Biochemical and Immunological Evaluation of Recombinant CS6-Derived Subunit Enterotoxigenic Escherichia coli Vaccine Candidates

Abstract

CS6, a prevalent surface antigen expressed in nearly 20% of clinical enterotoxigenic Escherichia coli (ETEC) isolates, is comprised of two major subunit proteins, CssA and CssB. Using donor strand complementation, we constructed a panel of recombinant proteins of 1 to 3 subunits that contained combinations of CssA and/or CssB subunits and a donor strand, a C-terminal extension of 16 amino acids that was derived from the N terminus of either CssA or CssB. While the entire panel of recombinant proteins could be obtained as soluble, folded proteins, it was observed that the proteins possessing a heterologous donor strand, derived from the CS6 subunit different from the C-terminal subunit, had the highest degree of physical and thermal stability. Immunological characterization of the proteins, using a murine model, demonstrated that robust anti-CS6 immune responses were generated from fusions containing both CssA and CssB. Proteins containing only CssA were weakly immunogenic. Heterodimers, i.e., CssBA and CssAB, were sufficient to recapitulate the anti-CS6 immune response elicited by immunization with CS6, including the generation of functional neutralizing antibodies, as no further enhancement of the response was obtained with the addition of a third CS6 subunit. Our findings here demonstrate the feasibility of including a recombinant CS6 subunit protein in a subunit vaccine strategy against ETEC.

The TLR4 agonist adjuvant SLA-SE promotes functional mucosal antibodies against a parenterally delivered ETEC vaccine

“…Antigen and adjuvant doses were selected from our previous published and unpublished works. 12,27 Peripheral blood was collected on Day 21 by retro-orbital bleed. Central blood was collected on Day 35 by cardiac puncture from mice under deep anesthesia.…”

Evaluation of the reactogenicity, adjuvanticity and antigenicity of LT(R192G) and LT(R192G/L211A) by intradermal immunization in mice

“…Our ETEC vaccine strategy incorporates both the potential for toxin neutralization and the inhibition of colonization through the disruption of bacterial adherence to the intestinal epithelium. For the latter, we generated recombinant subunit antigens from ETEC colonization factors (CFs) such as CfaE, the minor fimbrial adhesin of the CFA/I CF [12] and the dimeric fusion CssBA, which contains the CssA and CssB subunits of CS6 [13]. These recombinant antigens remain immunogenic and able to promote functional antibodies, capable of inhibiting bacterial binding to red blood cells in the case of class 5 CF-derived antigens (i.e.…”

Preclinical optimization of an enterotoxigenic Escherichia coli adjuvanted subunit vaccine using response surface design of experiments

“…An effective ETEC vaccine should promote immunity against several CFs to allow for sufficiently broad coverage against a range of clinical strains 48 . The addition of dmLT to the formulation can elicit anti-LT immunity as well as enhance the response against different CF-based vaccine candidates, as has been recently demonstrated 38,49 . Using the same dose optimization approach, the next step will be to select the dose of other CF components, such as the recently described antigen coli surface antigen 6 fimbrial subunit B and A protein heterodimer, which elicits immunity to coli surface antigen 6 49 , to ensure immunity to all targeted CFs and also to avoid cross-antigen interference.…”

“…The addition of dmLT to the formulation can elicit anti-LT immunity as well as enhance the response against different CF-based vaccine candidates, as has been recently demonstrated 38,49 . Using the same dose optimization approach, the next step will be to select the dose of other CF components, such as the recently described antigen coli surface antigen 6 fimbrial subunit B and A protein heterodimer, which elicits immunity to coli surface antigen 6 49 , to ensure immunity to all targeted CFs and also to avoid cross-antigen interference. These steps will benefit from the selection methods used in the present study and could be further leveraged by recent findings that anti-CfaEB IgG1 titres could predict anti-CF functionality measured by HAI, at least for class 5a antigens 38 .…”