Fig 1.
Multiple replication kinetics of infections in PER.C6 cells with a cell density of 107 cells/ml at an MOI of 1–2, at 30°C and 37°C, harvested at 0–48 hours post infection.
Panel A) Average and standard deviation of two (n = 2) replication kinetic curves of the Brunenders strain versus 3 selected clones (clone A, B and C) derived after passage with impaired growth at 37°C. Panel B) Average and standard deviation of three (n = 3) independent infections of Brunenders and the CAVA backbone, which contained all mutations from Clones A, B and C combined. Panel C) Average and standard deviation of three (n = 3) independent infections of the Brunenders strain versus the CAVA vaccine strains (CAVA-1 Mahoney, CAVA-2 MEF-1 and CAVA-3 Saukett).
Fig 2.
Schematic overview of the viruses described here and their incorporated mutations.
Black vertical lines represent the synonymous CAVA mutations whilst red vertical lines represent non-synonymous CAVA mutations, dispersed over the poliovirus genome; a detailed description of the individual mutations is given in S1 Table. 5’UTR = 5’ Untranslated Region, 3’UTR = 3’Untranslated Region.
Fig 3.
Electron micrographs of two representative cells per panel after infection of suspension PER.C6 cells with a cell density of 107 cells/ml at an MOI of 1, harvested between 24–48 hours post infection.
Panel A) PBS (Mock) infected cells, Panel B) Mahoney infection at 37°C, Panel C) CAVA-1 Mahoney at 37°C, Panel D) CAVA-1 Mahoney at 30°C.
Table 1.
Infectious titers and in vivo neurovirulence after intra cerebral inoculation of the CAVA vaccine strains as compared to the cIPV and Sabin strains.
Fig 4.
Quantification of poliovirus infectious units (by infectious titer determination, Panel A), viral RNA levels (by RTqPCR, Panel B) and viral proteins (by Western blot, Panel C) after infection of suspension PER.C6 cells with a cell density of 107 cells/ml at an MOI of 1 at 30°C and 37°C, harvested between 0–48 hours post infection.
Viruses used were Brunenders (WT), Brunenders with the CAVA mutations in the IRES (IRES), Brunenders with the CAVA mutations in the Non-Structural proteins (NS), the CAVA-1 Mahoney (C1) vaccine strain and the CAVA backbone virus (CBB); Control (Ctrl) is an uninfected control. Data depict one representative infection (n = 1) measured once for infectivity and (n = 3) times for viral RNA and protein levels. Error bars represent standard deviation from the mean and one representative of three independent western blots is shown.
Fig 5.
Replication kinetics of poliovirus infection of suspension PER.C6cells with a cell density of 107 cells/ml at an MOI of 1 at 30°C and 37°C harvested between 0–48 hours post infection.
Viruses used were Brunenders, Brunenders—with 14 CAVA mutations, MEF-1, MEF-1 –with 14 CAVA mutations, Sabin 3, Sabin 3 –with 14 CAVA mutations and the CAVA backbone virus (see overview of all viruses in Fig 2). The Sabin 3/Sabin 3–14 infection was performed independently from the Brunenders/Brunenders-14 and MEF-1/MEF-1-14 infections. However, the control taken along (CAVA-backbone) was similar for both experiments.
Fig 6.
Reversion of the temperature sensitive phenotype by stepwise increase of the infection temperature.
Serial passage was performed using CAVA-1 Mahoney in suspension PER.C6 cells infected at a cell density of 107 cells/ml at low MOI (0.01) and harvested at 3–4 days post infection. Temperature was gradually increased (33–37°C) or temperature was kept constant at 30°C for the control viruses. Panel A depicts the viral titers at each passage when titrated and incubated at 30°C or 37°C for the two independent experiments (n = 2). Panel B lists the reverting CAVA mutations of the viruses at passage number 6 where the nucleotide number refers to the position from the start of the viral genome.
Fig 7.
In vivo immunogenicity of the CAVA vaccine strains as compared to cIPV.
Groups of ten (n = 10) rats were immunized with a full dose (FD: 100% 40:8:32 DU/dose or 150% 60:12:48 DU/dose) or a 1:2, 1:4 or 1:16 dilution of the full dose. Poliovirus type 1, 2 and 3-specific neutralizing antibody titers were determined by Sabin Virus Neutralizing Assay at day 21 post immunization. Each dot represents one individual animal; the connected line represents the geometric mean at each dose. Relative potency estimates and 95% confidence intervals of the difference between the CAVA vaccine strain and cIPV reference based on the number of seroconverting animals are depicted in the table, horizontal dotted line represents the seroconversion limit for each assay.
Fig 8.
Secondary RNA structure prediction of Domain II and Domain VI of the IRES in Brunenders and CAVA using the MFOLD program developed by M. Zuker.
Circled nucleotides (at positions 133, 142, 146, and 163 in domain II and at positions 597, 609 in domain VI) represent nucleotide changes between CAVA and Brunenders. The last remaining CAVA IRES mutation (nt579) lies outside of any IRES domains and in the spacer region between Domains V and VI. After serial passage at increasing temperature (Fig 6) nucleotide 127 (indicated by square box) mutated in both passaging experiments (n = 2) from U to C, forming a C–G base pair with CAVA mutation nt 163. The CAVA mutations induce a change in predicted secondary structure and increased free energy (ΔG) for domain II, whilst for domain VI only the free energy is affected.