Assessing anti-rabies vaccine response in humans: A rapid and high-throughput adaptable, pseudovirus-based neutralization assay as an alternative to rapid fluorescent focus inhibition test (RFFIT).

BACKGROUND: Rabies is a fatal, but vaccine-preventable disease. The conventional 'gold standard' recommended for estimating anti-rabies vaccine immune response is Rapid Fluorescent Focus Inhibition Test (RFFIT). Here, we describe a pseudovirus-based neutralization assay (PVNT) as an alternative method to detect the neutralizing antibody response. METHODOLOGY/PRINCIPAL FINDINGS: We used a recombinant Vesicular Stomatitis Virus (rVSV)-based pseudotyping system in HEK293 cells transfected with rabies G glycoprotein to produce high titre pseudoviruses. These were confirmed by neutralization with anti-G monoclonal antibodies and a reference serum, as well as immunofluorescence-based detection of pseudovirus infection. A secreted alkaline phosphatase (SEAP) reporter was included to make the assay high-throughput adaptable with readouts possible using a conventional ELISA reader. A PVNT was developed that had a dynamic range of OD405 from 0.10 to 2.00, and was used to test a set of RFFIT-validated 71 human serum samples. Results of PVNT and RFFIT showed significant concordance with a Pearson's correlation value of 0.8555. In samples with border-line RFFIT positive values (a RFFIT titre in the range of 0.5-1.8 IU/mL), concordance was low, with the PVNT results tending to be negative. PVNT assay had a specificity of 100% when compared to RFFIT. The assay also had 100% positive predicative value indicating its robustness for use in the evaluation for the presence of neutralizing antibodies against a near 100% fatal disease such as rabies. CONCLUSIONS/SIGNIFICANCE: Our results show that PVNT is a reproducible, sensitive and specific assay with an objective and documentable readout. The assay is faster with a turnaround time of less than 24h and can be automated for large scale screening. Importantly, it can be performed without the restrictions in handling live rabies virus. PVNT provides a reliable alternative to RFFIT for monitoring vaccine efficacy and immunity status, making it a valuable tool in rabies control programs.