Serological Assays to Measure Rabies Antibody Response in Equine Serum Samples.

Overview

• This research developed safer and more accessible laboratory methods to measure rabies antibody levels in horse blood samples, which can improve rabies diagnosis and vaccine evaluation without the need for high-level biosafety labs.

Background

• Rabies is a deadly viral disease caused by the rabies virus (RV), leading to nearly 60,000 deaths worldwide annually, mainly affecting low-income communities.
• Transmission to humans mostly occurs via bites from infected dogs, although other mammals can carry RV, but rarely transmit it.
• Rabies has an almost 100% fatality rate once symptoms appear.
• Despite this, vaccination and immunization—both pre- and post-exposure—are effective preventative measures.
• The high cost of rabies prophylaxis and treatment limits access for many individuals at risk.
• Globally, about 29 million rabies vaccinations are administered yearly, which imposes a large financial burden.
• Measuring rabies antibodies in serum is important to monitor immunity and vaccine efficacy but typically requires handling live rabies virus in Biosafety Level 3 (BSL-3) labs, limiting accessibility.

Research Goals

• To establish safer, reliable, and quantitative assays compatible with lower biosafety levels (BSL-2) for detecting anti-rabies antibodies in equine (horse) serum samples.
• To develop a platform that could facilitate rabies serology testing and vaccine evaluation without the need for live rabies virus culture.

Methodology

• Developed a replication-deficient vesicular stomatitis virus (VSV) pseudovirus engineered to express the rabies virus glycoprotein (RV-G), which is critical for the virus’s ability to infect cells and induce immune recognition.
• The pseudovirus was purified and tested for infectivity by measuring luciferase enzyme activity, an indicator of successful infection.
• Demonstrated that the pseudovirus can infect various mammalian cell lines, although infectivity varied among different types.
• Used hyper-immunized equine serum samples to assess antibody responses.
• Developed and applied a multiplexed Luminex immunoassay capable of quantifying rabies-binding antibodies in these serum samples.
• Performed neutralization assays using the pseudovirus to evaluate functional neutralizing antibodies, which prevent virus infection.

Key Findings

• The pseudovirus showed robust, measurable luciferase activity confirming it could successfully infect cells and serve as a surrogate for live rabies virus in assays.
• Strong positive correlation (correlation coefficient ρ > 0.9, p-value < 0.001) was found between the antibody titers measured by the Luminex binding assay and neutralizing antibody titers measured by the pseudovirus-based neutralization assay.
• This correlation indicates that the Luminex assay reliably estimates protective antibody levels similar to the neutralization assay.
• Both assays can be conducted under BSL-2 conditions, representing a safer and more accessible alternative to traditional methods requiring BSL-3 facilities.

Implications and Applications

• The developed assays provide a safe, quantitative platform for measuring rabies-specific antibody responses, especially useful in resource-limited settings where BSL-3 labs are unavailable.
• Equine serum testing is relevant because horses can be used to produce therapeutic antibodies or antiserum for rabies treatment.
• These methods support improved rabies vaccine testing, quality control, and surveillance by enabling easier and faster antibody assessment.
• The approach could potentially reduce the costs and risks associated with rabies serology, helping expand monitoring and immunization programs globally.
• A safer, pseudovirus-based neutralization assay also enables broader research into rabies immunology without the hazards of using live virus.

Conclusion

• This study successfully created a BSL-2 compatible pseudovirus-based platform alongside a multiplexed immunoassay to measure rabies antibodies in horse serum.
• These tools offer a practical, safer alternative to traditional live-virus assays, with strong correlation between binding and neutralizing antibody measurements.
• Overall, this work advances rabies serological evaluation and vaccine development with important public health and economic benefits.