Reliable quantification of neutralizing antibodies (nAb) against human adenovirus type 55 (HAdV-55) is critical for the evaluation of emerging vaccine candidates. While the plaque reduction neutralization test (PRNT) is currently the reference standard, its utility for large-scale studies is limited by low throughput, labor-intensive plaque counting, and prolonged assay times. In this study, we established and analytically validated a microneutralization assay based on cytopathic effect (MN-CPE) as a scalable alternative for HAdV-55-specific nAb quantification. Comparative performance analysis revealed that both assays maintain high dilution linearity, with coefficients of determination (R²) of 0.988 for MN-CPE and 0.9926 for PRNT. Relative accuracy assessments using high-, middle-, and low-titer reference sera demonstrated acceptable responses across the dynamic range. Notably, the MN-CPE assay allowed for the definition of a negative-control acceptance range, providing a distinct statistical advantage over PRNT, where negative-control values were consistently zero. Furthermore, both assays successfully detected HAdV-55-specific nAbs in immunized cynomolgus macaques, with no cross-reactivity observed against other HAdV types such as HAdV-4. These findings indicate that the MN-CPE assay is analytically comparable to PRNT and serves as a practical, relatively high-capacity alternative for HAdV-55 neutralization testing in clinical and preclinical vaccine research.

The 2015 Zika virus (ZIKV) outbreak in Brazil and its global spread underscored the urgent need for effective and broadly protective vaccines. While C57BL/6 and BALB/c mice are widely used in preclinical vaccine research, direct comparisons of their ability to elicit ZIKV-specific neutralizing antibodies (nAbs) remain limited. This study aimed to systematically evaluate and compare the immunogenic potential of these two common mouse strains across diverse vaccine platforms, focusing on their capacity to generate functional neutralizing antibody responses. We assessed nAb and IgG responses following four vaccination strategies: (1) DNA vaccine encoding prMEΔTM followed by E protein domain III boost, (2) recombinant EΔTM protein expressed using baculovirus system, (3) formalin-inactivated ZIKV, and (4) live ZIKV. Although both strains generated detectable ZIKV- and E protein-specific IgG, the magnitude and quality of responses varied by vaccine platform and strain. Notably, C57BL/6 mice consistently mounted significantly higher nAb titers than BALB/c mice across all immunization groups, including subunit- and whole-virus-based vaccines. In contrast, BALB/c mice showed lower or undetectable nAb responses, despite comparable or higher total IgG levels in some cases. These findings show that host genetic background is a critical determinant of vaccine-induced neutralization and underscore the importance of selecting appropriate animal models in ZIKV vaccine development. C57BL/6 mice, due to their robust nAb responses, represent a reliable model for evaluating vaccine immunogenicity. Conversely, the limited nAb responses in BALB/c mice position them as a potential low-responder model, offering a stringent system to test the potency and breadth of protective immunity under suboptimal conditions.