Comparable immunogenicity from murine blood collection methods in intranasal gonococcal vaccination with ACP and MtrE supports refinement of preclinical vaccine studies.

Preclinical vaccine studies often rely on antibody titers and functional assays to assess immunogenicity, but the impact of blood collection methods on these immunological outcomes remains poorly characterized. Retro-orbital (RO) bleeding is commonly used in murine models but raises animal welfare concerns, prompting the adoption of saphenous vein (SV) sampling as a less invasive alternative. Here, we compared RO and SV blood collection methods in mice immunized intranasally with Neisseria gonorrhoeae vaccine candidates—Adhesin Complex Protein (ACP) and Multiple Transferable Resistance E protein (MtrE)—formulated with or without the CpG adjuvant. Epitope mapping confirmed that both antigens contain surface-accessible B- and T-cell epitopes, supporting their potential for inducing protective immunity. Across 45 comparisons of serum antibody subclasses, only three total IgG differences between RO and SV were statistically significant, indicating high concordance between methods. Human serum bactericidal activity titers were similarly robust across both sampling techniques. In lysozyme inhibition assays, pooled SV sera from rACP + CpG-immunized mice restored significantly more human lysozyme activity compared to rACP alone, although individual-level analyses revealed no significant differences, despite a trend favoring CpG. These results demonstrate that SV blood collection yields immunologically comparable data to RO, while offering improved animal welfare. Adoption of SV sampling may enhance standardization, data quality, and ethical compliance in preclinical vaccine research.