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  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
  • C07K14/08—RNA viruses
  • C07K14/18—Togaviridae; Flaviviridae
  • C07K14/1816—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus), border disease virus
  • C07K14/1825—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
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  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
  • C12N7/04—Inactivation or attenuation; Producing viral sub-units
  • C12N7/06—Inactivation or attenuation by chemical treatment
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
  • A61K2039/5252—Virus inactivated (killed)
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  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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  • A61K2039/5254—Virus avirulent or attenuated
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
  • A61K2039/5258—Virus-like particles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55505—Inorganic adjuvants
• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55516—Proteins; Peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
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  • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
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  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/24011—Flaviviridae
  • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
  • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/24011—Flaviviridae
  • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
  • C12N2770/24151—Methods of production or purification of viral material
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  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/24011—Flaviviridae
  • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
  • C12N2770/24161—Methods of inactivation or attenuation
  • C12N2770/24163—Methods of inactivation or attenuation by chemical treatment
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the disclosure relates to methods for the purification of viruses for use in vaccines.
• Protamine was originally isolated from the sperm of salmon and other species of fish but is now produced primarily through recombinant biotechnology. It is a highly cationic peptide that binds to negatively charged molecules such as nucleic acids to form a stable ion pair. Its use in removing host cell nucleic acid is well document.
• aspects of the invention provide processes for the purification of infectious virus particles comprising the steps of (a) providing a crude harvest (a) comprising virus particles and impurities, wherein the impurities are generated from growing said virus particles on a cell substrate; (b) reducing impurities from the crude harvest (a) by precipitation with an agent comprising a protamine salt, preferably a protamine sulphate, to obtain a virus preparation (b); and further purifying the virus preparation (b) by method or methods selecting for size of the virus particles, such as e.g. a sucrose density gradient centrifugation to obtain a virus preparation (c) comprising the infectious virus particles.
• the concentration of protamine sulphate in step (b) is about 1 to 10 mg/ml, more preferably about 1 to 5 mg/ml, more preferably about 1 to 2 mg/ml. In one embodiment, the concentration of protamine sulphate in step (b) is about 2 mg/mL. In one embodiment, the concentration of protamine sulphate is 1.2 to 1.8 mg/ml, more preferably 1.4 to 1.6 mg/ml. In a preferred embodiment, the concentration of protamine sulphate in step (b) is about 1.6 mg/ml or about 2 mg/ml.
• the residual host cell DNA of the virus preparation (e) is less than 1 ⁇ g/mL, especially less than 900, 800, 700, 600, 500, 400, 300 or 200 ng/mL, preferably less than 100 ng/mL. In a preferred embodiment, the residual host cell DNA of the virus preparation (c) is less than 10 ng/mL. In some embodiments, the residual host cell protein of the final virus preparation (c) is less than 10 ⁇ g/mL, especially less than 9, 8, 7, 6, 5, 4, 3 or 2 ⁇ g/mL, preferably less than 1 ⁇ g/mL. In a preferred embodiment, the residual host cell protein of the virus preparation (c) is less than 100 ng/mL.
• the residual non-infectious virus particles of the final virus preparation (c) is less than 10 ⁇ g/mL, especially less than 9, 8, 7, 6, 5, 4, 3 or 2 ⁇ g/mL, preferably less than 1 ⁇ g/mL. In a preferred embodiment, the residual non-infectious virus particles of the virus preparation (c) is less than 100 ng/mL.
• the residual protamine is less than 1 ⁇ g/mL, especially less than 900, 800, 700, 600, 500, 400, 300 or 200 ng/mL, preferably less than 100 ng/mL, more preferably is below the detection limit of HPLC, in particular below the detection limit in the final drug substance.
• the PS content is tested by HPLC or size exclusion chromatography (SEC). For example, HPLC is validated for PS determination in JEV sucrose gradient pool samples as a routine release assay and is very sensitive (i.e., LOQ 3 ⁇ g/mL; LOD 1 ⁇ g/mL). In the current invention, PS content in in virus DS samples was ⁇ LOD.
• the HPLC assessment of PS content can be performed on a Superdex Peptide 10/300GL column (GE: 17-5176-01) using 30% Acetonitrile, 0,1% Trifluoroacetic acid as solvent with a flow rate of 0,6 ml/min at 25°C and detection at 214 nm.
• MS mass spectrometry
• the residual PS levels in a virus preparation are tested by MS or other such highly sensitive method, e.g., nuclear magnetic resonance (NMR).
• residual PS as well as fragments and/or breakdown products of PS, can be detected at trace amounts, such as levels as low as, for example, 10 6 , 10 7 or 10 8 molecules per typical sample load.
• the PS levels are tested in the sucrose gradient pool.
• the PS levels are tested in the drug product.
• the PS levels are tested in the drug substance.
• the crude harvest (a) comprising the virus particles and impurities is subjected to one or more pre-purification step(s) prior to step (b).
• the one or more pre- purification step(s) comprises digesting host cell genomic DNA in the crude harvest (a) comprising the virus particles and impurities by enzymatic treatment.
• the one or more pre- purification step(s) comprises filtration, ultrafiltration, concentration, buffer exchange and/or diafiltration.
• the one or more pre-purification steps is filtration using a filter having a pore size equal to or less than 1 ⁇ . In some embodiments, the filter has a pore size equal to or less than 0.2 ⁇ .
• the filter has a pore size of 0.2 ⁇ .
• the concentration and/or ultra/diafiltration and/or buffer exchange is performed by tangential flow filtration (TFF).
• ultra/diafiltration of the crude harvest (a) comprising the virus particles and impurities is performed using a hollow fiber membrane having a cut-off of equal to or less than 300 kDa.
• the hollow fiber membrane has a cut-off of 100 kDa.
• the virus particle is a live virus, a chimeric virus, an attenuated live virus, a modified live virus, or a recombinant live virus.
• the virus particles of the invention may by optionally inactivated.
• the virus particle is an attenuated form of the virus particle.
• the virus may have reduced infectivity, virulence, and/or replication in a host, as compared to a wild-type virus.
• the virus is a mutated or modified virus, for example the nucleic acid of the virus may contain at least one mutation relative to the wild-type virus.
• the virus is a recombinant live virus, meaning a virus that is generated recombinantly and may contain nucleic acid from different sources.
• the virus particle is a live virus, an attenuated live virus, a modified live virus, or a recombinant live virus.
• the virus belongs to a virus family selected from the group consisting of Paramyxoviridae, Orthomyxoviridae, Flaviviridae, Filoviridae, Arenaviridae, Rhabdoviridae, and Coronaviridae.
• the virus belongs to a virus family selected from the group consisting of Togaviridae (being live or inactivated), such as alphaviruses, or Flaviviridae (being live or inactivated).
• the virus is a virus of the family Flaviviridae, i.e. a flavivirus.
• the virus is a Zika virus or Yellow Fever virus.
• the virus is a Zika virus.
• the Zika virus is a Zika virus from the Asian lineage.
• the relative reduction of impurity of the final virus preparation relative to the liquid medium (a) comprising the virus particles and impurities is in a range from 60 to 95%. In some embodiments, the residual impurity of the final virus preparation is less than 1%.
• the filtration of step in (b)(ii) of claim 1 is performed using a filter having a pore size equal to or greater than 1 ⁇ .
• the filter has a pore size equal to or greater than 0.2 ⁇ .
• the filter has a pore size of 0.2 ⁇ .
• the virus is propagated in a cell line selected from the group consisting of an EB66 cell line, a Vero cell line, a Vero-aHis 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, a MDSK cell line, a chicken embryonic fibroblast cell line, a duck cell line, and a diploid avian cell line.
• said cell line is a duck cell line.
• said cell line is a diploid avian cell line.
• said cell line is EB66 cell line.
• said cell line is a Vero cell line.
• the composition is a vaccine.
• the composition or vaccine is directed against Chikungunya virus.
• the composition or vaccine is directed against a flavivirus.
• the composition or vaccine is directed against Yellow Fever virus.
• the composition or vaccine is directed against Zika virus such as e.g. a Zika virus of the Asian lineage.
• compositions comprising the virus particles obtainable by any of the processes described herein for treating and/or preventing a viral infection.
• the viral infection is caused by Chikungunya virus.
• the viral infection is caused by a flavivirus.
• the viral infection is caused by Yellow Fever virus.
• the viral infection is caused by Zika virus such as e.g. a Zika virus of the Asian lineage.
• the attenuated form of ChikV is derived from the LR2006-OPY1 ChikV infectious clone (La Reunion isolate). In some embodiments, the attenuated form of ChikV is the A5nsP3 mutant as described by Hallengard et al. (Novel Attenuated Chikungunya Vaccine Candidates Elicit Protective Immunity in C57BL/6 mice (2014) Journal of Virology 88(5):2858-2866) or an immunogenic variant thereof.
• the immunogenic variant of the A5nsP3 ChikV mutant is herein defined as having at least 80% sequence identity to the nucleotide sequence of the A5nsP3 mutant sequence as provided by SEQ ID NO: 77, especially at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NO: 77.
• the Zika virus is derived from the Asian lineage.
• the Zika virus is a Zika virus as described partially or fully in Sequence section of this application, i.e. any of sequences SEQ ID Nos 2 to 69 or 78, in particular all partly or fully described Zika viruses of the Asian lineages or an immunogenic variant thereof.
• the immunogenic variants of the Zika virus or Zika virus of the Asian lineages are herein defined as having at least 80%> sequence identity to the nucleotide sequence of the sequences described in any of sequences SEQ ID Nos 2 to 69 or 78, especially at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%> sequence identity.
• the process of the invention results in an enrichment of infectious virus particles from the crude harvest comprising infectious virus particles and non-infectious virus particles and other virus products such that the enrichment of the infectious virus particles is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, preferably at least 80%, especially 85% relative to the total virus particle content of the crude harvest (a) comprising the virus particles and impurities.
• the residual impurity of the final virus preparation with respect to all impurities in the crude harvest is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, preferably less than 5% as determined by SEC-HPLC (Size Exclusion Chromatography - HPLC).
• the filtration step of the virus preparation (b) after contact with the solid-phase matrix is performed using a filter having a pore size equal to or greater than 1 ⁇ .
• the filter has a pore size equal to or greater than 0.2 ⁇ .
• the filter has a pore size of about 0.2 ⁇ , such as 0.22 ⁇ .
• the Zika virus, or Chikungunya virus is propagated in a cell line selected from the group consisting of an EB66 cell line, a Vero cell line, a Vero-aHis 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, a MDSK cell line, a chicken embryonic fibroblast cell line, a duck cell line, and a diploid avian cell line.
• said cell line is a duck cell line.
• said cell line is a diploid avian cell line.
• said cell line is EB66 cell line.
• said cell line is a Vero cell line.
• the composition is a vaccine.
• the vaccine is administered to the subject once, twice or three or more times. In a preferred embodiment, the vaccine is administered once or twice. In a preferred embodiment, the vaccine is administered only once.
• the herein disclosed in vivo data regarding immunogenicity of the inactivated Zika virus vaccine of the current invention indicates that the virus is surprisingly potently immunogenic and also highly cross- protective (very similar immunogenicity in African and Asian strains). Data indicate that immunogenicity was unexpectedly higher than the recently reported inactivated Zika virus vaccine candidate (Larocca, et. al, 2016, Nature doi: 10.1038/naturel 8952.). Inactivated viruses are among the safest vaccines and especially preferred for delivery to populations where safety is especially concerning, such as pregnant women, children and immunocompromised individuals, which makes the herein disclosed inactivated Zika virus particularly suitable. Obtaining a high titer of inactivated virus is a challenge in the field.
• compositions comprising the virus particles obtainable by any of the processes described herein for treating and/or preventing a Chikungunya virus infection.
• Figure 1 Average distance tree (by % identity, nt), complete genomes.
• Figure 2 Neighbor joining tree (by % identity, nt), complete genomes.
• Figure 3 Pairwise alignment- Jalview (% identity, nt), complete genomes.
• Figure 4 Average distance tree (by % identity, aa), E-protein.
• Figure 5 Neighbor joining tree (by % identity, aa), E-protein.
• Figure 6 Pairwise alignment- Jalview (% identity, aa), E-protein.
• Figure 7 Alignment (shading: % identity, aa), E-protein.
• Figure 8 An example of virus particle maturation in the host cell. As observed in flaviviruses, full maturation of the particles requires proteolytic cleavage of the precursor membrane glycoprotein (prM) by the host protease furin. Not all prM molecules are cleaved, resulting in the release of mature, mosaic or immature-like conformations from the cells. Mosaic and immature forms are generally not infectious— only mature virions are infective and have hemagglutinin (HA) / TCID50 activity. ( Figure adapted from Plevka, et al., Maturation of flaviviruses starts from one or more icosahedrally independent nucleation centres, EMBO reports (2011) 12, 602-606).
• prM membrane glycoprotein
• FIG. 9 CHIKV schematic genome, including non-structural and structural proteins (labeled "CHIKV”) as well as a schematic representation of the A5nsP3 attenuated Chikungunya virus used to exemplify the purification process of the current invention (labeled "A5nsP3").
• the black triangle indicates the approximate location of the deletion in the nsP3 coding region.
• Figure 10 Flow-chart showing an exemplary downstream A5nsP3 CHIK virus purification process from the crude harvest to formulation of the (vaccine) drug substance, a preferred embodiment of the process of the invention.
• FIG 11 Absorbance at 214 nm, 260 nm and 280 nm of individual sucrose gradient centrifugation (SGC) fractions of a representative purification run of the process of the invention (A); the SEC-HPLC analysis of the final pooled fractions containing purified infectious attenuated A5nsP3 ChikV virus particles (B); and a silver-stained SDS-PAGE gel showing the protein content of the virus preparation following different steps of the process of the invention (defined in the table below the figure) (C).
• the SGC purified pool consisting of SGC fractions F7-F11 is shown in lane 12.
• Figure 12 SEC area (mAU*min; right axis) and TCID 50 results (log TCID50/mL; left axis) of attenuated A5nsP3 ChikV production harvests before and after PS treatment.
• the grey portions of the bars indicate large losses in SEC area following PS treatment, but no corresponding change in the total number of infectious particles (indicated by black portions of the bars) (A); SEC profile of virus preparation before and after PS addition, showing a complete removal of large size virus aggregates by PS treatment as well as a reduction in host cell proteins (HCP) and LMW impurities (B).
• HCP host cell proteins
• B LMW impurities
• Figure 13 Electron micrographs of attenuated A5nsP3 ChikV harvest before and after PS treatment.
• Figure 14 Preparation of the sucrose gradient.
• Figure 15 Comparison of four different sucrose gradient centrifugation experiments performed to empirically determine the optimal combination of sucrose layers for CHIKV purification.
• the CHIKV content in the gradient fractions was determined by SEC.
• the sucrose content in the gradient fractions was determined by refractometry (comparing the value of the refractive index of the sucrose solution to that of sucrose standard curve the concentration of sucrose solution can be determined with good accuracy, this is also referred to as "Brix" scale that is calibrated to give the percentage (w/w) of sucrose dissolved in water, i.e. "°Bx").
• Protamine sulphate (PS) was determined by SEC.
• PS is separated within the sucrose gradient alongside host cell derived residual contaminants and was therefore used to assess the quality of CHIKV separation from residual contaminants in the tested gradients.
• CHIKV load material containing 10 % sucrose was loaded on top of a two layer system consisting of a 50 % (w/w) sucrose bottom layer and a second 35 % (w/w) sucrose layer. Determination of sucrose content in the fractions showed the formation of a linear gradient. SEC showed concentration of CHIKV within a sucrose concentration range from 40 -30 % (w/w) sucrose. PS SEC showed acceptable separation of PS from CHIKV, however a slight overlap is still present.
• C CHIKV load material containing 10 % sucrose was loaded on top of a two layer system consisting of a 50 % (w/w) sucrose bottom layer and a second 25 % (w/w) sucrose layer.
• sucrose content in the fractions showed the formation of a linear gradient.
• SEC showed concentration of CHIKV within a sucrose concentration range from 40 -30 % (w/w) sucrose.
• PS SEC showed a good separation of PS from CHIKV.
• D CHIKV load material containing 10 % sucrose was loaded on top of a three layer system consisting of a 50 % (w/w) sucrose bottom layer as well as a 35 % and a 15 % (w/w) sucrose layer.
• Determination of sucrose content in the fractions showed the formation of a linear gradient and SEC showed concentration of CHIKV within a sucrose concentration range from 40 -30 %(w/w) sucrose.
• PS SEC showed a very good separation of PS and residual contaminants from CHIKV.
• the combination of 3 layers shown in Figure 16D
• Figure 16 Relative amounts of attenuated A5nsP3 ChikV particles and other components by SEC-HPLC analysis at the different steps of the process of the invention including, from top to bottom: crude harvest (a); 10X concentrated harvest; diafiltrated concentrated harvest; PS treated material; CC700-treated material and SGC purified pool.
• FIG 17 An exemplary downstream virus purification process from the crude harvest to formulation of the drug substance (vaccine), a preferred embodiment of the process of the invention (A).
• a flow-chart of an exemplary virus inactivation process is shown in (B).
• FIG 18 PS treatment results in selective removal of Zika virus aggregates and Vero HCP and LMW impurities (SEC-HPLC of 30x concentrated Zika Virus harvest day 5).
• Figure 19 SEC-HPLC of individual 30x concentrated Zika harvest prior PS treatment at different time points.
• Figure 20 SEC-HPLC of individual 30x concentrated Zika harvest post PS treatment at different time points.
• Figure 21 Representative SDS-PAGE from the sucrose gradient harvest of a Zika purification is shown.
• Figure 22 Correlation between JEV Antigen content in NIV analysed by ELISA and SEC-HPLC (Dionex Ultimate 3000, Superose 6 column).
• Figure 23 Comparison of JEV and ZikaV harvest schedules/yields.
• FIG. 24 SEC-HPLC elution profile of ZikaV NIV. Data were processed on Dionex Ultimate 3000 / Superose 6 Increase column. Both panels are from the same chromatogram. The upper graph is the complete elution profile; the lower graph is an enlargement of the ZIKAV elution peak.
• FIG. 25 SEC-MALLS analysis of inactivated ZikaV.
• Figure 26 Cumulative particle size distribution of Zika NIV.
• Figure 27 Graphical representation of the neutralization of the Zika virus H/PF/2013 with pooled mouse sera. The number of plaques without serum was set to 100%. The EC50 was calculated with the 3- parameter method.
• Figure 28 Graphical representation of the neutralization of the Zika virus MR766 with pooled mouse sera. The number of plaques without serum was set to 100%. The EC50 was calculated with the 3- parameter method.
• Figure 29 Change in SEC profile of Yellow fever virus peak after PS addition according to the invention showing a complete removal of large size aggregates and LMW impurities.
• infectious virus particles i.e., mature, functional virus particles, e.g. flavivirus particles (Yellow Fever, Zika Virus, Japanese Encephalitis virus, Dengue virus) and/or alphavirus particles (Chikungunya virus).
• flavivirus particles Yellow Fever, Zika Virus, Japanese Encephalitis virus, Dengue virus
• alphavirus particles Chikungunya virus
• the processes disclosed are characterized by the removal of undesired by-products of virus production on host cells, such as non-infectious virus particles and aggregated and immature virus by-products.
• the processes provided herein allow the production of highly -purified virus preparations comprising mostly infectious virus particles.
• Protamines are small arginine-rich nuclear proteins, present in high amounts in the sperm of fish, which have an important role in DNA packaging during spermatogenesis.
• Protamine sulfate (or "protamine” or “PS") can form a stable ion pair with heparin and is thus commonly used during certain surgeries when the anti -coagulation effect of heparin is no longer needed.
• protamine sulfate administered alone can also have a weak anticoagulant effect (“Protamine sulfate”.
• Protamine Sulphate is additionally routinely used in biotechnology applications such as DNA precipitation (e.g., removal of host cell DNA from cell culture processes), purification of DNA binding proteins and retroviral-mediated gene transfer.
• Protamine is obtained from salmon sperm or produced recombinantly and is used as a sulphate salt.
• the four major peptides which constitute almost the entire nitrogen-containing material in salmon protamine, have been fully characterized and found to be polypeptides of 30-32 amino acids in length, of which 21— 22 residues are arginine.
• the average molecular mass is in the range of 4250 Da for the following sequence: PRRRRSSSRP VRRRRRPRVS RRRRGGRR RR (SEQ ID NO: 1).
• protamine is also referred to as protamine salt, or preferably protamine sulphate.
• the present invention relates to the use of protamine sulphate (PS) in a process of purification of a live virus, wherein the protamine sulphate facilitates the removal of impurities from a crude virus harvest, including non-infectious virus particles and aggregates.
• PS protamine sulphate
• virus production in the host cell can result in the release of virus products which are not mature, and non-infectious particles, which can also be considered impurities according to the present invention.
• the present invention also relates to the enrichment of infectious virus particles from a crude harvest containing a mixture of virus particles and other viral products in various stages of maturation.
• protamine sulphate can follow crude cell lysis or any further step after cell lysis (e.g. including after a pre -purification with filtration, chromatography etc) wherein the virus particles are further enriched or concentrated and/or other impurities are removed and/or buffer components are exchanged.
• the further steps may comprise filtration or concentration of the crude cell lysate.
• the protamine sulphate may comprise the sequence PRRRRSSSRP VRRRRRPRVS RRRRRRGGRR RR (SEQ ID NO: 1) or a variant thereof wherein the amino acid sequence comprises from 28-35 amino acids, preferably 29-34, more preferably 30-33 amino acids, most preferably 31 or 32 amino acids.
• the protamine sulphate preferably comprises at least 19 arginine residues, more preferably at least 20 arginine residues, more preferably at least 21 arginine residues, even more preferably at least 22 residues, most preferably 20 or 21 arginine residues.
• protamine salt herein shall serve to encompass natural variations on SEQ ID NO: 1, preferably, but not limited to, the protamine sulphate forms.
• the process according to the current invention may also comprise the use of a sucrose gradient, preferably an optimized sucrose gradient.
• the sucrose gradient is preferably optimized for the removal of protamine sulfate, also for the removal of immature viral particles or other viral particles which are noninfectious or host cell proteins or nucleic acids (DNA, RNA, mRNA, etc) or other host cell debris.
• the optimized sucrose gradient comprises at least two, at least three, at least four layers of sucrose solutions with different densities.
• the virus preparation to be purified is provided in a sucrose solution which has a density of about 8%, about 9%, about 10%, about 11%, about 12% sucrose (w/w), preferably about 10%>.
• one sucrose solution in the gradient has a density of about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%> sucrose (w/w), preferably about 50%.
• one sucrose solution in the gradient has a density of about 30%, about 31%, about 32%, about 33%>, about 34%, about 35%, about 36%, about 37%>, about 38%>, about 39%>, about 40% sucrose (w/w), preferably about 35%>.
• one sucrose solution in the gradient has a density of about 10%>, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%> sucrose (w/w), preferably about 15% sucrose.
• the sucrose gradient comprises three layers of sucrose solutions of about 50%>, about 35%o and about 15% (w/w) sucrose and the virus composition to be purified is contained in about 10% (w/w) sucrose. Because the invention provided for means to not only test for host cell DNA but also immature viral particles, the skilled person in the art is able to more precisely optimize the sucrose gradient for most efficient purification and include additional tools such as PRNT assay to monitor purification success.
• the process comprising the use of protamine sulphate of the invention can be applied to purification of any virus for use in pharmaceutical compositions, for example, for a pharmaceutical composition such as a vaccine where it is important that the virus is in its infectious form.
• the virus to be purified may be any live virus, any live attenuated virus or any live chimeric virus, preferably a live wild type virus such as a Zika virus of the Asian lineage.
• the virus particle is also be later inactivated.
• the virus is inactivated with formaldehyde.
• the produced Zika virus is derived from the Asian lineage (which includes the strains found in South America and all strains derived from any Asian lineage). In some other embodiments, the produced Zika virus is a Zika virus as described in the Sequence section of this application (SEQ ID NO: 2 to 69 or 78).
• the live attenuated Chikungunya virus is the protective ChikV-ICRESl- A5nsP3 described by Hallengard et al. (Novel Attenuated Chikungunya Vaccine Candidates Elicit Protective Immunity in C57BL/6 mice (2014) J. Virology, 88(5):2858-2866).
• the ChikV genome carries a positive-sense single-stranded RNA genome of 11 Kb containing two open reading frames encoding nonstructural proteins (nsPl to nsP4) and structural proteins (C, E3, E2, 6K, and El), respectively (see Fig. 9, top construct).
• the A5nsP3 ChikV mutant was shown to be infectious, highly immunogenic and protective against challenge with wild type ChikV (Hallengard, et al, supra and Hallengard, et al, Prime-Boost Immunization Strategies against Chikungunya Virus (2014) J. Virology, 88(22): 13333-13343).
• the live attenuated Chikungunya virus may be a variant of the ChikV-ICRESl-A5nsP3 attenuated mutant virus.
• FIG 10 Chikungunya virus
• Figure 17A Zika virus
• Degrees Brix is the sugar content of an aqueous solution.
• One degree Brix is 1 gram of sucrose in 100 grams of solution and represents the strength of the solution as percentage by mass.
• °Bx corresponds to the sucrose content in % (w/w), eg. 45 °Bx equals 45 % (w/w) sucrose.
• Oligoname gene- Pair restriction sites (lower case) primer
• size [bp] specific
• Oligoname gene- Pair restriction sites (lower case) primer) size [bp] specific
• Oligoname gene- Pair restriction sites (lower case) primer
• size [bp] specific
• nucleic acid sequences of the genomes of Zika viruses that may be used in the methods, compositions, and/or vaccines described herein.

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