Supporting Information

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Fig. 4. Comparison of longitudinal population divergence during experimental infection with EIAV_PV. Genetic distances of the deduced amino acid sequences were calculated by using Distance software with Kimura correction (GCG Wisconsin Package of software). Distances were calculated from aligned gp90 regions longitudinal isolates. Calculated distances were plotted as the divergence from the inoculating strain, EIAV_PV. The days after infection are indicated below the x axis in blue. (-), mean divergence; I, II, III, IV, V, VI, fever episodes; Inapparent, inapparent stage of disease; È, population subclones chosen for variant challenge strain development.

Fig. 5. Evolutionary analyses of the longitudinal isolates derived from experimental infection with EIAV_PV. A phylogenetic tree of aligned plasma and tissue immune suppression (inapparent stage) and febrile deduced amino acid sequences (gp90 regions) were constructed by the neighbor-joining method from Kimura-corrected distances with the optimality criterion set to distance. The tree was rooted to EIAV_PV. Bootstrap values were determined over 1,000 iterations and are indicated at the nodes of the branches. Branch lengths are proportional to the distance existing between the sequences. IS, immune suppression; Å, population subclones chosen for variant challenge strain development.

Fig. 7. Neutralization profiles of proviral variant strains with serum from animals experimentally infected with EV0, EV6, or EV13. Variant proviral strains were tested for in vivo pathogenecity through experimental infections of ponies. At 6 months after infection, serum from two representative animals (labeled A and B) was tested in our standard neutralization assay. The mean reciprocal dilutions of serum that neutralized 50% of input EV0, EV6, or EV13 as measured in an infectious center assay are presented. The line (─) denotes the cut off (≥25) value for valid 50% neutralization titers.

Fig. 8. Clinical and virological profiles of EV0 challenge strain vaccine trial group. (A) Eight EIAV-naïve ponies were vaccinated with 10³ TCID₅₀ EIAV_D9 i.m. (#Vax#). Rectal temperature (red line, right y axis) and platelet counts (blue dashed line, first left y axis) were followed daily for up to 370 days (x axis) after the first vaccine dose. Quantification of the virus load (¨, second left y axis) was performed on viral RNA extracted from plasma at periodic time points before and after virulent virus challenge 7 months after the first vaccination with 10³ TCID₅₀ EV0, I.M (#Challenge). (B) Six EIAV-naïve ponies were also challenged with 10³ TCID₅₀ EV0, i.m. (#Challenge). A 14-day period of immune suppression with 0.1mg/kg dexamethasone is indicated by a pink shaded box. Febrile episodes were defined by a achieving a combination of two to three features such as: rectal temperature >39°C in conjunction with thrombocytopenia (platelet decrease of ≥70,000/µl of whole blood), EIAV viral load ≥10⁵ as well as other clinical signs of EIA. Diagnostic results of viral strain for each vaccinated animal are indicated in each respective panel above the date of analysis by either a V (vaccine strain) or a C (challenge strain): lower case letters represent the minor population if both vaccine and challenge strain viruses were detected. Pink box, animal killed due to severe disease; gray box, animal killed as endpoint of study.

Fig. 9. Clinical and virological profiles of EV6 challenge strain vaccine trial group. (A) Eight EIAV-naïve ponies were vaccinated with 10³ TCID₅₀ EIAV_D9 I.M. (#Vax#). Rectal temperature (red line, right y axis) and platelet counts (blue dashed line, first left y axis) were followed daily for up to 370 days (x axis) after the first vaccine dose. Quantification of the virus load (¨, second left y axis) was performed on viral RNA extracted from plasma at periodic time points before and after virulent virus challenge 7 months after the first vaccination with 10³ TCID₅₀ EV6, I.M (#Challenge). (B) Six EIAV-naïve ponies were also challenged with 10³ TCID₅₀ EV6, I.M (#Challenge). A 14-day period of immune suppression with 0.1mg/kg dexamethasone is indicated by a pink shaded box. Febrile episodes were defined by a achieving a combination of two to three features such as: rectal temperature >39°C in conjunction with thrombocytopenia (platelet decrease of ≥70,000/µl of whole blood), EIAV viral load ≥10⁵ as well as other clinical signs of EIA. Diagnostic results of viral strain for each vaccinated animal are indicated in each respective panel above the date of analysis by either a V (vaccine strain) or a C (challenge strain): lower-case letters represent the minor population if both vaccine and challenge strain viruses were detected. Pink box, animal killed due to severe disease;gray box, animal killed as endpoint of study.

Fig. 10. Clinical and virological profiles of EV13 challenge strain vaccine trial group. (A) Eight EIAV-naïve ponies were vaccinated with 10³ TCID₅₀ EIAV_D9 I.M. (#Vax#). Rectal temperature (red line, right y axis) and platelet counts (blue dashed line, first left y axis) were followed daily for up to 370 days (x axis) after the first vaccine dose. Quantification of the virus load (¨, second left y axis) was performed on viral RNA extracted from plasma at periodic time points prior to and after virulent virus challenge 7 months after the first vaccination with 10³ TCID₅₀ EV13, I.M (#Challenge). (B) Six EIAV-naïve ponies were also challenged with 10³ TCID₅₀ EV13, I.M (#Challenge). Febrile episodes were defined by a achieving a combination of two to three features such as: rectal temperature >39°C in conjunction with thrombocytopenia (platelet decrease of ≥70,000/µl of whole blood), EIAV viral load ≥10⁵ as well as other clinical signs of EIA. Diagnostic results of viral strain for each vaccinated animal are indicated in each respective panel above the date of analysis by either a V (vaccine strain) or a C (challenge strain): lower-case letters represent the minor population if both vaccine and challenge strain viruses were detected. Pink box, animal killed due to severe disease; gray box, animal killked as endpoint of study.

Table 4. Trial outcome

NA, not applicable; DPC, days after challenge; C, challenge strain (dominant); V, vaccine strain (dominant); c, challenge strain; v, vaccine strain.

*Pony was killed at this date because of severe disease.