AB5-Type Toxin as a Pentameric Scaffold in Recombinant Vaccines against the Japanese Encephalitis Virus

Abstract

Japanese encephalitis virus (JEV) is an enveloped icosahedral capsid virus with a prime neutralizing epitope present in E protein domain III (EDIII). E dimers are rearranged into a five-fold symmetry of icosahedrons. Cholera toxin B (CTB) and heat-labile enterotoxin B (LTB) of AB5-type toxin was used as the structural scaffold for emulating the pentameric axis of EDIII. We produced homo-pentameric EDIII through the genetic fusion of LTB or CTB in E. coli without recourse to additional refolding steps. Harnessing an RNA-mediated chaperone further enhanced the soluble expression and pentameric assembly of the chimeric antigen. The pentameric assembly was validated by size exclusion chromatography (SEC), non-reduced gel analysis, and a GM1 binding assay. CTB/LTB−EDIII chimeric antigen triggered high neutralizing antibodies against the JEV Nakayama strain after immunization in mice. Altogether, our proof-of-principle study creating a JEV-protective antigen via fusion with an AB5-type toxin as both a pentameric scaffold and a built-in adjuvant posits the bacterially produced recombinant chimeric antigen as a cost-effective alternative to conventional inactivated vaccines against JEV.

Efficacy of genotype-matched vaccine against re-emerging genotype V Japanese encephalitis virus

“…The estimated sizes of the constructs determined by SEC closely matched the pentamer sizes observed on SDS-PAGE (Figure S2). GM1 ganglioside serves as the receptor for CTB, and the efficient binding of CTB to GM1 indicates that CTB retains its native pentameric conformation [ 25 , 37 , 38 ]. Thus, to ascertain that our CTB-conjugated vaccine antigen forms a pentameric structure, we conducted a GM1 binding assay.…”

“…Contrary to these apprehensions, even genotypically mismatched immunizations did not worsen infections but instead offered partial protection. In addition, our recombinant antigens showed enhanced cellular immunity compared to commercial vaccines, likely a result of CTB conjugation [ 25 , 44 ]. Research indicates that antigen-specific T-cell responses may mitigate ADE and improve protective efficacy of JEV vaccine [ 45 , 46 ].…”

“…Indeed, several attempts at vaccine development using the EDIII of JEV have been successful in preventing experimental encephalitis [ 21–25 ]. In our earlier study, we also developed a vaccine candidate that presents five EDIII monomers derived from the GIII Nakayama using cholera toxin B subunit (CTB) as a pentameric scaffold [ 25 ]. CTB naturally forms a pentamer and possesses inherent adjuvanticity, making it a useful structural component for designing nanoparticle vaccines.…”

“…In the current study, we utilize Escherichia coli for expression system. However, EDIII expressed in the E. coli expression system is insoluble, requiring an additional step for solubilization [ 22 , 23 , 25 ]. To achieve soluble expression of CTB-EDIII in E. coli , we employed the RNA-mediated chaperone technique called “Chaperna,” (chaperone + RNA) which can mitigate structural misfolding [ 27–29 ].…”