vaccine and further validating the application of the MEFA vaccinology platform for multivalent vaccine development.

Keywords: CFA MEFA-II; ETEC (enterotoxigenic E. coli); MecVax; diarrhea; vaccine.

Fig 1

Mouse serum antigen-specific IgG titers (log₁₀) from the group intramuscularly immunized with MecVax (white boxes), “MecVax + CFA MEFA-II” (MecVax supplemented with CFA MEFA-II; gray boxes), CFA MEFA-II (white boxes with diagonal lines), or phosphate buffer saline (PBS) (dark boxes); dmLT adjuvant was included in four groups. ELISAs with 2HB plates coated with 100 ng recombinant protein CfaB, CooA, CotA, CstH, CsaB, CsfA, CssA, CsvA, CswA, CsuA, CsbA, LngA, or cholera toxin (CT, Sigma) or CoStar plates with 10 ng STa-ovalbumin conjugates were used to titrate anti-CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS12, CS14, CS17, CS21, LT, or STa IgG antibodies in twofold serum dilutions (1:400 to 1:102,400) from each mouse in a group. Antibody titers (in log₁₀) were presented in means and standard deviations. * and ** indicate a P-value less than 0.05 and 0.001, respectively.

Fig 2

Results of antibody inhibition assay to show mouse serum antibodies inhibited adherence of E. coli (CS1, CS2) or ETEC strains expressing the adhesins targeted by MecVax (CFA/I, CS1–CS6) and CFA MEFA-II (CS7, CS12, CS14, CS17, and CS21) to Caco-2 cells. Adherent (to Caco-2 cells) bacteria expressing CFA/I, CS1, CS2, CS3, CS4/CS6, CS5/CS6, CS6, CS7, CS12/CS20, CS14, CS17, or CS21 adhesin, after treated with mouse sera from the group injected with PBS (dark boxed), MecVax (white boxes), “MecVax + CFA MEFA-II” (gray boxes), or CFA MEFA-II (white boxes with diagonal lines), were counted (CFUs; in means and standard deviations) and converted to percentages by referring CFUs from cells treated with control mouse sera as 100%. *** indicates a P value less than 0.0001.

Fig 3

Results of antibody neutralization activities against STa or CT enterotoxicity, by using T-84 cells and a cyclic GMP or AMP EIA kit. Intracellular cGMP (left) or cAMP (right) concentrations (nM; in means and standard deviations) in T-84 cells after incubation with toxin STa (2 ng) or CT (30 ng) which were pre-treated with mouse sera pooled from the group IM immunized with PBS control (black box), MecVax (white box), or “MecVax + CFA MEFA-II” (gray box). Intracellular cGMP or cAMP levels of T-84 cells incubated with culture medium (no toxin and no sera) were used as the baseline (a box with horizontal lines). *** indicates a P-value less than 0.0001.