The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4–9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV.
Response to and monitoring of viral outbreaks can be efficiently focused when rapid, quantitative, kinetic information provides the location and the number of infected individuals. Environmental surveillance traditionally provides information on location of populations with contagious, infected individuals since infectious poliovirus is excreted whether infections are asymptomatic or symptomatic. Here, we describe development of rapid (1 week turnaround time, TAT), quantitative RT-PCR of poliovirus RNA extracted directly from concentrated environmental surveillance samples to infer the number of infected individuals excreting poliovirus. The quantitation method was validated using data from vaccination with bivalent oral polio vaccine (bOPV). The method was then applied to infer the weekly number of excreters in a large, sustained, asymptomatic outbreak of wild type 1 poliovirus in Israel (2013) in a population where >90% of the individuals received three doses of inactivated polio vaccine (IPV). Evidence-based intervention strategies were based on the short TAT for direct quantitative detection. Furthermore, a TAT shorter than the duration of poliovirus excretion allowed resampling of infected individuals. Finally, the method documented absence of infections after successful intervention of the asymptomatic outbreak. The methodologies described here can be applied to outbreaks of other excreted viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), where there are (1) significant numbers of asymptomatic infections; (2) long incubation times during which infectious virus is excreted; and (3) limited resources, facilities, and manpower that restrict the number of individuals who can be tested and re-tested.
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