The development of ETVAX may provide a valuable roadmap for development of other improved inactivated whole cell enteric vaccines, and I. Khalil, personal communication. A first-generation precursor of the current ETVAX candidate was a whole cell vaccine consisting of formalin-inactivated ETEC bacteria expressing CFA/I and CS1 to CS5, combined with cholera toxin B-subunit (CTB), which is highly homologous to the B-subunit (LTB) of LT enterotoxin produced by approximately 66% of ETEC strains. This 2-dose vaccine candidate was found to be immunogenic in children and adults in endemic areas. Furthermore, it protected adult travelers against moderate/severe diarrhea. In these studies, an anti-CTB serum IgA titer > 360 when trial participants arrived in Guatemala or Mexico was found to be associated with a reduced risk of developing moderate to severe ETEC traveler's diarrhea (TD) ( Table 1 ), suggesting that level of anti-toxoid IgA antibody may be a marker for effective vaccination in travelers. In pediatric studies, a full dose of the vaccine caused vomiting in 6-17 months old Bangladeshi children; but a quarter dose was found to be safe. The vaccine did not confer protection in 6-24 months old Egyptian children, but both vaccinated and unvaccinated children experienced mainly mild disease during the study period and the impact on moderate/severe diarrhea could not be evaluated. These data were reviewed by a WHO panel, which recommended overexpression of antigens on the cells comprising the vaccine and inclusion of an adjuvant in the formulation to help improve vaccine immunogenicity in the target age-group.
ETVAX is now undergoing clinical trials as a second generation multivalent oral ETEC vaccine-containing inactivated E. coli strains over-expressing colonization factor antigens CFA/I, CS3, CS5 and CS6 (the latter was not included in the first-generation vaccine) at significantly higher levels than in naturally occurring cells. In addition, it was also noted that formalin inactivation of the CS6 expressing ETEC was detrimental to CS6 immunogenicity, and this was overcome in the second-generation formulation by phenol inactivation of the CS6 strain. The current generation vaccine is administered together with the more LT-like toxoid LCTBA. To further enhance the immunogenicity of the vaccine, it was combined with the double mutant labile toxin of ETEC (dmLT) adjuvant. When tested in Swedish adults (ISRCTN91363076), ETVAX with or without dmLT was found to be safe and to induce significant fecal SIgA responses as well as IgA antibody-secreting cell (ASC) responses against all CFs and LTB. Addition of 10 ug dmLT to the vaccine significantly enhanced ALS responses only to the CS6 component. ETVAX also induced long-lasting immunological memory in Swedish adults (; ISRCTN27096290). Subsequent results demonstrated that ETVAX induced IgA antibody responses that cross-react with CFs belonging to the same CF families, possibly expanding the coverage of the vaccine. These successful results have led to clinical evaluation of ETVAX in a large phase 1/2 trial in adults and lower age groups (5 years to 6 months) in Bangladesh.
Adults: The safety of ETVAX alone or together with dmLT adjuvant was evaluated in Bangladeshi adults to provide the basis for studies in Bangladeshi children and infants [; NCT02531802]. The Bangladeshi adults received two oral doses of ETVAX with or with-out dmLT adjuvant or placebo in the initial part of a randomized, double-blind, placebo-controlled, age-descending, dose-escalation trial. This was the first clinical trial of the second generation ETVAX vaccine conducted in an ETEC endemic country. Two full doses of ETVAX, both when administered alone and with 10 ug dmLT adjuvant, were safe and well tolerated in the healthy Bangladeshi adults tested, confirming previous safety data from studies in Swedish adults. This vaccine was highly immunogenic in Bangladeshi adults, inducing intestine-derived ASC responses in all, and plasma antibody responses in a majority, of the vaccine recipients to all primary vaccine antigens (four CFs and LTB). Addition of dmLT adjuvant to the vaccine had no apparent effect on the ALS responses in the adults in this study. In contrast, in adult Swedes, 10 ug of dmLT significantly enhanced ASC responses to CS6, which is the CF present in the lowest amount in the vaccine.
Both the Swedish and Bangladeshi trials demonstrated that the oral ETVAX vaccine is safe in adults and induces strong mucosal as well as systemic immune responses against key vaccine antigens. These findings have provided a base for further evaluation of this vaccine in descending age groups in children and infants.
Children and infants: Healthy children in one of three age groups (24-59 months, 12-23 months, and 6-11 months) were randomly assigned with block randomization to receive either ETVAX, with or without dmLT, or placebo (; NCT02531802). ETVAX (half [5.5 x 1010 cells, 500 ug LCTBA], quarter [2.5 x 1010 cells, 250 ug LCTBA], or eighth [1.25 x 1010 cells, 125 ug LCTBA] adult dose), with or without dmLT adjuvant (2.5 mug, 5.0 mug, or 10.0 mug), or placebo were administered orally in two doses 2 weeks apart. The primary endpoint was safety and tolerability, assessed in all children who received at least one dose of vaccine. Antibody responses to vaccine antigens, defined as at least a two-times increase in antibody levels between baseline and post-immunization, were assessed as secondary endpoints.
No solicited adverse events occurred that were greater than moderate in severity, and most were mild. The most common solicited event was vomiting which appeared related to dose and age. The addition of dmLT did not modify the safety profile. Mucosal IgA antibody responses in lymphocyte secretions were detected against all primary vaccine antigens (CFA/I, CS3, CS5, CS6, and the LCTBA toxoid) in most participants in the two older age groups, whereas such responses to four of the five antigens were less frequent and of lower magnitude in infants aged 6-11 months than in older children.
Mucosal antibody response frequencies were consistently higher in the adjuvanted vaccine group when >= 3, 4 or 5 antigens were considered ( Table 2 ). Seventy-eight (56%) of 139 infants aged 6-11 months who were vaccinated developed mucosal responses against at least three of the vaccine antigens versus 14 (29%) of 49 of the infants given placebo. Addition of the adjuvant dmLT enhanced the magnitude, breadth, and kinetics (based on number of responders after the first dose of vaccine) of immune responses in infants. A similar positive dmLT effect was observed on the mucosal antibody response in the 12-23 month age group in this study when the antigenic breath of the responses was considered. In more in-depth immunological studies, the inclusion of dmLT in the vaccine formulation improved expression of B cell memory markers and T cell responses to key ETEC antigens as well. Subsequent findings showed that ETVAX induced mucosal and systemic immune responses against O78 LPS (present in three of the CS expressing strains in the vaccine) in all age groups and that dmLT improved intestinal immune responses among infants. These observations may have implications for more successful use of dmLT with other oral vaccines, like cholera (OCV), which have experienced difficulty in inducing protective O antigen antibody responses in children and infants under 5 years of age. Also, the enhancement of protein colonization factors and 078 LPS responses in infants and young children suggests in the case of OCV that including dmLT in the formulation may make single dose vaccine strategies more protective, thereby extending available stockpiles of this vaccine.
The Swedish and Bangladeshi studies showed that ETVAX could induce strong intestine-derived and/or fecal immune responses in a majority of vaccinated adults and in different age groups, including infants, in Bangladesh. This trial assessed for the first time the safety and immunogenicity of ETVAX in young children and infants in a low and middle income country (LMIC) and was the first analysis of dmLT adjuvant with a vaccine in this population in a LMIC. Immune responses were improved in infants aged 6-11 months by administering dmLT with the vaccine. Importantly, this study showed that occasional mild to moderate vomiting can be reduced without loss of immunogenicity by reducing the dose of vaccine, even when dmLT was included in the formulation.