Virus purification

1. A method for separating infectious Chikungunya virus particles from non-infectious Chikungunya virus particles comprising precipitating the non-infectious virus particles with protamine.

2. The method according to claim 1 , wherein said protamine precipitation also facilitates the separation of infectious Chikungunya virus particles from host cell proteins and/or low molecular weight materials.

3. A method for purifying infectious Chikungunya virus particles, comprising the steps of

i) providing a crude harvest (a) comprising infectious Chikungunya virus particles, non-infectious Chikungunya virus particles, and impurities, wherein the impurities are generated from growing said virus particles on a cell substrate;

ii) contacting said crude harvest (a) with an agent comprising protamine to obtain a Chikungunya virus preparation (b) comprising infectious Chikungunya virus particles, wherein the enrichment of infectious Chikungunya virus particles in the virus preparation (b) relative to total Chikungunya virus particles in the crude harvest (a) is in the range of at least 50% to 95%.

4. The method according to claim 3 , wherein said Chikungunya virus preparation (b) is further purified by one or more size exclusion methods selected from

i) sucrose density gradient centrifugation,

ii) a solid-phase matrix packed in a column comprising a ligand-activated core and an inactive shell comprising pores, wherein the pores comprise a molecular weight cut-off that excludes the virus particles from entering the ligand-activated core, and wherein a molecule smaller than the molecular weight cut-off of the pores can enter the ligand-activated core, and

iii) size exclusion chromatography;

to obtain a final Chikungunya virus preparation (c) comprising the infectious Chikungunya virus particles, less than 100 ng/mL residual host cell DNA, less than 1 μg/mL residual host cell protein, and less than 1 μg/mL Chikungunya virus particle aggregates.

5. The method according to claim 4 , wherein the final Chikungunya virus preparation (c) comprises less than 10 ng/mL residual host cell DNA and less than 100 ng/mL residual host cell protein.

6. The method according to claim 3 , wherein said crude harvest (a) is subjected to one or more pre-purification step(s) prior to step ii), wherein the one or more pre-purification step(s) comprise

a) filtration using a filter having a pore size equal to or less than 0.2 μm,

b) digestion of host cell genomic DNA by enzymatic treatment, and

c) ultra/diafiltration using a hollow fiber membrane having a pore size equal to or greater than 100 kDa.

7. The method according to claim 3 , wherein the concentration of protamine is between 0.5 mg/mL and 3 mg/mL.

8. The method according to claim 4 , wherein the infectious Chikungunya virus particles in said final Chikungunya virus preparation (c) are enriched by at least 50% to 95% relative to total Chikungunya virus particles in crude harvest (a).

9. The method according to claim 4 , wherein said Chikungunya virus preparation (c) comprises less than 10% impurities.

10. The method according to claim 3 , wherein said infectious Chikungunya virus particles are propagated in a cell line selected from the group consisting of an EB66 cell line, a Vero cell line, a Vero-αHis cell line, a HeLa cell line, a HeLa-S3 cell line, a 293 cell line, a PC12 cell line, a CHO cell line, a 3T3 cell line, a PerC6 cell line, an MDSK cell line, a chicken embryonic fibroblast cell line, a duck cell line, and a diploid avian cell line.

11. The method according to claim 3 , wherein said infectious Chikungunya virus particles are selected from the group consisting of a live virus, an attenuated live virus, a chimeric virus, a modified live virus, and a recombinant live virus.

12. The method according to claim 4 , further comprising a step iv) comprising inactivating the Chikungunya virus preparation (c).

13. The method according to claim 12 , wherein said inactivating step iv) is performed using formaldehyde.

14. The method according to claim 3 , wherein said Chikungunya virus is a Chikungunya virus comprising a deletion mutation in the non-structural protein 3 provided by SEQ ID NO: 77 or an immunogenic variant thereof, wherein said immunogenic variant is defined as having at least 80% sequence identity to SEQ ID NO: 77.

15. The method according to claim 3 , where said protamine is selected from the group comprising a protamine salt, a protamine sulphate, and a recombinant protamine sulphate.

16. A composition for immunization against a Chikungunya virus infection, wherein said composition comprises a Chikungunya virus comprising a deletion mutation in the non-structural protein 3 provided by SEQ ID NO: 77 or an immunogenic variant thereof, wherein said immunogenic variant is defined as having at least 80% sequence identity to SEQ ID NO: 77; and wherein at least 50% of the Chikungunya virus particles in the composition are in the size range of 20-40 nm.

17. A composition for immunization against a Chikungunya virus infection, wherein said composition comprises a Chikungunya virus comprising a deletion mutation in the non-structural protein 3 provided by SEQ ID NO: 77 or an immunogenic variant thereof, wherein said immunogenic variant is defined as having at least 80% sequence identity to SEQ ID NO: 77; and wherein at least 50% of the Chikungunya virus particles comprised in the composition are infectious Chikungunya virus particles.

18. The composition for immunization against a Chikungunya virus infection according to claim 16 , wherein the composition is a vaccine.

19. The composition for immunization against a Chikungunya virus infection according to claim 17 , wherein the composition is a vaccine.