Development and internal analytical validation of plaque reduction neutralization test for clinical trial application...

Kyasanur Forest Disease (KFD) is a tick-borne viral hemorrhagic fever endemic in the Western Ghats of India. The previous KFD vaccine was discontinued in 2022 due to waning immunity and suboptimal protection. A new vaccine candidate developed by the Indian Council of Medical Research in partnership with Indian Immunologicals Ltd. has progressed to clinical development, necessitating accurate measurement of virus-specific neutralizing antibodies. This study outlines the development, optimization, and internal analytical validation of a plaque reduction neutralization test (PRNT) for KFD virus using BHK-21 cells, following ICH Q2(R2) guidelines. The acceptance criteria were: assay cut-off (mean + 2SD of negative sera ≤1:10), precision (CV ≤20%), linearity (R² ≥0.98), and dilution agreement (log₁₀ difference <0.301). Optimized parameters were 5 × 10⁶ cells/well, 1.1% CMC overlay, 72 h incubation, and virus dilution of 10^-3·⁶ TCID₅₀/mL producing 40 ± 5 plaques/well. The virus stock titre was 10⁷·² TCID₅₀/mL with no significant loss after three freeze-thaw cycles. The assay was evaluated using KFDV-positive (n = 24), heterologous flavivirus-positive (DENV, JEV, WNV, ZIKV; n = 39), and negative sera (n = 107). The operational cutoff based on mean + 2SD was ≥1:10. At ≥1:10 and ≥1:40, the specificity against heterologous flaviviruses sera panel was 61.5% (95% CI 45.9-75.1%) and 100% (95% CI, 91.0-100%) respectively. Inter-assay precision demonstrated CVs of 11.64% and 16.26% for positive controls across two analysts. Linearity was excellent (R² = 0.998) across 1:10-1:5120. Dilution agreement, tested using four sera at 1:4, 1:16, and 1:64, showed mean recovery of 103.8% (SD 17.3%), with all diluted observations meeting the two-fold criterion. Limitations include requirement of BSL-3 laboratory, modest heterologous sample sizes, single-site validation, and absence of long-term stability studies. The validated PRNT is suitable for measuring vaccine-induced neutralizing antibody responses in clinical trials; broader epidemiological applications would require additional validation.