Evaluation of Multivalent Enterotoxigenic Escherichia coli Vaccine Candidate MecVax Antigen Dose-Dependent Effect in a Murine Model

Abstract

There are no licensed vaccines against enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea and travelers' diarrhea. Recently, protein-based vaccine candidate MecVax was demonstrated to induce functional antibodies against both ETEC toxins (heat-stable toxin [STa] and heat-labile toxin [LT]) and seven ETEC adhesins (CFA/I and CS1 to CS6) and to protect against ETEC clinical diarrhea or intestinal colonization preclinically. Those studies used intraperitoneal, intramuscular, and intradermal routes, and a dose range for MecVax protein antigens, toxoid fusion 3xSTa_N12S-mnLT_R192G/L211A, and adhesin CFA/I/II/IV MEFA has not been investigated. Here, we further characterized MecVax broad immunogenicity, utilizing a subcutaneous route, and examined vaccine dose-dependent antibody response effects and also antibody functional activities against ETEC enterotoxicity and bacterial adherence. Data showed that mice immunized subcutaneously with MecVax developed robust IgG responses to seven ETEC adhesins (CFA/I, as well as CS1 to CS6) and two toxins (STa and LT). At a subcutaneous dose of 25, 20, or 10 μg or at an intramuscular dose of 12, 6, or 3 μg, MecVax induced similar levels IgG responses to the targeted toxins and adhesins, and these antibodies exhibited equivalent functional activities against ETEC toxin enterotoxicity and bacterial adherence. Once the intramuscular dose was decreased to 1 μg, vaccine-induced antibodies were significantly reduced and no longer neutralized STa enterotoxicity. The results indicated that MecVax administered subcutaneously is broadly immunogenic and, at an intramuscular dose of 3 μg, can induce functional antitoxin and anti-adhesin antibodies in mice, providing instructive information for future vaccine dose studies in humans and accelerating MecVax vaccine development. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is a leading cause of children's diarrhea and the most common cause of travelers' diarrhea. ETEC infections are responsible for >200 million diarrhea clinical cases and near 100,000 deaths annually. Currently, there are no licensed vaccines for ETEC diarrhea. The protein-based vaccine candidate MecVax unprecedentedly targets two ETEC toxins (STa and LT, produced by all ETEC strains) and seven ETEC adhesins (CFA/I, as well as CS1 to CS6, associated with >60% of ETEC clinical diarrhea cases) and has been demonstrated to be broadly immunogenic and cross protective; as such, it represents a potentially effective multivalent vaccine against ETEC-associated children's and travelers' diarrhea. This study further confirmed MecVax broad immunogenicity and evaluated the vaccine antigen dose effect on the induction of antigen-specific antibody responses in mice and on antibody functional activities against ETEC toxin enterotoxicity and bacterial adherence, yielding useful information for future human volunteer studies and the development of MecVax as an effective ETEC vaccine.

Keywords: ETEC; MecVax; diarrhea; dose-dependent effect; dose-dependent study; enterotoxigenic Escherichia coli; vaccine.

FIG 1

IgG titers (log₁₀) from serum samples of the mice subcutaneously immunized with MecVax composed of 25 μg of CFA/I/II/IV MEFA protein and 25 μg of toxoid fusion 3xSTa_N12S-mnLT_R192G/L211A protein (●) or PBS as the control (○). dmLT adjuvant (0.2 μg) was used in the immunized group. Bars indicate antibody mean titers and standard deviations.

FIG 2

IgG (log₁₀) titers from serum samples of the mice intramuscularly immunized with MecVax at different doses of CFA/I/II/IV MEFA and toxoid fusion 3xSTa_N12S-mnLT_R192G/L211A. Four groups of mice intramuscularly immunized with MecVax at various antigen doses—12 μg (♦), 6 μg (▾), 3 μg (▴), or 1 μg (■)—another group with PBS (●) served as the control. We included 0.2 μg of dmLT adjuvant in the four immunization groups. Each dot represents an IgG titer from a mouse in each group. Asterisks (*, **, and ***) represent P values of <0.05, <0.01, and <0.001, respectively. Bars indicate antibody titer means and standard deviations.

FIG 3

Intracellular cGMP or cAMP levels (nM [pmol/mL]) in T-84 cells to show the antibody neutralization activity against STa and CT enterotoxicity. T-84 cells exposed to STa or CT (LT homologue) pretreated with the serum sample from mice intramuscularly immunized with MecVax at different doses (■) or the control mice (□) were measured for cGMP or cAMP concentrations. T-84 cells incubated with culture medium (no toxin and no serum) were used to show T-84 cell baseline cGMP or cAMP. Boxes and bars indicate means and standard deviations of cGMP or cAMP levels. Asterisks (** and ***) indicate P values of <0.01 and <0.001, respectively.

FIG 4

Antibody adherence inhibition assays were performed to show the number of ETEC or E. coli bacteria (CFU [%]) adherent to Caco-2 cells after incubation with serum samples of mice intramuscularly immunized with MecVax (■; at different doses) or PBS as the control (□). E. coli expressing CS1 or CS2 adhesin and ETEC isolates producing CFA/I, CS3, CS4/CS6, CS5/CS6, or CS6 adhesin were treated with mouse serum samples from the group intramuscularly immunized with MecVax at a dose of 12, 6, 3, 1, or 0 μg and then incubated with Caco-2 cells. Bacteria adhered to Caco-2 cells were collected, plated, and counted for CFU after overnight growth. Boxes and bars represent means and standard deviations of bacterial adherence (in %). Asterisks (*, **, and ***) indicate P values of <0.05, <0.01, and <0.001, respectively.