A pseudovirus-based neutralizing antibody assay for measuring neutralizing antibody against rabies virus
ABSTRACT To establish a laboratory detection assay for a human neutralizing antibody (NAb) against rabies virus and to evaluate its efficiency in human serum samples. A pseudovirus pXN2‑RABV‑G harboring the RABV G gene was applied. The precision, robustness, linearity, stability, inclusivity, and limit of detection of the assay were evaluated. The consistency and correlation of the assay with ELISA and rapid fluorescent focus inhibition test (RFFIT) were analyzed. A pseudovirus-based neutralizing antibody (pVNA) detection system based on the pXN2‑RABV‑G was successfully developed and showed broad robustness (culture media: χ2 = 4.00, P > 0.05, cell passages: χ2 = 0.25, P > 0.05), precision (independent detection: P > 0.05), lower/upper limit of linearity (0.09 to 93.73 IU/mL, R2 > 0.95), and stability (P > 0.05). The limit of detection (LOD) was 2.63 IU/mL (95% CI: 1.81–3.84), expressed as the lowest human sera antibody level reliably distinguishable from background signals, and the coefficient of variation was less than 20%. Compared with the commercial ELISA and the gold standard RFFIT assay, the kappa values were 0.74 and 0.82, respectively, and the results were highly consistent. Linear correlation analysis revealed a strong correlation between these variables (R2 > 0.90). A total of 103 recipients from 2019 to 2023 were included in the serological testing and the following analysis. NAb titers were significantly correlated with vaccine dose (P < 0.001) and the most recent vaccination time (P < 0.001). The geometric mean titer (GMT) of the 5-dose vaccination group was greater than that of the 1-dose, 2-dose, 3-dose, and 4-dose groups (3.15 vs 3.19 vs 6.18 vs 12.21 vs 13.40 IU/mL). The established pVNA assay for human rabies NAb detection has a detection capability similar to that of the gold standard RFFIT, but it has the advantages of reduced biosafety requirements, laboratory activity, and time, which highlight its safety, high efficiency, and feasibility for screening and monitoring rabies virus antibodies after vaccination. IMPORTANCE Rabies is a lethal disease with no effective clinical treatment. Vaccination is the only way to prevent it routinely, which makes post-vaccination antibody testing essential. The current standard test requires a highly equipped laboratory, biosafety measures, and is time-consuming. This study develops a safe, efficient test for detecting rabies neutralizing antibodies using pseudovirus technology. It is comparable to the gold standard test in accuracy but easier to implement in routine labs, which enables broader use in screening and monitoring antibodies post-vaccination. This advantage addresses a key gap in rabies prevention, reducing the uncertainty for individuals with immunodeficiency who are nonresponsive to vaccination and at risk of rabies exposure. Rabies is a lethal disease with no effective clinical treatment. Vaccination is the only way to prevent it routinely, which makes post-vaccination antibody testing essential. The current standard test requires a highly equipped laboratory, biosafety measures, and is time-consuming. This study develops a safe, efficient test for detecting rabies neutralizing antibodies using pseudovirus technology. It is comparable to the gold standard test in accuracy but easier to implement in routine labs, which enables broader use in screening and monitoring antibodies post-vaccination. This advantage addresses a key gap in rabies prevention, reducing the uncertainty for individuals with immunodeficiency who are nonresponsive to vaccination and at risk of rabies exposure.