The Use of Next-Generation Sequencing for the Quality Control of Live-Attenuated Polio Vaccines.

BACKGROUND: Next-generation sequencing (NGS) analysis was compared to the current MAPREC (mutational analysis by polymerase chain reaction and restriction enzyme cleavage) assay for quality control of live-attenuated oral polio vaccine (OPV). METHODS: MAPREC measures reversion of the main OPV attenuating mutations such as uracil (U) to cytosine (C) at nucleotide 472 in the 5' noncoding region of type 3 OPV. Eleven type 3 OPV samples were analyzed by 8 laboratories using their in-house NGS method. RESULTS: Intraassay, intralaboratory, and interlaboratory variability of NGS 472-C estimates across samples and laboratories were very low, leading to excellent agreement between laboratories. A high degree of correlation between %472-C results by MAPREC and NGS was observed in all laboratories (Pearson correlation coefficient r = 0.996). NGS estimates of sequences at nucleotide 2493 with known polymorphism among type 3 OPV lots also produced low assay variability and excellent between-laboratory agreement. CONCLUSIONS: The high consistency of NGS data demonstrates that NGS analysis can be used as high-resolution test alternative to MAPREC, producing whole-genome profiles to evaluate OPV production consistency, possibly eliminating the need for tests in animals. This would be very beneficial for the quality assessment of next-generation polio vaccines and, eventually, for other live-attenuated viral vaccines.

Differences in Antigenic Structure of Inactivated Polio Vaccines Made From Sabin Live-Attenuated and Wild-Type Poliovirus Strains: Impact on Vaccine Potency Assays.

BACKGROUND: Following the declaration of wild-type 2 poliovirus eradication in 2015, the type 2 component was removed from the live-attenuated oral polio vaccine (OPV). This change implies a need to improve global coverage through routine immunization with inactivated polio vaccine (IPV), to ensure type 2 immunity. Several manufacturers use Sabin OPV strains for IPV production (sIPV), rather than the usual wild-type strains used for conventional IPV (cIPV). However, in contrast to cIPV, potency assays for sIPV have not been standardized, no international references exist, and no antigen units have been defined for a sIPV human dose. Thus, sIPV products from different manufacturers cannot be compared, and the relationship between antigenicity and immunogenicity of sIPV is not well understood. METHODS: A collaborative study was conducted in which laboratories used different methods to measure the antigen content of a set of sIPV and cIPV samples with an aim to identify a suitable reference for sIPV products. RESULTS: The study revealed differences in the reactivity of antibody reagents to cIPV and sIPV products. CONCLUSIONS: Homologous references are required to measure the antigen content of IPV products consistently. The first World Health Organization international standard for sIPV was established, with new, specific Sabin D-antigen units assigned.

Establishment of the 1st WHO International Standard for anti-EV71 serum (Human).

Enterovirus A71 (EV71) is the major causative agent of severe and fatal hand, foot and mouth disease. There is plenty of evidence that EV71 has circulated widely in the Western Pacific Region for the last twenty years. Vaccines against EV71 are already available or under development. A collaborative study to establish the 1st WHO International Standard for anti-EV71 serum (Human) was conducted to ensure that methods used to measure the serum neutralizing activity or antibody levels against EV71 are accurate, sensitive and reproducible. Two candidate samples as well as a third candidate reference containing low anti-EV71 antibody titre were produced from plasma samples donated by healthy individuals. All three serum samples exhibited good levels of neutralizing antibodies against a wide range of EV71 strains of various genotypes. The study showed that between laboratory variations in neutralization titres were significantly reduced when values were expressed relative to those of either of the two candidate sera. Sample 14/140 was established as the WHO 1st International Standard for anti-EV71 serum (human), 14/138 as its potential replacement and 13/238 as a WHO Reference Reagent, with assigned unitage of 1,000, 1090 and 300 International Units (IU) of anti-EV71 neutralizing antibodies per ampoule, respectively.

Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine.

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.