Adaptation of enterovirus to vero cells and vaccine formulations thereof

A method of adapting Enterovirus D68 to propagate in Vero cells to high titer comprising:

• (f) infecting the virus by adsorbing for about 60-120 minutes at 32-35° C.;
• (g) propagating the virus after addition of the maintenance media at 32-35° C.;
• (h) diluting the virus stock obtained from each passage at 1:3 to 1:10 dilution to infect the next batches of Vero cells for initial passages of the virus at 32-35° C.;
• (i) diluting the virus at 1:20-1:500 dilution to infect the Vero cells during later/subsequent passages at 32-35° C.;
• and
• (j) harvesting the virus during every passage when 90% or more cytopathic effect has been achieved or within 6 days of infection.

A method of inactivating Enterovirus D68 containing cDNA encoded from SEQ ID NO:

• 1 for immunization comprising:
• (a) sterile filtration of the harvested Enterovirus D68 virus;
• (b) removal of the host nucleic acid using nuclease treatment and concentrated by tangential flow filtration using 100 kDa filter;
• (c) inactivation using 1/2000- 1/4000 formalin for up to 3 weeks at temperature varying from 25-37° C. or with BPL at 4-25° C. for up to 120 hours or with 0.005% to 3% hydrogen peroxide at 25±5° C. for up to 6 hours;
• and
• (d) purification of inactivated antigen or purification before inactivation of the viral antigen by gel filtration using Sepharose CL-4B followed by anion exchange chromatography using DEAE resins or by double steps/two rounds of size exclusion chromatography or by cellufine sulphate followed by anion exchange chromatography or by mixed mode resins like CHT Type II (Biorad) alone or in combination with the other resin or combination of chromatography methods.