Multiplex PCR-based titration (MPBT) assay for determination of infectious titers of the three Sabin strains of live poliovirus vaccine

Abstract

BackgroundConventional assays to titrate polioviruses usually test serial dilutions inoculated into replicate cell cultures to determine a 50% cytopathic endpoint, a process that is both time-consuming and laborious. Such a method is still used to measure potency of live Oral Poliovirus Vaccine during vaccine development and production and in some clinical trials. However, the conventional method is not suited to identify and titrate virus in the large numbers of fecal samples generated during clinical trials. Determining titers of each of the three Sabin strains co-existing in Oral Poliovirus Vaccine presents an additional challenge.ResultsA new assay using quantitative multiplex polymerase chain reaction as an endpoint instead of cytopathic effect was developed to overcome these limitations. In the multiplex polymerase chain reaction-based titration assay, cell cultures were infected with serial dilutions of test samples, lysed after two-day incubation, and subjected to a quantitative multiplex one-step reverse-transcriptase polymerase chain reaction. All three serotypes of poliovirus were identified in single samples and titers calculated. The multiplex polymerase chain reaction-based titration assay was reproducible, robust and sensitive. Its lower limits of titration for three Sabin strains were 1–5 cell culture 50% infectious doses per ml. We prepared different combinations of three Sabin strains and compared titers obtained with conventional and multiplex polymerase chain reaction-based titration assays. Results of the two assays correlated well and showed similar results and sensitivity. Multiplex polymerase chain reaction-based titration assay was completed in two to 3 days instead of 10 days for the conventional assay.ConclusionsThe multiplex polymerase chain reaction-based titration (MPBT) is the first quantitative assay that identifies and titrates each of several different infectious viruses simultaneously in a mixture. It is suitable to identify and titrate polioviruses rapidly during the vaccine manufacturing process as a quality control test, in large clinical trials of vaccines, and for environmental surveillance of polioviruses. The MPBT assay can be automated for high-throughput implementation and applied for other viruses including those with no cytopathic effect.

Multiplex PCR-Based Neutralization (MPBN) Assay for Titers Determination of the Three Types of Anti-Poliovirus Neutralizing-Antibodies

“…The results summarized in Table 1 show that qmosRT-PCR was able to specifically identify and quantify each Sabin strain in all possible combinations. Consistent with our previous studies [18,19,21], qmosRT-PCR was found to be very specific for the identification and quantification of the different Sabin strains. To study the growth kinetics of Sabin strains in cells infected with the same virus concentration as used in the neutralization tests, monolayers of HEp-2C cells were prepared in 96-well plates and infected with 100 CCID 50 /per well of a mixture of the three Sabin strains.…”

“…This communication describes the development of a multiplex PCR-based neutralization (MPBN) assay for anti-poliovirus antibodies titration that uses a quantitative multiplex one-step RT-PCR (qmosRT-PCR) [18] as a read-out instead of CPE used in the conventional assay. Recently, we successfully used a similar approach to develop a multiplex PCR-based titration (MPBT) assay for the simultaneous titration of the three poliovirus serotypes [19].…”

Development of a Quantitative One-Step RT-PCR Method for the Detection of Sabin 2 Virus Contamination in a Novel Oral Poliovirus Vaccine Type 2

“…Sabin 2 and the recovered nOPV2c1-295 viruses were titrated by MPBT assay [ 27 ] with small modifications; only primers and probe for poliovirus type 2 were used.…”

Hypothetical emergence of poliovirus in 2020: part 1. Consequences of policy decisions to respond using nonpharmaceutical interventions

“…Recent experience with poliovirus diagnostic advances further supports the feasibility of such a sequence of events (e.g. detection of emerging vaccine-related polioviruses by deep sequencing [62], numerous algorithms for detection and ITD of polioviruses by various RT-PCR approaches [63][64][65][66]). We assume that upon detection of a nWPV paralytic disease cluster and initial signals from molecular testing of a common genetic signature, China would enact a containment strategy characterized by testing, contact tracing, ramping up treatment capacity, and social distancing measures in the affected block upon recognition of the infectious disease, similar to what occurred for SARS-CoV-2.…”