Development of a new oral poliovirus vaccine for the eradication end game using codon deoptimization

Abstract

Enormous progress has been made in global efforts to eradicate poliovirus, using live-attenuated Sabin oral poliovirus vaccine (OPV). However, as the incidence of disease due to wild poliovirus has declined, vaccine-derived poliovirus (VDPV) has emerged in areas of low-vaccine coverage. Coordinated global cessation of routine, type 2 Sabin OPV (OPV2) use has not resulted in fewer VDPV outbreaks, and continued OPV use in outbreak-response campaigns has seeded new emergences in low-coverage areas. The limitations of existing vaccines and current eradication challenges warranted development of more genetically stable OPV strains, most urgently for OPV2. Here, we report using codon deoptimization to further attenuate Sabin OPV2 by changing preferred codons across the capsid to non-preferred, synonymous codons. Additional modifications to the 5' untranslated region stabilized known virulence determinants. Testing of this codon-deoptimized new OPV2 candidate (nOPV2-CD) in cell and animal models demonstrated that nOPV2-CD is highly attenuated, grows sufficiently for vaccine manufacture, is antigenically indistinguishable from Sabin OPV2, induces neutralizing antibodies as effectively as Sabin OPV2, and unlike Sabin OPV2 is genetically stable and maintains an attenuation phenotype. In-human clinical trials of nOPV2-CD are ongoing, with potential for nOPV strains to serve as critical vaccine tools for achieving and maintaining polio eradication.

Keywords: Live attenuated vaccines; Policy and public health in microbiology; Vaccines; Virology.

Fig. 1. Codon-deoptimized wild- and Sabin-derived strains of type 2 poliovirus exhibit reduced replicative fitness.

Type 2 WPV strain MEF-1 was codon deoptimized by modifying synonymous codons at 20 to 100% of possible CpG_2-3 sites within the capsid region. a Plaque morphology of codon-deoptimized MEF-1 on HeLa cell monolayers after 65 h at 37 °C. b Mean plaque area of codon-deoptimized MEF-1 viruses as compared to Sabin OPV2 and nOPV2-CD on HeLa cell monolayers after 65 h at 37 °C. c Plaque morphology of Sabin OPV2 and nOPV2-CD on HeLa cell monolayers after 65 h at 37 °C. Representative images and measurements of n = 2 biologically independent replicates showing similar results shown. Scale bar = 1 cm.

Fig. 2. nOPV2-CD exhibits similar growth properties and temperature sensitivity as Sabin OPV2 but delivers more RNA per PFU.

Vero cells were infected with Sabin OPV2 or nOPV2-CD at an MOI of 0.1. a Viral titers were determined at the indicated times post-infection at 33 °C via plaque assay. b Time to reach 100% cytopathic effect (CPE) at 33 °C and the corresponding viral titers at that time were determined via plaque assay. c Viral titers were measured via plaque assay after incubation of infected Vero cells at the indicated temperatures for 48 h. d Plaque morphology on Vero cell monolayers after 65 h. Scale bar = 1 cm. e Ratio of viral RNA copy number to PFU was established via qRT-PCR and plaque assay. Error bars indicate mean ± SD. n = 3 to 4 biologically independent replicates showing similar results. All P values were determined by unpaired two-tailed t-tests. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Fig. 3. The antigenicity profile of nOPV2-CD parallels that of Sabin OPV2.

The antigenicity of Sabin OPV2 and nOPV2-CD was determined via non-competitive sandwich ELISA using monoclonal antibodies specific for native structural conformations of four key antigenic sites on the virion particle. Dose-dependent reactivity to these four antibodies correlates with antigenicity. a Antigenic site 1 (MAb 433). b Antigenic site 2a (MAb 1247). c Antigenic site 2b (MAb 1037). d Antigenic site 3b (MAb 1050). Error bars indicate mean ± SD. n = 3 biologically independent replicates showing similar results.

Fig. 4. nOPV2-CD elicits a dose-dependent immune response comparable to that of Sabin OPV2.

Juvenile interferon-receptor knockout mice expressing the human poliovirus receptor were inoculated i.p. with either Sabin OPV2 or nOPV2-CD at a range of doses. Neutralizing antibody titers were determined in sera at day 21 post-infection. Human sera collected from a polio-immunized individual served as a positive assay control. Titers for individual animals are shown, with error bars indicating standard error of the mean (SEM). n = 10 weight- and sex-matched mice per group. All P-values were determined by a two-tailed Mann-Whitney U-test. *P = 0.0230.

Fig. 5. Growth of nOPV2-CD remains stable during serial passage in Vero cells.

Vero cells were infected with Sabin OPV2 or nOPV2-CD at an MOI of 0.1 and incubated at 37 °C. After 10 h, virus was harvested, viral titers determined via plaque assay, and the amount of virus necessary to infect a subsequent round of Vero cells at MOI 0.1 calculated. A total of ten serial passages was completed, with the mean viral titer at the indicated passages shown. Error bars indicate mean ± SEM. n = 3 biologically independent replicates showing similar results. Viral stocks were derived from the tenth serial passage in Vero cells and used as inoculum for mouse neurovirulence testing (Table 1).