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. IMPORTANCE There 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.

E nterotoxigenic Escherichia coli (ETEC) strains that produce heterogeneous adhesins [colonization factor antigen (CFA) or coli surface antigen (CS)] and two types of enterotoxins [heat-labile toxin (LT) and heat-stable toxin (STa)], are one of the top four causes of diarrhea in children living in developing countries (children's diarrhea) and the most common course of diarrhea in international travelers (travelers' diarrhea) (1)(2)(3)(4).Currently, there is no vaccine licensed against ETEC-associated children's diarrhea or travelers' diarrhea.An effective ETEC vaccine can potentially save about a hundred thousand lives and prevent over 200 million clinical diarrhea cases annually (5)(6)(7).Additionally, a protective ETEC vaccine would reduce the use of antibiotics to treat patients with severe infections from ETEC strains which continuously acquire antibiotic resistance, thus helping to combat the emergence of antimicrobial resistance and superbugs (8,9).
One key challenge in developing effective vaccines for ETEC is the heterogeneity among ETEC strains (pathovars).ETEC strains possess different profiles of virulence factors, with various combinations of adhesins and toxins.There are over 25 immuno logically heterogeneous adhesins, including CFA and CS, produced by ETEC strains to attach bacteria to host receptors and colonize small intestines.These ETEC strains produce one or two types of enterotoxins, LT, and STa and deliver toxins to intesti nal epithelial cells to elevate intracellular cyclic adenosine monophosphate (cAMP) or guanosine monophosphate (cGMP) levels and cause fluid hyper-secretion into the gut lumen.Epidemiological and systematic prevalence studies revealed that the ETEC strains expressing seven adhesins, CFA/I and CS1-CS6 (and STa and/or LT toxin), are estimated to cause two-thirds of the ETEC-associated clinical cases as well as the moderate-to-severe cases (10)(11)(12).However, ETEC prevalence can shift chronically and geographically (10,13).ETEC strains producing other adhesins including CS7, CS12, CS14, CS17, and CS21 (and one or both ETEC toxins) also play a significant role in causing clinical diarrhea and moderate-to-severe diarrheal cases (10,11,14).
There are a few ETEC vaccine candidates currently under preclinical or clinical investigation, but past ETEC vaccine research and development efforts mostly target a few of the seven most prevalent adhesins (CFA/I and CS1-CS6) in addition to toxin LT but barely STa (15).Among them, ETVAX, a killed whole-cell ETEC vaccine candidate, composed of four inactivated strains to express four adhesins (CFA/I, CS3, CS5, and CS6) and supplemented with a recombinant CT B /LT B chimeric protein (16), was demonstrated to be tolerated and induce moderate cross-reactive immunity in adults and children (17)(18)(19).However, ETVAX does not carry antigens to protect against STa, the ETEC toxin that plays a more important role in causing children's diarrhea and travelers' diarrhea (20,21).In contrast to the conventional cellular and acellular vaccine approaches, a novel epitope-and structure-based vaccinology platform named multiepitope-fusionantigen (MEFA) was developed recently and used to construct a multivalent ETEC vaccine candidate MecVax to target all of the seven most important ETEC adhesins and both ETEC toxins (22)(23)(24)(25).MecVax is composed of two polyvalent protein immunogens that present heterogeneous functional epitopes (from seven ETEC adhesins or two ETEC toxins) on a backbone and mimic epitope native antigenicity.We have demonstrated that MecVax not only induces functional antibodies against the seven targeted adhesins (CFA/I and CS1-CS6) and two toxins (STa, LT) but also protects against ETEC intestinal colonization and clinical diarrhea in animal models (23,24,26,27).More recently, by using the same MEFA platform, we generated another polyvalent MEFA protein (termed as CFA MEFA-II) to target five remaining important ETEC adhesins (CS7, CS12, CS14, CS17, and CS21) and showed that this CFA MEFA-II protein elicits functional antibodies against the five targeted ETEC adhesins (28,29).
An ideal ETEC vaccine would cover the important ETEC adhesins and both ETEC toxins and protect against all cases of ETEC-associated children's diarrhea and travelers' diarrhea.MecVax can become a pan-ETEC vaccine if it can further expand coverage and induce functional antibodies against the 12 important ETEC adhesins (CFA/I, CS1-CS6, CS7, CS12, CS14, CS17, and CS21) and two ETEC toxins (STa, LT).In this study, we combined three polyvalent proteins, by supplementing MecVax with adhesin MEFA-II, intramuscularly immunized mice, examined mouse antigen-specific antibody responses, and measured mouse antibody functional activities against ETEC bacterial adherence and toxin enterotoxicity.Results showed that the immunized mice developed robust antibody responses to all target antigens, 12 ETEC adhesins and 2 ETEC toxins.Moreover, the induced antibodies were functional, as shown by the inhibition of adherence of bacteria that express any of the target adhesins and neutralization of both toxins.While the current study is limited to immunogenicity and antibody functional activity assessment and future studies of preclinical and clinical efficacy evaluation are needed, data from this study suggest the potential of MecVax combined with CFA MEFA-II for developing a more broadly protective vaccine against ETEC-associated diarrhea.

Mouse antibodies derived from 'MecVax + CFA MEFA-II' neutralized the enterotoxicity of both ETEC toxins (STa, CT)
Sera from the mice IM immunized with "MecVax + CFA MEFA-II" neutralized STa and CT enterotoxicity, shown by the prevention of STa or CT enterotoxicity from elevating intracellular cGMP or cAMP in T-84 cells (Fig. 3).T-84 cell intracellular cGMP levels were 39 ± 3.4 (nM) after cells were incubated with STa toxin (2 ng) pre-mixed with the sera from the mice immunized with "MecVax + CFA MEFA-II." In contrast, significantly greater cGMP levels (266 ± 17.1 nM; P < 0.0001) were detected in the T-84 cells that were incubated with STa pre-treated with the control mouse sera.The baseline intracellular cGMP levels (T-84 cells in tissue culture medium) were 24 ± 3.7 nM.
The intracellular cAMP levels in the cells incubated with CT (cholera toxin, a homolog of LT, 30 ng) pre-mixed with the sera from the mice immunized with "MecVax + CFA MEFA-II" were 76 ± 2.0 nM.These levels were significantly lower than the cAMP in the cells treated with CT only (394 ± 50.1 nM; P < 0.0001).Because dmLT adjuvant was administered to all the mice including the control group and dmLT induces neutralizing anti-LT antibodies, the cAMP concentrations in the cells treated with CT and the control mouse sera were 124 ± 10.3 nM, which were higher than the levels in the cells treated with sera of the mice immunized with "MecVax + CFA MEFA-II" though the increasing was not significant.The baseline cAMP levels in T-84 cells (in tissue culture medium, no serum or toxin) were 12 ± 0.4 nM.

Mouse serum antibodies derived from "MecVax + CFA MEFA-II" or MecVax equally neutralized STa or CT enterotoxicity
The sera from the mice IM immunized with MecVax or "MecVax + CFA MEFA-II" showed similar levels of neutralizing activities against STa or CT enterotoxicity (Fig. 3), indicat ing that toxin-specific antibody neutralization activities from MecVax (which carries toxin antigens) were not compromised after being combined with CFA MEFA-II protein antigen.The cGMP concentrations in T-84 cells incubated with STa and the sera from the group immunized with MecVax were 38 ± 2.6 nM, which is nearly identical to the cGMP levels in the cells treated with the toxin and the sera of the mice immunized with "MecVax + CFA MEFA-II" (39 ± 3.4 nM).Similarly, the cAMP in the T-84 cells treated with CT and the sera from the group immunized with MecVax (45 ± 4.4 nM) did not differ significantly from the cAMP levels in the cells treated with CT and sera of the group immunized with "MecVax + CFA MEFA-II" (76 ± 2.0 nM; P = 0.54).

DISCUSSION
Data from this study indicated that mice intramuscularly immunized with MecVax supplemented with polyvalent antigen CFA MEFA-II ("MecVax + CFA-MEFA-II") developed functional antibodies unprecedentedly to 12 ETEC adhesins (CFA/I, CS1-CS7, CS12, CS14, CS17, and CS21) and 2 toxins (STa, LT).These 12 adhesins (together with one or both enterotoxins) are expressed by ETEC strains causing more than 86% of ETEC clinical diarrheal cases and moderate-to-severe cases.A vaccine that induces protective immunity against adherence to these 12 ETEC adhesins can potentially protect against ETEC-associated moderate-to-severe cases and a vast majority of clinical diarrheal cases.
Results from this study indicate that the third protein antigen CFA MEFA-II is antigenically compatible with the two proteins of MecVax.Mice IM immunized with "MecVax + CFA MEFA-II" developed similar levels of antibody responses to eight of the nine targeted antigens (CFA-I, CS2-CS6, STa, and LT) as the mice IM immunized with MecVax.Similarly, mice immunized with "MecVax and CFA MEFA-II" and the mice IM immunized with CFA MEFA-II developed similar levels of antibody responses to four of the five targeted antigens (CS7, CS14, CS17, and CS21).More importantly, antibod ies elicited by "MecVax + CFA MEFA-II" and the antibodies derived from MecVax had the same level of protection against adherence by ETEC strains expressing any of the seven target adhesins.Similarly, antibodies induced by "MecVax and CFA MEFA-II" and antibodies elicited by CFA MEFA-II equally inhibited the adherence of the five targeted adhesins.Additionally, the antitoxin antibodies derived from MecVax, alone or along with CFA MEFA-II, had the same level of neutralization activities against STa toxin, indicated by nearly identical levels of cGMP in T-84 cells after treatment with STa toxin and the sera from either immunization group ("MecVax + CFA MEFA-II" versus MecVax).The anti-LT antibody response and anti-LT (anti-CT) antibody neutralization activity were detected at the same levels from the group immunized with "MecVax and CFA MEFA-II" or the group immunized with MecVax alone, though the similarities were not conclusive in the current study because of the additive effect from adjuvant dmLT that also induces functional anti-LT antibodies.However, MecVax neutralization against CT enterotoxicity and protection from LT-mediated diarrhea were demonstrated previously (23)(24)(25).While protective anti-LT antibodies from dmLT adjuvant is an additive to MecVax for protection against LT-producing ETEC infection, future studies using a non-LT adjuvant will allow us to conclusively evaluate if anti-LT antibody response and neutralization activity against LT (CT) are compromised after MecVax is co-administered with CFA MEFA-II or another polyvalent protein antigen.
MecVax is a protein-based multivalent ETEC vaccine candidate that has been demonstrated to induce functional antibodies against the seven most important ETEC adhesins (CFA/I and CS1-CS6) and two ETEC toxins (STa, LT) (24,25,30).Moreover, MecVax is shown to protect against ETEC toxin-mediated clinical diarrhea in a pig challenge model and prevent over 99% of ETEC bacterial colonization in small intestines by ETEC strains expressing any of the seven targeted adhesins in a rabbit model (26,27).The adhesin-specific antibodies elicited by MecVax can potentially prevent over two-thirds of ETEC-associated clinical diarrheal cases since ETEC strains producing CFA/I and CS1-CS6 adhesins are estimated to be associated with about 65% of clinical cases (10,14).When supplemented with CFA MEFA-II which targets another five important ETEC adhesins (CS7, CS12, CS14, CS17, and CS21), MecVax expands its coverage to 12 ETEC adhesins, including all important ETEC adhesins associated with moderate-tosevere diarrheal cases.The functional antibodies against 12 adhesins can potentially improve MecVax efficacy from 65% to over 86% since CFA/I, CS1-CS7, CS12, CS14, CS17, and CS21 adhesins are expressed by 86.1% of STa-positive ETEC strains, 80.6% of STa-and/or LT-positive ETEC strains, and 47.3% of LT-positive ETEC strains (14).In addition, the toxin-specific antibodies elicited by MecVax neutralize STa and LT, the two enterotoxins, alone or together, are produced by all ETEC strains.This antitoxin immunity from the improved MecVax can provide supplemental (to the anti-adhesin immunity) protection against ETEC strains producing any of the 12 targeted adhesins and also independent protection against all the other ETEC strains that do not express the 12 targeted adhesins, further expanding vaccine protection against ETEC diarrhea.
It needs to be pointed out that the current study examined only the vaccine's broad immunogenicity and antibody in vitro functional activities against ETEC adherence and toxin excitotoxicity.Future studies with a rabbit colonization model and a pig passive protection model will allow us to evaluate the efficacy of the new MecVax preclinically.Surely, the controlled human infection model and clinical trials will eventually validate the broad immunogenicity and cross-protection of this protein-based injectable ETEC vaccine candidate.
The demonstration of no or negligent effects at antigen-specific immunogenicity and more importantly protective functions after multiple polyvalent protein antigens were combined and co-administered from this study not only signifies the feasibility of constructing a more effective ETEC vaccine but also paves the way toward future preparation of combination vaccines against different diseases.Combination vaccines can simplify the logistics of vaccine manufacture, storage, and administration, reducing vaccine costs and clinical expenses.Combination vaccines will become even more desirable as the expanded program on immunization especially for children is getting increasingly crowded because of the continuous introduction of new vaccines.We recently demonstrated that a polyvalent Shigella or cholera MEFA protein antigen is broadly immunogenic and cross-protective against heterogeneous serogroup or serotype strains (31,32).The encouraging data from the current study certainly enhance confidence for future research and development of combination vaccines against ETEC and Shigella and/or cholera (or other enteric pathogens), particularly since these pathogens infect the same populations, children in the endemic regions or countries as well as international travelers to these areas.
Holotoxin-structured double mutant LT (LT R192G/L211A ; dmLT) (33), which differs from the mnLT R192G/L211A component in the MecVax toxoid fusion antigen as this monomeric LT (mnLT) carries one A subunit mutant and one B subunit as a single peptide, 0.1 µg in 1 µL, was used as the adjuvant for four treatment groups in this study.

Mouse intramuscular immunization
Eight-week-old BALB/c female mice (Charles River Laboratories, Wilmington, MA, USA), eight per group, were included in intramuscular immunization.The injection site at the quadriceps femoris was shaved or clipped, wiped with 70% ethanol, and intramuscularly injected with antigen(s) or PBS premixed with dmLT adjuvant, in a total volume of 21 or 31 µL, with a 25-or 30-gauge needle.Two booster injections at the same dose of the primary were followed at an interval of 2 weeks on the alternative body side.All mice were observed daily for activities and abnormalities.
Blood samples (30-50 μL) were collected from each mouse before the primary and each booster from the lateral saphenous vein with an 18-or 22-gauge needle.Two weeks after the second booster, mice were anesthetized with CO 2 and euthanized with cervical dislocation.Blood samples (0.5 mL) were collected with cardiac puncture by ventral approach.Mouse sera were collected and stored at −20°C until use.
Mouse immunization protocol (#23060) was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Illinois at Urbana-Champaign; animal studies were supervised by an institutional attending veterinarian and staff.

Mouse serum antigen-antibody titration
Serum samples from each mouse were titrated in ELISAs for IgG antibody responses to the antigens targeted, 12 ETEC adhesins and 2 toxins.As we described previously (24,25,(27)(28)(29) Louis, MO, USA) or 10 ng STa-ovalbumin conjugates, in 100 µL bicarbonate/carbonate buffer, was coated to each well of a 2HB plate or a CoStar 96-well plate (Thermo Fisher Scientific), respectively.After overnight growth at 4°C, coated plates were washed with phosphate-buffered saline with Tween-20 (PBST) (0.05% Tween-20), blocked with 10% non-fat milk (in PBST) at 37°C for 1 h, and incubated with twofold serum dilutions (1:400 to 1:102,400) at 37°C for 1 h.Wells were washed with PBST and incubated with horse radish peroxidase (HRP)-conjugated goat-anti-mouse IgG (1:5,000, Bethyl Laboratories, Montgomery, TX, USA) at 37°C for 1 h.Wells were washed with PBST and PBS and then incubated with 3,3′,5,5′-tetramethylbenzidine microwell peroxidase substrate system 2C (Thermo Fisher Scientific).Optical density (OD) 650 readings were measured after 25 min at room temperature and subtracted with background values.The highest dilution given an OD over 0.3 was converted to a titer, at a scale of log 10 .
After incubation in a 5% CO 2 incubator at 37°C for 1 h, Caco-2 cells were gently washed with PBS to remove non-adherent bacteria, dislodged and lysed with 0.5% Triton (Sigma), and collected with centrifugation.The collected adherent ETEC bacteria were serially diluted, plated on LB plates, and counted for CFUs after overnight growth at 37°C.CFUs were converted to percentages by referring to the CFUs of the bacteria treated with the control mouse sera as 100%.

Mouse serum antibody neutralization against STa and CT enterotoxicity
Sera from mice in each treatment group were examined for neutralization activities against STa and CT enterotoxicity using T-84 cells (CCL-248, ATCC) and a cyclic GMP or AMP EIA kit (Enzo Life Sciences, Farmingdale, NY, USA).As we described previously (24,25,28,29), 30 µL mouse serum sample pooled from each group was mixed with 2 ng STa toxin (BEI Resources, ATCC) or 30 ng CT (Sigma; we increased CT from 10 ng to 30 ng in an attempt to differentiate neutralization activity against CT from antibodies from the vaccine antigen versus dmLT adjuvant) and incubated at room temperature for 30 min and then transferred to a well of a 24-well tissue culture plate (Falcon) containing 95%-100% confluent monolayered T-84 cells and incubated in a CO 2 incubator for 1 h (for cGMP) or 3 h (for cAMP).Cells were rinsed thoroughly with PBS to remove extracellular cGMP or cAMP, then dislodged, and lysed with 0.5% Triton to release intracellular cGMP or cAMP.The intracellular cGMP or cAMP levels (nM, picomole per mL) were measured by following the manufacturer's protocol (Enzo Life Science).T-84 cells in culture medium without treatment with mouse sera or toxin were also included in the assay to measure intracellular cGMP or cAMP baseline levels.

Statistical analysis
Data of antibody titers (in log 10 ) and antibody functional activities against ETEC bacterial adherence (CFUs in %) or toxin enterotoxicity (cGMP or cAMP nM) were presented in means and standard deviations and analyzed for differences between the control group and each immunization group or between the group immunized with "MecVax + CFA MEFA-II" and the group immunized with MecVax or CFA MEFA-II.One-way ANOVA (GraphPad Prism 10; San Diego, CA, USA) was used to analyze if differences between the two groups were significant, with the P value calculated from the post hoc test (Turkey's multiple comparisons test) of less than 0.05 to indicate a significant difference.

FIG 3
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.