Methods for inactivating Zika virus and related methods

Abstract

The present disclosure relates to methods for inactivating Zika virus, which can be used in vaccines and immunogenic compositions. The present disclosure also relates to a method for determining the completeness of inactivation of an arbovirus preparation and a method for determining the residual formaldehyde content in a pharmaceutical composition containing inactivated viruses.

Description

[0001] Statement Regarding Federally Sponsored Research

[0002] This invention was made with government support under Contract No. HHSO100201600015C with the Department of Health and Human Services, Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority. This invention was made in performance of a cooperative research and development agreement with the Centers for Disease Control and Prevention, an agency of the Department of Health and Human Services. The U.S. Government has certain rights in this invention. Technical Field

[0003] The present disclosure relates to a method for inactivating Zika virus that can be used in vaccines and immunogenic compositions. The present disclosure also relates to a method for determining the completeness of inactivation of an arbovirus preparation, and a method for determining the residual formaldehyde content in a pharmaceutical composition containing inactivated viruses. Background Art

[0004] Zika virus, a flavivirus classified with other mosquito-borne viruses such as yellow fever, dengue, West Nile and Japanese encephalitis within the Flaviviridae family, has spread rapidly since its introduction into Brazil in 2013, becoming a hemispheric epidemic. The virus has reached Central and North America, including the territories of the United States, and is now threatening the continental United States. In fact, the Zika virus strain PRVABC59 was isolated from the serum of a person who had traveled to Puerto Rico in 2015. The genome of this strain has been sequenced at least three times (see Lanciotti et al. Emerg. Infect. Dis. 2016 May;22(5):933-5 and GenBank Accession No. KU501215.1; GenBank Accession No. KX087101.3; and Yun et al. Genome Announc. 2016 Aug 18;4(4) and GenBank Accession No. ANK57897.1).

[0005] The virus was originally isolated in Uganda in 1947, first associated with human disease in 1952, and has been sporadically recognized as a cause of mild, self-limited febrile illness in Africa and Southeast Asia (Weaver et al. (2016) Antiviral Res. 130:69-80; Faria et al. (2016) Science. 352(6283):345-349). However, an outbreak occurred on the North Pacific island of Yap in 2007 and then spread across the Pacific between islands, causing a large outbreak in French Polynesia in 2013-2014, then spread to New Caledonia, the Cook Islands, and ultimately Easter Island. The Asian lineage virus was subsequently transferred to the Western Hemisphere through an uncertain route (Faria et al. (2016) Science. 352 (6283): 345-349). The virus can be transmitted by Aedes aegypti, A. albopictus, and possibly by A. hensilli and A. polynieseinsis in zoology (Weaver et al. (2016) Antiviral Res. 130: 69-80). In addition, it is believed that there may be other viral transmission vectors, and the virus can be transmitted by blood transfusion, placenta and / or sexual transmission.

[0006] In late 2015, a significant increase in fetal abnormalities (e.g., microcephaly) and Guillain-Barre syndrome (GBS) in areas with widespread Zika virus infection warned that Zika virus may be more deadly than initially thought, prompting the World Health Organization (WHO) to declare it a Public Health Emergency of International Concern (PHEIC) (Heymann et al. (2016) Lancet 387(10020):719-21). Although Zika virus poses a considerable threat to public health, there is currently no FDA-approved vaccine or treatment, and the only preventive measure for controlling Zika virus involves managing mosquito populations.

[0007] In recent efforts to characterize recombinant Zika virus to develop a potential vaccine, a non-human cell-adapted Zika virus was identified that has a mutation at position 330 in the viral envelope protein (Weger-Lucarelli et al. 2017. Journal of Virology). The authors of this study found that the full-length infectious cDNA clone of the Zika virus strain PRVABC59 was genetically unstable when amplified during cloning, and chose to split the viral genome to elucidate the observed instability, and developed and applied a dual plasmid system. However, a dual plasmid system for the development of a Zika vaccine is less desirable. Summary of the invention

[0008] Therefore, it is necessary to develop vaccines and immunogenic compositions that utilize genetically stable Zika virus to treat and / or prevent Zika virus infection. One option for vaccine development is to inactivate the whole virus and use the inactivated whole virus to vaccinate the subject. However, in the process of developing inactivated virus vaccines, the key safety guarantee is to make sure that there is no infectious virus in the drug or bulk drug. Developing an effective inactivation process and a sensitive analytical method to measure and determine whether infectious virions are still present is a key safety aspect for developing purified inactivated viruses derived from any wild-type virus but undoubtedly with pathogenic / encephalitis viruses that may cause fetal abnormalities. In addition, formaldehyde, which is known to be used for inactivating viruses, is genotoxic and carcinogenic, so it is important to monitor the residual formaldehyde level in bulk drugs and drugs, and regulatory authorities require manufacturers using formaldehyde as an inactivator to determine the residual formaldehyde content in drugs. Therefore, a sensitive method is needed for detecting residual formaldehyde in drugs or pharmaceutical compositions containing inactivated viruses such as inactivated Zika viruses.

[0009] The present disclosure is based, at least in part, on the surprising discovery that low concentrations of formaldehyde applied to the virus relatively briefly at room temperature can effectively inactivate Zika virus. Additionally, an assay was developed that allows for a high sensitivity determination of whether infectious virions are still present after inactivation. Finally, a method was developed that allows for the detection of low levels of residual formaldehyde in a final drug product or pharmaceutical composition.

[0010] Accordingly, certain aspects of the present disclosure relate to a method for inactivating a Zika virus preparation, the method comprising:

[0011] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation, wherein isolating the Zika virus preparation comprises one or more steps selected from the following:

[0012] (i) Depth filtration,

[0013] (ii) buffer exchange and / or dilution;

[0014] (iii) ion exchange chromatography; and

[0015] (b) treating the Zika virus preparation with formaldehyde, wherein the value obtained by multiplying the concentration of formaldehyde as measured in % (w / v) by the incubation time in formaldehyde as measured in days is between 0.025 and 0.5.

[0016] In some embodiments, the cell is a non-human cell or a Vero cell.

[0017] In some embodiments, the Zika virus preparation is obtained from an inoculum containing a heterogeneous population of Zika virus.

[0018] In some embodiments, the Zika virus preparation is obtained from a clinical isolate.

[0019] In some embodiments, the Zika virus preparation is obtained from a clonal isolate of Zika virus. The clonal isolate of Zika virus can be obtained by plaque purification. Prior to plaque purification, multiple cells can be inoculated with an inoculum containing a heterogeneous population of Zika virus.

[0020] Some aspects of the present disclosure relate to a method for inactivating a Zika virus preparation, the method comprising:

[0021] (a) obtaining Zika virus preparations from clinical isolates; and

[0022] (b) treating the Zika virus preparation with formaldehyde, wherein the value obtained by multiplying the concentration of formaldehyde as measured in % (w / v) by the incubation time in formaldehyde as measured in days is between 0.025 and 0.5.

[0023] Some aspects of the present disclosure relate to a method for inactivating a Zika virus preparation, the method comprising:

[0024] (a) obtaining a Zika virus preparation from an inoculum comprising a heterogeneous population of Zika viruses; and

[0025] (b) treating the Zika virus preparation with formaldehyde, wherein the value obtained by multiplying the concentration of formaldehyde as measured in % (w / v) by the incubation time in formaldehyde as measured in days is between 0.025 and 0.5.

[0026] In some embodiments, the Zika virus preparation is treated with formaldehyde at a concentration of 0.005% (w / v) to 0.02% (w / v).

[0027] In some embodiments, the Zika virus preparation is treated for eight to twelve days or ten days.

[0028] In some embodiments, the Zika virus preparation is treated at a temperature of 15°C to 30°C or 22°C.

[0029] The method may further comprise the step (c): determining the completeness of inactivation.

[0030] In some embodiments, step (c) comprises:

[0031] (i) inoculating cultured insect cells with the Zika virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing an insect cell supernatant;

[0032] (ii) inoculating cultured mammalian cells with the insect cell supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0033] (iii) determining whether the Zika virus preparation contains residual replicating virus that produces a cytopathic effect on the mammalian cells.

[0034] In some embodiments, the insect cell is selected from CCL-125 cells, Aag-2 cells, RML-12 cells, C6 / 36 cells, C7-10 cells, AP-61 cells, AtGRIP-1 cells, AtGRIP-2 cells, AtGRIP-3 cells, UM-AVE1 cells, Mos.55 cells, Sua1B cells, 4a-3B cells, Mos.42 cells, MSQ43 cells, LSB-AA695BB cells, NIID-CTR cells and TRA-171 cells, such as C6 / 36 cells.

[0035] In some embodiments, the first period of time is 3 to 7 days.

[0036] In some embodiments, the mammalian cell is selected from VERO cells, LLC-MK2 cells, MDBK cells, MDCK cells, ATCC CCL34 MDCK (NBL2) cells, MDCK 33016 (Deposit No. DSM ACC2219 as described in WO97 / 37001) cells, BHK21-F cells, HKCC cells and Chinese hamster ovary cells (CHO cells), such as VERO cells.

[0037] In some embodiments, the second period of time is 3 to 14 days.

[0038] The method may further comprise step (d): neutralizing the formaldehyde-treated Zika virus preparation with sodium metabisulfite, for example, neutralizing the formaldehyde-treated Zika virus preparation at least five days, at least seven days, at least nine days, at least 11 days, or at least 14 days after formaldehyde treatment.

[0039] The method may further comprise step (e): preparing a pharmaceutical composition comprising the inactivated Zika virus preparation.

[0040] In some embodiments, the Zika virus preparation is mixed with an adjuvant. The adjuvant can be selected from the group consisting of aluminum salts, toll-like receptor (TLR) agonists, monophosphatidyl lipid A (MLA), synthetic lipid A, lipid A mimetics or analogs, MLA derivatives, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligonucleotides, lipopolysaccharides (LPS) of Gram-negative bacteria, polyphosphazenes, emulsions, viral particles, cochleates, poly (lactide-co-glycolide) (PLG) microparticles, poloxamer particles, microparticles, liposomes, complete Freund's Adjuvant (CFA) and incomplete Freund's adjuvant (IFA).

[0041] In some embodiments, the adjuvant is an aluminum salt, such as aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate, or Alhydrogel85.

[0042] In some embodiments, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of one or more antigens in the Zika virus preparation are adsorbed to the adjuvant.

[0043] In some embodiments, the Zika virus comprises a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1, such as a Trp98Gly mutation in SEQ ID NO: 1.

[0044] In some embodiments, the Zika virus does not comprise a mutation in the envelope protein (E).In some embodiments, the sequence encoding the envelope protein is identical to the corresponding sequence in SEQ ID NO:2.

[0045] Some aspects of the present disclosure also relate to a pharmaceutical composition comprising an inactivated Zika virus obtainable by any of the methods described herein.

[0046] Some aspects of the present disclosure also relate to a pharmaceutical composition comprising inactivated Zika virus and having a residual formaldehyde content of less than 50 μg / ml. In some embodiments, the pharmaceutical composition is obtainable by the method of any one of claims 1 to 28.

[0047] Some aspects of the present disclosure also relate to a method for determining the completeness of inactivation of an arbovirus preparation, the method comprising the steps of:

[0048] (i) inoculating cultured insect cells with an arbovirus preparation that has undergone an inactivation step and incubating the insect cells for a first period of time, thereby producing an insect cell supernatant;

[0049] (ii) inoculating the cultured mammalian cells with the insect cell supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0050] (iii) determining whether the arbovirus preparation contains residual replicating virus that is cytopathic to the mammalian cells.

[0051] In some embodiments, the arbovirus is a flavivirus or an alphavirus. In some embodiments, the arbovirus is Zika virus, West Nile virus, Yellow Fever virus, Japanese Encephalitis virus, Tick-borne encephalitis virus, dengue virus, St. Louis Encephalitis virus, Chikungunya virus, O'nyong'nyong virus, or Mayaro virus.

[0052] In some embodiments, the arbovirus preparation is subjected to detergent, formalin, hydrogen peroxide, beta-propiolactone (BPL), diethylamine (BEI), acetylethyleneimine, methylene blue, or psoralen.

[0053] In some embodiments, the insect cell is selected from CCL-125 cells, Aag-2 cells, RML-12 cells, C6 / 36 cells, C7-10 cells, AP-61 cells, AtGRIP-1 cells, AtGRIP-2 cells, AtGRIP-3 cells, UM-AVE1 cells, Mos.55 cells, Sua1B cells, 4a-3B cells, Mos.42 cells, MSQ43 cells, LSB-AA695BB cells, NIID-CTR cells and TRA-171 cells, such as C6 / 36 cells.

[0054] In some embodiments, the first period of time is 3 to 7 days.

[0055] In some embodiments, the mammalian cell is selected from VERO cells, LLC-MK2 cells, MDBK cells, MDCK cells, ATCC CCL34 MDCK (NBL2) cells, MDCK 33016 (Deposit No. DSM ACC2219 as described in WO97 / 37001) cells, BHK21-F cells, HKCC cells and Chinese hamster ovary cells (CHO cells), such as VERO cells.

[0056] In some embodiments, the second period of time is 3 to 14 days.

[0057] In some embodiments, the method is capable of detecting less than 1.0 TCID 50 of arboviruses.

[0058] Some aspects of the present disclosure also relate to a method for determining the residual formaldehyde content in a pharmaceutical composition comprising inactivated viruses, the method comprising the following steps:

[0059] (a) providing a pharmaceutical composition comprising a virus that has been treated with formaldehyde;

[0060] (b) mixing the composition of (a) with phosphoric acid and 2,4-dinitrophenylhydrazine (DNPH), thereby providing a mixture;

[0061] (c) incubating the mixture of (b) under suitable conditions; and

[0062] (d) Analyzing the mixture for the presence of residual formaldehyde.

[0063] In some embodiments, the composition of (a) contains an adjuvant, which may be aluminum hydroxide. In some embodiments, the composition of (a) contains 0.1 mg / ml to 1.0 mg / ml aluminum hydroxide as an adjuvant.

[0064] In some embodiments, step (b) comprises mixing 50 parts of the composition of (a) with 1 part of 15 to 25% (v / v) phosphoric acid and 2.5 parts of 0.9 to 1.1 mg / ml DNPH.

[0065] In some embodiments, the composition of (a) is incubated with a mixture of phosphoric acid and 2,4-dinitrophenylhydrazine (DNPH) at room temperature. In some embodiments, the composition of (a) is incubated with a mixture of phosphoric acid and 2,4-dinitrophenylhydrazine (DNPH) for 10 to 30 minutes.

[0066] In some embodiments, the mixture of the composition of (a) and phosphoric acid and 2,4-dinitrophenylhydrazine (DNPH) is analyzed by HPLC, which may be reverse phase HPLC. In some embodiments, a mixture of water and acetonitrile (1:1, v / v) is used as the mobile phase in HPLC.

[0067] In some embodiments, the virus is an inactivated Zika virus. In some embodiments, the inactivated Zika virus has been treated with 0.01% (w / v) formaldehyde at 22° C. for 10 days. In some embodiments, the Zika virus comprises a mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, such as the Trp98Gly mutation in SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 Shown are bright-field microscopy images of Vero cell monolayers mock-infected (top) or infected with ZIKAV strain PRVABC59 (bottom).

[0069] Figure 2 TCID 50 Determined growth kinetics of ZIKAVPRVABC59P1 on Vero cell monolayers.

[0070] Figure 3 Demonstrated potency assay for Zika virus PRVABC59 P5 clone af (TCID 50 ).

[0071] Figure 4 Shown are bright field microscopy images depicting the cytopathic effect (CPE) of Zika virus PRVABC59 P6 clone af growing on Vero cell monolayers.

[0072] Figure 5 Demonstrated potency assay for Zika virus PRVABC59 P6 clone af (TCID 50 ).

[0073] Figure 6Shown is an amino acid sequence alignment comparing the Zika virus envelope glycoprotein sequence near residue 330 from Zika virus strains PRVABC59 P6e (SEQ ID NO:8) and PRVABC59 (SEQ ID NO:9) with several other flaviviruses (WNV (SEQ ID NO:10); JEV (SEQ ID NO:11); SLEV (SEQ ID NO:12); YFV (SEQ ID NO:13); DENV 1 16007 (SEQ ID NO:14); DENV 2 16681 (SEQ ID NO:15); DENV 3 16562 (SEQ ID NO:16); and DENV 4 1036 (SEQ ID NO:17)).

[0074] Figure 7 Shown is an amino acid sequence alignment comparing the Zika virus NS1 protein sequence near residue 98 from Zika virus strains PRVABC59 P6e (SEQ ID NO: 18) and PRVABC59 (SEQ ID NO: 19) with several other flaviviruses (WNV (SEQ ID NO: 20); JEV (SEQ ID NO: 21); SLEV (SEQ ID NO: 22); YFV (SEQ ID NO: 23); DENV 1 16007 (SEQ ID NO: 24); DENV 2 16681 (SEQ ID NO: 25); DENV 3 16562 (SEQ ID NO: 26); and DENV 4 1036 (SEQ ID NO: 27)).

[0075] Figure 8 The plaque phenotype of ZIKAV PRVABC59 P6 virus clone af compared to ZIKAV PRVABC59 P1 virus is shown.

[0076] Fig. 9 Shown are the average plaque sizes of ZIKAV PRVABC59 P6 virus clones compared to ZIKAV PRVABC59 P1 virus.

[0077] Fig.10 Shown are the growth kinetics of ZIKAV PRVABC59 P6 clone af in Vero cells under serum-free growth conditions.

[0078] Fig.11 A schematic diagram showing the steps taken to prepare PRVABC59 P6b and P6e formulations for use in immunization experiments.

[0079] Fig. 12AShown is the schedule for administration of vaccine formulations derived from ZIKAV PRVABC59 P6b and P6e clones to CD-1 mice. PBS was used as a placebo.

[0080] Fig. 12B Shows how Fig. 12A Serum ZIKAV neutralizing antibody titers in CD-1 mice immunized with vaccine formulations derived from ZIKAV PRVABC59P6b and P6e clones as described in. ZIKAV neutralizing antibody titers ZIKAV neutralizing antibody titers were determined by reporter virus particle (RVP) neutralization assay. The solid line represents the geometric mean of a group. The detection limit (1.93log 10 ) is represented by a dotted line.

[0081] Fig.13A Shown is the schedule for administration of vaccine formulations derived from ZIKAV PRVABC59 P6b and P6e clones to AG129 mice. PBS was used as a placebo.

[0082] Fig. 13B Shows how Fig.13A Serum ZIKAV neutralizing antibody titers in AG129 mice immunized with vaccine formulations derived from ZIKAV PRVABC59P6b and P6e clones as described in. The solid line represents the geometric mean of a group. Detection limit (1.30log 10 ) are indicated by dotted lines. Animals without detectable titers (<1.30) were assigned a titer of 0.5.

[0083] Fig.14 The mean weight of the AG129 test group after challenge is shown, expressed as a percentage of the starting weight. Error bars represent standard deviation.

[0084] Fig.15 Serum viremia of individual AG129 mice two days after challenge is shown, reported as PFU / mL. The solid line represents the mean of a group. Detection limit (2.0 log 10 ) is represented by a dotted line.

[0085] Fig.16 Shown is the survival analysis of the AG129 test group after challenge.

[0086] Fig.17 Shown are pre-challenge serum circulating ZIKAV neutralizing antibody (Nab) titers following passive transfer of pooled sera from vaccinated and challenged AG129 mice.

[0087] Fig.18 Mean body weights of passively transferred mice and control mice challenged with Zika virus are shown.

[0088] Fig.19 Serum viremia of individual AG129 mice three days after challenge is shown, reported as PFU / mL.

[0089] Fig. 20 Survival analysis of passively transferred mice and control mice challenged with Zika virus is shown.

[0090] Fig.21 demonstrated a correlation between ZIKAV neutralizing antibody titers and viremia observed in passively transferred mice.

[0091] Fig. 22 Shown is the survival analysis of AG129 mice using Kaplan Meier survival curve after infection with P6a and P6e Zika virus pre-MVS stocks.

[0092] Fig.23 Mean body weight after infection with P6a and P6e Zika virus pre-MVS stocks at the time of the invention is shown as a percentage of starting weight. The dashed line represents 100% of starting weight for reference.

[0093] Fig.24 Serum viremia of individual AG129 mice three days after infection with P6a and P6e Zika virus pre-MVS stocks is shown, reported in PFU / mL. The dashed line indicates the detection limit of the assay.

[0094] Fig.25 Compiled inactivation kinetic data are presented. The data compares infectivity (TCID50) versus RNA copies, as well as completeness of inactivation (COI) of four toxicology batches. These data demonstrate that the sensitivity of the COI assay exceeds that of the TCID50.

[0095] Fig.26 A comparison of the sensitivity of C6 / 36 and Vero in the assay as demonstrated by an input virus titer of 0.31 TCID50 is shown.

[0096] Fig. 27 Logistic regression analysis of CPE versus log TCID50 using C6 / 36 cells is shown, with the site including a 99% confidence interval around a target value of 0.01 TCID50 / well (-2 log TCID50 / well); the model predicts that 0.85% of the wells will be positive.

[0097] Fig.28Chromatograms of PBS (a) and PBS solutions containing 0.049 μg / mL (b), 0.098 μg / mL (c), 0.196 μg / mL (d), 0.491 μg / mL (e), 0.982 μg / mL (f), and 1.964 μg / mL (g) formaldehyde are shown. DETAILED DESCRIPTION

[0098] General Technology

[0099] The techniques and procedures described or referred to herein are generally better understood and routinely employed by those skilled in the art using conventional methods, such as the widely used methods described in the following: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel et al., eds., (2003)); Methods in Enzymology series (Academic Press): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual, 1996; Notebook (JE Cellis, 1998) Academic Press; Animal Cell Culture (RI Freshney, 1987); Introduction to Cell and Tissue Culture (JPMather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths and DG Newell, 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, 1993-8) J. Wiley and Sons; Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., ed., 1994); Current Protocols in Immunology (J. E. Coligan et al., ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, ed., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); and The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995).

[0100] Zika virus

[0101] Certain aspects of the present disclosure relate to a purified, inactivated whole Zika virus that can be used in a vaccine and / or immunogenic composition.

[0102] Zika virus (ZIKV) is a mosquito-borne flavivirus that was first isolated in 1947 from sentinel rhesus monkeys in the Zika Forest of Uganda. Since then, the virus has been isolated from humans in Africa and Asia, and in recent years in the Americas. It was found that ZIKV has two (possibly three) lineages: an African lineage (possibly independent East African and West African lineages) and an Asian lineage. Therefore, examples of suitable Zika viruses disclosed herein include, but are not limited to, viruses from African and / or Asian lineages. In some embodiments, the Zika virus is an African lineage virus. In some embodiments, the Zika virus is an Asian lineage virus. In addition, multiple strains within the African and Asian lineages of Zika virus have been previously identified. Any one or more suitable strains of Zika virus known in the art may be used in the present disclosure, including, for example, strains Mr 766, ArD 41519, IbH 30656, P6-740, EC Yap, FSS13025, ArD 7117, ArD 9957, ArD 30101, ArD30156, ArD 30332, HD 78788, ArD 127707, ArD 127710, ArD 127984, ArD 127988, ArD127994, ArD 128000, ArD 132912, 132915, ArD 141170, ArD 142623, ArD 149917, ArD149810, ArD 149938, ArD 157995, ArD 158084, ArD 165522, ArD 165531, ArA 1465, ArA27101, ArA 27290, ArA 27106, ArA 27096, ArA 27407, ArA 27433, ArA 506 / 96, ArA 975-99, Ara 982-99, ArA 986-99, ArA 2718, ArB 1362, Nigeria 68, Malaysia 66, Kedougou 84, Suriname, MR1429, PRVABC59, ECMN2007, DakAr41524, H / PF / 2013, R103451, 103344, 8375, JMB-185, ZIKV / H, Homo sapiens / Brazil / Natal / 2015, SPH2015, ZIKV / Hu / Chiba / S36 / 2016 and / or Cuba 2017. In some embodiments, the virus strain PRVABC59 is used in the present disclosure.

[0103] In some embodiments, an example of a Zika virus genome sequence is shown below as SEQ ID NO: 2:

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] In some embodiments, the Zika virus may comprise a genome sequence of GenBank Accession No. KU501215.1. In some embodiments, the Zika virus is from strain PRVABC59. In some embodiments, the genome sequence of GenBank Accession No. KU501215.1 comprises a sequence of SEQ ID NO:2. In some embodiments, the Zika virus may comprise a genome sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:2.

[0111] In some embodiments, the Zika virus may include at least one polypeptide encoded by the sequence of SEQ ID NO: 2. In some embodiments, the Zika virus may include at least one polypeptide having an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence encoded by the sequence of SEQ ID NO: 2.

[0112] Thus, in some embodiments, the inactivated Zika virus of the present disclosure can be used in any of the vaccines and / or immunogenic compositions disclosed herein. For example, the inactivated Zika virus of the present disclosure can be used to provide one or more antigens that can be used to treat or prevent Zika virus infection in a subject in need thereof and / or to induce an immune response, such as a protective immune response, against Zika virus in a subject in need thereof.

[0113] Zika virus for use in the present disclosure may be obtained from one or more cells in cell culture (eg, via plaque purification). Any suitable cell known in the art for producing Zika virus can be used, such as insect cells (e.g., mosquito cells, such as CCL-125 cells, Aag-2 cells, RML-12 cells, C6 / 36 cells, C7-10 cells, AP-61 cells, AtGRIP-1 cells, AtGRIP-2 cells, AtGRIP-3 cells, UM-AVE1 cells, Mos.55 cells, Sua1B cells, 4a-3B cells, Mos.42 cells, MSQ43 cells, LSB-AA695BB cells, NIID-CTR cells, TRA-171 cells, and cells from, for example, Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae) gambiae), Anopheles stephensi, Anopheles salbimus, Culex quinquefasciatus, Culex theileri, Culex tritaeniorhynchus, Culex bitaeniorhynchus and / or Toxorhynchites amboinensis), as well as mammalian cells (e.g., VERO cells (from monkey kidney), LLC-MK2 cells (from monkey kidney), MDBK cells, MDCK cells, ATCC CCL34MDCK (NBL2) cells, MDCK 33016 (deposited as DSM ACC No. 55729 as described in WO 97 / 37001). 2219) cells, BHK21-F cells, HKCC cells or Chinese hamster ovary cells (CHO cells). In some embodiments, Zika virus (e.g., Zika virus clone isolate) is produced by non-human cells. In some embodiments, Zika virus (e.g., Zika virus clone isolate) is produced by insect cells. In some embodiments, Zika virus (e.g., Zika virus clone isolate) is produced by mosquito cells. In some embodiments, Zika virus (e.g., Zika virus clone isolate) is produced by mammalian cells. In some embodiments, Zika virus (e.g., Zika virus clone isolate) is produced by VERO cells.

[0114] Zika virus has a positive single-stranded RNA genome encoding structural and non-structural polypeptides. The genome also contains non-coding sequences that play a role in viral replication in the 5' and 3' end regions. The structural polypeptides encoded by these viruses include but are not limited to capsid (C), precursor membrane (prM) and envelope (E). The non-structural (NS) polypeptides encoded by these viruses include but are not limited to NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5.

[0115] In certain embodiments, the Zika virus comprises a mutation in Zika virus nonstructural protein 1 (NS1).In some embodiments, the Zika virus contains a Trp98Gly mutation at position 98 of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO:1.

[0116] In some embodiments, the mutation is within the NS1 polypeptide. The amino acid sequence of a wild-type NS1 polypeptide from an exemplary Zika virus strain is shown below:

[0117] DVGCSVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEDGICGISSVSRMENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNELPHGWKAWGKSYFVRAAKTNNSFVVDGDTLKECPLKHRAWNSFLVEDHGFGVFHTSVWLKVREDYSL ECDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMKTCEWPKSHTLWTDGIEESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRKEPESNLVRSMVT(SEQ ID NO:1).

[0118] In some embodiments, the amino acid sequence of the NS1 polypeptide has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the NS1 polypeptide may be from the amino acid sequence encoded by the sequence of GenBank Accession No. KU501215.1 (SEQ ID NO: 2). In some embodiments, the amino acid sequence of the NS1 polypeptide may be amino acid positions 795 to 1145 of the amino acid sequence encoded by the sequence of GenBank Accession No. KU501215.1. In some embodiments, the amino acid sequence of the NS1 polypeptide may be from the Zika virus strain PRVABC59.

[0119] "Sequence identity", "sequence identity %", "identity %", "identity %" or "sequence alignment" means the comparison of a first amino acid sequence to a second amino acid sequence, or the comparison of a first nucleic acid sequence to a second nucleic acid sequence, and is calculated as a percentage based on the comparison. The result of this calculation may be referred to as "identity percentage" or "ID percentage".

[0120] Generally, sequence alignment can be used to calculate sequence identity by one of two different methods. In the first method, mismatches at a single position and spaces at a single position are both counted as non-identical positions in the final sequence identity calculation. In the second method, mismatches at a single position are counted as non-identical positions in the final sequence identity calculation; however, spaces at a single position are not counted as non-identical positions (ignored) in the final sequence identity calculation. In other words, in the second method, spaces are ignored in the final sequence identity calculation. The difference between these two methods, i.e., spaces at a single position are counted as non-identical positions versus spaces ignored, may cause the variability of the sequence identity values ​​between the two sequences.

[0121] In some embodiments, sequence identity is determined by a program that generates an alignment, and by counting mispairing at a single position and a vacancy at a single position as non-identical positions in the final sequence identity calculation to calculate identity. For example, the program Needle (EMBOS) performs the algorithm of Needleman and Wunsch (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), and by first generating an alignment between a first sequence and a second sequence, then counting the number of identical positions on the alignment length, then dividing the number of identical residues by the alignment length, then multiplying this number by 100, generating a sequence identity % to calculate the sequence identity under each default setting [sequence identity % = (number of identical residues / alignment length) × 100)].

[0122] Sequence identity can be calculated by pairwise alignment of the two sequences over their entire length, thus showing the entire length of the first and second sequences ("overall sequence identity"). For example, the program Needle (EMBOSS) generates such alignments; % sequence identity = (number of identical residues / alignment length) x 100)].

[0123] Sequence identity can be calculated by pairwise alignments showing only local regions of the first or second sequence ("local identity"). For example, the program Blast (NCBI) generates such alignments; % sequence identity = (number of identical residues / alignment length) x 100)].

[0124] The sequence alignment is preferably generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, pp. 443-453). Preferably, the program "NEEDLE" (The European Molecular Biology Open Software Suite (EMBOSS)) and the program default parameters (gap opening = 10.0, gap extension = 0.5 and matrix for proteins = EBLOSUM62, and matrix for nucleotides = EDNAFULL) are used together. The sequence identity can then be calculated by showing the two sequences over their full length, thus showing the alignment of the full length of the first sequence and the second sequence ("overall sequence identity"). For example: sequence identity % = (number of identical residues / alignment length) × 100)].

[0125] In some embodiments, the mutation occurs at one or more amino acid positions within the NS1 polypeptide. In some embodiments, the mutation occurs at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1 using a pairwise alignment algorithm. In some embodiments, the mutation at position 98 is a tryptophan to glycine substitution.

[0126] In some embodiments, the Zika virus comprises a mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1. The position corresponding to position 98 of SEQ ID NO: 1 can be determined by aligning the amino acid sequence of the NS1 protein with SEQ ID NO: 1 using a pairwise alignment algorithm. The amino acid residues corresponding to the tryptophan residue at position 98 of SEQ ID NO: 1 in viruses other than the Zika virus are shown in the Figure 7 , wherein these residues are boxed. In some embodiments, the mutation at position 98 is a tryptophan to glycine substitution. In some embodiments, the mutation at position 98 is a tryptophan to glycine substitution at position 98 of SEQ ID NO: 1. In some embodiments, the mutation at position 98 is a tryptophan to glycine substitution at a position corresponding to position 98 of SEQ ID NO: 1 when aligned with SEQ ID NO: 1 using a pairwise alignment algorithm.

[0127] In some embodiments, the Zika virus contains a mutation in the NS1 protein, and at least one mutation in one or more of the C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 viral proteins. In some embodiments, the Zika virus contains a mutation in the NS1 protein, and does not contain at least one mutation in one or more of the C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 viral proteins. In some embodiments, the Zika virus contains a mutation in the NS1 protein and does not contain at least one mutation in the envelope protein E. In some embodiments, the whole inactivated virus contains at least one mutation in the Zika virus nonstructural protein 1 (NS1), and does not include a mutation in the Zika virus envelope protein E (Env). In some embodiments, the Zika virus contains a mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, and does not contain any mutation in the envelope protein E. In some embodiments, the whole inactivated Zika virus contains a mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, and does not include a mutation in the Zika virus envelope protein E (Env). In some embodiments, the whole inactivated virus contains at least one mutation in the Zika virus nonstructural protein 1 (NS1), and the sequence encoding the envelope protein is identical to the corresponding sequence in SEQ ID No. 2. In some embodiments, the Zika virus contains a mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, and the sequence encoding the envelope protein is identical to the corresponding sequence in SEQ ID NO. 2. In some embodiments, the whole inactivated Zika virus contains a mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, and the sequence encoding the envelope protein is identical to the corresponding sequence in SEQ ID NO: 2. In some embodiments, the whole inactivated Zika virus contains a tryptophan to glycine substitution at position 98 of SEQ ID NO:1, or at a position corresponding to position 98 of SEQ ID NO:1, and the sequence encoding the envelope protein is identical to the corresponding sequence in SEQ ID NO:2.

[0128] In some embodiments, the Zika virus contains at least one mutation that enhances genetic stability compared to a Zika virus lacking the at least one mutation. In some embodiments, the Zika virus contains at least one mutation that enhances viral replication compared to a Zika virus lacking the at least one mutation. In some embodiments, the Zika virus contains at least one mutation that reduces or otherwise inhibits, for example, undesirable mutations in the envelope protein E (Env) of the Zika virus.

[0129] In the above embodiments of the present disclosure, an exemplary pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm, which uses default parameters (e.g., gap open penalty=10.0, gap extension penalty=0.5, using the EBLOSUM62 scoring matrix). The algorithm is preferably implemented in the needle tool in the EMBOSS program package.

[0130] In some embodiments, inactivated Zika virus can be used in vaccines and immunogenic compositions. For example, inactivated Zika virus can be used to treat or prevent Zika virus infection in a subject in need thereof and / or to induce an immune response, such as a protective immune response, against Zika virus in a subject in need thereof.

[0131] Production of vaccines and immunogenic compositions

[0132] Other aspects of the present disclosure relate to Zika virus vaccines and immunogenic compositions containing purified inactivated whole virus, such as a Zika virus having a mutation that is a tryptophan to glycine substitution at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1 as described herein. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, wherein the Zika virus is derived from strain PRVABC59. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, wherein the Zika virus is derived from strain PRVABC59 comprising a genomic sequence according to SEQ ID NO: 2. In one embodiment, vaccines and immunogenic compositions contain plaque-purified cloned Zika virus isolates. Such vaccines and immunogenic compositions can be used, for example, to treat or prevent Zika virus infection in a subject in need thereof and / or to induce an immune response, such as a protective immune response, against Zika virus in a subject in need thereof.

[0133] The production of vaccines and / or immunogenic compositions of the present disclosure includes the growth of Zika virus. Growth in cell culture is a method for preparing vaccines and / or immunogenic compositions of the present disclosure. Cells used for virus growth can be cultured in suspension or under adherent conditions.

[0134] Cell lines suitable for growing at least one virus of the present disclosure include, but are not limited to, insect cells (e.g., mosquito cells as described herein, VERO cells (from monkey kidney), horse, cow (e.g., MDBK cells), sheep, dog (e.g., MDCK cells from dog kidney, ATCC CCL34 MDCK (NBL2) or MDCK 33016, deposit number DSM ACC 2219 as described in WO 97 / 37001), cat, and rodent (e.g., hamster cells, e.g., BHK21-F, HKCC cells, or Chinese hamster ovary cells (CHO cells)), and can be obtained from a variety of developmental stages including, for example, adult, newborn, fetal, and embryonic. In certain embodiments, the cells are immortalized (e.g., PERC.6 cells, as described in WO 01 / 38362 and WO 02 / 40665, and as deposited under ECACC accession number 96022940). In a preferred embodiment, mammalian cells are utilized and may be selected from and / or derived from one or more of the following non-limiting cell types: fibroblasts (e.g., skin, lung), endothelial cells (e.g., aorta, coronary artery, lung, blood vessel, skin microvessel, umbilicus), hepatocytes, keratinocytes, immune cells (e.g., T cells, B cells, macrophages, NK, dendritic), breast cells (e.g., epithelial), smooth muscle cells (e.g., blood vessel, aorta, coronary artery, artery, uterus, bronchus, cervix, retinal pericytes), melanocytes, neural cells (e.g., astrocytes), prostate cells (e.g., epithelial, smooth muscle), kidney cells (e.g., epithelial, mesangial, proximal tubules), skeletal cells (e.g., chondrocytes, osteoclasts, osteoblasts), muscle cells (e.g., myoblasts, skeletal muscle cells, smooth muscle cells, bronchial), hepatocytes, retinal blasts, and stromal cells. WO 97 / 37000 and WO 97 / 37001 describe the production of animal cells and cell lines capable of growth in suspension and in serum-free medium and which can be used to produce and replicate viruses. In one embodiment, the cells used to grow at least one virus are Vero cells.

[0135] Culture conditions for the above cell types are known and described in multiple publications. Alternatively, media, supplements, and conditions are commercially available, for example, as described in the catalog of Cambrex Bioproducts (East Rutherford, NJ) and in other literature.

[0136] In certain embodiments, the cells used in the methods described herein are cultured in serum-free and / or protein-free medium. If the medium does not contain any additives from serum of human or animal origin, it is referred to as serum-free medium in the context of the present disclosure. It should be understood that protein-free means a culture in which cell proliferation occurs without proteins, growth factors, other protein additives, and non-serum proteins, but may optionally include proteins such as trypsin or other proteases that may be required for viral growth. Cells grown in such cultures naturally contain proteins themselves.

[0137] Known serum-free culture media include Iscove's medium, Ultra-CHO medium (BioWhittaker) or EX-CELL (JRH Bioscience). Commonly used serum-containing culture media include Eagle's Basal Medium (BME) or Minimum Essential Medium (MEM) (Eagle, Science, 130, 432 (1959)) or Dulbecco's Modified Eagle Medium (DMEM or EDM), which are usually used with up to 10% fetal bovine serum or similar additives. Optionally, Minimum Essential Medium (MEM) (Eagle, Science, 130, 432 (1959)) or Dulbecco's Modified Eagle Medium (DMEM or EDM) can be used without any serum-containing supplements. Protein-free media such as PF-CHO (JHR Bioscience), chemically defined media such as ProCHO 4CDM (BioWhittaker) or SMIF 7 (Gibco / BRL Life Technologies), and mitogenic peptides such as Primactone, Pepticase or HyPep.TM. (all from Quest International) or lactalbumin hydrolysates (Gibco and other manufacturers) are also well known in the art. Media additives based on plant hydrolysates have the particular advantage of excluding contamination by viruses, mycoplasmas or unknown infectious agents.

[0138] Cell culture conditions (temperature, cell density, pH, etc.) vary over a wide range due to the suitability of the cell lines employed in accordance with the present disclosure, and may be tailored to the requirements of a specific viral strain.

[0139] Methods for propagating viruses in cultured cells generally include the following steps: inoculating the cultured cells with the virus strain to be cultured; culturing the infected cells for a period of time required for virus propagation, for example as determined by virus titer or antigen expression (e.g., between 24 hours and 168 hours after inoculation); and collecting the propagated virus. In some embodiments, the virus is collected via plaque purification. The cultured cells are inoculated with the virus (measured by PFU or TCID50) to a cell ratio of 1:500 to 1:1, preferably 1:100 to 1:5. The virus is added to a cell suspension or applied to a cell monolayer, and the virus is adsorbed on the cells at 25°C to 40°C, preferably 28°C to 38°C for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, but usually less than 300 minutes. The infected cell culture (e.g., monolayer) can be removed by harvesting the supernatant (without cells), freezing and thawing, or by enzymatic action to increase the virus content of the harvested culture supernatant. The fluid of harvest is then inactivated or frozen for storage. The cultured cells can be infected at a multiplicity of infection ("MOI") of about 0.0001 to 10, preferably 0.002 to 5, more preferably 0.001 to 2. More preferably, cells are infected at an MOI of about 0.01. During infection, the ratio of culture medium to cell culture vessel area can be lower than during cell culture. Keeping this ratio low can allow the virus to infect cells as much as possible. The supernatant of infected cells can be harvested 30 to 60 hours after infection or 3 to 10 days after infection. In certain preferred embodiments, the supernatant of infected cells is harvested 3 to 7 days after infection. More preferably, the supernatant of infected cells is harvested 3 to 5 days after infection. In some embodiments, protease (such as trypsin) can be added during cell culture to allow viral release, and protease can be added at any suitable stage during culture. Alternatively, in certain embodiments, the supernatant of infected cell culture can be harvested and can be separated or otherwise purified from the supernatant virus.

[0140] The viral inoculum and viral culture preferably do not contain (i.e. have been tested and given a negative result for contamination with) herpes simplex virus, respiratory syncytial virus, parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus, reovirus, polyomavirus, bisRNA virus, circovirus and / or parvovirus (WO 2006 / 027698).

[0141] In the case where the virus has been grown on a cell line, it is standard practice to minimize the amount of residual cell line DNA in the final vaccine to minimize any carcinogenic activity of the host cell DNA. Standard purification procedures such as chromatography can be used to remove contaminating DNA during vaccine preparation. Removal of residual host cell DNA can be enhanced by nuclease treatment, such as by using deoxyribonucleases. A convenient method disclosed in the reference (Lundblad (2001) Biotechnology and Applied Biochemistry 34: 195-197, Guidance for Industry: Bioanalytical Method Validation. USDepartment of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Veterinary Medicine (CVM). May 2001) involves a two-step treatment, first using a deoxyribonuclease (e.g., Benzonase), which can be used during viral growth, and then using a cationic detergent (e.g., CTAB), which can be used during virion disruption. It can also be removed by treatment with β-propiolactone. In one embodiment, contaminating DNA is removed by universal nuclease treatment of the culture supernatant.

[0142] Antigen production

[0143] Zika virus can be produced and / or purified or otherwise isolated by any suitable method known in the art. In one embodiment, the antigen of the present disclosure is a purified inactivated whole Zika virus.

[0144] In some embodiments, inactivated viruses can be produced as described above in the section entitled "Production of Vaccines and Immunogenic Compositions."

[0145] In certain embodiments, the Zika virus of the present disclosure can be produced by culturing non-human cells. Cell lines suitable for producing the Zika virus of the present disclosure may include insect cells (e.g., any of the mosquito cells described herein). Cell lines suitable for producing the Zika virus of the present disclosure may also be cells of mammalian origin, and include, but are not limited to: VERO cells (from monkey kidney), horses, cows (e.g., MDBK cells), sheep, dogs (e.g., MDCK cells from canine kidneys, ATCCCCL34 MDCK (NBL2) or MDCK 33016, such as the deposit number DSM ACC 2219 described in WO 97 / 37001), cats, and rodents (e.g., hamster cells, such as BHK21-F, HKCC cells, or Chinese hamster ovary cells (CHO cells)), and may be obtained from multiple developmental stages including, for example, adult, newborn, fetus, and embryo. In certain embodiments, the cells are immortalized (eg, PERC.6 cells, as described in WO 01 / 38362 and WO 02 / 40665, and as deposited under ECACC Accession No. 96022940). In a preferred embodiment, mammalian cells are utilized and may be selected from and / or derived from one or more of the following non-limiting cell types: fibroblasts (e.g., skin, lung), endothelial cells (e.g., aorta, coronary artery, lung, vascular, skin microvascular, umbilicus), hepatocytes, keratinocytes, immune cells (e.g., T cells, B cells, macrophages, NK, dendritic), breast cells (e.g., epithelial), smooth muscle cells (e.g., vascular, aorta, coronary artery, artery, uterine, bronchial, cervical, retinal pericytes), melanocytes, neural cells (e.g., astrocytes), prostate cells (e.g., epithelial, smooth muscle), kidney cells (e.g., epithelial, mesangial, proximal tubules), skeletal cells (e.g., chondrocytes, osteoclasts, osteoblasts), muscle cells (e.g., myoblasts, skeletal muscle cells, smooth muscle cells, bronchial), hepatocytes, retinal blasts, and stromal cells. WO 97 / 37000 and WO 97 / 37001 describe the production of animal cells and cell lines capable of growth in suspension and in serum-free medium and can be used to produce viral antigens. In certain embodiments, non-human cells are cultured in serum-free medium. In certain embodiments, the Zika virus of the present disclosure can be produced by culturing Vero cells.

[0146] Virus inactivation

[0147] Certain embodiments of the present disclosure relate to Zika virus vaccines and / or immunogenic compositions containing purified inactivated Zika virus. The term "inactivated Zika virus" as used herein is intended to include Zika virus that has been treated with an inactivation method, such as with an effective amount of formalin. Specifically, an inactivated Zika virus can be obtained from / from the following method, wherein the Zika virus is treated with formaldehyde in an amount of about 0.01% w / v at a temperature of 20° C. to 24° C. for 10 days. The inactivated Zika virus is no longer able to infect host cells that may have been infected by the Zika virus that has not yet been inactivated. In one embodiment, the inactivated Zika virus is no longer able to infect VERO cells and exerts a cytopathic effect on VERO cells.

[0148] The term "purified Zika virus" means that the Zika virus has been subjected to a purification method as described below. Purified Zika virus has a lower content of host cell proteins such as Vero cell proteins and host cell DNA such as Vero cell DNA than unpurified Zika virus. The purity of the purified Zika virus can be determined by size exclusion chromatography. The main peak of the purified Zika virus in size exclusion chromatography can exceed 85% of the total area under the curve in size exclusion chromatography, or exceed 90% of the total area under the curve in size exclusion chromatography, or exceed 95% of the total area under the curve in size exclusion chromatography. Such results are considered to be "purified" Zika virus.

[0149] Thus, the term "purified inactivated whole Zika virus" refers to a Zika virus obtainable / obtained from a method wherein the purified Zika virus is treated with formaldehyde in an amount of 0.01% w / v for 10 days at a temperature of 20°C to 24°C and provides a main peak that accounts for at least 85% of the total area under the curve in size exclusion chromatography. In some embodiments, therefore, the term "purified inactivated whole Zika virus" refers to a Zika virus obtainable / obtained from a method wherein the purified Zika virus is treated with formaldehyde in an amount of 0.01% w / v for 10 days at a temperature of 20°C to 24°C and provides a main peak that accounts for at least 90% of the total area under the curve in size exclusion chromatography. In some embodiments, therefore, the term "purified inactivated whole Zika virus" refers to a Zika virus obtainable / obtained by a method wherein the purified Zika virus is treated with formaldehyde in an amount of 0.01% w / v at a temperature of 20°C to 24°C for 10 days and provides a major peak that accounts for at least 95% of the total area under the curve in size exclusion chromatography. In certain embodiments, the purified inactivated whole Zika virus is a clonal isolate obtained / obtainable by plaque purification.

[0150] It is known in the art that the virus is inactivated or killed to destroy its ability to infect mammalian cells but not to destroy the secondary, tertiary or quaternary structure and immunogenic epitopes of the virus. Such methods include chemical and physical methods. Suitable for the method of inactivating the virus include but are not limited to being processed with an effective amount of one or more agents selected from the following: detergent, formalin (also referred to as "formaldehyde" herein), hydrogen peroxide, β-propiolactone (BPL), diethylamine (BEI), acetylethyleneimine, heat, electromagnetic radiation, x-ray radiation, gamma radiation, ultraviolet (ultraviolet radiation), UV-A radiation, UV-B radiation, UV-C radiation, methylene blue, psoralen, carboxyfullerene (C60), hydrogen peroxide and any combination of any of them. As mentioned above, for the purpose of the present application, the terms "formalin" and "formaldehyde" are used interchangeably. When formaldehyde concentration is mentioned herein, it refers to formaldehyde concentration rather than formalin concentration. Thus, "a formaldehyde concentration of 0.01% (w / v)" means 0.01% (w / v) formaldehyde, and this concentration does not need to be further corrected for the formaldehyde concentration in the formalin stock solution (which typically contains 37% formaldehyde by mass). For example, such a formaldehyde concentration in a viral preparation can be obtained by diluting formalin to a working solution having a formaldehyde content of 1.85% (w / v), and then further diluting to the required concentration when mixing with a viral preparation, such as a Zika virus preparation.

[0151] In certain embodiments of the present disclosure, at least one virus is chemically inactivated. The agent for chemical inactivation and the method for chemical inactivation are well-known in the art and are described herein. In some embodiments, at least one virus is chemically inactivated with one or more of BPL, hydrogen peroxide, formalin or BEI. In certain embodiments in which at least one virus is chemically inactivated with BPL, the virus may contain one or more modifications. In some embodiments, the one or more modifications may include modified nucleic acids. In some embodiments, modified nucleic acids are alkylated nucleic acids. In other embodiments, the one or more modifications may include modified polypeptides. In some embodiments, modified polypeptides contain modified amino acid residues, including one or more of modified cysteine, methionine, histidine, aspartic acid, glutamic acid, tyrosine, lysine, serine and threonine.

[0152] In certain embodiments where at least one virus is chemically inactivated with formalin, the inactivated virus may contain one or more modifications. In some embodiments, the one or more modifications may include modified polypeptides. In some embodiments, the one or more modifications may include cross-linked polypeptides. In certain embodiments where at least one virus is chemically inactivated with formalin, the vaccine or immunogenic composition also includes formalin. In certain embodiments where at least one virus is chemically inactivated with BEI, the virus may contain one or more modifications. In some embodiments, the one or more modifications may include modified nucleic acids. In some embodiments, modified nucleic acids are alkylated nucleic acids.

[0153] In certain embodiments where at least one virus is chemically inactivated with formalin, any remaining unreacted formalin can be neutralized with sodium pyrosulfite, dialyzed, and / or buffer exchange can be performed to remove remaining unreacted formalin. In some embodiments, sodium pyrosulfite is added in excess. In some embodiments, the solution can be mixed using a mixer such as a static mixer in series, and then filtered or further purified (e.g., using a cross-flow filtration system).

[0154] Certain embodiments of the present disclosure relate to a method for inactivating a Zika virus preparation. In some embodiments, the method comprises:

[0155] (a) isolating a Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce a Zika virus preparation, wherein isolating the Zika virus preparation comprises one or more steps selected from the following:

[0156] (i) Depth filtration,

[0157] (ii) buffer exchange and / or dilution;

[0158] (iii) ion exchange chromatography; and

[0159] (b) treating the Zika virus preparation with formaldehyde, wherein the value obtained by multiplying the concentration of formaldehyde as measured in % (w / v) by the incubation time in formaldehyde as measured in days is between 0.025 and 0.5.

[0160] For example, when the formaldehyde concentration is 0.01% (w / v) and the incubation time under formaldehyde is 10 days, the numerical result of multiplying the formaldehyde concentration by the incubation time under formaldehyde is 0.01×10=0.1.

[0161] In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.05 to 0.25. In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.075 to 0.15. In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.1.

[0162] In some embodiments, the formaldehyde concentration is between 0.005% (w / v) and 0.02% (w / v). In some embodiments, the formaldehyde concentration is between 0.0075% (w / v) and 0.015% (w / v). In some embodiments, the formaldehyde concentration is 0.01% (w / v).

[0163] In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.025 to 0.5 and the formaldehyde concentration is 0.005% (w / v) to 0.02% (w / v). In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.025 to 0.5 and the formaldehyde concentration is 0.0075% (w / v) to 0.015% (w / v). In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.025 to 0.5 and the formaldehyde concentration is 0.01% (w / v).

[0164] In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.05 to 0.25 and the formaldehyde concentration is 0.005% (w / v) to 0.02% (w / v). In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.05 to 0.25 and the formaldehyde concentration is 0.0075% (w / v) to 0.015% (w / v). In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.05 to 0.25 and the formaldehyde concentration is 0.01% (w / v).

[0165] In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days has a numerical result of 0.075 to 0.15 and the formaldehyde concentration is 0.005% (w / v) to 0.02% (w / v). In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days has a numerical result of 0.075 to 0.15 and the formaldehyde concentration is 0.0075% (w / v) to 0.015% (w / v). In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days has a numerical result of 0.075 to 0.15 and the formaldehyde concentration is 0.01% (w / v).

[0166] In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.1 and the formaldehyde concentration is 0.005% (w / v) to 0.02% (w / v). In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.1 and the formaldehyde concentration is 0.0075% (w / v) to 0.015% (w / v). In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.1 and the formaldehyde concentration is 0.01% (w / v).

[0167] In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days has a numerical result of 0.025 to 0.5 and the incubation time under formaldehyde is eight to twelve days. In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days has a numerical result of 0.025 to 0.5 and the incubation time under formaldehyde is nine to eleven days. In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days has a numerical result of 0.025 to 0.5 and the incubation time under formaldehyde is ten days.

[0168] In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde is between 0.05 and 0.25 and the incubation time under formaldehyde is between eight and twelve days. In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde is between 0.05 and 0.25 and the incubation time under formaldehyde is between nine and eleven days. In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde is between 0.05 and 0.25 and the incubation time under formaldehyde is ten days.

[0169] In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days has a numerical result of 0.075 to 0.15 and the incubation time under formaldehyde is eight to twelve days. In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days has a numerical result of 0.075 to 0.15 and the incubation time under formaldehyde is nine to eleven days. In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days has a numerical result of 0.075 to 0.15 and the incubation time under formaldehyde is ten days.

[0170] In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.1 and the incubation time under formaldehyde is eight to twelve days. In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.1 and the incubation time under formaldehyde is nine to eleven days. In some embodiments, the formaldehyde concentration as measured in % (w / v) multiplied by the incubation time under formaldehyde measured in days is 0.1 and the incubation time under formaldehyde is ten days.

[0171] In some embodiments, the cell is a non-human cell. Suitable non-human mammalian cells include, but are not limited to, VERO cells, LLC-MK2 cells, MDBK cells, MDCK cells, ATCC CCL34 MDCK (NBL2) cells, MDCK33016 (DSM ACC 2219 as described in WO97 / 37001), BHK21-F cells, HKCC cells, and Chinese hamster ovary cells (CHO cells). In some embodiments, the mammalian cell is a Vero cell.

[0172] In certain embodiments of the method, the Zika virus preparation is treated with formalin at a temperature ranging from about 2°C to about 42°C. For example, the Zika virus preparation can be treated with formalin at a temperature ranging from about 2°C to about 42°C, about 2°C to about 8°C, about 15°C to about 37°C, about 17°C to about 27°C, about 20°C to about 25°C, or at a temperature of about 2°C, about 4°C, about 8°C, about 10°C, about 15°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 37°C, or about 42°C. In some embodiments, the Zika virus preparation is treated with formalin at a temperature of 15°C to 30°C. In some embodiments, the Zika virus preparation is treated with formalin at a temperature of 18°C ​​to 25°C. In some embodiments, the Zika virus preparation is treated with formalin at room temperature. In some embodiments, the Zika virus preparation is treated with formalin at a temperature of 22°C.

[0173] In some embodiments, the Zika virus preparation is treated with formalin for at least about 1 day. For example, the Zika virus preparation is treated with formalin for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days or more. In some embodiments, the Zika virus preparation is treated with formalin for at least about 9 days. In some embodiments, the Zika virus preparation is treated with formalin for at least about 11 days. In some embodiments, the Zika virus preparation is treated with formalin for at least about 14 days. In some embodiments, the Zika virus preparation is treated with formalin for at least about 20 days. In some embodiments, the Zika virus preparation is treated with formalin for at least about 30 days. In some embodiments, the Zika virus preparation is treated with formalin for eight to twelve days. In some embodiments, the Zika virus preparation is treated with formalin for nine to eleven days. In some embodiments, the Zika virus preparation is treated with formalin for ten days.

[0174] In the middle of the inactivation treatment period, the mixture of virus preparation and formalin can be filtered to remove aggregates. After filtering, the mixture of virus preparation and formalin is transferred to a new container and further treated with formalin until the inactivation treatment period ends. In some embodiments, if the entire formalin treatment period is eight to twelve days, then after formalin treatment four to six days, the mixture of virus preparation and formalin is filtered. In some embodiments, if the entire formalin treatment period is nine to eleven days, then after formalin treatment five to six days, the mixture of virus preparation and formalin is filtered. In some embodiments, if the entire formalin treatment period is ten days, then after formalin treatment five days, the mixture of virus preparation and formalin is filtered. A suitable filter for this step is a 0.2 μm filter.

[0175] In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w / v) formalin at a temperature of 15°C to 30°C for eight to twelve days. In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w / v) formalin at a temperature of 15°C to 30°C for nine to eleven days. In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w / v) formalin at a temperature of 15°C to 30°C for ten days. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w / v) formalin at a temperature of 15°C to 30°C for eight to twelve days. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w / v) formalin at a temperature of 15°C to 30°C for nine to eleven days. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w / v) formalin at a temperature of 15°C to 30°C for ten days. In some embodiments, the Zika virus preparation is treated with 0.01% (w / v) formalin at a temperature of 15°C to 30°C for eight to twelve days. In some embodiments, the Zika virus preparation is treated with 0.01% (w / v) formalin at a temperature of 15°C to 30°C for nine to eleven days. In some embodiments, the Zika virus preparation is treated with 0.01% (w / v) formalin at a temperature of 15°C to 30°C for ten days.

[0176] In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w / v) formalin at a temperature of 18°C ​​to 25°C for eight to twelve days. In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w / v) formalin at a temperature of 18°C ​​to 25°C for nine to eleven days. In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w / v) formalin at a temperature of 18°C ​​to 25°C for ten days. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w / v) formalin at a temperature of 18°C ​​to 25°C for eight to twelve days. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w / v) formalin at a temperature of 18°C ​​to 25°C for nine to eleven days. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w / v) formalin at a temperature of 18°C ​​to 25°C for ten days. In some embodiments, the Zika virus preparation is treated with 0.01% (w / v) formalin at a temperature of 18°C ​​to 25°C for eight to twelve days. In some embodiments, the Zika virus preparation is treated with 0.01% (w / v) formalin at a temperature of 18°C ​​to 25°C for nine to eleven days. In some embodiments, the Zika virus preparation is treated with 0.01% (w / v) formalin at a temperature of 18°C ​​to 25°C for ten days.

[0177] In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w / v) formalin at a temperature of 22°C for eight to twelve days. In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w / v) formalin at a temperature of 22°C for nine to eleven days. In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w / v) formalin at a temperature of 22°C for ten days. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w / v) formalin at a temperature of 22°C for eight to twelve days. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w / v) formalin at a temperature of 22°C for nine to eleven days. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w / v) formalin at a temperature of 22°C for ten days. In some embodiments, the Zika virus preparation is treated with 0.01% (w / v) formalin at a temperature of 22°C for eight to twelve days. In some embodiments, the Zika virus preparation is treated with 0.01% (w / v) formalin at a temperature of 22°C for nine to eleven days. In some embodiments, the Zika virus preparation is treated with 0.01% (w / v) formalin at a temperature of 22°C for ten days.

[0178] It is believed that the inactivated whole Zika virus preparation is obtainable from / is obtained from a method in which the Zika virus is treated with formaldehyde in an amount in the range of about 0.02% w / v at a temperature of 22° C. for 14 days. In some embodiments, it is believed that the inactivated whole Zika virus preparation is obtainable from / is obtained from a method in which the Zika virus is treated with formaldehyde in an amount of about 0.01% w / v at a temperature of 22° C. for 10 days.

[0179] In some embodiments, the method further comprises neutralizing unreacted formalin with an effective amount of sodium metabisulfite. In some embodiments, the effective amount of sodium metabisulfite is in the range of about 0.01 mM to about 100 mM. For example, sodium metabisulfite can be added at an effective concentration of about 0.01 mM to about 100 mM, about 0.1 mM to about 50 mM, about 0.5 mM to about 20 mM, or about 1 mM to about 10 mM, or at a concentration of about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 75 mM, or about 100 mM. In some embodiments, formalin is neutralized with about 2 mM sodium metabisulfite.

[0180] In some embodiments, the Zika virus preparation is treated with hydrogen peroxide. In some embodiments, the Zika virus preparation is treated with hydrogen peroxide at a concentration ranging from 0.1 to 3% or 0.1 to 1% at any temperature from 20°C to 30°C for 5 to 120 minutes. In some embodiments, the Zika virus preparation is treated with hydrogen peroxide at a final concentration of 0.01% for 60 minutes or less.

[0181] In some embodiments, the method includes (a) isolating a Zika virus preparation from one or more cells cultured in vitro for producing a virus preparation; (b) purifying the virus preparation by one or more purification steps; (c) treating the virus preparation with an effective amount of formalin; (d) neutralizing the virus preparation with an effective amount of sodium metabisulfite; and (e) preparing a pharmaceutical composition comprising inactivated Zika virus. Any method of purifying a virus preparation known in the art can be used to separate Zika virus, including but not limited to using cross-flow filtration (CFF), multimodal chromatography, size exclusion chromatography, cation exchange chromatography, and / or anion exchange chromatography. In some embodiments, the virus preparation is separated by cross-flow filtration (CFF). In some embodiments, the virus preparation is highly purified to an amount of about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more.

[0182] In some embodiments, the Zika virus can be selected from the group of strains consisting of Mr 766, ArD 41519, IbH 30656, P6-740, EC Yap, FSS13025, ArD 7117, ArD 9957, ArD 30101, ArD30156, ArD 30332, HD 78788, ArD 127707, ArD 127710, ArD 127984, ArD 127988, ArD127994, ArD 128000, ArD 132912, 132915, ArD 141170, ArD 142623, ArD 149917, ArD149810, ArD 149938, ArD 157995, ArD 158084, ArD 165522, ArD 165531, ArA 1465, ArA27101, ArA 27290, ArA 27106, ArA 27096, ArA 27407, ArA 27433, ArA 506 / 96, ArA 975-99, Ara 982-99, ArA 986-99, ArA 2718, ArB 1362, Nigeria 68, Malaysia 66, Kédougou 84, Suriname, MR1429, PRVABC59, ECMN2007, DakAr41524, H / PF / 2013, R103451, 103344, 8375, JMB-185, ZIKV / H, Homo sapiens / Brazil / Natal / 2015, SPH2015, ZIKV / Hu / Chiba / S36 / 2016, Thailand SVO127 / 14, Philippines COC C0740, Brazil Fortaleza 2015 and Cuba 2017.

[0183] In certain embodiments, the Zika virus includes a mutation in the Zika virus nonstructural protein 1 (NS1). In some embodiments, the Zika virus contains a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus, the inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, wherein the Zika virus is derived from the strain PRVABC59. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus, the inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, wherein the Zika virus is derived from the strain PRVABC59 comprising a genomic sequence according to SEQ ID NO: 2. In some embodiments, the vaccine or immunogenic composition comprises purified inactivated whole Zika virus that differs from strain PRVABC59 in having a Trp98Gly mutation at position 98 of SEQ ID NO:1.

[0184] Vaccines and / or immunogenic compositions of the present disclosure containing one or more antigens from at least one inactivated Zika virus can be used to treat or prevent Zika virus infection in a subject in need thereof and / or to induce an immune response, e.g., a protective immune response, against Zika virus in a subject in need thereof.

[0185] The method for inactivating a Zika virus preparation may further comprise step (c) as described below: determining completeness of inactivation.

[0186] Determine completeness of inactivation

[0187] Other aspects of the present disclosure relate to methods for determining the completeness of inactivation of an arbovirus preparation by using sequential infection of two different cell types. The method has an unexpectedly low limit of detection (LOD) compared to assays using only one cell type and compared to other methods such as the TCID50 method. In addition, the method avoids the use of animals to determine the infectivity of the inactivated virus.

[0188] The method for determining the completeness of inactivation of an arboviral preparation comprises the following steps:

[0189] (i) inoculating cultured insect cells with an arbovirus preparation that has undergone an inactivation step and incubating the insect cells for a first period of time, thereby producing an insect cell supernatant;

[0190] (ii) inoculating the cultured mammalian cells with the insect cell supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0191] (iii) Determine whether the virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0192] Arboviruses are viruses transmitted to humans by arthropods. They include viruses from the genus Flavivirus, Togavirus and Bunyavirus. The arbovirus preparations examined by the methods disclosed herein contain arboviruses that can infect mammalian cells, particularly Vero cells, and cause cytopathic effects on these cells. In some embodiments, the arbovirus is selected from Zika virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, dengue virus, St. Louis encephalitis virus, tick-borne encephalitis virus, Chikungunya virus, Oneonian virus or Mayaro virus. In some embodiments, the arbovirus is Zika virus.

[0193] In some embodiments, the Zika virus can be selected from the group of strains consisting of Mr 766, ArD 41519, IbH 30656, P6-740, EC Yap, FSS13025, ArD 7117, ArD 9957, ArD 30101, ArD30156, ArD 30332, HD 78788, ArD 127707, ArD 127710, ArD 127984, ArD 127988, ArD127994, ArD 128000, ArD 132912, 132915, ArD 141170, ArD 142623, ArD 149917, ArD149810, ArD 149938, ArD 157995, ArD 158084, ArD 165522, ArD 165531, ArA 1465, ArA27101, ArA 27290, ArA 27106, ArA 27096, ArA 27407, ArA 27433, ArA 506 / 96, ArA 975-99, Ara982-99, ArA 986-99, ArA 2718, ArB 1362, Nigeria 68, Malaysia 66, Kédougou 84, Suriname, MR1429, PRVABC59, ECMN2007, DakAr41524, H / PF / 2013, R103451, 103344, 8375, JMB-185, ZIKV / H, Homo sapiens / Brazil / Natal / 2015, SPH2015, ZIKV / Hu / Chiba / S36 / 2016, Thailand SVO127 / 14, Philippines COC C0740, Brazil Fortaleza 2015 and Cuba 2017.

[0194] In certain embodiments, the Zika virus includes a mutation in the Zika virus nonstructural protein 1 (NS1). In some embodiments, the Zika virus contains a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus, the inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, wherein the Zika virus is derived from the virus strain PRVABC59. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus, the inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, wherein the Zika virus is derived from the virus strain PRVABC59 comprising a genomic sequence according to SEQ ID NO: 2. In some embodiments, the vaccine or immunogenic composition comprises purified inactivated whole Zika virus, which differs from strain PRVABC59 in having a Trp98Gly mutation at position 98 of SEQ ID NO:1.

[0195] The cultured insect cells are inoculated with the arbovirus preparation by adding the arbovirus preparation to an insect cell culture containing the insect cells and a growth medium. The inoculated insect cells are then incubated with the arbovirus preparation under suitable conditions for a first period of time. In some embodiments, the first period of time is three to seven days. In some embodiments, the first period of time is five to seven days. In some embodiments, the first period of time is six days. Thus, in some embodiments, the inoculated insect cells are incubated with the arbovirus preparation for three to seven days. In some embodiments, the inoculated insect cells are incubated with the arbovirus preparation for five to seven days. In some embodiments, the inoculated insect cells are incubated with the arbovirus preparation for six days. During the incubation period, any live virus will be secreted into the insect cell supernatant.

[0196] The insect cells used can be any insect cells that can be infected by the insect-borne virus being studied and whose viability is not changed by virus infection. Insect cells are selected so that the virus has no cytopathic effect on the cells. Suitable insect cells include, but are not limited to, CCL-125 cells, Aag-2 cells, RML-12 cells, C6 / 36 cells, C7-10 cells, AP-61 cells, AtGRIP-1 cells, AtGRIP-2 cells, AtGRIP-3 cells, UM-AVE1 cells, Mos.55 cells, Sua1B cells, 4a-3B cells, Mos.42 cells, MSQ43 cells, LSB-AA695BB cells, NIID-CTR cells, and TRA-171 cells. In some embodiments, the insect cell is a C6 / 36 cell.

[0197] Then, the insect cell supernatant produced by incubating the insect cells with the arbovirus preparation is used to inoculate the cultured mammalian cells. For inoculation, the insect cell supernatant is transferred to the mammalian cells and incubated with the mammalian cells for 60 to 120 minutes or 80 to 100 minutes or 90 minutes. After inoculation, the cell culture medium is added and the mammalian cells are incubated with the insect cell supernatant under suitable conditions for a second period of time. In some embodiments, the second period of time is 3 to 14 days. In some embodiments, the second period of time is five to twelve days. In some embodiments, the second period of time is six to ten days. In some embodiments, the second period of time is seven to nine days. In some embodiments, the second period of time is eight days. Therefore, in some embodiments, the inoculated mammalian cells are incubated with the insect cell supernatant for 3 to 14 days. In some embodiments, the inoculated mammalian cells are incubated with the insect cell supernatant for five to twelve days. In some embodiments, the inoculated mammalian cells are incubated with the insect cell supernatant for seven to nine days. In some embodiments, the inoculated mammalian cells are incubated with the insect cell supernatant for eight days. During the incubation period, any live virus will exert a cytopathic effect on the mammalian cells. During the incubation period, any residual replicating virus will exert a cytopathic effect on mammalian cells such as Vero cells.

[0198] The mammalian cell used can be any mammalian cell that can be infected by the arbovirus being studied and on which the virus exerts a cytopathic effect. Suitable mammalian cells include, but are not limited to, VERO cells, LLC-MK2 cells, MDBK cells, MDCK cells, ATCC CCL34 MDCK (NBL2) cells, MDCK 33016 (Deposit No. DSM ACC 2219 as described in WO97 / 37001) cells, BHK21-F cells, HKCC cells, and Chinese hamster ovary cells (CHO cells). In some embodiments, the mammalian cell is a Vero cell.

[0199] In some embodiments, the method for determining the completeness of inactivation of an arboviral preparation comprises the following steps:

[0200] (i) inoculating C6 / 36 cells with an arbovirus preparation that has undergone an inactivation step and incubating the C6 / 36 cells for a first period of time, thereby producing a C6 / 36 cell supernatant;

[0201] (ii) inoculating the cultured mammalian cells with the C6 / 36 cell supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0202] (iii) Determine whether the virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0203] In some embodiments, the method for determining the completeness of inactivation of an arboviral preparation comprises the following steps:

[0204] (i) inoculating cultured insect cells with an arbovirus preparation that has undergone an inactivation step and incubating the insect cells for a first period of time, thereby producing an insect cell supernatant;

[0205] (ii) inoculating Vero cells with the insect cell supernatant produced in (i) and incubating the Vero cells for a second period of time; and

[0206] (iii) Determine whether the virus preparation contains residual replicating virus that is cytopathic to Vero cells.

[0207] In some embodiments, the method for determining the completeness of inactivation of an arboviral preparation comprises the following steps:

[0208] (i) inoculating C6 / 36 cells with an arbovirus preparation that has undergone an inactivation step and incubating the C6 / 36 cells for a first period of time, thereby producing a C6 / 36 cell supernatant;

[0209] (ii) inoculating Vero cells with the C6 / 36 cell supernatant produced in (i) and incubating the Vero cells for a second period of time; and

[0210] (iii) Determine whether the virus preparation contains residual replicating virus that is cytopathic to Vero cells.

[0211] In some embodiments, the method for determining the completeness of inactivation of a Zika virus preparation comprises the following steps:

[0212] (i) inoculating C6 / 36 cells with an arbovirus preparation that has undergone an inactivation step and incubating the C6 / 36 cells for a first period of time, thereby producing a C6 / 36 cell supernatant;

[0213] (ii) inoculating Vero cells with the C6 / 36 cell supernatant produced in (i) and incubating the Vero cells for a second period of time; and

[0214] (iii) Determine whether the virus preparation contains residual replicating virus that is cytopathic to Vero cells.

[0215] In some embodiments, the method for determining the completeness of inactivation of a Zika virus preparation comprises the following steps:

[0216] (i) inoculating C6 / 36 cells with an arbovirus preparation that has undergone an inactivation step and incubating the C6 / 36 cells for three to seven days, thereby producing a C6 / 36 cell supernatant;

[0217] (ii) inoculating Vero cells with the C6 / 36 cell supernatant produced in (i) and incubating the Vero cells for a second period of time; and

[0218] (iii) Determine whether the virus preparation contains residual replicating virus that is cytopathic to Vero cells.

[0219] In some embodiments, the method for determining the completeness of inactivation of a Zika virus preparation comprises the following steps:

[0220] (i) inoculating C6 / 36 cells with an arbovirus preparation that has undergone an inactivation step and incubating the C6 / 36 cells for a first period of time, thereby producing a C6 / 36 cell supernatant;

[0221] (ii) inoculating Vero cells with the C6 / 36 cell supernatant produced in (i) and incubating the Vero cells for 3 to 14 days; and

[0222] (iii) Determine whether the virus preparation contains residual replicating virus that is cytopathic to Vero cells.

[0223] In some embodiments, the method for determining the completeness of inactivation of a Zika virus preparation comprises the following steps:

[0224] (i) inoculating C6 / 36 cells with an arbovirus preparation that has undergone an inactivation step and incubating the C6 / 36 cells for three to seven days, thereby producing a C6 / 36 cell supernatant;

[0225] (ii) inoculating Vero cells with the C6 / 36 cell supernatant produced in (i) and incubating the Vero cells for 3 to 14 days; and

[0226] (iii) Determine whether the virus preparation contains residual replicating virus that is cytopathic to Vero cells.

[0227] In some embodiments, the method for determining the completeness of inactivation of a Zika virus preparation comprises the following steps:

[0228] (i) inoculating C6 / 36 cells with an arbovirus preparation that has undergone an inactivation step and incubating the C6 / 36 cells for six days, thereby producing a C6 / 36 cell supernatant;

[0229] (ii) inoculating Vero cells with the C6 / 36 cell supernatant produced in (i) and incubating the Vero cells for eight days; and

[0230] (iii) Determine whether the virus preparation contains residual replicating virus that is cytopathic to Vero cells.

[0231] At the end of the second period of time, it is determined whether the viral preparation has a cytopathic effect on the mammalian cells. Cytopathic effect is any change in the cell structure caused by viral invasion, infection and germination from the cell during viral replication. In the method of the present disclosure, if the cells are cultured in a culture medium containing phenol red, the cytopathic effect is determined by the color of the culture medium changing from pink to orange or yellow, or by microscopic examination of the mammalian cells. If microscopic examination of the mammalian cells shows that the cells are rounded, begin to leave the tissue culture container (plate, well or flask) or clear from the tissue culture plate / flask, then it is considered that there is a cytopathic effect. Other signs of cytopathic effect include fusion of adjacent cells to form syncytia and the appearance of nuclear inclusions or cytoplasmic inclusions.

[0232] As discussed above, the method disclosed herein has an extremely low detection limit. Using this method, it is possible to detect less than 1.0 TCID 50 In some embodiments, less than 0.8 TCID 50 In some embodiments, less than 0.5 TCID 50In some embodiments, less than 0.2 TCID 50 In some embodiments, less than 0.1 TCID 50 of virus content.

[0233] The above method for determining completeness of inactivation can be used for any method for inactivating an arbovirus. In one embodiment, the method for inactivating an arbovirus preparation comprises:

[0234] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0235] (b) treating the arbovirus preparation with 0.005% to 0.02% w / v formaldehyde;

[0236] (c) Completeness of inactivation is determined by:

[0237] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0238] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0239] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0240] In one embodiment, a method for inactivating an arboviral agent comprises:

[0241] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0242] (b) treating the arbovirus preparation with 0.0075% to 0.015% w / v formaldehyde;

[0243] (c) Completeness of inactivation is determined by:

[0244] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0245] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0246] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0247] In one embodiment, a method for inactivating an arboviral agent comprises:

[0248] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0249] (b) treating the arbovirus preparation with 0.01% w / v formaldehyde;

[0250] (c) Completeness of inactivation is determined by:

[0251] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0252] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0253] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0254] In one embodiment, a method for inactivating an arboviral agent comprises:

[0255] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0256] (b) treating the arbovirus preparation with 0.005% to 0.02% w / v formaldehyde for a period of eight to twelve days;

[0257] (c) Completeness of inactivation is determined by:

[0258] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0259] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0260] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0261] In one embodiment, a method for inactivating an arboviral agent comprises:

[0262] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0263] (b) treating the arbovirus preparation with 0.0075% to 0.015% w / v formaldehyde for a period of eight to twelve days;

[0264] (c) Completeness of inactivation is determined by:

[0265] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0266] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0267] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0268] In one embodiment, a method for inactivating an arboviral agent comprises:

[0269] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0270] (b) treating the arbovirus preparation with 0.01% w / v formaldehyde for a period of eight to twelve days;

[0271] (c) Completeness of inactivation is determined by:

[0272] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0273] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0274] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0275] In one embodiment, a method for inactivating an arboviral agent comprises:

[0276] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0277] (b) treating the arbovirus preparation with 0.005% to 0.02% w / v formaldehyde for a period of nine to eleven days;

[0278] (c) Completeness of inactivation is determined by:

[0279] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0280] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0281] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0282] In one embodiment, a method for inactivating an arboviral agent comprises:

[0283] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0284] (b) treating the arbovirus preparation with 0.0075% to 0.015% w / v formaldehyde for a period of nine to eleven days;

[0285] (c) Completeness of inactivation is determined by:

[0286] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0287] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0288] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0289] In one embodiment, a method for inactivating an arboviral agent comprises:

[0290] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0291] (b) treating the arbovirus preparation with 0.01% w / v formaldehyde for a period of nine to eleven days;

[0292] (c) Completeness of inactivation is determined by:

[0293] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0294] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0295] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0296] In one embodiment, a method for inactivating an arboviral agent comprises:

[0297] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0298] (b) treating the arbovirus preparation with 0.005% to 0.02% w / v formaldehyde for a period of ten days;

[0299] (c) Completeness of inactivation is determined by:

[0300] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0301] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0302] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0303] In one embodiment, a method for inactivating an arboviral agent comprises:

[0304] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0305] (b) treating the arbovirus preparation with 0.0075% to 0.015% w / v formaldehyde for a period of ten days;

[0306] (c) Completeness of inactivation is determined by:

[0307] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0308] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0309] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0310] In one embodiment, a method for inactivating an arboviral agent comprises:

[0311] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0312] (b) treating the arbovirus preparation with 0.01% w / v formaldehyde for a period of ten days;

[0313] (c) Completeness of inactivation is determined by:

[0314] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0315] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0316] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0317] In some embodiments, the method for inactivating an arboviral agent comprises:

[0318] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0319] (b) treating the arbovirus preparation with 0.1% to 3% hydrogen peroxide at a temperature of 20° C. to 30° C. for 5 to 120 minutes;

[0320] (c) Completeness of inactivation is determined by:

[0321] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0322] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0323] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0324] In some embodiments, the method for inactivating an arboviral agent comprises:

[0325] (a) isolating an arboviral preparation from one or more in vitro cultured cells, wherein the cells are used to produce an arboviral preparation;

[0326] (b) treating the arbovirus preparation with 0.01% hydrogen peroxide at a temperature of 20°C to 30°C for 60 minutes;

[0327] (c) Completeness of inactivation is determined by:

[0328] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0329] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0330] (iii) Determine whether the arbovirus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0331] The above method for determining completeness of inactivation can be used for any method for inactivating Zika virus. In one embodiment, the method for inactivating a Zika virus preparation comprises:

[0332] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0333] (b) treating the Zika virus preparation with 0.005% to 0.02% w / v formaldehyde;

[0334] (c) Completeness of inactivation is determined by:

[0335] (i) inoculating cultured insect cells with the Zika virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0336] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0337] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0338] In one embodiment, a method for inactivating a Zika virus preparation comprises:

[0339] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0340] (b) treating the Zika virus preparation with 0.005% to 0.02% w / v formaldehyde;

[0341] (c) Completeness of inactivation is determined by:

[0342] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0343] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0344] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0345] In one embodiment, a method for inactivating a Zika virus preparation comprises:

[0346] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0347] (b) treating the Zika virus preparation with 0.0075% to 0.015% w / v formaldehyde;

[0348] (c) Completeness of inactivation is determined by:

[0349] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0350] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0351] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0352] In one embodiment, a method for inactivating a Zika virus preparation comprises:

[0353] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0354] (b) treating the Zika virus preparation with 0.01% w / v formaldehyde;

[0355] (c) Completeness of inactivation is determined by:

[0356] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0357] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0358] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0359] In one embodiment, a method for inactivating a Zika virus preparation comprises:

[0360] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0361] (b) treating the Zika virus preparation with 0.005% to 0.02% w / v formaldehyde for a period of eight to twelve days;

[0362] (c) Completeness of inactivation is determined by:

[0363] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0364] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0365] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0366] In one embodiment, a method for inactivating a Zika virus preparation comprises:

[0367] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0368] (b) treating the Zika virus preparation with 0.0075% to 0.015% w / v formaldehyde for a period of eight to twelve days;

[0369] (c) Completeness of inactivation is determined by:

[0370] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0371] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0372] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0373] In one embodiment, a method for inactivating a Zika virus preparation comprises:

[0374] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0375] (b) treating the Zika virus preparation with 0.01% w / v formaldehyde for a period of eight to twelve days;

[0376] (c) Completeness of inactivation is determined by:

[0377] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0378] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0379] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0380] In one embodiment, a method for inactivating a Zika virus preparation comprises:

[0381] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0382] (b) treating the Zika virus preparation with 0.005% to 0.02% w / v formaldehyde for a period of nine to eleven days;

[0383] (c) Completeness of inactivation is determined by:

[0384] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0385] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0386] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0387] In one embodiment, a method for inactivating a Zika virus preparation comprises:

[0388] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0389] (b) treating the Zika virus preparation with 0.0075% to 0.015% w / v formaldehyde for a period of nine to eleven days;

[0390] (c) Completeness of inactivation is determined by:

[0391] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0392] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0393] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0394] In one embodiment, a method for inactivating a Zika virus preparation comprises:

[0395] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0396] (b) treating the Zika virus preparation with 0.01% w / v formaldehyde for a period of nine to eleven days;

[0397] (c) Completeness of inactivation is determined by:

[0398] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0399] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0400] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0401] In one embodiment, a method for inactivating a Zika virus preparation comprises:

[0402] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0403] (b) treating the Zika virus preparation with 0.005% to 0.02% w / v formaldehyde for a period of ten days;

[0404] (c) Completeness of inactivation is determined by:

[0405] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0406] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0407] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0408] In one embodiment, a method for inactivating a Zika virus preparation comprises:

[0409] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0410] (b) treating the Zika virus preparation with 0.0075% to 0.015% w / v formaldehyde for a period of ten days;

[0411] (c) Completeness of inactivation is determined by:

[0412] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0413] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0414] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0415] In one embodiment, a method for inactivating a Zika virus preparation comprises:

[0416] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0417] (b) treating the arbovirus preparation with 0.01% w / v formaldehyde for a period of ten days;

[0418] (c) Completeness of inactivation is determined by:

[0419] (i) inoculating cultured insect cells with the virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0420] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0421] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0422] In some embodiments, a method of inactivating a Zika virus preparation comprises:

[0423] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0424] (b) treating the Zika virus preparation with 0.1% to 3% hydrogen peroxide at a temperature of 20°C to 30°C for 5 to 120 minutes;

[0425] (c) Completeness of inactivation is determined by:

[0426] (i) inoculating cultured insect cells with the Zika virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0427] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0428] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0429] In some embodiments, a method of inactivating a Zika virus preparation comprises:

[0430] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0431] (b) treating the Zika virus preparation with 0.01% hydrogen peroxide at a temperature of 20°C to 30°C for 60 minutes;

[0432] (c) Completeness of inactivation is determined by:

[0433] (i) inoculating cultured insect cells with the Zika virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0434] (ii) inoculating mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0435] (iii) Determine whether the Zika virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0436] In some embodiments, a method of inactivating a Zika virus preparation comprises:

[0437] (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation;

[0438] (b) treating the Zika virus preparation with 0.05% formalin at a temperature of 20°C to 30°C, for example 22°C, for seven days;

[0439] (c) Completeness of inactivation is determined by:

[0440] (i) inoculating cultured insect cells with the Zika virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing a supernatant;

[0441] (ii) inoculating the cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and

[0442] (iii) Determine whether the virus preparation contains residual replicating virus that is cytopathic to mammalian cells.

[0443] In some embodiments, the cell is a non-human cell. Suitable non-human mammalian cells include, but are not limited to, VERO cells, LLC-MK2 cells, MDBK cells, MDCK cells, ATCC CCL34 MDCK (NBL2) cells, MDCK33016 (DSM ACC 2219 as described in WO97 / 37001), BHK21-F cells, HKCC cells, and Chinese hamster ovary cells (CHO cells). In some embodiments, the mammalian cell is a Vero cell.

[0444] Pharmaceutical composition

[0445] Other aspects of the present disclosure relate to pharmaceutical compositions comprising inactivated Zika virus obtainable by the methods disclosed herein. These pharmaceutical compositions have particularly low levels of residual formaldehyde. Without wishing to be bound by any particular theory, it is hypothesized that low levels of residual formaldehyde reduce the risk of a subject developing side effects.

[0446] The term "residual formaldehyde content" refers to the amount of formaldehyde still present in the pharmaceutical composition after the Zika virus has been inactivated and the formulation has been neutralized and optionally subjected to one or more further purification or filtration steps. According to the US Pharmacopeia, the upper limit for residual formaldehyde in vaccines containing inactivated bacteria or viruses is 0.02%, equivalent to 100 μg / ml formaldehyde.

[0447] The residual formaldehyde content can be determined by any method known to the skilled person. One suitable method is described in EMEA, VICH Topic GL25, Biologicals: Testing of residual formaldehyde, April 30, 2002, and involves the use of Methylbenzothiazolone hydrazone hydrochloride (MBTH). Other methods include acetylacetone titration, ferric chloride titration and basic fuchsin test. A particularly suitable method is described herein.

[0448] Pharmaceutical compositions are in a form that can be administered to a subject and typically contain one or more pharmaceutically acceptable excipients.

[0449] The residual formaldehyde content in the pharmaceutical composition is less than 50 μg / ml. In one embodiment, the residual formaldehyde content in the pharmaceutical composition is less than 45 μg / ml, less than 40 μg / ml, less than 35 μg / ml, less than 30 μg / ml, less than 25 μg / ml, less than 20 μg / ml, less than 15 μg / ml or less than 10 μg / ml. In one embodiment, the residual formaldehyde content in the pharmaceutical composition is less than 9.5 μg / ml, less than 9 μg / ml, less than 8.5 μg / ml, less than 8 μg / ml, less than 7.5 μg / ml, less than 7 μg / ml, less than 6.5 μg / ml, less than 6 μg / ml, less than 5.5 μg / ml, less than 5 μg / ml, less than 4.5 μg / ml, less than 4 μg / ml, less than 3.5 μg / ml, less than 3 μg / ml, less than 2.5 μg / ml, less than 2 μg / ml, less than 1.5 μg / ml, less than 1 μg / ml, or less than 0.5 μg / ml. In one embodiment, the residual formaldehyde content in the pharmaceutical composition is less than 0.5 μg / ml.

[0450] Method for determining residual formaldehyde content

[0451] Other aspects of the present disclosure relate to a method for determining the residual formaldehyde content in a pharmaceutical composition comprising inactivated viruses, the method comprising the following steps:

[0452] (a) providing a composition comprising a virus that has been treated with formaldehyde;

[0453] (b) mixing the composition of (a) with phosphoric acid and 2,4-dinitrophenylhydrazine (DNPH), thereby providing a mixture;

[0454] (c) incubating the mixture of (b) under suitable conditions; and

[0455] (d) Analyzing the mixture for the presence of residual formaldehyde.

[0456] The use of DNPH as a detection reagent provides the following advantages: (1) high sensitivity, (2) UV detection of the derivatized formaldehyde, and (3) sample preparation in a single step without heating. The method of the present invention is particularly suitable for detecting residual formaldehyde in vaccines containing adjuvants such as aluminum hydroxide. The method is validated according to the International Conference on Harmonization (ICH) Q2 guidelines in terms of specificity, linearity, accuracy, repeatability, robustness and stability.

[0457] The composition comprising a virus that has been treated with formaldehyde may also include an adjuvant. In one embodiment, the adjuvant is aluminum hydroxide. In one embodiment, the composition comprising a virus that has been treated with formaldehyde comprises 0.1 mg / ml to 1.0 mg / ml aluminum hydroxide as an adjuvant. In one embodiment, the composition comprising a virus that has been treated with formaldehyde comprises 0.2 mg / ml to 0.9 mg / ml aluminum hydroxide as an adjuvant. In one embodiment, the composition comprising a virus that has been treated with formaldehyde comprises 0.3 mg / ml to 0.8 mg / ml aluminum hydroxide as an adjuvant. In one embodiment, the composition comprising a virus that has been treated with formaldehyde comprises 0.3 mg / ml to 0.7 mg / ml aluminum hydroxide as an adjuvant. In one embodiment, the composition comprising a virus that has been treated with formaldehyde comprises 0.3 mg / ml to 0.6 mg / ml aluminum hydroxide as an adjuvant. In one embodiment, the composition comprising a virus that has been treated with formaldehyde comprises 0.3 mg / ml to 0.5 mg / ml aluminum hydroxide as an adjuvant. In one embodiment, the composition comprising a virus that has been treated with formaldehyde comprises 0.4 mg / ml aluminum hydroxide as an adjuvant.

[0458] In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde are mixed with 1 part of 15 to 25% (v / v) phosphoric acid and 2.5 parts of 0.9 to 1.1 mg / ml DNPH. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde are mixed with 1 part of 20% (v / v) phosphoric acid and 2.5 parts of 1.0 mg / ml DNPH. In some embodiments, 1 ml of a composition comprising a virus that has been treated with formaldehyde is mixed with 20 μl of 20% (v / v) phosphoric acid and 50 μl of 1.0 mg / ml DNPH.

[0459] In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 18° C. to 30° C. In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 20° C. to 25° C. In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 22° C.

[0460] In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH for 10 to 30 minutes. In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH for 15 to 25 minutes. In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH for 20 minutes.

[0461] In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 18° C. to 30° C. for 10 to 30 minutes. In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 18° C. to 30° C. for 15 to 25 minutes. In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 18° C. to 30° C. for 20 minutes.

[0462] In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 20° C. to 25° C. for 10 to 30 minutes. In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 20° C. to 25° C. for 15 to 25 minutes. In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 20° C. to 25° C. for 20 minutes.

[0463] In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 22° C. for 10 to 30 minutes. In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 22° C. for 15 to 25 minutes. In some embodiments, the composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 22° C. for 20 minutes.

[0464] In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde are incubated with a mixture of 1 part 20% phosphoric acid and 2.5 parts 1.0 mg / ml DNPH at a temperature of 18° C. to 30° C. for 10 to 30 minutes. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde are incubated with a mixture of 1 part 20% phosphoric acid and 2.5 parts 1.0 mg / ml DNPH at a temperature of 18° C. to 30° C. for 15 to 25 minutes. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde are incubated with a mixture of 1 part 20% phosphoric acid and 2.5 parts 1.0 mg / ml DNPH at a temperature of 18° C. to 30° C. for 20 minutes.

[0465] In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde are incubated with a mixture of 1 part 20% phosphoric acid and 2.5 parts 1.0 mg / ml DNPH at a temperature of 20° C. to 25° C. for 10 to 30 minutes. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde are incubated with a mixture of 1 part 20% phosphoric acid and 2.5 parts 1.0 mg / ml DNPH at a temperature of 20° C. to 25° C. for 15 to 25 minutes. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde are incubated with a mixture of 1 part 20% phosphoric acid and 2.5 parts 1.0 mg / ml DNPH at a temperature of 20° C. to 25° C. for 20 minutes.

[0466] In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde are incubated with a mixture of 1 part 20% phosphoric acid and 2.5 parts 1.0 mg / ml DNPH at a temperature of 22° C. for 10 to 30 minutes. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde are incubated with a mixture of 1 part 20% phosphoric acid and 2.5 parts 1.0 mg / ml DNPH at a temperature of 22° C. for 15 to 25 minutes. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde are incubated with a mixture of 1 part 20% phosphoric acid and 2.5 parts 1.0 mg / ml DNPH at a temperature of 22° C. for 20 minutes.

[0467] After incubation, the mixture of the composition comprising the virus treated with formaldehyde and phosphoric acid and DNPH can be analyzed by any suitable method. In one embodiment, after incubation, the mixture of the composition comprising the virus treated with formaldehyde and phosphoric acid and DNPH is analyzed by HPLC. In one embodiment, after incubation, the mixture of the composition comprising the virus treated with formaldehyde and phosphoric acid and DNPH is analyzed by reversed-phase HPLC. In one embodiment, the part of reversed-phase HPLC column is selected from C18, n-butyl (n-butal), n-octyl, phenyl and cyanopropyl. In one embodiment, the part of reversed-phase HPLC column is C18. In one embodiment, a mixture of water and acetonitrile (1: 1, v / v) is used as the mobile phase in reversed-phase HPLC. In one embodiment, the detection wavelength is 360nm.

[0468] In one embodiment, the present disclosure provides a method for determining the residual formaldehyde content in a pharmaceutical composition comprising inactivated viruses, the method comprising the following steps:

[0469] (a) providing a composition comprising a virus that has been treated with formaldehyde;

[0470] (b) mixing 50 parts of the composition of (a) with 1 part of 20% phosphoric acid and 2.5 parts of 1 mg / ml 2,4-dinitrophenylhydrazine (DNPH), thereby providing a mixture;

[0471] (c) incubating the mixture of (b) at room temperature for 20 minutes; and

[0472] (d) The mixture was analyzed for the presence of residual formaldehyde by reverse phase HPLC using a C18 column and a mixture of water and acetonitrile (1:1, v / v) as the mobile phase.

[0473] In one embodiment, the present disclosure provides a method for determining the residual formaldehyde content in a pharmaceutical composition comprising inactivated Zika virus, the method comprising the following steps:

[0474] (a) providing a composition comprising a Zika virus that has been treated with formaldehyde;

[0475] (b) mixing 50 parts of the composition of (a) with 1 part of 20% phosphoric acid and 2.5 parts of 1 mg / ml 2,4-dinitrophenylhydrazine (DNPH), thereby providing a mixture;

[0476] (c) incubating the mixture of (b) at room temperature for 20 minutes; and

[0477] (d) The mixture was analyzed for the presence of residual formaldehyde by reverse phase HPLC using a C18 column and a mixture of water and acetonitrile (1:1, v / v) as the mobile phase.

[0478] Adjuvant

[0479] Other aspects of the present disclosure relate to Zika virus vaccines and / or immunogenic compositions containing one or more antigens from at least one Zika virus described herein and one or more adjuvants. In some embodiments, the vaccine and / or immunogenic composition contains a purified inactivated whole virus, such as a Zika virus having a mutation of a tryptophan to glycine substitution at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1 as described herein, and one or more adjuvants. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus, the inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, and one or more adjuvants, wherein the Zika virus is derived from strain PRVABC59. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, and one or more adjuvants, wherein the Zika virus is derived from strain PRVABC59 comprising a genomic sequence according to SEQ ID NO: 2. In one embodiment, the vaccine and immunogenic composition contain a plaque-purified cloned Zika virus isolate and one or more adjuvants. Such adjuvanted vaccines and / or immunogenic compositions disclosed herein can be used to treat or prevent Zika virus infection in a subject in need thereof and / or to induce an immune response, such as a protective immune response, against Zika virus in a subject in need thereof.

[0480] Various methods for achieving the adjuvant effect of vaccines are known and can be used in combination with the Zika virus vaccines and / or immunogenic compositions disclosed herein. General principles and methods are described in detail in "The Theory and Practical Application of Adjuvants", 1995, Duncan E. Stewart-Tull (ed.), John Wiley & Sons Ltd, ISBN 0-471-95170-6 and "Vaccines: New Generation Immunological Adjuvants", 1995, Gregoriadis G et al. (eds.), Plenum Press, New York, ISBN 0-306-45283-9.

[0481] Exemplary adjuvants may include, but are not limited to, aluminum salts, calcium phosphates, toll-like receptor (TLR) agonists, monophosphatidyl lipid A (MLA), MLA derivatives, synthetic lipid A, lipid A mimetics or analogs, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligonucleotides, lipopolysaccharides (LPS) of Gram-negative bacteria, polyphosphazenes, emulsions (oil emulsions), chitosan, vitamin D, stearoyl or octadecyl tyrosine, viral particles, lipid rolls, poly (lactide-co-glycolide) (PLG) microparticles, poloxamer particles, microparticles, liposomes, complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA). In some embodiments, the adjuvant is an aluminum salt.

[0482] In some embodiments, the adjuvant includes at least one of alum, aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate, and Alhydrogel 85. In some embodiments, it has been found that the aluminum salt adjuvant of the present disclosure increases the antigen adsorption of the Zika virus vaccine and / or immunogenic composition of the present disclosure. Thus, in some embodiments, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the antigen is adsorbed to the aluminum salt adjuvant.

[0483] Certain embodiments of the present disclosure include a method for preparing a Zika virus vaccine or immunogenic composition with an adjuvant, the method comprising (a) mixing a vaccine or immunogenic composition with an aluminum salt adjuvant, wherein the vaccine or immunogenic composition includes one or more antigens from at least one Zika virus described herein; and (b) incubating the mixture under suitable conditions for a period of time ranging from about 1 hour to about 24 hours (e.g., about 16 hours to about 24 hours), wherein at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the antigens are adsorbed to the aluminum salt adjuvant. In certain embodiments of the method, at least one Zika virus is a Zika virus comprising a non-human cell adaptation mutation (e.g., a non-human cell adaptation mutation in protein NS1, such as a Trp98Gly mutation). In some embodiments, at least one Zika virus is a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, wherein the Zika virus is derived from strain PRVABC59. In some embodiments, the Zika virus is a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, wherein the Zika virus is derived from strain PRVABC59 comprising a genomic sequence according to SEQ ID NO: 2.

[0484] Virus purification

[0485] Other aspects of the present disclosure relate to methods for purifying Zika virus. In some embodiments, the method includes inoculating multiple cells with an inoculum containing a Zika virus colony, and obtaining a Zika virus clone isolate from one or more of the inoculated cells by plaque purification. In some embodiments, the cell is a non-human cell (e.g., an insect cell, a mammalian cell, etc.). In some embodiments, the cell is an insect cell (e.g., any of the mosquito cells / cell lines described herein). In some embodiments, the cell is a mammalian cell (e.g., any of the mammalian cells / cell lines described herein). In some embodiments, the mammalian cell is a monkey cell. In some embodiments, the mammalian cell is a Vero cell.

[0486] In some embodiments, the Zika virus colony is heterogeneous, that is, it comprises two or more genotypes. The two or more genotypes are different from each other in at least one nucleotide. In some embodiments, the Zika virus colony comprises a Zika virus clinical isolate (e.g., from virus strain PRVABC59) and / or one or more Zika viruses previously passaged in cell culture. The Zika virus clinical isolate is obtained from a sample of a patient infected with Zika virus. In some embodiments, plaque purification (e.g., as described herein) allows to substantially and / or completely separate (genetically homogeneous) cloned isolates from a heterogeneous virus colony. In some embodiments, monkey cells come from VERO cell lines (e.g., VERO 10-87 cells). In some embodiments, the inoculum comprises human serum. In some embodiments, the inoculum comprises one or more adventitious agents (e.g., one or more contaminating viruses). In some embodiments, plaque purification (e.g., as described herein) allows to substantially and / or completely purify (genetically homogeneous) cloned purified strains so that it does not contain one or more adventitious factors.

[0487] In some embodiments, methods for isolating and / or purifying a Zika virus clone are described comprising one or more (e.g., one or more, two or more, three or more, four or more, five or more, etc.) additional plaque purifications of a Zika virus clonal isolate. In some embodiments, methods for isolating and / or purifying a Zika virus clonal isolate are described comprising passaging a Zika virus clonal isolate one or more (e.g., one or more, two or more, three or more, four or more, five or more, etc.) in a cell culture (e.g., in insect cells such as a mosquito cell line and / or in mammalian cells such as a VERO cell line).

[0488] Other aspects of the present disclosure relate to methods for purifying Zika virus for preparing vaccines or immunogenic compositions. In some embodiments, the method includes one or more (e.g., one or more, two or more, three or more, four or more, five or more, or six or more) steps (in any order, including the following order): depth filtration of a sample or preparation containing Zika virus; buffer exchange and / or dilution (e.g., by cross-flow filtration (CFF)) of a sample containing Zika virus to produce a retentate; binding a sample containing Zika virus to an ion exchange membrane (e.g., an anion exchange membrane, a cation exchange membrane) to produce a binding portion, wherein the binding portion contains Zika virus, and eluting the binding portion from the ion exchange membrane; treating a sample containing Zika virus with an effective amount of any chemical inactivator described herein; neutralizing a sample containing chemically inactivated Zika virus with sodium pyrosulfite; and / or purifying a neutralized sample containing chemically inactivated Zika virus (e.g., by cross-flow filtration (CFF)). In some embodiments, the method includes the following steps: (a) passing a sample containing Zika virus through a first depth filter to produce a first eluate, wherein the first eluate contains Zika virus; (b) buffer exchanging and / or diluting the first eluate by cross flow filtration (CFF) to produce a first retentate, wherein the first retentate contains Zika virus; (c) binding the first retentate to an ion exchange membrane to produce a first bound portion, wherein the first bound portion contains Zika virus, and eluting the first bound portion from the ion exchange membrane to produce a second eluate, wherein the second eluate contains Zika virus; (d) passing the second eluate through a second depth filter to produce a second retentate, wherein the second retentate contains Zika virus; (e) treating the second retentate with an effective amount of a chemical inactivator; (f) neutralizing the treated second retentate with sodium metabisulfite; and (g) purifying the neutralized second retentate by cross flow filtration (CFF).

[0489] In the method of the present invention, the Zika virus preparation is separated from one or more in vitro cultured cells by one or more steps selected from depth filtration, buffer exchange and / or dilution and ion exchange chromatography, wherein the cells are used to produce the Zika virus preparation. In one embodiment, the Zika virus preparation is separated from one or more in vitro cultured cells by the steps of depth filtration, buffer exchange and / or dilution and ion exchange chromatography, wherein the cells are used to produce the Zika virus preparation. In one embodiment, the Zika virus preparation is separated from one or more in vitro cultured cells by the steps of depth filtration, buffer exchange and / or dilution and ion exchange chromatography, wherein the cells are used to produce the Zika virus preparation, wherein the steps are performed in the order of depth filtration, buffer exchange and / or dilution and ion exchange chromatography.

[0490] Depth filtration means using a porous filter media where particles are retained within the media rather than just on the surface of the media.

[0491] In one embodiment, the step of buffer exchange and / or dilution of the sample comprising the Zika virus preparation involves cross-flow filtration. In cross-flow filtration, also known as tangential flow filtration, the feed stream passes tangentially across the filter surface rather than into the filter.

[0492] In one embodiment, the step of ion exchange chromatography relates to anion exchange chromatography. In one embodiment, anion exchange chromatography uses an anion exchange membrane comprising a quaternary ammonium ligand. In some embodiments, by stepwise elution, for example, using 250mM NaCl, 500mM NaCl and 750mM NaCl to elute the virus from the anion exchange membrane.

[0493] Formulation of vaccines and / or immunogenic compositions

[0494] Other aspects of the present disclosure relate to the preparation of the vaccine and / or immunogenic composition of the present disclosure, which contains one or more antigens from the Zika virus described herein. In some embodiments, the Zika virus is a purified inactivated whole Zika virus. In some embodiments, the purified inactivated whole Zika virus comprises a mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1. In some embodiments, the purified inactivated whole Zika virus comprises a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1. In some embodiments, the purified inactivated whole Zika virus comprises a Trp98Gly mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1, wherein the Zika virus is derived from strain PRVABC59. In some embodiments, the purified inactivated whole Zika virus comprises a Trp98Gly mutation at position 98 of SEQ ID NO:1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising a genomic sequence according to SEQ ID NO:2.

[0495] Such vaccines and / or immunogenic compositions of the present disclosure containing one or more antigens from the Zika virus described herein can be used to treat or prevent Zika virus infection in a subject in need thereof and / or to induce an immune response, e.g., a protective immune response, against Zika virus in a subject in need thereof.

[0496] Typically, the vaccines and / or immunogenic compositions of the present disclosure are prepared as injectables in the form of liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquids prior to injection may also be prepared. Such preparations may also be emulsified or produced in the form of dry powders. The active immunogenic ingredients are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, sucrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if necessary, the vaccine or immunogenic composition may contain auxiliary substances, such as wetting agents or emulsifiers, pH buffers, or adjuvants that enhance the efficacy of the vaccine or immunogenic composition.

[0497] Vaccines or immunogenic compositions can be routinely administered parenterally, by injection, such as subcutaneously, transdermally, intradermally, subdermally or intramuscularly. Other formulations suitable for other modes of administration include suppositories and oral, oral, intranasal, buccal, sublingual, intraperitoneal, intravaginal, anal and intracranial preparations in some cases. For suppositories, traditional adhesives and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed by a mixture containing an active ingredient in the range of 0.5% to 10% or even 1-2%. In certain embodiments, a low melting point wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted, and the Zika virus vaccine and / or immunogenic composition described herein is uniformly dispersed, for example, by stirring. The molten uniform mixture is then poured into a mold of suitable size, and allowed to cool and solidify.

[0498] The vaccine and / or immunogenic composition of the present disclosure can be administered in a manner compatible with the administration of the formulation and in an amount that will be effective and immunogenic for treatment. The amount to be administered depends on the subject to be treated, including, for example, the ability of the individual's immune system to initiate an immune response and the degree of protection required. Suitable dosage ranges may include, for example, about 0.1 μg to about 100 μg of purified inactivated whole Zika virus. The amount of purified inactivated Zika virus can be determined by Bradford assay (Bradford et al. (1976) Anal. Biochem. 72: 248-254) using a determined amount of recombinant Zika envelope protein to establish a standard curve to determine.

[0499] Suitable schedules for initial administration and booster shots also vary, but typically involve an initial administration followed by vaccinations or other administrations.

[0500] The mode of application can vary widely. Any conventional method for administering a vaccine or immunogenic composition is applicable. These methods include oral administration, parenteral administration, injection, etc. on a solid physiologically acceptable matrix or in a physiologically acceptable dispersion. The dosage of a vaccine or immunogenic composition will depend on the route of administration and can vary according to the age of the person to be vaccinated and the formulation of the antigen. A vaccine or immunogenic composition can have a unit dose volume of more than 0.5 mL, 0.5 mL or less than 0.5 mL, as described herein. For example, its application volume can be 0.25 mL.

[0501] To control tonicity, physiological salts, such as sodium salts, are preferably included. Sodium chloride (NaCl) is preferred and may be present in an amount between 1 and 20 mg / ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, and the like.

[0502] The vaccines and / or immunogenic compositions of the present disclosure may include one or more buffers. Typical buffers include: phosphate buffer; Tris buffer; borate buffer; succinate buffer; histidine buffer (especially with aluminum hydroxide adjuvant); or citrate buffer. The content of the buffer is typically in the range of 5-20 mM.

[0503] The pH of the vaccine or immunogenic composition is generally between 5.0 and 8.5 or between 5.0 and 8.1, and more typically between 6.0 and 8.5, such as between 6.0 and 8.0, between 6.5 and 8.0, between 6.5 and 7.5, between 7.0 and 8.5, between 7.0 and 8.0, or between 7.0 and 7.8. Thus, the manufacturing method of the present disclosure may include a step of adjusting the pH of the bulk vaccine prior to packaging.

[0504] The vaccine or immunogenic composition is preferably sterile. It is preferably non-pyrogenic, for example containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. It is preferably gluten-free.

[0505] In certain embodiments, the vaccine and / or immunogenic composition of the present disclosure may include an effective concentration of detergent. In some embodiments, the effective amount of detergent may include but is not limited to about 0.00005% v / v to about 5% v / v or about 0.0001% v / v to about 1% v / v. In certain embodiments, the effective amount of detergent is about 0.001% v / v, about 0.002% v / v, about 0.003% v / v, about 0.004% v / v, about 0.005% v / v, about 0.006% v / v, about 0.007% v / v, about 0.008% v / v, about 0.009% v / v or about 0.01% v / v. Without wishing to be bound by theory, detergent helps maintain the vaccine and / or immunogenic composition of the present disclosure in a dissolved state, and helps prevent the vaccine and / or immunogenic composition from aggregating.

[0506] Suitable detergents include, for example, polyoxyethylene sorbitan ester surfactants (known as 'Tweens'), octoxynol (e.g., octoxynol-9 (Triton X 100) or t-octylphenoxypolyethoxyethanol), hexadecene trimethylammonium bromide ('CTAB'), and sodium deoxycholate. The detergent may be present in only trace amounts. Other remaining components in trace amounts may be antibiotics (e.g., neomycin, kanamycin, polymyxin B). In some embodiments, the detergent contains polysorbate. In some embodiments, the effective concentration of the detergent includes a range of about 0.00005% v / v to about 5% v / v.

[0507] The vaccine and / or immunogenic composition is preferably stored between 2°C and 8°C. Ideally, it is protected from direct light. The antigen and emulsion are typically mixed, although they may initially be presented in a kit of separate components that are mixed just prior to use. The vaccine and / or immunogenic composition will generally be in aqueous form when administered to a subject.

[0508] Methods of the present disclosure

[0509] Other aspects of the present disclosure relate to methods of treating or preventing Zika virus in a subject in need thereof and / or inducing an immune response to Zika virus in a subject in need thereof using the vaccines and / or immunogenic compositions described herein, which contain purified, inactivated whole Zika virus, e.g., a Zika virus having a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1, as described herein, that is a tryptophan to glycine substitution). Other aspects of the present disclosure relate to methods of treating or preventing Zika virus in a subject in need thereof and / or inducing an immune response to Zika virus in a subject in need thereof using the vaccines and / or immunogenic compositions described herein, which contain purified, inactivated whole Zika virus having a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1, that is a tryptophan to glycine substitution. Other aspects of the disclosure relate to methods of treating or preventing Zika virus in a subject in need thereof and / or inducing an immune response to Zika virus in a subject in need thereof using the vaccines and / or immunogenic compositions described herein, the vaccines and / or immunogenic compositions containing a purified, inactivated whole Zika virus having a mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1 that is a tryptophan to glycine substitution, wherein the Zika virus is derived from strain PRVABC59. Other aspects of the disclosure relate to methods of treating or preventing Zika virus in a subject in need thereof and / or inducing an immune response to Zika virus in a subject in need thereof using the vaccines and / or immunogenic compositions described herein, the vaccines and / or immunogenic compositions containing a purified, inactivated whole Zika virus having a mutation at position 98 of SEQ ID NO: 1 or at a position corresponding to position 98 of SEQ ID NO: 1 that is a tryptophan to glycine substitution, wherein the Zika virus is derived from strain PRVABC59 comprising a genomic sequence according to SEQ ID NO: 2.

[0510] In some embodiments, the present disclosure relates to methods for treating or preventing Zika virus infection in a subject in need thereof, by administering to the subject a purified, inactivated whole Zika virus, e.g., a Zika virus having a mutation of a tryptophan to glycine substitution at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1, as described herein.

[0511] In some embodiments, the present disclosure relates to methods for treating or preventing Zika virus infection in a subject in need thereof, by administering to the subject a therapeutically effective amount of a vaccine and / or immunogenic composition of the present disclosure, the vaccine and / or immunogenic composition containing a purified, inactivated whole Zika virus having a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1, that is a tryptophan to glycine substitution. In some embodiments, the present disclosure relates to methods for treating or preventing Zika virus infection in a subject in need thereof, by administering to the subject a therapeutically effective amount of a vaccine and / or immunogenic composition of the present disclosure, the vaccine and / or immunogenic composition containing a purified, inactivated whole Zika virus having a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1, that is a tryptophan to glycine substitution, wherein the Zika virus is derived from strain PRVABC59. In some embodiments, the present disclosure relates to methods for treating or preventing Zika virus infection in a subject in need thereof, by administering to the subject a therapeutically effective amount of a vaccine and / or immunogenic composition of the present disclosure, the vaccine and / or immunogenic composition containing a purified, inactivated whole Zika virus having a mutation of a tryptophan to glycine substitution at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1, wherein the Zika virus is derived from strain PRVABC59 comprising a genomic sequence according to SEQ ID NO: 2.

[0512] In some embodiments, the present disclosure relates to methods for inducing an immune response to Zika virus in a subject in need thereof, by administering to the subject a therapeutically effective amount of a vaccine and / or immunogenic composition of the present disclosure, the vaccine and / or immunogenic composition containing a purified, inactivated whole Zika virus, e.g., a Zika virus having a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1, that is a tryptophan to glycine substitution as described herein). In some embodiments, the present disclosure relates to methods for inducing an immune response to Zika virus in a subject in need thereof, by administering to the subject a therapeutically effective amount of a vaccine and / or immunogenic composition of the present disclosure, the vaccine and / or immunogenic composition containing a purified, inactivated whole Zika virus having a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1, that is a tryptophan to glycine substitution, wherein the Zika virus is derived from strain PRVABC59. In some embodiments, the present disclosure relates to methods for inducing an immune response to Zika virus in a subject in need thereof, by administering to the subject a therapeutically effective amount of a vaccine and / or immunogenic composition of the present disclosure, said vaccine and / or immunogenic composition containing a purified, inactivated whole Zika virus having a mutation of a tryptophan to glycine substitution at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1, wherein the Zika virus is derived from strain PRVABC59 comprising a genomic sequence according to SEQ ID NO: 2.

[0513] In some embodiments, the administering step induces a protective immune response against Zika virus in the subject. In some embodiments, the subject is a human. In some embodiments, the subject is pregnant or intends to become pregnant.

[0514] In some embodiments, the step of applying includes applying one or more times. Application can be by single dose schedule or multiple dose (initial immunization-reinforcement) schedule. In multiple dose schedule, various dosages can be given by the same or different approach, such as parenteral initial immunization and mucosal reinforcement, mucosal initial immunization and parenteral reinforcement, etc. Typically, it is given by the same approach. Multiple dosages are typically administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 16 weeks, etc.). Assuming that two dosages are separated by 25-30 days (e.g., 28 days) is particularly useful.

[0515] The method of the present disclosure includes administering a therapeutically effective amount or an immunogenic amount of a Zika virus vaccine and / or an immunogenic composition of the present disclosure. A therapeutically effective amount or an immunogenic amount may be an amount of a vaccine and / or an immunogenic composition of the present disclosure that will induce a protective immune response in an uninfected, infected, or unexposed subject to which it is administered. Such responses will generally cause secretory, cellular, and / or antibody-mediated immune responses to the vaccine to be manifested in the subject. Typically, such responses include, but are not limited to, one or more of the following effects; production of antibodies from any of the immunological classes of, for example, immunoglobulins A, D, E, G, or M; proliferation of B and T lymphocytes; providing activation, growth, and differentiation signals for immune cells; expansion of helper T cells, inhibitory T cells, and / or cytotoxic T cells.

[0516] Preferably, the therapeutically effective amount or immunogenic amount is sufficient to treat or prevent disease symptoms. The exact amount required will vary depending on, among other things: the subject being treated; the age and general condition of the subject to be treated; the ability of the subject's immune system to synthesize antibodies; the degree of protection desired; the severity of the condition being treated; the specific Zika virus antigen selected and its mode of administration. An appropriate therapeutically effective amount or immunogenic amount is readily determined by one of ordinary skill in the art. The therapeutically effective amount or immunogenic amount will be within a relatively wide range that can be determined by routine experimentation.

[0517] The present disclosure will be more fully understood by reference to the following examples, which, however, should not be considered to limit any aspect or scope of the present disclosure in any way.

[0518] Example

[0519] Example 1: Generation of cloned Zika virus strains

[0520] This example describes the generation of a Zika virus (ZIKAV) strain with a known research history.

[0521] Materials and Methods

[0522] Maintenance of Vero cells

[0523] A vial of WHO Vero 10-87 cells was rapidly thawed in a water bath and inoculated directly into a T-75 cm plate at 36°C + / - 2°C, 5% CO2. 2 19 mL of pre-warmed DMEM (Dulbecco's modified minimum essential medium) containing penicillin-streptomycin, 40 mM L-glutamine and 10% FBS was placed in the flask. The cells were grown to confluence and subcultured using TrypE. This flask was expanded into two T-185 cm 2 Flasks, grow to confluence and subculture to 31×T-185cm 2flask and grown until the cells reached 100% confluence. The cells were harvested by trypsinization, centrifuged at 800 × g for 10 min, and resuspended in DMEM containing 10% FBS and 10% DMSO at a concentration of 1.9 × 10 7 A vial of Vero cells was rapidly thawed and revived to T-75 cm as described above. 2 These cells were subcultured twice to culture at 13×T-185 cm 2 After trypsinization, cells were centrifuged at 800 × g and resuspended in freezing medium (DMEM containing 10% FBS and 10% DMSO) at a concentration of 4.68 × 10 5 The cell bank was aliquoted into cryovials.

[0524] Vero cells were grown and maintained in DMEM containing penicillin-streptomycin, L-glutamine, and 10% FBS (cDMEM-10%-FBS). TryplExpress was used to maintain cells and perform trypsinization. Two days before virus adsorption, 4-5×10 cells / mL of 3 mL cDMEM-10%-FBS were added to 6-well plates. 5 cells / well or T-25cm 2 7 × 10 in 5 mL cDMEM-10%-FBS in flask 5 cells or 1 × 10 cells in 0.1 mL cDMEM-10%-FBS in a 96-well plate 4 The incubator was monitored daily to maintain the indicated temperature. Vero cell lines were stored in liquid nitrogen.

[0525] Plaque assay

[0526] Virus titer was determined by plaque titration in fresh confluent monolayers of Vero cells grown in 6-well plates. Frozen aliquots were thawed and a series of ten-fold dilutions were made in cDMEM-0%-FBS in 96-well plates. Diluted viruses were maintained on ice before inoculating Vero cell monolayers. During the assay, growth medium was aspirated from the 6-well plates and 100 μL of each virus dilution was added to the wells. Virus was adsorbed for 60 minutes at 36°C ± 2°C, 5% CO2, with frequent (every 10 minutes) shaking of the plates to prevent cell sheets from drying. After virus adsorption, 4 mL of the first agarose overlay (1X cDMEM-2%-FBS+0.8% agarose) maintained at 40-41°C was added to each well. Agarose was solidified at room temperature for 30 minutes, and then the plates were inverted and incubated at 36°C + / 2°C, 5% CO2 for 4-6 days. On day 4, 2 mL of a second agarose overlay containing 160 μg / mL neutral red vital dye was added. Plaques were visually inspected on days 5 and 6.

[0527] Quantification of virus by TCID50 assay

[0528] Virus titer is also determined by titration in fresh confluent monolayers of Vero cells grown in 96-well plates. Frozen aliquots are thawed and a series of ten-fold dilutions are performed on aliquots in cDMEM-2%-FBS diluent in 96-well plates. Diluted viruses are maintained on ice before inoculating Vero cell monolayers. During the assay, growth medium is aspirated from the 96-well plates and 100 μL of each virus dilution is added to the wells. The plates are incubated at 36°C + / 2°C, 5% CO2 for 5 days. 50% tissue culture infection dose (TCID50) titer is calculated using the Reed / Muench method.

[0529] Test Items

[0530] Zika virus strain PRVABC59 (a 0.5 mL vial on dry ice) was obtained from the Centers for Disease Control and Prevention (CDC). The identity of the Zika virus was confirmed by RT-PCR. The virus strain was negative for alphavirus and mycoplasma contamination by PCR. This information is summarized in Table 1.

[0531] Table 1: PRVABC59 virus strain information

[0532]

[0533]

[0534] Sequencing

[0535] RNA was extracted from the stable viral harvest of each isolate using the QIAampViral RNA Mini Spin kit according to the manufacturer's protocol. RNA extracted from each isolate was used to establish and amplify 6 cDNA fragments covering the entire Zika virus genome. The size and purity of the amplified cDNA fragments were analyzed on a 1% agarose / TBE gel and subsequently gel purified using the Qiagen Quick Gel Extraction Kit. Automatic sequencing reactions were performed using an ABI 3130XL Gene Analysis Sequencer. Sequencing data were analyzed using Lasergene SeqMan software.

[0536] result

[0537] A ZIKAV strain with a known research history associated with the current ZIKAV outbreak in the Americas was sought. For this purpose, the ZIKAV strain PRVABC59 was selected. To generate a well-characterized virus suitable for growth in Vero cells, ZIKAVPRVABC59 was first amplified in Vero cells (P1).

[0538] Vero cells (T-175 cm 2 ) flasks were infected with 4 mL cDMEM-0%-FBS at an MOI of 0.01. Virus was adsorbed to the monolayer at 36°C ± 2°C, 5% CO2 for 60 minutes, and then 20 mL cDMEM-0%-FBS was applied at 36°C ± 2°C, 5% CO2 for virus amplification. The cell layer was monitored for cytopathic effect (CPE) every day after inoculation ( Figure 1 ). After 96 hours, the supernatant was harvested by collecting the culture medium and clarified by centrifugation (600×g, 4° C., 10 minutes). The harvest was stabilized by adding trehalose to a final concentration of 18% w / v. The bulk was aliquoted into 0.5 mL cryovials and stored at -80° C.

[0539] The presence of infectious virus in stabilized P1 harvests on Vero cell monolayers was analyzed by TCID50 assay. Growth kinetics were monitored by taking aliquots daily starting at 0 hours. Peak titers ( Figure 2 ).

[0540] P1 material was plaque purified by titering the harvest from day 3 on a 6-well monolayer of Vero cells. Plaques were visually inspected on day 6 and 10 plaques to be isolated were identified by drawing a circle around the distinct and individual plaques on the bottom of the plastic plate. Plaques were picked by extracting the agarose plug using a sterile wide bore pipette while breaking the bottom of the well and washing with cDMEM-10%-FBS. The agarose plug was added to 0.5 mL cDMEM-10%-FBS, spun, labeled PRVABC59 P2a-j and placed in an incubator at 36°C ± 2°C, 5% CO2 overnight.

[0541] Three plaques (PRVABC59 P2a-c) were carried forward for additional purification. Each isolate was plated in duplicate onto fresh 6-well monolayers of Vero cells without solvent. The P2 / P3 switch was plaque purified and labeled PRVABC59 P3a-j.

[0542] Six plaques (PRVABC59 P3a-f) were carried forward for the final round of purification. Each isolate was plated in duplicate onto fresh 6-well monolayers of Vero cells without solvent. The P3 / P4 switch was plaque purified and labeled PRVABC59 P4a-j.

[0543] Six plaques from P4 plaque purification (PRVABC59 P4a-f) were plated at T-25 cm 2 Blind passage on Vero cell monolayers in flasks. Dilute each selected plaque in 2 mL cDMEM-0%-FBS - allow 1 mL to adsorb for 1 hour at 36°C ± 2°C, 5% CO2; another 1 mL is stabilized with trehalose (18% v / v final) and stored at <-60°C. After virus adsorption, cDMEM-0%-FBS is added to each flask and grown for 4 days at 36°C ± 2°C, 5% CO2. Harvest the virus supernatant, clarify by centrifugation (600×g, 4°C, 10 minutes), stabilize in 18% trehalose, and aliquot and store at <-60°C. The P5 seeds are tested for Zika virus potency by TCID50 ( Figure 3 ).

[0544] T-175cm 2 Each of the six PRVABC59 clones (P5a-f) was infected with confluent Vero cell monolayers in 4 mL cDMEM-0%-FBS at an MOI of 0.01. Virus was allowed to adsorb for 60 minutes at 36°C + / 2°C, 5% CO2, and then 20 mL cDMEM-0%-FBS was added to each flask and allowed to grow at 36°C + / 2°C, 5% CO2. The health and CPE of the Vero cell monolayers were monitored daily. As indicated ( Figure 4), and virus was harvested on days 3 and 5. Harvests of the P6 strain from days 3 and 5 were pooled, stabilized with 18% trehalose, aliquoted and stored at < -60°C.

[0545] Each of the six clones of PRVABC59 (P6a-f) was tested for Zika virus potency in vitro ( Figure 5 The potency was determined by two different methods, TCID50 and plaque titration. The CPE was visually inspected (microscope) and by measuring the difference in absorbance (A) of wells showing CPE (yellow) compared to red (no CPE). 560 -A 420 ) to calculate TCID50. Plates were read on a plate reader and applied to the same calculation (absorbance) as for plates read with a microscope. TCID50 values ​​were quite similar between the two scoring techniques, while the values ​​obtained by plaque titration were lower.

[0546] An overview of P6 virus generation and characterization is shown in Table 2 below.

[0547] Table 2: Overview of virus passage and characterization used to generate clonal ZIKAV strains

[0548]

[0549]

[0550] An isolated Zika virus clone was sought that closely resembled the envelope glycoprotein sequence of the original isolate, since the envelope protein of flaviviruses is the dominant immunogenic part of the virus. PRVABC59 clones P6a, P6c, P6d, and P6f contained a G→T mutation at nucleotide 990 (G990T) in the envelope region, resulting in an amino acid mutation of Val→Leu at envelope residue 330, while the envelope genes of PRVABC59 clones P6b and P6e were identical relative to the reference virus strain (GenBank reference KU501215.1) (Tables 3 and 4). Figure 6 ).

[0551] Table 3: Sequencing of PRVABC59 P6 clones

[0552]

[0553]

[0554] Then, two clones lacking mutations in the Zika envelope sequence were subjected to complete genome sequencing. The sequencing results are summarized in Table 3 above. Sequence analysis revealed a T→G substitution at nucleotide 292 in the NS1 region of both clones, resulting in a Trp→Gly mutation at residue 98 of NS1. The NS1 W98G mutation is located in a wrap-around loop of the wing domain of ZIKAV NS1 that is associated with membrane association, interaction with envelope proteins, and possibly formation of hexameric NS1. While other tryptophan residues (W115, W118) are highly conserved among flaviviruses, W98 is not ( Figure 7 ). Interestingly, however, 100% conservation of the W98 residue was observed in 11 different ZIKAV strains, including those from African and Asian lineages. The mutations identified in each strain are summarized in Table 4.

[0555] Table 4: Summary of mutations identified in PRVABC59 P6 clones

[0556]

[0557]

[0558] Phenotypic analysis of the ZIKAV PRVABC59 P6 stock was performed to characterize the ZIKAV clones. Figure 8 As shown in Fig. 9 As quantified in Figure 2, each clonal isolate consisted of a relatively homogeneous population of large-sized plaques, compared to the P1 virus, which had a mixed population of large and small plaques. These data indicate successful isolation of a single ZIKAV clone.

[0559] Then, the growth kinetics of ZIKAVPRVABC59P6 clones in Vero cells were analyzed. 50 Each ZIKAV P6 clone / cell infected Vero cells. Virus supernatant samples were taken daily and measured by TCID 50 The peak titers of all P6 clones occurred between days 3 and 4 (approximately 9.0 log 10 TCID 50 / mL). There was no significant difference in the growth kinetics of various P6 clones ( Fig.10 ).

[0560] In summary, the result shows that Zika virus seed is successfully produced. The seed selection needs to understand the growth history, kinetics, yield, genotype and phenotype of the virus. Importantly, the clone separation of Zika virus strain allows successful virus purification, so that it does not contain contaminating factors (for example, the extraneous factors that may exist in the parent human isolate). Interestingly, three times of plaque purification successfully select the virus (strain P6a-f) of adapting to Vero cell quickly, and these strains can be well replicated in serum-free Vero cell culture, wherein strain P6a, c, d and f have mutations in viral envelope protein, and strain p6b and p6e obtain mutations in viral NS1 protein (viral envelope is not modified). In addition, the strain adapted to Vero can make the subsequent viral passages bred by these strains grow and manufacture efficiently and reproducibly. Without wishing to be bound by theory, the Env-V330L mutation observed in virus strains P6a, c, d and f may be the result of in vitro adaptation, because mutations at Env 330 were also observed when passaged in Vero cells (Weger-Lucarelli et al. 2017. Journal of Virology). Because envelope protein is the dominant immunogenic epitope of Zika virus, virus strains containing Vero adaptive mutations in Env may negatively affect vaccine immunogenicity. Without wishing to be bound by theory, adaptive mutations in protein NS1 not only appear to enhance viral replication, but also reduce or otherwise inhibit undesirable mutations in envelope protein E (Env) of Zika virus, for example. In addition, it is known that NS1 is bound to envelope protein during the viral life cycle. This mutation (NS1W98G) may be associated with the ability of NS1 to associate with viruses during downstream processing and possibly co-purify. It is also known that NS1 is immunogenic and may be associated with the immune response to vaccines.

[0561] Example 2: Preclinical immunogenicity and efficacy of purified inactivated Zika virus vaccines (PIZV) derived from P6b and P6e strains

[0562] The following examples describe the preclinical immunogenicity and efficacy of inactivated Zika virus vaccines (PIZV) derived from P6b and P6e strains in CD1 and AG129 mice. As described in Example 1, six clones were generated from the epidemic-related PRVABC59 strain, and two (P6b and P6e) were selected for further preclinical immunogenicity and efficacy studies.

[0563] Materials and Methods

[0564] Purification, inactivation and formulation of Zika virus vaccines

[0565] Large quantities of inactivated ZIKAV vaccines suitable for preclinical immunogenicity and efficacy studies were generated and characterized. Viruses were amplified from P6b and P6e strains by infecting flasks of confluent Vero cells at an MOI of 0.01. The viruses were allowed to adsorb for 1 hour at 36°C ± 2°C / 5% CO2. After adsorption, 20 mL of cDMEM-0%-FBS was added to each flask and grown for five days at 36°C ± 2°C / 5% CO2. Cell supernatants were harvested on days 3 and 5 post-infection and purified of cell debris by centrifugation.

[0566] For each isolate, the purified supernatant is pooled, stabilized and stored at <-60°C in DMEM containing 18% trehalose. The purified viral supernatant collected is thawed in a 37°C water bath and treated with omnipotent nuclease overnight at 4°C. After omnipotent nuclease treatment, each sample is applied to a Sartorius PP3 depth filter. After depth filtration, each sample is applied to a Centricon Plus-70 tangential flow filtration (tangential flowfiltration, TFF) device. The retentate is buffer exchanged, diluted and applied to Sartorius SartobindQIEXNano. Each sample is applied to a second Sartorius SartobindQ IEXNano and eluted with 250mM, 500mM and 750mM NaCl using a 3-step elution method. After MonoQ chromatography and dilution, each 250 mM eluate was applied to a Centricon Plus-70 cross-flow filtration (CFF) device for buffer exchange, diluted to 35 mL with PBS and stored at 2-8°C.

[0567] For formalin inactivation, freshly prepared 1% formaldehyde was added dropwise to each purified sample under slow swirling to obtain a final formaldehyde concentration of 0.02%. The samples were incubated at room temperature (about 22°C) for 14 days with daily inversion. Formaldehyde was neutralized with sodium pyrosulfite for 15' at room temperature and then applied to a Centricon Plus-70 tangential flow filtration (TFF) device. The buffer was changed four times by adding 50mL of bulk drug buffer (10mM NaH2PO4, 50mM NaCl, 6% sucrose, pH 7.4). Each sample was then diluted to 15mL with bulk drug buffer, sterilized using a 0.2m syringe filter, aliquoted into sterile stoppered glass vials (0.5mL per vial) and frozen at <-60°C.

[0568] Virus inactivation was confirmed by TCID50 assay and double infectivity assay. Briefly, the bulk drug sample was applied to C6 / 36 cells and amplified for 6 days. The supernatant from C6 / 36 cells was applied to Vero cells and CPE was monitored for 8 days. To prepare the drug, vials of PIZV bulk drug were thawed, pooled according to sample type, and diluted to 1 μg / mL or 10 μg / mL in PBS with or without Alhydrogel (Brenntag; 0.5 mg / mL final, 0.050 mg per dose) and incubated overnight at 2-8°C with gentle agitation. The resulting drug batch was then aliquoted into sterile stoppered glass vials and stored at 2-8°C until use. Fig.11 An overview of the steps used to prepare a drug product is provided.

[0569] Mouse immunization and challenge

[0570] For immunogenicity studies, six-week-old male and female Swiss-ICR (CD-1) mice were divided into 6 groups (n=10 / group). On day 0, mice in groups 1-5 were vaccinated with 0.1 mL of the vaccine by intramuscular (im) route (2×0.05 mL injections). Mice in group 6 were inoculated with PBS as a placebo control. On days 28 and 56, mice were boosted with the same dose and vaccine type as on day 0. Blood samples were collected on day -1 (pre-immunization), day 27 (primary immunization), day 42 (boost 1), and day 70 (boost 2).

[0571] For immunogenicity and efficacy studies, four-week-old male and female AG129 mice were divided into 7 groups (n=5 / group). On day 0, mice in groups 1-6 were vaccinated with 0.1 mL of the vaccine via the intramuscular (im) route (2×0.05 mL injections). Mice in group 7 were vaccinated with PBS as a placebo control. On day 28, mice were boosted with the same dose and vaccine type as on day 0. Blood samples were collected from the tail vein on day -1 (pre-immunization), day 27 (primary immunization), and day 55 (boost). At the time of euthanasia, mice were bled via cardiac puncture under deep anesthesia with isoflurane (terminal). On day 56, mice were intraperitoneally injected with 10 4 Mice were challenged with plaque forming units (PFU) of ZIKAV PRVABC59.

[0572] Serum transfer

[0573] Serum was collected from PIZV-inoculated and challenged AG129 mice and frozen after pooling (Groups 1, 2, 4, and 5 of Table 6). The serum pool was thawed and test articles were produced by diluting the serum pool three times in PBS. Placebo was produced using 3-fold dilutions of AG129 normal mouse serum in PBS.

[0574] The test article was administered as a 0.1 mL intraperitoneal injection to AG129 mice (control mice were administered an equal volume of placebo article). 4 Animals were challenged with plaque-forming units of the Zika virus strain PRVABC59.

[0575] On day -11 (before immunization), the blood volume allowed by weight was collected from ten mice in whole blood by tail bleeding. On the 1st day (initial circulation Nab) and the 4th day (viremia), whole blood was collected from each mouse by tail bleeding. The terminal bleeding after the fatal attack was carried out by cardiac puncture under deep anesthesia to obtain a larger volume, and then euthanasia was imposed by cervical dislocation. The blood samples were collected in microtainer SST serum separation gel tubes and coagulated for at least 30 minutes, then separated by centrifugation (10,000 × g, 2 minutes) The serum was frozen at -80 ° C.

[0576] Plaque Reduction Neutralization Test

[0577] Neutralizing antibody titers were determined by plaque reduction neutralization test (PRNT) as previously described (see, e.g., Osorio et al. Lancet Infect Dis. 2014 Sep;14(9):830-8).

[0578] Reporter Virus Particle (RVP) Neutralization Assay

[0579] Neutralizing antibody titers were analyzed by titrating serum samples with a constant amount of Zika RVP in Vero cells grown in 96-well plates. The RVP contains the prME protein of Zika (strain SPH2012) and a dengue-based Renilla luciferase reporter. Briefly, sera were heat inactivated at 56°C for 30 minutes, diluted, and then incubated with RVP at 37°C. The serum / RVP mixture was then mixed with Vero cells and incubated at 37°C ± 2°C / 5% CO2 for 72 hours and then detected with luciferase substrate. Data were analyzed using JMP11 nonlinear 4-parameter analysis, normalized to a positive chase control, and the effective dose 50% (EC 50 ).

[0580] Unless indicated to the contrary, all additional experimental procedures were performed as described in Example 1 above.

[0581] result

[0582] To assess the immunogenicity of candidate PIZVs in 6-week-old male and female CD-1 mice, groups of CD-1 mice (N=10 / group) were immunized by the intramuscular route with 0.1 μg (+ alum), 1.0 μg (+ alum) doses of vaccines derived from the ZIKAV PRVABC69 P6b or P6e strains. To assess the need for adjuvants, one group of animals was vaccinated with 0.1 μg of vaccine derived from P6e and lacking alum adjuvant. Vaccinations were given on days 0, 28, and 56, with group 6 receiving PBS as a placebo control ( Fig. 12A and Table 5).

[0583] Table 5: PIZV preparation and challenge in CD-1 mice

[0584] Group Virus strain Dosage (μg) Alum(μg) N 1 P6b 0.1 0.50 10 2 P6b 1.0 0.50 10 3 P6e 0.1 0.50 10 4 P6e 1.0 0.50 10 5 P6e 0.1 - 10 6 Placebo (PBS) - - 10

[0585] After vaccination, serum samples collected after the primary (day 27), second (day 40), and third (day 70) immunizations were tested for ZIKAV-specific neutralizing antibodies by RVP neutralization assay ( Fig. 12B Twenty-seven days after the first dose, a mild neutralizing antibody response was observed in mice vaccinated with PIZV derived from either clone containing alum, compared with the PBS placebo control group. Importantly, this response increased significantly after the second immunization (day 40), but was no longer enhanced after the third immunization (day 70). No neutralizing antibody response was observed in mice vaccinated with the unadjuvanted vaccine ( Fig. 12B ).

[0586] To assess the immunogenicity and protective efficacy of candidate PIZVs, groups of 4-week-old AG129 mice (n=5 / group) were immunized intramuscularly on days 1 and 28 with 0.1 μg (+alum), 1.0 μg (+alum), or 0.1 μg (-alum) of vaccines derived from ZIKAV PRVABC59 P6b or P6e stock solutions ( Fig.13A and Figure 6 ).

[0587] Table 6: PIZV preparation and challenge in AG129 mice

[0588] Group gender Virus strain Dosage (μg) Alum(μg) N 1 F P6b 0.1 0.50 5 2 F P6b 1.0 0.50 5 3 F P6b 0.1 - 5 4 M P6e 0.1 0.50 5 5 M P6e 1.0 0.50 5 6 M P6e 0.1 - 5 7 M Placebo (PBS) - - 5

[0589] After vaccination, on day 56, 104 Vaccinated mice and control mice were challenged intraperitoneally with PFU ZIKAV PRVABC59 (low passage). Serum samples collected after primary (D27) and secondary (D55) immunizations were tested for ZIKAV-specific neutralizing antibody responses ( Fig. 13B And Table 7). Only the groups that received high doses of the vaccine with alum adjuvant (Groups 2 and 5) elicited neutralizing antibody responses after a single immunization, and the responses increased significantly after boosting. In contrast, the groups that received low or high doses of the vaccine with alum adjuvant produced high neutralizing antibody responses after the second dose. After receiving two doses of the vaccine, there was no statistical difference between the groups of mice that received the vaccine with alum adjuvant, regardless of the dose or source from the P6 clone.

[0590] Table 7: ZIKAV-specific neutralizing antibody responses

[0591]

[0592] All groups were also monitored for mortality, morbidity, and weight loss within 21 days after the attack. Viremia was detected after the attack and quantified by plaque titration. As assessed by plaque reduction neutralization test (PRNT) assay and similar secondary neutralization assays, mice (Groups 1, 2, 4, and 5) vaccinated with low or high doses of candidate PIZV formulated with alum completely avoided fatal ZIKAV attacks (Table 8). No weight loss or clinical signs of disease were observed in the vaccinated mice, no detectable infectious viremia was observed in the three days after the attack, and all mice vaccinated with low or high doses of antigen + alum adjuvant survived to 21 days after the attack (Table 8). Figure 14-16 ). In contrast, challenges with all untreated mice resulted in high viremia on day 2 post-challenge and morbidity / death between days 10 and 18 post-challenge (median survival = D13). In addition, challenges with mice vaccinated with a low dose of a vaccine derived from strain P6b without alum adjuvant resulted in high viremia on day 2 post-challenge and a median survival of days similar to that of the placebo control group, while mice vaccinated with a low dose of a vaccine derived from clone e without alum adjuvant were still partially protected, with a median survival of 19 days. These results suggest that immunization under alum is more effective, that secondary immunizations may be required, and that low doses are equally effective as high doses.

[0593] Table 8: Serum neutralizing antibody titers

[0594]

[0595] In addition, the presence of NS1 in vaccine bulk drugs (DS) produced by whole inactivated P7b and P7e viruses (from another passage of P6b and P6e strains, respectively) was tested. A sandwich ELISA was performed using plates pre-coated with monoclonal antibodies reactive to both the Asian and African lineages of Zika virus NS1 but not cross-reactive with dengue NS1. Repeated 2-fold, 4-fold, 8-fold, 16-fold and 32-fold dilutions of DS were prepared and compared to a standard curve using recombinant purified NS1 in duplicate at a concentration of 0-8 ng / mL. Dilutions of DS buffer alone were prepared in duplicate as negative controls. Bound NS1 was detected with anti-NS1 HRP-conjugate, and the absorbance (A) of the wells with DS buffer alone was compared. 450 -A 630 ) minus the absorbance measured in the wells containing the matched DS samples. The results of the sandwich ELISA are shown below in Table 9. Interestingly, NS1 was observed to be co-purified with the vaccine drug substance preparation, suggesting that viral NS1 can be an immunogenic component of a whole inactivated virus vaccine.

[0596] Table 9: NS1 ELISA

[0597]

[0598] We then tested the threshold of neutralizing antibodies (Nab) required for protection against wild-type Zika virus challenge following passive antibody transfer (Tables 10A and B).

[0599] Table 10A: Design of passive transfer study in AG129 mice

[0600]

[0601]

[0602] Table 10B: Timing of passive transfer studies in AG129 mice

[0603]

[0604] Pooled sera from vaccinated and challenged AG129 mice were serially diluted 3-fold in PBS and injected intraperitoneally into 7 groups (N=5 / group) of 5-6 week old AG129 mice. Pre-immune AG129 mouse sera were used as placebo controls (Group 8). After passive transfer (approximately 16-19 hours later), whole blood was collected from each mouse and serum was separated by centrifugation before virus challenge to determine circulating neutralizing antibody titers ( Fig.17). Immediately upon virus challenge, the groups of mice (referred to as Group 1, Group 2, Group 3, Group 4, Group 5, Group 6, Group 7, Group 8) had mean log10 neutralizing antibody titers of 2.69, 2.26, 1.72, 1.30, <1.30, <1.30, <1.30, <1.30, respectively.

[0605] Twenty-four hours after passive transfer of ZIKV nAb, mice were intraperitoneally treated with 10 4 pfu ZIKV PRVABC59 challenge. After challenge, animals were weighed daily and disease signs were monitored 1-3 times a day for 28 days. Based on the symptoms, each animal was given a clinical score (Table 11). Animals that were moribund and / or showed obvious neurological signs (clinical score ≥ 2) were humanely euthanized and counted as non-surviving animals.

[0606] Table 11: Description of clinical scores given, while monitoring morbidity and mortality

[0607]

[0608]

[0609] Disease signs began to appear nine days after challenge in the control group (Group 8) and Groups 5-7, with corresponding weight loss ( Fig.18 Three days after challenge, whole blood was collected from each animal and serum was separated by centrifugation. Serum samples were analyzed for the presence of infectious ZIKV using a plaque titration assay ( Fig.19 The average infection titer of mice in groups 1-8 (log 10 pfu / mL) were 1.66, 2.74, 4.70, 4.92, 7.24, 7.54, 7.54, and 7.46, respectively. Importantly, compared with control mice, groups 1-4 had detectable levels of ZIKV neutralizing antibodies (≥1.30 log 10 ) had statistically significantly lower levels (102.5 to 106.0 times lower titers) of viremia (p = 0.0001, 0.0003, 0.0007, and 0.0374). These results indicate that detectable levels of ZIKV neutralizing antibodies (≥ 1.30 log10) reduce viremia in a dose-dependent manner.

[0610] The median survival days of mice in Groups 1-8 were: indeterminate, day 17, day 17, day 13, day 11, day 11, day 11, and day 10, respectively. Fig. 20Importantly, the survival curves of the groups of mice with detectable ZIKV neutralizing antibody titers (Groups 1-4) were statistically different from those of the control group (Group 8) (p = 0.0019, 0.0019, 0.0019, 0.0153, respectively). These results suggest that detectable levels of ZIKV neutralizing antibodies (≥1.30 log 10 ) delayed the onset of disease in a dose-dependent manner.

[0611] Finally, the ZIKV neutralizing antibody titer of each animal was plotted against its corresponding viremia titer and subjected to linear regression analysis. A highly negative correlation was observed between the ZIKV neutralizing antibody titer and the viremia level on day 3 after challenge ( Fig.21 ). A summary of the passive transfer study results is shown in Table 12 below.

[0612] Table 12: Summary of passive transfer results

[0613]

[0614]

[0615] Although the group of mice that received ZIKAV neutralizing antibodies in this experiment did not completely avoid lethal ZIKAV challenge, it demonstrated a dose-dependent reduction in viremia levels and delayed disease onset in the group of mice with detectable levels of circulating ZIKAV neutralizing antibody titers.

[0616] In summary, the preclinical data from two studies of CD-1 and AG129 mice show that PIZV derived from a separate and well-characterized viral clone is immunogenic and can prevent the attack of wild-type ZIKAV. Importantly, low and high vaccine doses induce similar neutralizing antibody responses after two doses, and provide similar levels of prevention against lethal ZIKAV attacks. Interestingly, mice vaccinated with candidate PIZV without adjuvant also show partial avoidance of ZIKAV attacks. Vaccine antiserum significantly reduces viremia in passively immunized AG129 mice, and prolongs survival for lethal ZIKAV attacks. These results also demonstrate that the candidate PIZV fully characterized in the highly ZIKAV susceptible AG129 mouse model is highly effective against ZIKAV infection.

[0617] In addition, it was found that the sequence of PRVABC59 (from PRVABC59 P6e) at the 7th generation was genetically identical to that of the 6th generation. This is surprising because flaviviruses are generally considered to be genetically unstable. PRVABC59 P6e was selected as the pre-main virus seed, in part because it was genetically stable over 7 generations. Without wishing to be bound by theory, it is believed that this enhanced genetic stability may be due to a single amino acid substitution (W98G) in the wing domain of NS1, as this is the only mutation observed in the PRVABC59 P6 genome adapted to Vero cells. In addition, genetic stability and uniformity are advantageous because they reduce the variability of subsequent strains that can be used for vaccine formulation and increase the reproducible production of these strains.

[0618] Example 3: Preclinical evaluation of the phenotype of P6a and P6e virus strains

[0619] Materials and Methods

[0620] AG129 mice (deficient in interferon α / β and γ receptors) are susceptible to ZIKV infection and disease including severe brain lesions. AG129 mice aged 14 weeks were infected intraperitoneally for 10 4 and 10 3 pfu ZIKV passage 6 clones a and e.

[0621] Mice were weighed daily (up to 28 days) and monitored for clinical signs of disease (weight loss, ruffled fur, hunched posture, lethargy, limb weakness, partial / complete paralysis). In addition, viremia was analyzed by plaque titration of serum samples collected three days after challenge as described in Example 1.

[0622] result

[0623] Pre-infection with MVS P6e caused 100% mortality (median survival time = 12.5 days), while pre-infection with MVS P6a caused only 33% mortality (median survival time = undetermined) ( Fig. 22 ). Consistent with this, mice infected with pre-MVS P6e showed greater weight loss compared with mice infected with PRVABC59 P6a (3). No statistical differences in mean group viremia levels were found between groups of mice infected with PRVABC59 P6a or P6e ( Fig.24 These data suggest that growth kinetics alone may not be the key determinant (as both strains produced similar viremias, and similar peak titers in vitro) and that the characteristics of the envelope protein may be important for virulence (wild-type strains) and immunogenicity (inactivated candidate viruses).

[0624] Example 4: Inactivation Completeness Assay to Determine Inactivation Efficacy

[0625] A dual infectivity assay, also known as the completeness of inactivation (COI) assay, was developed to determine the potency of formalin inactivation (0.01% formaldehyde) and potential residual infectious viral activity of purified inactivated Zika virus (PIZV) bulk drug substance (BDS).

[0626] Sample preparation: Four purified inactivated Zika vaccine (PIZV) batches (Tox batches 1-4) of clone e as described above were made by growing in Vero cells. The supernatant from 4 daily harvests (about 4000 mL in total) was purified by chromatography, followed by the addition of formaldehyde to a final concentration of 0.01% w / v, and inactivated at 22°C for 10 days. During the inactivation period, process control (IPC) samples were removed from bulk drug substance (BDS) every day for characterization and analysis. The daily IPC samples were neutralized with sodium pyrosulfite and dialyzed into DMEM (virus growth medium). The samples contained purified inactivated Zika virus. On the last day of inactivation, the remaining volume of BDS samples was not neutralized, but processed with TFF to remove formaldehyde and the buffer was exchanged into PBS.

[0627] Inactivation completeness assay (COI): The COI assay is used to analyze the inactivation efficacy in daily IPC samples to understand the inactivation kinetics and final BDS. For the highest sensitivity, two cell lines, Vero and C6 / 36, were initially used in this assay to detect potential live viruses in IPC and DS samples. When Zika virus infects Vero cells in the presence of a growth medium containing phenol red, the byproducts of cell death cause the pH to drop. Therefore, the color of the culture medium changes from red / pink to yellow, indicating that this acidic change has occurred in the pH of the culture medium. This phenomenon is caused by apoptosis and cytopathic effect (CPE), which refers to the observed changes in the cell structure of host cells caused by viral invasion, infection, and germination from cells during viral replication. Ultimately, although C6 / 36 mosquitoes and Vero cells are both permissive cell lines for infection, Zika virus infection only kills Vero cells in vitro. Therefore, Vero cells are used as indicator cell lines for determination. In contrast, C6 / 36 cells derived from the natural host carrier of Zika virus do not exhibit CPE and do not dissolve after Zika infection. The medium did not change color and the viability of the C6 / 36 cells was unchanged.

[0628] Therefore, the assay is divided into two parts: the first part of the assay allows parallel amplification of possible live viral particles on 96-well plates of two susceptible cell lines for six days. The second step of the assay involves transferring the supernatant of the 96-well plate (including the particles that may be amplified) to a 6-well plate containing a monolayer of Vero cells and incubating for another 8 days to allow viral infection and cytopathic effects to appear on the Vero cells. Any CPE observed is confirmed using an optical microscope.

[0629] Although detailed using 96-well plates in the first part of the assay, culturing C6 / 36 cells, and six-well plates in the second part of the assay, culturing Vero cells to observe cytopathic effects, the assay is easily scaled up according to the following table:

[0630]

[0631]

[0632] It is evident that during the expansion period, the sample volume per cm2 container was kept constant for part 1 and the same virus infection conditions were maintained in part 2.

[0633] COI Assay Controls: Titer and back titration controls for this assay were performed using Vero indicator cells and were expressed as TCID 50 Scoring was performed in a 96-well format where wells were scored as positive based on a change in media color from pink to yellow as a surrogate for the presence of cell death or CPE.

[0634] Virus titer control test: Two independent replicates of a control virus (PRVABC59) with a known titer were serially diluted 10-fold in medium containing 2% FBS, and 100 μL of each dilution was added to four wells of a 96-well plate containing Vero cells. The plate was incubated for 5 days, and then the wells containing CPE were recorded and the virus titer was calculated using the Reed-Meunch method.

[0635] Virus back titration control test: A control virus of known titer was serially diluted to 200 TCID50. Two independent replicates of the 200 TCID50 control virus were serially diluted 2-fold in medium containing 2% FBS, and 100 μL of each dilution was added to four wells of a 96-well plate containing Vero cells. The cells were incubated for 5 days, and then the wells containing CPE were recorded and the virus titer was calculated using the Reed-Meunch method.

[0636] Detailed COI plan:

[0637] 1. First part of the assay: Two days before the addition of the samples, Vero (1.4E +05 cells / mL) and Aedes aegypti C6 / 36 (4E +05Cells were seeded in 96-well plates at 4% cells / ml. Vero cells were cultured in DMEM + 10% final FBS + 2% L-glutamine + 1% penicillin / streptomycin at 37°C. C6 / 36 cells were cultured in DMEM + 10% FBS + 2% L-glutamine + 1% penicillin / streptomycin + 1% non-essential amino acids at 28°C.

[0638] 2. Dilute 200 TCID50 control virus (prepared in the virus back titration control test) or three independent replicates of DS samples in medium containing 2% FBS (5-fold and 10-fold dilutions).

[0639] 3. Inoculate the cells in the 96-well plate with the samples. Before infection of the cell monolayer in the 96-well plate, spin the samples to break up any possible aggregation. Apply 100 μL of each dilution to each of the 5 wells in two separate 96-well plates containing Vero and C6 / 36 cells, respectively.

[0640] 4. For each cell type, include culture medium alone in another well as a negative CPE control.

[0641] 5. Incubate the plates for 6 days at a temperature appropriate to the cell line.

[0642] 6. Second part of the determination: To further amplify live virus on permissive cell lines and visualize by CPE, the entire volume of each 96-well supernatant from Vero and C6 / 36 cells was transferred to individual wells of a 6-well plate of Vero cells. Inoculation was performed for 90 minutes with shaking at 15-minute intervals.

[0643] 7. Medium containing 2% FBS was added to the wells and the plates were incubated for an additional 8 days for subsequent testing of the amplified samples as a function of CPE. If any replicate of the DS showed CPE at the end of day 8, the inactivation was considered incomplete.

[0644] 7. Visually assess the presence of live / replicating virions by plaque or CPE formation on susceptible cell monolayers after transfer to 6-well plates and incubation for 8 days to allow viral replication. The % CPE score in the 6-well plates at the end of the assay is calculated as follows:

[0645] - Each 6-well plate of Vero cells was checked for CPE by visual inspection of the colorimetric change followed by confirmation of CPE by inspection under an inverted light microscope.

[0646] - Each 6-well plate represents one replicate (5 wells plus one well containing medium alone) of the DS dilutions prepared in the 5-fold and 10-fold dilutions described above.

[0647] Therefore, the %CPE for each replicate reflects the number of wells with CPE out of a total of 5 wells per sample (a total of 120 wells were used for each assay).The average %CPE and standard deviation were calculated based on three replicates for each dilution.

[0648] Results: Daily samples were analyzed in each of Tox batch numbers 1-4 as shown in the table below.

[0649] Table A: Inactivation kinetics, Tox batch number 1

[0650]

[0651]

[0652] Table B: Inactivation kinetics, Tox batch number 2

[0653] sample Transfer Average %CPE STDV 1:10 Day -1 Vero to Vero 100 0 1:10 Day 0 Vero to Vero 100 0 1:10 Day 1 Vero to Vero 100 0 1:10 Day 2 Vero to Vero 0 0 1:10 Day 3 Vero to Vero 0 0 1:10 Day 4 Vero to Vero 0 0 1:10 Day 7 Vero to Vero 0 0 1:10 Day 8 Vero to Vero 0 0 1:10 Day 9 Vero to Vero 0 0 1:10 Day 10 Vero to Vero 0 0 100TCID50 / mL Vero to Vero 100 0 1:10 Day -1 C6 / 36 to Vero 100 0 1:10 Day 0 C6 / 36 to Vero 100 0 1:10 Day 1 C6 / 36 to Vero 100 12 1:10 Day 2 C6 / 36 to Vero 13.3 12 1:10 Day 3 C6 / 36 to Vero 0 0 1:10 Day 4 C6 / 36 to Vero 0 0 1:10 Day 7 C6 / 36 to Vero 0 0 1:10 Day 8 C6 / 36 to Vero 0 0 1:10 Day 9 C6 / 36 to Vero 0 0 1:10 Day 10 C6 / 36 to Vero 0 0 100TCID50 / mL C6 / 36 to Vero 100 0

[0654] Table C: Inactivation kinetics, Tox batch number 3

[0655]

[0656]

[0657] Table D: Inactivation kinetics, Tox batch number 4

[0658]

[0659]

[0660] Compiled inactivation kinetics data: COI data for samples from four toxicology batches were compared to infectious potency (TCID50) and RNA copies determined as described above. RNA copies were determined by purifying nucleic acid from the samples and amplifying Zika RNA using an RT-PCR kit with serotype-specific primers. Fig.25 The results shown in show that the sensitivity of the COI assay significantly exceeds TCID50.

[0661] Performance characteristics of COI assay - Accuracy: Target dilutions (TCID50 / well) and their corresponding CPE ratios were used to determine relative accuracy. For Vero cells, there was a statistically significant linear relationship between the observed and expected positive CPE ratios. The slope of the line correlating the observed and expected results was 0.92, with a 95% confidence interval (CI) of 0.83 to 1.01, overlapping 1, indicating 100% accuracy. For C6 / 36 cells, there was a statistically significant linear relationship between the observed and expected positive CPE. The slope of the line correlating the observed and expected results was 0.88, with a 95% confidence interval (CI) of 0.80 to 0.95, indicating that a slight bias (5-20%) was seen with this cell line. Both cell lines showed satisfactory accuracy (relative).

[0662] COI assay performance characteristics - limit of detection (LoD): Assess the assay sensitivity of C6 / 36 to Vero and Vero to Vero flat plates. As described above, the ratio of positive CPE observed in all wells coated with each titer dilution was fitted using least squares regression, with coating starting at 10.00TCID50 / well down to a lower titer of 0.08TCID50. In addition, for each dilution in the flat plate, a negative control (0.00TCID50 / well) was included. CPE was scored for each dilution on C6 / 36 to Vero and Vero to Vero flat plates. A clear relationship between CPE and log input virus titer was seen. This shows a logarithmic (S-shaped) relationship between the ratio of CPE-positive wells relative to the log10 concentration of TCID50 / well, as well as a 99% lower confidence limit and a 99% upper confidence limit. At -2log10 concentration (=0.01TCID50 / hole), the model based on and accounting for all fixed and random source variations in the identification data predicts 0.85%, or 0.01 when rounded at 0.01TCID50 / hole, with a 99% confidence lower limit of 0.42%. Because the 99% confidence lower limit does not include zero, the probability that 0.85% CPE holes may be generated by 0TCID50 / hole (i.e., due to noise) is almost immeasurable (<1%). This establishes an assay detection limit of at least 0.01TCID50 / hole for the assay (i.e., the minimum amount of live Zika particles detectable in the sample). That is, when rounded, 1 of 60 holes will be CPE positive, or assuming these parameters, the minimum theoretical proportion of CPE positive detectable in 60 holes will be 1.67% or 0.0167.

[0663] Comparing the relative sensitivity of the cell types (C363 and Vero), C6 / 36 demonstrated that lower dilutions of viral titer could be detected compared to Vero cells, as shown in Figure 2. Fig.26As shown; at the same virus input level (0.31TCID50), the proportion of CPE-positive wells of C6 / 36 was higher than that of Vero cells.

[0664] The lowest virus input value used during this determination and identification period is 0.02TCID50 (-1.61log TCID50). Using the fitting curve of C6 / 36 cells, this produces 0.035 or 3.5% (1 in 28 holes) of the hole that is scored as CPE positive. If the curve is inferred for the minimum operating level of 0.167 or 1.6%, then this is equal to the input level of 0.015TCID50 (-1.82logTCID50). However, when determining the lowest level of the infectious virus reflected by the positive CPE result that can be detected, it is necessary to consider the influence of the determination variance of transmission. This noise is produced by the working stock solution of the input virus. The comparison of the target TCID50 and the back titration calculation show that the TCID50 of the working stock virus exhibits a standard deviation (SD) of 85TCID50 / mL when the target is a reserve TCID50 / mL concentration of 200, which is derived from a mean value of 213. Calculating %CV is about 40%, which deviates from about +7%. This noise can be factored into the logarithmic regression model to generate confidence intervals around the target value of the virus dilution. At a target value of 0.01 TCID50 / well, a model based on and accounting for all fixed and random sources in the identification data at both sites predicts that 0.86% of the wells will be CPE positive (1 in 60 wells). Because the 99% lower confidence limit does not include zero, the probability that 0.85% CPE positive wells may be generated by 0 TCID50 / well due to noise is almost incalculable (<1%) ( Fig. 27 ). This established the following assay detection limit for the assay: 0.01 TCID50 / well was the lowest amount of live Zika particles detectable in the sample.

[0665] Performance Characteristics of the COI Assay - Range: The range of the assay is 0.01 TCID50 / well to 4.5 TCID50 / well and is defined as the range of input virus that elicits a CPE positive proportion score greater than 0% but less than 100%.

[0666] Conclusion: Analysis of four Tox revealed complete inactivation after 10 days of incubation in 0.01% formaldehyde at room temperature. In all batches produced, inactivation was achieved by day 3-4 as measured by the COI assay. The COI assay is more sensitive than TCID50 potency or RNA measurements; increased sensitivity was also observed by LoD.

[0667] Example 5: Determination of residual formalin content in a pharmaceutical composition

[0668] 1. Materials and Methods

[0669] 1.1 Materials

[0670] Formaldehyde standard solution (in methanol) (982 μg / mL), DNPH, HPLC grade acetonitrile, and phosphoric acid were purchased from Wako Pure Chemicals (Tokyo, Japan). Distilled water for diluting phosphoric acid was obtained from Otsuka Pharmaceutical (Tokushima, Japan). Aluminum hydroxide gel was obtained from Brenntag (Frederikssund, Denmark). 2% (corresponding to 10 mg / mL aluminum). PBS is prepared in-house, and the Zika vaccine drug product containing aluminum hydroxide gel is manufactured as described below. Zika virus is purified by various techniques after harvesting. After inactivation with formaldehyde, the virus is concentrated and the buffer is exchanged for PBS by filtration. The bulk drug substance is diluted with PBS and formulated with aluminum hydroxide gel (0.4 mg / mL aluminum) to form the final drug product.

[0671] 1.2 HPLC conditions

[0672] A Waters HPLC system equipped with a UV detector (Milford, USA) and a reverse phase column (YMC-Pack ODS-A, 4.6 mm × 250 mm, 5 μm (Kyoto, Japan)) was used. A mixture of water and acetonitrile (1:1, v / v) was used as the mobile phase, the detection wavelength was set at 360 nm, and the flow rate was 1.0 mL / min. The column temperature and injection volume were 25 ° C and 50 μL, respectively.

[0673] 1.3 Sample preparation

[0674] The vaccine drug (1.2 mL) was centrifuged at 15000 rpm for 10 minutes, and the supernatant (1 mL) was transferred to a 2 mL HPLC glass vial purchased from Waters (Milford, USA). Then, 20 μL of 20% (v / v) phosphoric acid and 50 μL of 1.0 mg / mL DNPH solution in acetonitrile were added, and the mixture was stirred and allowed to stand at room temperature for 20 minutes before injection.

[0675] 1.4 Method Validation

[0676] The method was validated based on sample specificity, linearity, accuracy, repeatability, intermediate precision, robustness and stability according to ICH Q2 guidelines. In the accuracy study, Zika vaccine drug substance and aluminum hydroxide gel solution were spiked with a specific amount of formaldehyde and the samples were mixed thoroughly by vortexing and then tested according to the procedure described in Section 2.3.

[0677] 2. Results and Discussion

[0678] 2.1 Linearity and specificity

[0679] Six standard solutions of formaldehyde (0.049, 0.098, 0.196, 0.491, 0.982, and 1.964 μg / mL) were prepared by diluting with PBS. Then, 20% (v / v) phosphoric acid and 1 mg / mL DNPH in acetonitrile were added to each solution and the mixture was stirred for 2 h. Fig.28 The corresponding chromatogram is shown in Figure 1. Obviously, the peak area at 10.4 minutes shows linearity with the following regression equation: y=1075730x+11731 (where y is the peak area at 10.4 minutes and x is the formaldehyde concentration in μg / mL) (correlation coefficient: 0.9998), which indicates that it is attributed to HCHO-DNPH (i.e., formaldehyde derivatized with DNPH). In addition, the peak at 5.8 minutes is attributed to DNPH because this peak is detected in all samples with DNPH added. Therefore, the HCHO-DNPH peak area is used to evaluate linearity and accuracy after subtracting the background peak area in PBS.

[0680] 2.2 Accuracy and Precision (Repeatability)

[0681] The effect of aluminum hydroxide adjuvant was evaluated by recovery studies, which were performed by adding 0.05 μg / mL formaldehyde to three samples of aluminum hydroxide in PBS (0.1, 0.4 and 1.0 mg / mL aluminum) in the absence of vaccine bulk drug. The average recovery was 102% (n=3), 100% (n=3) and 100% (n=3), respectively, with low relative standard deviation (RSD) values ​​(Table 13). The RSD of the accuracy data was calculated to evaluate repeatability, and it was found to be 1.0%, which indicates that aluminum amounts up to 1.0 mg / mL do not interfere with the recovery of formaldehyde.

[0682] Table 13 Accuracy and repeatability evaluated using aluminum hydroxide samples spiked with 0.05 μg / mL formaldehyde

[0683]

[0684] The accuracy of the method was evaluated by recovery studies conducted by spiking the Zika vaccine drug product with aluminum hydroxide adjuvant with three concentrations of formaldehyde (0.05, 0.10 and 1.00 μg / mL) and the average recovery results are shown in Table 14. The RSD of the accuracy data was calculated to assess repeatability and was found to be 3.7%, indicating that the Zika vaccine drug product formulated with aluminum hydroxide did not interfere with the recovery of formaldehyde between 0.05 and 1.00 μg / mL.

[0685] Table 14 Accuracy and reproducibility of Zika vaccine drug product evaluation using added formaldehyde containing aluminum hydroxide

[0686]

[0687]

[0688] 2.4 Stability

[0689] The robustness of the method was evaluated to determine how the formaldehyde concentration in the sample was affected by changes in the experimental parameters during sample preparation. The concentrations of DNPH and phosphoric acid were selected as monitoring parameters in this study in view of the effect on the efficacy of derivatization. The effects were examined by varying the concentrations of DNPH and phosphoric acid by ±0.1 mg / mL and ±5%, respectively. Formaldehyde was determined under each condition in two development drug product batches, and the results shown in Table 15 indicate that changes in DNPH and phosphoric acid concentrations had no significant effect on the determination of formaldehyde.

[0690] Table 15 Robustness of the method

[0691]

[0692] (*) Required conditions of the method Sequence Listing <110> Takeda Vaccine Co., Ltd. The United States of America, represented by the Secretary of Health and Human Services <120> Methods for inactivating Zika virus and related methods <130> 741256 <150> US 62 / 592995 <151> 2017-11-30 <150> PCT / US2018 / 059227 <151> 2018-11-05 <160> 27 <170> PatentIn version 3.5 <210> 1 <211> 351 <212> PRT <213> Zika virus <400> 1 Asp Val Gly Cys Ser Val Asp Phe Ser Lys Lys Glu Thr Arg Cys Gly 1 5 10 15 Thr Gly Val Phe Val Tyr Asn Asp Val Glu Ala Trp Arg Asp Arg Tyr 20 25 30 Lys Tyr His Pro Asp Ser Pro Arg Arg Leu Ala Ala Ala Val Lys Gln 35 40 45 Ala Trp Glu Asp Gly Ile Cys Gly Ile Ser Ser Val Ser Arg Met Glu 50 55 60 Asn Ile Met Trp Arg Ser Val Glu Gly Glu Leu Asn Ala Ile Leu Glu 65 70 75 80 Glu Asn Gly Val Gln Leu Thr Val Val Val Gly Ser Val Lys Asn Pro 85 90 95 Met Trp Arg Gly Pro Gln Arg Leu Pro Val Pro Val Asn Glu Leu Pro 100 105 110 His Gly Trp Lys Ala Trp Gly Lys Ser Tyr Phe Val Arg Ala Ala Lys 115 120 125 Thr Asn Asn Ser Phe Val Val Asp Gly Asp Thr Leu Lys Glu Cys Pro 130 135 140 Leu Lys His Arg Ala Trp Asn Ser Phe Leu Val Glu Asp His Gly Phe 145 150 155 160 Gly Val Phe His Thr Ser Val Trp Leu Lys Val Arg Glu Asp Tyr Ser 165 170 175 Leu Glu Cys Asp Pro Ala Val Ile Gly Thr Ala Val Lys Gly Lys Glu 180 185 190 Ala Val His Ser Asp Leu Gly Tyr Trp Ile Glu Ser Glu Lys Asn Asp 195 200 205 Thr Trp Arg Leu Lys Arg Ala His Leu Ile Glu Met Lys Thr Cys Glu 210 215 220 Trp Pro Lys Ser His Thr Leu Trp Thr Asp Gly Ile Glu Glu Ser Asp 225 230 235 240 Leu Ile Ile Pro Lys Ser Leu Ala Gly Pro Leu Ser His His Asn Thr 245 250 255 Arg Glu Gly Tyr Arg Thr Gln Met Lys Gly Pro Trp His Ser Glu Glu 260 265 270 Leu Glu Ile Arg Phe Glu Glu Cys Pro Gly Thr Lys Val His Val Glu 275 280 285 Glu Thr Cys Gly Thr Arg Gly Pro Ser Leu Arg Ser Thr Thr Ala Ser 290 295 300 Gly Arg Val Ile Glu Glu Trp Cys Cys Arg Glu Cys Thr Met Pro Pro 305 310 315 320 Leu Ser Phe Arg Ala Lys Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg 325 330 335 Pro Arg Lys Glu Pro Glu Ser Asn Leu Val Arg Ser Met Val Thr 340 345 350 <210> 2 <211> 10675 <212> DNA <213> Zika virus <400> 2 gttgttgatc tgtgtgaatc agactgcgac agttcgagtt tgaagcgaaa gctagcaaca 60 gtatcaacag gttttatttt ggatttggaa acgagagttt ctggtcatga aaaacccaaa 120 aaagaaatcc ggaggattcc ggattgtcaa tatgctaaaa cgcggagtag cccgtgtgag 180 cccctttggg ggcttgaaga ggctgccagc cggacttctg ctgggtcatg ggcccatcag 240 gatggtcttg gcgattctag cctttttgag attcacggca atcaagccat cactgggtct 300 catcaataga tggggttcag tggggaaaaa agaggctatg gaaacaataa agaagttcaa 360 gaaagatctg gctgccatgc tgagaataat caatgctagg aaggagaaga agagacgagg 420 cgcagatact agtgtcggaa ttgttggcct cctgctgacc acagctatgg cagcggaggt 480 cactagacgt gggagtgcat actatatgta cttggacaga aacgatgctg gggaggccat 540 atcttttcca accacattgg ggatgaataa gtgttatata cagatcatgg atcttggaca 600 catgtgtgat gccaccatga gctatgaatg ccctatgctg gatgaggggg tggaaccaga 660 tgacgtcgat tgttggtgca acacgacgtc aacttgggtt gtgtacggaa cctgccatca 720 caaaaaaggt gaagcacgga gatctagaag agctgtgacg ctcccctccc attccaccag 780 gaagctgcaa acgcggtcgc aaacctggtt ggaatcaaga gaatacacaa agcacttgat 840 tagagtcgaa aattggatat tcaggaaccc tggcttcgcg ttagcagcag ctgccatcgc 900 ttggcttttg ggaagctcaa cgagccaaaa agtcatatac ttggtcatga tactgctgat 960 tgccccggca tacagcatca ggtgcatagg agtcagcaat agggactttg tggaaggtat 1020 gtcaggtggg acttgggttg atgttgtctt ggaacatgga ggttgtgtca ccgtaatggc 1080 acaggacaaa ccgactgtcg acatagagct ggttacaaca acagtcagca acatggcgga 1140 ggtaagatcc tactgctatg aggcatcaat atcagacatg gcttctgaca gccgctgccc 1200 aacacaaggt gaagcctacc ttgacaagca atcagacact caatatgtct gcaaaagaac 1260 gttagtggac agaggctgggg gaatggatg tggacttttt ggcaaaggga gcctggtgac 1320 atgcgctaag tttgcatgct ccaagaaaat gaccgggaag agcatccagc cagagaatct 1380 ggagtaccgg ataatgctgt cagttcatgg ctcccagcac agtgggatga tcgttaatga 1440 1500 agccgaagcc accctgggg gttttggaag cctaggactt gattgtgaac cgaggacagg 1560 ccttgacttt tcagatttgt attacttgac tatgaataac aagcactggt tggttcacaa 1620 ggagtggttc cacgacattc cattaccttg gcacgctggg gcagacaccg gaactccaca 1680 ctggaacaac aaagaagcac tggtagagtt caaggacgca catgccaaaa ggcaaactgt 1740 cgtggttcta gggagtcaag aaggagcagt tcacacggcc cttgctggag ctctggaggc 1800 tgagatggat ggtgcaaagg gaaggctgtc ctctggccac ttgaaatgtc gcctgaaaat 1860 ggataaactt agattgaagg gcgtgtcata ctccttgtgt actgcagcgt tcacattcac 1920 caagatcccg gctgaaacac tgcacgggac agtcacagtg gaggtacagt acgcagggac 1980 agatggacct tgcaaggttc cagctcagat ggcggtggac atgcaaactc tgaccccagt 2040 tgggaggttg ataaccgcta accccgtaat cactgaaagc actgagaact ctaagatgat 2100 gctgggaactt gatccaccat ttggggactc ttacattgtc ataggagtcg gggagaagaa 2160 gatcacccac cactggcaca ggagtggcag caccattgga aaagcatttg aagccactgt 2220 gagaggtgcc aagagaatgg cagtcttggg agacacagcc tgggactttg gatcagttgg 2280 aggcgctctc aactcattgg gcaagggcat ccatcaaatt tttggagcag ctttcaaatc 2340 attgtttgga ggaatgtcct ggttctcaca aattctcatt ggaacgttgc tgatgtggtt 2400 gggtctgaac acaaagaatg gatctatttc ccttatgtgc ttggccttag ggggagtgtt 2460 gatcttctta tccacagccg tctctgctga tgtggggtgc tcggtggact tctcaaagaa 2520 ggagacgaga tgcggtacag gggtgttcgt ctataacgac gttgaagcct ggagggacag 2580 gtacaagtac catcctgact ccccccgtag attggcagca gcagtcaagc aagcctggga 2640 agatggtatc tgcgggatct cctctgtttc aagaatggaa aacatcatgt ggagatcagt 2700 agaaggggag ctcaacgcaa tcctggaaga gatggagtt siactgacgg tcgttgtggg 2760 atctgtaaaa aacccatgt ggagaggtcc acagagattg cccgtgcctg tgaacgagct 2820 gccccacggc tggaggctt gggggaaatc gtatttcgtc agagcagcaa agacaataa 2880 cagctttgtc gtggatggtg acacactgaa ggaatgccca ctcaacata gagcatggaa 2940 cagctttctt gtggaggatc atgggttcgg ggtatttcac actagtgtct ggctcaggt 3000 taggagagat tattcattag agtgtgatcc agccgttatt ggaacagctg ttaagggaaa 3060 ggaggctgta cacagtgatc taggctactg gattgagagt gagagaatg acacatggag 3120 gctgaagagg gcccatctga tcgagatgaa aacatgtgaa tggccaagt cccacacatt 3180 gtggacagat ggaatagaag agagtctct gatcataccc aagtctttag ctgggccact 3240 cagccatcac ataccagag agggctacag gacccaatg aaagggccat ggcacagtga 3300 agagcttgaa attcggttg aggaatgccc agcactaag gtccacgtgg aggaaacatg 3360 tggaacaaga ggaccatctc tgagatcac cactgcaagc ggaagggtga tcgaggatg 3420 gtgctgcagg gagtgcacaa tgccccact gtcgttccgg gctaaagatg gctgttggta 3480 tggaatggag ataggccca ggaaagaacc agaaagcaac ttagtaaggt caatggtgac 3540 tgcaggatca actgatcaca tggaccactt ctcccttgga gtgcttgtga tcctgctcat 3600 ggtgcaggaa gggctgaaga agaatgac cacaaagatc atcataagca catcaatggc 3660 agtgctggta gctatgatcc tgggaggatt ttcaatgagt gacctggcta agcttgcaat 3720 3780 gatagcggca ttcaaagtca gaccagcgtt gctggtatct ttcatcttca gagctaattg 3840 gacaccccgt gaaagcatgc tgctggcctt ggcctcgtgt cttttgcaaa ctgcgatctc 3900 cgccttggaa ggcgacctga tggttctcat caatggtttt gctttggcct ggttggcaat 3960 acgagcgatg gttgttccac gcactgataa catcaccttg gcaatcctgg ctgctctgac 4020 accactggcc cggggcacac tgcttgtggc gtggagagca ggccttgcta cttgcgggg 4080 gtttatgctc ctctctctga agggaaaagg cagtgtgaag aagaacttac catttgtcat 4140 ggccctggga ctaaccgctg tgaggctggt cgaccccatc aacgtggtgg gactgctgtt 4200 gctcacaagg agtgggaagc ggagctggcc ccctagcgaa gtactcacag ctgttggcct 4260 gatatgcgca ttggctggag ggttcgccaa ggcagatata gagatggctg ggcccatggc 4320 cgcggtcggt ctgctaattg tcagttacgt ggtctcagga aagagtgtgg acatgtacat 4380 tgaaagagca ggtgacatca catgggaaaa agatgcggaa gtcactggaa acagtccccg 4440 gctcgatgtg gcgctagatg agagtggtga tttctccctg gtggaggatg acggtccccc 4500 catgagagag atcatactca aggtggtcct gatgaccatc tgtggcatga acccaatagc 4560 catacccttt gcagctggag cgtggtacgt atacgtgaag actggaaaaa ggagtggtgc 4620 tctatgggat gtgcctgctc ccaaggaagt aaaaaagggg gagaccacag atggagtgta 4680 cagagtaatg actcgtagac tgctaggttc aacacaagtt ggagtgggag ttatgcaaga 4740 gggggtcttt cacactatgt ggcacgtcac aaaaggatcc gcgctgagaa gcggtgaagg 4800 gagacttgat ccatactggg gagatgtcaa gcaggatctg gtgtcatact gtggtccatg 4860 gaagctagat gccgcctggg atgggcacag cgaggtgcag ctcttggccg tgccccccgg 4920. agagagagcg aggaacatcc agactctgcc cggaatattt aagacaaagg atggggacat tggagcggtt gcgctggatt acccagcagg aacttcagga tctccaatcc tagacaagtg tgggagagtg atggcttt atggcaatgg ggtcgtgatc aaaaacggga gttatgttag tgccatcacc caagggagga gggaggaga gactcctgtt gagtgcttcg agccctcgat 5160 gctgaagaag aagcagctaa ctgtcttaga cttgcatcct ggagctgggga aaaccaggag agttcttcct gaatagtcc gtgaagccat aaaaacaaga ctccgtactg tgatcttagc tccaaccagg gttgtcgctg ctgaatgga ggaggccctt agagggcttc cagtgcgtta 5400. gcagtcaatg tcacccactc tggacaga atcgtcgact taatgtgcca tgccaccttc acttcacgtc tactacagcc aatcagagtc cccaactata atctgtatat 5520. gcccacttca cagatccctc aagtatagca gcaagaggat acatttcaac aagggttgag atgggcgagg cggctgccat cttcatgacc gccacgccac caggaacccg 5580. tgacgcattt ccggactcca actcaccaat tatggacacc gaagtggaag tcccagagag agcctggagc tcaggctttg attgggtgac ggatcattct ggaaaaacag tttggtttgt 5700. tccaagcgtg aggaacggca atgagatcgc agcttgtctg acaaaggctg gaaaacgggt catacagctc agcagaaaga cttttgagac agagttccag aaaacaaaac atcaagagtg ggactttgtc gtgacaactg acatttcaga gatgggcgcc aactttaag ctgaccgtgt catagattcc aggagatgcc taaagccggt catacttgat ggcgagagag tcattctggc 5940 tggacccatg cctgtcacac atgccagcgc tgcccagagg agggggcgca taggcagga tcccaacaaa cctggagatg agtatctgta tggaggtggg tgcgcagaga ctgacgaaga 6060 ccatgcacac tggcttgaag cagaatgct ccttgacaat atttacctcc aagatggcct catagcctcg ctctatcgac ctgaggccga caaagtagca gccattgagg gagagttcaa gcttaggacg gagcaaagga agacctttgt ggaactcatg aaaagaggag atcttcctgt 6240 ttggctggcc tatcaggttg catctgccgg grandfather acagataga gatggtgctt tgatggcacg accaacaaca ccataatgga agacagtgtg ccggcagagg tgtggaccag 6360 acacggagag aaaagagtgc tcaaaccgag gtggatggac gccagagttt gttcagatca 6420 tgcggccctg aagtcattca aggagtttgc cgctgggaaa agaggagcgg cttttggagt 6480 gatggaagcc ctgggaacac tgccaggaca catgacagag agattccagg aagccattga 6540 caacctcgct gtgctcatgc gggcagagac tggaagcagg ccttacaaag ccgcggcggc 6600 ccaattgccg gagaccctag agaccataat gcttttgggg ttgctgggaa cagtctcgct 6660 gggaatcttc ttcgtcttga tgaggaacaa gggcataggg aagatgggct ttggaatggt 6720 gactcttggg gccagcgcat ggctcatgtg gctctcggaa attgagccag ccagaattgc 6780 atgtgtcctc attgttgtgt tcctattgct ggtggtgctc atacctgagc cagaaaagca 6840 aagatctccc caggacaacc aaatggcaat catcatcatg gtagcagtag gtcttctggg 6900 cttgattacc gccaatgaac tcggatggtt ggagagaaca aagagtgacc taagccatct 6960 aatgggaagg agagaggagg gggcaaccat aggattctca atggacattg acctgcggcc 7020 agcctcagct tgggccatct atgctgcctt gacaactttc attaccccag ccgtccaaca 7080 tgcagtgacc acctcataca acaactactc cttaatggcg atggccacgc aagctggagt 7140 gttgtttggc atgggcaaag ggatgccatt ctacgcatgg gactttggag tcccgctgct 7200 aatgataggt tgctactcac aattaacacc cctgacccta atagtggcca tcattttgct 7260 cgtggcgcac tacatgtact tgatcccagg gctgcaggca gcagctgcgc gtgctgccca 7320 gaagagaacg gcagctggca tcatgaagaa ccctgttgtg gatggaatag tggtgactga 7380 cattgacaca atgacaattg acccccaagt ggagaaaaag atgggacagg tgctactcat 7440 agcagtagcc gtctccagcg catactgtc gcggaccgcc tgggggtggg gggaggctgg 7500 ggctctgatc acagccgcaa cttccacttt gtgggaaggc tctccgaaca agtactggaa 7560 ctcctctaca gccacttcac tgtgtaacat ttttagggga agttacttgg ctggagcttc 7620 tctaatctac acagtaacaa gaaacgctgg cttggtcaag agacgtgggg gtggaacagg 7680 agagaccctg ggagagaaat ggaaggcccg cttgaaccag atgtcggccc tggagttcta 7740 ctcctacaaa aagtcaggca tcaccgaggt gtgcagaga gaggcccgcc gcgccctcaa ggacggtgtg gcaacggggag gccatgctgt gtcccgagga agtgcaaagc tgagatggtt 7860. ggtggagcgg ggatacctgc agccctatgg aaaggtcatt gatcttggat gtggcagagg 7920 gggctggagt tactacgtcg ccaccatccg caaagttcaa gaagtgaaag gatacacaaa aggaggccct ggtcatgaag aacccgtgtt ggtgcaaagc tatgggtgga acatagtccg 8040. tcttaagagt ggggtggacg tctttcatat ggcggctgag ccgtgtgaca cgttgctgtg 8100 tgacataggt gagtcatcat ctagtcctga agtggaaga gcacggacgc tcagagtcct ctccatggtg ggggattggc ttgaaaaag accaggagcc ttttgtata aagtgttgtg 8220. cccatacacc agcactatga tggaaaccct ggagcgactg cagcgtaggt atgggggagg 8280. actggtcaga gtgccactct cccgcaactc tacacatgag atgtactggg tctctggagc gaaaagcaac accataaaaa gtgtgtccac cacgagccag ctcctcttgg ggcgcatgga cgggcctagg aggccagtga aatatgagga ggatgtgaat ctcggctctg gcacgcgggc 8460 tgtggtaagc tgcgctgaag ctcccacat gagatcatt ggtaccgca ttgaaggat 8520 ccgcagtgag cacgcggaaa cgtggttctt tgacgagac cacccatata ggacatgggc 8580 ttaccatgga agctatgagg ccccacaca agggtcagcg tcctctta taaacggggt 8640 tgtcaggctc ctgtcaaac cctgggatgt ggtgactgga gtcacaggaa tagccatgac 8700 cgacaccaca ccgtatggtc agcaaggt ttcaggaa aaagtggaca ctagggtgcc 8760 agacccccaa gaaggcactc gtcaggttat gagcatgtc tctcctggt tgtggaaaga 8820 gctaggcaaa cacaacggc cacgagtctg caccaaagaa gagttcatca acaaggttcg 8880 tagcaatgca gcattagggg caatttga agaggaaaaa gagtggaga ctgcagtgga 8940 agctgtgaac gatccaggt tctgggctct agtggacaag gaagagagc accacctgag 9000 aggagtgc cagagctgtg tgtacacat gatgggaaaa aggaaaaga aaaaggga 9060 atttggaaag gccaagggca gccgcgccat ctggtatatg tggctagggg ctagatttct 9120 agagttcgaa gcccttggat tctgaacga ggatcactgg atgggag agaaccagg 9180 aggtggtgtt gaagggctgg gattacaaag actcggatat gtcctagaag agatgagtcg 9240 tataccagga ggaaggatgt atgcagatga cactgctggc tgggacaccc gcattagcag 9300 gtttgatctg gagaatgaag ctctaatcac caaccaaatg gagaaagggc acagggcctt 9360 ggcattggcc ataatcaagt acacatacca aaacaaagtg gtaaaggtcc ttagaccagc 9420 tgaaaaaggg aaaacagtta tggacattat ttcgagacaa gaccaaaggg ggagcggaca 9480 agttgtcact tacgctctta acacatttac caacctagtg gtgcaactca ttcggaatat 9540 ggaggctgag gaagttctag agatgcaaga cttgtggctg ctgcggaggt cagagaaagt 9600 gaccaactgg ttgcagagca acggatggga taggctcaaa cgaatggcag tcagtggaga 9660 tgattgcgtt gtgaagccaa ttgatgatag gtttgcacat gccctcaggt tcttgaatga 9720 tatgggaaaa gttaggaagg acacacaaga gtggaaaccc tcaactggat gggacaactg 9780 ggaagaagtt ccgttttgct cccaccactt caacaagctc catctcaagg acgggaggtc 9840 cattgtggtt ccctgccgcc accaagatga actgattggc cgggcccgg tctctccagg 9900 ggcgggatgg agcatccggg agactgcttg cctagcaaaa tcatatgcgc aaatgtggca 9960 gctcctttat ttccacagaa gggacctccg actgatggcc aatgccattt gttcatctgt 10020 gccagttgac tgggttccaa ctgggagaac tacctggtca atccatggaa agggagaatg 10080 gatgaccact gaagacatgc ttgtggtgtg gaagagagtg tggattgagg agaacgacca 10140 catggagaac aagaccccag ttacgaaatg gacagacatt ccctatttgg gaaaaaggga 10200 agacttgtgg tgtggatctc tcatagggca cagaccgcgc accacctggg ctgagaacat 10260 taaaaacaca gtcaacatgg tgcgcaggat cataggtgat gaagaaaagt acatggacta 10320 cctatccacc caagttcgct acttgggtga agaagggtct acacctggag tgctgtaagc 10380 accaatctta atgttgtcag gcctgctagt cagccacagc ttgggggaaag ctgtgcagcc 10440 tgtgacccccc ccaggagaag ctgggaaacc aagcctatag tcaggccgag aacgccatgg 10500 cacggaagaa gccatgctgc ctgtgagccc ctcagaggac actgagtcaa aaaaccccac 10560 gcgcttggag gcgcaggatg ggaaaagaag gtggcgacct tccccaccct tcaatctggg 10620 gcctgaactg gagatcagct gtggatctcc agaagaggga ctagtggtta gagga 10675 <210> 3 <211> 20 <212> DNA <213> Artificial <220> <223> Oligonucleotide 1 (CpG 1826) <400> 3 tccatgacgt tcctgacgtt 20 <210> 4 <211> 18 <212> DNA <213> Artificial <220> <223> Oligonucleotide 2 (CpG 1758) <400> 4 tctcccagcg tgcgccat 18 <210> 5 <211> 30 <212> DNA <213> Artificial <220> <223> Oligonucleotide 3 <400> 5 accgatgacg tcgccggtga cggcaccacg 30 <210> 6 <211> twenty four <212> DNA <213> Artificial <220> <223> Oligonucleotide 4 (CpG 2006) <400> 6 tcgtcgtttt gtcgttttgt cgtt 24 <210> 7 <211> 20 <212> DNA <213> Artificial <220> <223> Oligonucleotide 5 (CpG 1668) <400> 7 tccatgacgt tcctgatgct 20 <210> 8 <211> 31 <212> PRT <213> Zika virus <400> 8 Phe Thr Lys Ile Pro Ala Glu Thr Leu His Gly Thr Val Thr Val Glu 1 5 10 15 Val Gln Tyr Ala Gly Thr Asp Gly Pro Cys Lys Val Pro Ala Gln 20 25 30 <210> 9 <211> 31 <212> PRT <213> Zika virus <400> 9 Phe Thr Lys Ile Pro Ala Glu Thr Leu His Gly Thr Val Thr Val Glu 1 5 10 15 Val Gln Tyr Ala Gly Thr Asp Gly Pro Cys Lys Val Pro Ala Gln 20 25 30 <210> 10 <211> 31 <212> PRT <213> West Nile Virus <400> 10 Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr Val Val Leu Glu 1 5 10 15 Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys Lys Val Pro Ile Ser 20 25 30 <210> 11 <211> 31 <212> PRT <213> Japanese Encephalitis Virus <400> 11 Phe Ala Lys Asn Pro Ala Asp Thr Gly His Gly Thr Val Val Ile Glu 1 5 10 15 Leu Thr Tyr Ser Gly Ser Asp Gly Pro Cys Lys Ile Pro Ile Val 20 25 30 <210> 12 <211> 31 <212> PRT <213> Saint Louis Encephalitis Virus <400> 12 Phe Ser Lys Asn Pro Ala Asp Thr Gly His Gly Thr Val Ile Val Glu 1 5 10 15 Leu Gln Tyr Thr Gly Ser Asn Gly Pro Cys Arg Val Pro Ile Ser 20 25 30 <210> 13 <211> 30 <212> PRT <213> Yellow Fever Virus <400> 13 Phe Val Lys Asn Pro Thr Asp Thr Gly His Gly Thr Val Val Met Gln 1 5 10 15 Val Lys Val Ser Lys Gly Ala Pro Cys Arg Ile Pro Val Ile 20 25 30 <210> 14 <211> 31 <212> PRT <213> Dengue virus <400> 14 Leu Glu Lys Glu Val Ala Glu Thr Gln His Gly Thr Val Leu Val Gln 1 5 10 15 Val Lys Tyr Glu Gly Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser 20 25 30 <210> 15 <211> 31 <212> PRT <213> Dengue virus <400> 15 Val Val Lys Glu Ile Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg 1 5 10 15 Val Gln Tyr Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu 20 25 30 <210> 16 <211> 31 <212> PRT <213> Dengue virus <400> 16 Leu Lys Lys Glu Val Ser Glu Thr Gln His Gly Thr Ile Leu Ile Lys 1 5 10 15 Val Glu Tyr Lys Gly Glu Asp Ala Pro Cys Lys Ile Pro Phe Ser 20 25 30 <210> 17 <211> 31 <212> PRT <213> Dengue virus <400> 17 Ile Asp Lys Glu Met Ala Glu Thr Gln His Gly Thr Thr Val Val Lys 1 5 10 15 Val Lys Tyr Glu Gly Ala Gly Ala Pro Cys Lys Val Pro Ile Glu 20 25 30 <210> 18 <211> 29 <212> PRT <213> Zika virus <400> 18 Ser Val Lys Asn Pro Met Gly Arg Gly Pro Gln Arg Leu Pro Val Pro 1 5 10 15 Val Asn Glu Leu Pro His Gly Trp Lys Ala Trp Gly Lys 20 25 <210> 19 <211> 29 <212> PRT <213> Zika virus <400> 19 Ser Val Lys Asn Pro Met Trp Arg Gly Pro Gln Arg Leu Pro Val Pro 1 5 10 15 Val Asn Glu Leu Pro His Gly Trp Lys Ala Trp Gly Lys 20 25 <210> 20 <211> 29 <212> PRT <213> West Nile Virus <400> 20 Lys Gln Glu Gly Met Tyr Lys Ser Ala Pro Lys Arg Leu Thr Ala Thr 1 5 10 15 Thr Glu Lys Leu Glu Ile Gly Trp Lys Ala Trp Gly Lys 20 25 <210> twenty one <211> 29 <212> PRT <213> Japanese Encephalitis Virus <400> twenty one Lys Pro Val Gly Arg Tyr Arg Ser Ala Pro Lys Arg Leu Ser Met Thr 1 5 10 15 Gln Glu Lys Phe Glu Met Gly Trp Lys Ala Trp Gly Lys 20 25 <210> twenty two <211> 29 <212> PRT <213> Saint Louis Encephalitis Virus <400> twenty two Glu Asp Pro Lys Tyr Tyr Lys Arg Ala Pro Arg Arg Leu Lys Lys Leu 1 5 10 15 Glu Asp Glu Leu Asn Tyr Gly Trp Lys Ala Trp Gly Lys 20 25 <210> twenty three <211> 29 <212> PRT <213> Yellow Fever Virus <400> twenty three Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro Phe Ser Arg Ile 1 5 10 15 Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp Gly Lys 20 25 <210> twenty four <211> 29 <212> PRT <213> Dengue virus <400> twenty four Asp Val Ser Gly Ile Leu Ala Gln Gly Lys Lys Met Ile Arg Pro Gln 1 5 10 15 Pro Met Glu His Lys Tyr Ser Trp Lys Ser Trp Gly Lys 20 25 <210> 25 <211> 29 <212> PRT <213> Dengue virus <400> 25 Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg Pro Gln 1 5 10 15 Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys 20 25 <210> 26 <211> 29 <212> PRT <213> Dengue virus <400> 26 Asp Ile Thr Gly Val Leu Glu Gln Gly Lys Arg Thr Leu Thr Pro Gln 1 5 10 15 Pro Met Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys 20 25 <210> 27 <211> 29 <212> PRT <213> Dengue virus <400> 27 Asp Val Lys Gly Val Leu Thr Lys Gly Lys Arg Ala Leu Thr Pro Pro 1 5 10 15 Val Asn Asp Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys 20 25

Claims

1. A method for determining the residual formaldehyde content in a pharmaceutical composition comprising inactivated Zika virus, the method comprising the following steps: (a) providing a composition comprising a Zika virus that has been treated with formaldehyde and an adjuvant, wherein the adjuvant is aluminum hydroxide having an aluminum concentration of 0.1 mg / ml to 1.0 mg / ml; (b) mixing 50 parts of the composition of (a) with 1 part of 15 to 25% (v / v) phosphoric acid and 2.5 parts of 0.9 to 1.1 mg / ml 2,4-dinitrophenylhydrazine (DNPH), thereby providing a mixture; (c) incubating the mixture of (b) at 20°C-25°C for 10 to 30 minutes; and (d) The mixture was analyzed by reverse phase HPLC for the presence of residual formaldehyde.

2. The method of claim 1, wherein a mixture of water and acetonitrile in a volume ratio of 1:1 is used as the mobile phase in HPLC.

3. The method of claim 1 or 2, wherein the inactivated Zika virus has been treated with 0.01% (w / v) formaldehyde at 22°C for 10 days.

4. A method for inactivating a Zika virus preparation, the method comprising: (a) isolating the Zika virus preparation from one or more in vitro cultured cells, wherein the cells are used to produce the Zika virus preparation, wherein isolating the Zika virus preparation comprises one or more steps selected from the following: (i) Depth filtration, (ii) buffer exchange and / or dilution; (iii) ion exchange chromatography; (b) treating the Zika virus preparation with formaldehyde, wherein the numerical result of the formaldehyde concentration measured in % (w / v) multiplied by the incubation time in formaldehyde measured in days is 0.1; (c) preparing a pharmaceutical composition comprising the inactivated Zika virus preparation, wherein the inactivated Zika virus preparation is mixed with an adjuvant, the adjuvant is aluminum hydroxide, and the pharmaceutical composition comprises aluminum hydroxide at an aluminum concentration of 0.1 mg / ml to 1.0 mg / ml; and (d) determining the residual formaldehyde content in the pharmaceutical composition according to the method of any one of claims 1 to 3.

5. The method of claim 4, wherein the cell is a non-human cell.

6. The method of claim 4 or 5, wherein the cells are Vero cells.

7. The method of any one of claims 4 to 6, wherein the Zika virus preparation is obtained from an inoculum containing a heterogeneous population of Zika viruses.

8. The method of any one of claims 4 to 7, wherein the Zika virus preparation is obtained from a clinical isolate.

9. The method of any one of claims 4 to 8, wherein the Zika virus preparation is obtained from a clonal isolate of Zika virus.

10. The method of claim 9, wherein the Zika virus clonal isolate is obtained by plaque purification.

11. The method of claim 10, wherein the plurality of cells are inoculated with an inoculum containing a heterogeneous population of Zika virus prior to plaque purification.

12. A method for inactivating a Zika virus preparation, the method comprising: (a) obtaining Zika virus preparations from clinical isolates; (b) treating the Zika virus preparation with formaldehyde, wherein the value obtained by multiplying the concentration of formaldehyde measured in % (w / v) by the incubation time in formaldehyde measured in days is 0.1; (c) preparing a pharmaceutical composition comprising the inactivated Zika virus preparation, wherein the inactivated Zika virus preparation is mixed with an adjuvant, the adjuvant is aluminum hydroxide, and the pharmaceutical composition comprises aluminum hydroxide at an aluminum concentration of 0.1 mg / ml to 1.0 mg / ml; and (d) determining the residual formaldehyde content in the pharmaceutical composition according to the method of any one of claims 1 to 3.

13. A method for inactivating a Zika virus preparation, the method comprising: (a) obtaining a Zika virus preparation from an inoculum containing a heterogeneous population of Zika viruses; (b) treating the Zika virus preparation with formaldehyde, wherein the value obtained by multiplying the concentration of formaldehyde measured in % (w / v) by the incubation time in formaldehyde measured in days is 0.1; (c) preparing a pharmaceutical composition comprising the inactivated Zika virus preparation, wherein the inactivated Zika virus preparation is mixed with an adjuvant, the adjuvant is aluminum hydroxide, and the pharmaceutical composition comprises aluminum hydroxide at an aluminum concentration of 0.1 mg / ml to 1.0 mg / ml; and (d) determining the residual formaldehyde content in the pharmaceutical composition according to the method of any one of claims 1 to 3.

14. The method of any one of claims 4 to 13, wherein the Zika virus preparation is treated with formaldehyde at a concentration of 0.005% (w / v) to 0.02% (w / v).

15. The method of any one of claims 4 to 14, wherein the Zika virus preparation is treated with formaldehyde for eight to twelve days.

16. The method of any one of claims 4 to 15, wherein the Zika virus preparation is treated with formaldehyde for ten days.

17. The method of any one of claims 4 to 16, wherein the Zika virus preparation is treated with formaldehyde at a temperature of 15°C to 30°C.

18. The method of any one of claims 4 to 17, wherein the Zika virus preparation is treated with formaldehyde at a temperature of 22°C.

19. The method of any one of claims 4 to 18, further comprising the step of determining the completeness of inactivation after step (b) and before step (c).

20. The method of claim 19, wherein the step of determining completeness of inactivation comprises: (i) inoculating cultured insect cells with the Zika virus preparation treated according to step (b) and incubating the insect cells for a first period of time, thereby producing an insect cell supernatant; (ii) inoculating cultured mammalian cells with the insect cell supernatant produced in (i) and incubating the mammalian cells for a second period of time; as well as (iii) determining whether the Zika virus preparation contains residual replicating virus that produces a cytopathic effect on the mammalian cells.

21. The method of claim 20, wherein the insect cells are selected from CCL-125 cells, Aag-2 cells, RML-12 cells, C6 / 36 cells, C7-10 cells, AP-61 cells, At GRIP-1 cells, At GRIP-2 cells, AtGRIP-3 cells, UM-AVE1 cells, Mos.55 cells, Sua1B cells, 4a-3B cells, Mos.42 cells, MSQ43 cells, LSB-AA695BB cells, NIID-CTR cells and TRA-171 cells.

22. The method of claim 20 or 21, wherein the first period of time is 3 to 7 days.

23. The method of any one of claims 20 to 22, wherein the mammalian cell is selected from VERO cells, LLC-MK2 cells, MDBK cells, MDCK cells, BHK21-F cells, HKCC cells and Chinese hamster ovary cells (CHO cells).

24. The method of any one of claims 20 to 22, wherein the mammalian cells are selected from ATCC CCL34MDCK (NBL2) cells and MDCK 33016 cells.

25. The method of any one of claims 20 to 24, wherein the second period of time is 3 to 14 days.

26. The method according to any one of claims 4 to 25, further comprising a step after step (b) and before step (c): neutralizing the formaldehyde-treated Zika virus preparation with sodium metabisulfite.

27. The method of claim 26, wherein the formaldehyde-treated Zika virus preparation is neutralized at least five days, at least seven days, at least nine days, at least 11 days, or at least 14 days after formaldehyde treatment.

28. The method of any one of claims 4 to 27, wherein at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of one or more antigens in the Zika virus preparation is adsorbed to the adjuvant.

29. A pharmaceutical composition comprising inactivated Zika virus and having a residual formaldehyde content of less than 50 μg / ml, wherein the residual formaldehyde content is determined by the method of any one of claims 1 to 3, and wherein the pharmaceutical composition further comprises an adjuvant, the adjuvant being aluminum hydroxide having an aluminum concentration of 0.1 mg / ml to 1.0 mg / ml.

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