Development of a micro-neutralization assay for ebolaviruses using a replication-competent vesicular stomatitis hybrid virus and a quantitative PCR readout

Abstract

Development of vaccines against highly pathogenic viruses that could also be used as agents of bioterrorism is both a public health issue and a national security priority. Methods that can quantify neutralizing antibodies will likely be crucial in demonstrating vaccine effectiveness, as most licensed viral vaccines are effective due to their capacity to elicit neutralizing antibodies. Assays to determine whether antibodies are neutralizing traditionally involve infectious virus, and the assay most commonly used is the plaque-reduction neutralization test (PRNT). However, when the virus is highly pathogenic, this assay must be done under the appropriate level of containment; for tier one select agents, such as Ebola virus (EBOV), it is performed under Biological Safety Level 4 (BSL-4) conditions. Developing high-throughput neutralization assays for these viruses that can be done in standard BSL-2 laboratories should facilitate vaccine development. Our approach is to use a replication-competent hybrid virus whose genome carries the envelope gene from the pathogenic virus on the genetic backbone of a non-pathogenic virus, such as vesicular stomatitis virus (VSV). We have generated hybrid VSVs carrying the envelope genes for several species of ebolavirus. The readout for infectivity is a one-step reverse transcriptase quantitative PCR (RT-qPCR), an approach that we have used for other viruses that allows robustness and adaptability to automation. Using this method, we have shown that neutralization can be assessed within 6-16h after infection. Importantly, the titers obtained in our assay with two characterized antibodies were in agreement with titers obtained in other assays. Finally, although in this paper we describe the VSV platform to quantify neutralizing antibodies to ebolaviruses, the platform should be directly applicable to any virus whose envelope is compatible with VSV biology.

Pseudovirus rVSVΔG-ZEBOV-GP Infects Neurons in Retina and CNS, Causing Apoptosis and Neurodegeneration in Neonatal Mice

“…To determine whether the disease caused by rVSVΔG-ZEBOV-GP was driven by the GP, we compared their disease progression with that of mice that received similar inoculums of the parental VSV (Indiana strain), rVSVΔG-ZEBOV-GPΔMUCIN, a VSV pseudovirus that expresses the ZEBOV GP with a deleted mucin domain, or rVSVΔG-RESTON-GP, in which the G of VSV is replaced by the Reston Ebola GP (Lee et al, 2017). P3 mice inoculated with the parental VSV strain succumbed 3–5 days post-challenge, whereas mice inoculated with rVSVΔG-RESTON-GP showed a weight gain similar to uninfected mice and survived the challenge (Figure 1B).…”

“…The rVSVΔG-ZEBOV-GPΔMUC virus was a gift from Dr. Gerardo Kaplan. The rVSVΔG-RESTON-GP virus was a gift from Dr. Keith Peden (Lee et al, 2017). Viral stocks were passaged in Vero E6 cells to produce a master stock and stored at −80°C.…”

A micro-PRNT for the detection of Ross River virus antibodies in mosquito blood meals: A useful tool for inferring transmission pathways

“…We developed a micro-PRNT [19,20] to suit small sample volumes. In this alternative approach, we exploit the fact that vertebrate antibodies persist within mosquito blood meals for some time after the mosquito has fed on a seropositive host.…”

A micro-PRNT for the detection of Ross River virus antibodies in mosquito blood meals: a useful tool for inferring transmission pathways

“…It does not need prior knowledge of host origin but typically requires large quantities of sera or tissue; substantially larger than a typical mosquito blood meal (ca 3 µl [17]). We developed a micro-PRNT [18, 19] to suit small sample volumes.…”