Comparative immunogenicity study of quartet and bifurcated tetravalent dengue virus envelope domain III displayed virus-like particle vaccine candidates in BALB/c mice.

Developing an effective dengue vaccine requires targeting all four dengue virus (DENV) serotypes while ensuring both safety and efficacy. Our research aimed to address this by developing DENV vaccine candidates strategically designed to display Envelope Domain III (EDIII) antigens on a DENV capsid virus-like particle (VLP). We engineered tetravalent EDIII constructs, designed either as a quartet (qE) or bifurcated (bE) arrangement, and expressed them alongside DENV type 2 capsid (C2) in silkworm larvae, followed by affinity purification. These purified EDIII constructs (qE and bE) were then covalently linked to C2 using SpyTag/SpyCatcher (SpT/SpC) isopeptide coupling. Successful tetravalent display was confirmed through Western blot and immuno-electron microscopy. Subsequent immunogenicity evaluation in BALB/c mice demonstrated that both EDIII-displayed VLPs, C2-bE and C2-qE, elicited immune responses without any significant difference between them. However, the C2-bE consistently exhibited the strongest neutralization capacity across all serotypes, with significantly better neutralization activity against serotype 2 compared to both bE and C2-qE. In contrast, serotype 4 consistently elicited the lowest immune response and neutralization potential across all vaccine groups. The observed dominance of IgG1 antibodies suggests a predominant Th2-type immune response in immunized BALB/c mice. Overall, these findings indicate that both C2-bE and C2-qE effectively induce immune responses and produce neutralizing antibodies, warranting further investigation.