The selection of naturally stable candidate foot-and-mouth disease virus vaccine strains for East Africa

Figures

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  Fig. 1. Accelerated thermostability assessment of candidate FMDV vaccine strains. Thermostability of the four candidate vaccine strains (O/ETH/29/2008, A/ETH/9/2008, SAT1/KEN/80/2010 and SAT2/ETH/65/2009) was confirmed by exposure to an elevated temperature prior to thermofluor analyses. An equal amount of control virus, with lower thermostability and belonging to the same serotype (O/ETH/26/2011, A/KEN/28/2008, SAT1/ETH/3/2007 and SAT2/2/2007, respectively), was analysed alongside each candidate strain. Virus strains belonging to the O and A serotypes were exposed to 45 °C, SAT1 to 38 °C and SAT2 to 43 °C for the indicated period before being tested by thermofluor assay. The loss of a dissociation peak, as observed for each control virus after 1 h heat treatment, indicates the absence of intact capsid due to its dissociation. Assays were conducted in triplicate.

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  Fig. 2. Storage assessment of inactivated vaccine antigens. Unformulated, inactivated candidate vaccine strains (O/ETH/29/2008, A/ETH/9/2008, SAT1/KEN/80/2010 and SAT2/ETH/65/2009) were stored at +4 °C for 15 months, during which the integrity of their capsid was analysed by thermofluor assay (see Table 3). Day 1 and month 15 thermofluor analyses are shown to facilitate comparison of the respective dissociation peaks at the beginning and end of the storage period, respectively. Thermofluor assays were performed in triplicate.

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  Fig. 3. Production of monovalent vaccinate sera. Separate groups of five calves were vaccinated with one of the candidate FMD vaccines (O/ETH/29/2008, A/ETH/9/2008, SAT1/KEN/80/2010 and SAT2/ETH/65/2009) on day 0 and boosted on day 14 post vaccination; blood samples were collected 0, 7-, 14-, and 21-days post vaccination and assayed to determine homologous mean virus neutralizing-antibody titres (VNT (log10)). Error bars indicate standard deviation.

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  Supplementary Fig. 1. FMDV strain origin. Schematic representation of the 37 African FMDV strains (see Table 1) belonging to the O, A, SAT1 and SAT2 serotypes that were screened for thermostability. East African countries are shaded grey and Egypt (North Africa) is unshaded.

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  Supplementary Fig. 2. Thermofluor-based screen for candidate FMDV strains with improved thermostability. The 37 FMDV strains listed in Table 1 were screened by thermofluor assay to determine the temperature at which the viral genome is released as a result of capsid dissociation. The thermofluor plots for each of the O, A. SAT1 and SAT2 candidate vaccine strains are shown. Four non-African FMDV belonging to the A serotype (A/IRAQ/22/1995, A/IRAN/1/2005, A/ARG/1/2000 and A/SAU/1995) are included in the respective plot as thermostable virus controls. Table 1 lists the respective T r values and thermofluor assays were conducted in triplicate.

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  Supplementary Fig. 3. Estimation of intact antigen loss following long-term storage at 4 °C. Unformulated, inactivated candidate vaccine strains (O/ETH/29/2008, A/ETH/9/2008, SAT1/KEN/80/2010 and SAT2/ETH/65/2009) were stored at +4 °C for 15 months, during which the integrity of their capsid was assessed by thermofluor assay (see Table 3 and Figure 2). To estimate the loss of intact SAT1 antigen over the storage period, the respective dissociation peaks generated using day 1 and month 15 stored antigen were compared with peaks generated using different amounts of virus under the same assay conditions. Negligible losses for stored intact O, A and SAT2 antigens, and a 20% reduction of intact SAT1 antigen was estimated. Thermofluor assays were performed in triplicate.

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  Supplementary Fig. 4. Comparison of the external capsid amino acid sequences of candidate vaccine strains with Eastern Africa FMDV reference antigen panel. (A). On-line software (NGphylogeny.fr) was used to analyse the amino acid sequences of the VP1-3 external capsid proteins from each of the four candidate vaccine strains (O/ETH/29/2008, A/ETH/9/2008, SAT1/KEN/80/2010 and SAT2/ETH/65/2009) alongside the respective Eastern African FMDV reference strains (4 for each of the O, A, SAT1 and SAT2 serotypes) selected by the World Reference Laboratory for Foot-and-Mouth Disease (WRL-FMD). (B) Percent identity of VP1-3 amino acids from each of the four candidate vaccine strains and the respective reference strain determined using on-line software (MUSCLE).