Methods for inactivating Zika virus and related methods
阅读说明：本技术 用于将寨卡病毒灭活的方法和相关方法 (Methods for inactivating Zika virus and related methods ) 是由 J.A.利文古德 H.吉布勒 H.迪安 T.佐藤 R.拉奥 J.马克斯 M.里昂 A. 于 2018-11-30 设计创作，主要内容包括：本公开涉及用于将寨卡病毒灭活的方法,所述寨卡病毒可用于疫苗和免疫原性组合物中。本公开还涉及一种用于确定虫媒病毒制剂的灭活完全性的方法和一种用于确定包含灭活病毒的药物组合物中的残余甲醛含量的方法。(The present disclosure relates to methods for inactivating Zika virus, which may be used in vaccines and immunogenic compositions. The 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 comprising an inactivated virus.)
1. A method for inactivating a zika virus preparation, the method comprising:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, 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 group consisting of:
(i) the filter paper is subjected to deep filtration,
(ii) buffer exchange and/or dilution;
(iii) ion exchange chromatography; and
(b) the Zika virus preparation was treated with formaldehyde, wherein the value of the concentration of formaldehyde as measured in% (w/v) multiplied by the incubation time under formaldehyde as measured in days resulted in 0.025 to 0.5.
2. The method of claim 1, wherein the cell is a non-human cell.
3. The method of claim 1 or 2, wherein the cell is a Vero cell.
4. The method of any one of the preceding claims, wherein the Zika virus preparation is obtained from an inoculum containing a heterogeneous population of Zika viruses.
5. The method of any one of the preceding claims, wherein said Zika virus preparation is obtained from a clinical isolate.
6. The method of any one of the preceding claims, wherein the Zika virus preparation is obtained from a Zika virus clone isolate.
7. The method of claim 6, wherein said clones of Zika virus are obtained by plaque purification.
8. The method of claim 7, wherein the plurality of cells is inoculated with an inoculum containing a heterogeneous population of Zika virus prior to plaque purification.
9. A method for inactivating a zika virus preparation, the method comprising:
(a) obtaining a Zika virus preparation from a clinical isolate; and
(b) the Zika virus preparation was treated with formaldehyde, wherein the value of the concentration of formaldehyde as measured in% (w/v) multiplied by the incubation time under formaldehyde as measured in days resulted in 0.025 to 0.5.
10. 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; and
(b) the Zika virus preparation was treated with formaldehyde, wherein the value of the concentration of formaldehyde as measured in% (w/v) multiplied by the incubation time under formaldehyde as measured in days resulted in 0.025 to 0.5.
11. The method of any one of the preceding claims, wherein the Zika virus preparation is treated with formaldehyde at a concentration of 0.005% (w/v) to 0.02% (w/v).
12. The method of any one of the preceding claims, wherein the Zika virus preparation is treated with formaldehyde for eight to twelve days.
13. The method of any one of the preceding claims, wherein the Zika virus preparation is treated with formaldehyde for ten days.
14. The method of any one of the preceding claims, wherein the Zika virus preparation is treated with formaldehyde at a temperature of 15 ℃ to 30 ℃.
15. The method of any one of the preceding claims, wherein the Zika virus preparation is treated with formaldehyde at a temperature of 22 ℃.
16. The method of any one of the preceding claims, further comprising step (c): the completeness of inactivation was determined.
17. The method of claim 16, wherein step (c) 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) (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
(iii) determining whether said Zika virus preparation contains residual replication virus that produces a cytopathic effect on said mammalian cells.
18. The method of claim 17, wherein the insect cell is selected from the group consisting of a CCL-125 cell, Aag-2 cell, RML-12 cell, C6/36 cell, C7-10 cell, AP-61 cell, a.t.grip-1 cell, a.t.grip-2 cell, a.t.grip-3 cell, UM-AVE1 cell, mos.55 cell, Sua1B cell, 4a-3B cell, mos.42 cell, MSQ43 cell, LSB-AA695BB cell, NIID-CTR cell, and TRA-171 cell, e.g., C6/36 cell.
19. The method of claim 17 or 18, wherein the first period of time is 3 to 7 days.
20. The method of any one of claims 17 to 19, wherein the mammalian cells are selected from VERO cells, LLC-MK2 cells, MDBK cells, MDCK cells, ATCC CCL34 MDCK (NBL2) cells, MDCK 33016 (accession number DSMACC 2219 as described in WO 97/37001) cells, BHK21-F cells, HKCC cells and chinese hamster ovary cells (CHO cells), e.g., VERO cells.
21. The method of any one of claims 17 to 20, wherein the second period of time is from 3 to 14 days.
22. The method of any one of the preceding claims, further comprising step (d): the formaldehyde-treated Zika virus preparation was neutralized with sodium metabisulfite.
23. The method of claim 22, wherein said 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.
24. The method of any one of the preceding claims, further comprising step (e): preparing a pharmaceutical composition comprising the inactivated Zika virus preparation.
25. The method of claim 24, wherein said Zika virus preparation is mixed with an adjuvant.
26. The method of claim 25, wherein the adjuvant is selected from the group consisting of: aluminum salts, toll-like receptor (TLR) agonists, Monophosphoryl 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).
27. The method of claim 25, wherein the adjuvant is an aluminum salt, such as aluminum phosphate, aluminum hydroxide, aluminum potassium sulfate, and Alhydrogel 85.
28. The method of any one of claims 25-27, 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 the one or more antigens in the Zika virus preparation are adsorbed to the adjuvant.
29. The method of any one of the preceding claims, wherein 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.
30. The method of claim 29, wherein the mutation is a Trp98Gly mutation in SEQ ID No. 1.
31. The method of claim 29 or 30, wherein said Zika virus does not comprise a mutation in the envelope protein (E).
32. The method of claim 31, wherein the sequence encoding the envelope protein is identical to the corresponding sequence in SEQ ID No. 2.
33. A pharmaceutical composition comprising inactivated zika virus obtainable by the method of any one of claims 1 to 32.
34. A pharmaceutical composition comprising inactivated zika virus and having a residual formaldehyde content of less than 50 μ g/ml.
35. The pharmaceutical composition of claim 34, obtainable by the method of any one of claims 1 to 32.
36. A method for determining the completeness of inactivation of an arbovirus preparation, the method comprising the steps of:
(i) inoculating the cultured insect cells with the arbovirus preparation that has undergone the inactivation step and incubating the insect cells for a first period of time, thereby producing an insect cell supernatant;
(ii) (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
(iii) determining whether said arbovirus preparation contains residual replication virus that produces a cytopathic effect on said mammalian cells.
37. The method of claim 36, wherein the arbovirus is a flavivirus or alphavirus.
38. The method of claim 36 or 37, wherein 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, Anonene Niang virus, or Maurera virus.
39. The method of any one of claims 36 to 38, wherein the arbovirus preparation is subjected to detergent, formalin, hydrogen peroxide, beta-propiolactone (BPL), Binary Ethylamine (BEI), acetylethyleneimine, methylene blue, or psoralen treatment.
40. The method of any one of claims 36 to 39, wherein the insect cell is selected from the group consisting of CCL-125 cells, Aag-2 cells, RML-12 cells, C6/36 cells, C7-10 cells, AP-61 cells, A.t.GRIP-1 cells, A.t.GRIP-2 cells, A.t.GRIP-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, e.g., C6/36 cells.
41. The method of any one of claims 36-40, wherein the first period of time is 3 to 7 days.
42. The method of any one of claims 36 to 41, wherein the mammalian cells are selected from VERO cells, LLC-MK2 cells, MDBK cells, MDCK cells, ATCC CCL34 MDCK (NBL2) cells, MDCK 33016 (accession number DSMACC 2219 as described in WO 97/37001) cells, BHK21-F cells, HKCC cells and Chinese hamster ovary cells (CHO cells), e.g., VERO cells.
43. The method of any one of claims 36 to 42, wherein the second period of time is 3 to 14 days.
44. The method of any one of claims 36 to 43, wherein the method is capable of detecting less than 1.0TCID50The arbovirus of (1).
45. A method for determining residual formaldehyde content in a pharmaceutical composition comprising an inactivated virus, the method comprising the steps of:
(a) providing a composition comprising a virus that has been treated with formaldehyde;
(b) mixing the composition of (a) with phosphoric acid and 2, 4-Dinitrophenylhydrazine (DNPH), thereby providing a mixture;
(c) incubating the mixture of (b) under suitable conditions; and
(d) the mixture was analyzed for the presence of residual formaldehyde.
46. The method of claim 45, wherein said composition of (a) contains an adjuvant.
47. The method of claim 46, wherein the adjuvant is aluminum hydroxide.
48. The method of any one of claims 45 to 47, wherein the composition of (a) contains 0.1 to 1.0mg/ml aluminum hydroxide as adjuvant.
49. The method of any one of claims 45-48, wherein 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 1.1mg/ml DNPH.
50. The method of any one of claims 45 to 49, wherein the composition of (a) is incubated with the mixture of phosphoric acid and 2, 4-Dinitrophenylhydrazine (DNPH) at room temperature.
51. The method of any one of claims 45 to 50, wherein the composition of (a) is incubated with the mixture of phosphoric acid and 2, 4-Dinitrophenylhydrazine (DNPH) for 10 to 30 minutes.
52. The method of any one of claims 45-51, wherein the mixture of the composition of (a) and phosphoric acid and 2, 4-Dinitrophenylhydrazine (DNPH) is analyzed by HPLC.
53. The method of claim 52, wherein the HPLC is reverse phase HPLC.
54. The process of claim 53, wherein a mixture of water and acetonitrile (1:1, v/v) is used as the mobile phase in HPLC.
55. The method of any one of claims 45 to 54, wherein the virus is an inactivated Zika virus.
56. The method of claim 55, wherein said inactivated Zika virus has been treated with 0.01% (w/v) formaldehyde at 22 ℃ for 10 days.
57. The method of claim 55 or 56, wherein 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.
Technical Field
The present disclosure relates to methods for inactivating Zika virus (Zika virus) useful in vaccines and immunogenic compositions. The 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 comprising an inactivated virus.
Background
The flavivirus (flavivirus) within the family of Flaviviridae (Flaviviridae) classified along with other mosquito-borne viruses (e.g., yellow fever virus, dengue virus (dengue), West Nile virus (West Nile), and japanese encephalitis virus), which has spread rapidly since its introduction into brazil in 2013, became a hemispheric epidemic. This virus has reached both central and north america, including the united states territory, and is now threatening the continental united states. In fact, the Zika virus strain PRVABC59 was isolated from sera from a person who had traveled 2015 to Puerto Rico (Puerto Rico). The Genome of this strain has been sequenced at least three times (see Lanciotti et al emery. Infect. Dis.2016, 5.5; 22(5):933-5 and GenBank accession number KU 501215.1; GenBank accession number KX 087101.3; and Yun et al Genome Announc.2016, 8.18.8.4 (4) and GenBank accession number ANK 57897.1).
This virus was originally isolated in Uganda (Uganda) in 1947, was first associated with human disease in 1952, and has been sporadically recognized as the cause of mild self-limiting febrile diseases in Africa and southeast Asia (Weaver et al (2016) Antiviral Res.130: 69-80; Faria et al (2016) science.352(6283): 345-349). However, in 2007, one outbreak occurred in the Atalanta North Pacific Island (the North Pacific Island of Yap), followed by spread across the Pacific ocean between Islands, causing a broad outbreak in 2013-2014 French Bolirnia (French Polynesia), followed by spread to New Kelidonia (New Caledonia), Cockstrin Islands (Cook Islands), and finally to Easter Island (Easter Island). Asian lineage viruses were then transferred to the Western hemisphere via an undefined pathway (Faria et al (2016) science.352(6283): 345-349). Viruses are transmitted zoologically by aedes aegypti (Aedesaegypti), aedes albopictus (a. albopictus) and possibly by aedes huricini (a. henseli) and aedes bornesis (a. polynieesensis) (Weaver et al (2016) Antiviral res 130: 69-80). In addition, it is contemplated that other viral transmission vectors may be present, and that the virus may be transmitted by blood transfusion, via placenta, and/or by sexual transmission.
By the end of 2015, fetal abnormalities (e.g., microcephaly) and Guillain-Barre syndrome (GBS) have increased significantly in widespread areas of Zika virus infection, alerting that Zika virus may be more lethal than originally thought, which prompted the World Health Organization (WHO) to declare it an emergent Public Health event of International Concern (Public Health initiative of International conn., 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 preventative measure for control of Zika virus involves the management of mosquito populations.
In recent efforts to characterize recombinant Zika virus to develop potential vaccines, a non-human cell-adapted Zika virus was identified that had a mutation at position 330 in the viral envelope protein (Weger-Lucarilli et al 2017.Journal of Virology). The authors of this study found that the full-length infectious cDNA clone of zika virus strain PRVABC59 was genetically unstable when amplified during cloning, and chose to split the viral genome to elucidate the observed instability, develop and apply a two-plasmid system. However, the dual plasmid system for the development of the Zika vaccine is less desirable.
Disclosure of Invention
Accordingly, there is a need to develop vaccines and immunogenic compositions for treating and/or preventing Zika virus infection using genetically stable Zika viruses. One option for vaccine development is to inactivate whole virus and vaccinate a subject with the inactivated whole virus. However, in the development of inactivated virus vaccines, a key safety guarantee is the assurance that the drug or drug substance is free of infectious virus. An efficient inactivation process and sensitive assays were developed to measure and determine whether infectious virions still present a critical safety aspect for developing purified inactivated virus derived from any wild-type virus but clearly having pathogenic/encephalitis virus that may cause fetal abnormalities. Furthermore, formaldehyde, which is known to be useful for inactivating viruses, is genotoxic and carcinogenic, and therefore it is important to monitor residual formaldehyde levels in bulk drugs and pharmaceuticals, and regulatory authorities require manufacturers who use formaldehyde as an inactivating agent to determine residual formaldehyde content in pharmaceuticals. Therefore, there is a need for a sensitive method for detecting residual formaldehyde in a pharmaceutical product or pharmaceutical composition containing an inactivated virus, such as inactivated Zika virus.
The present disclosure is based, at least in part, on the following discoveries: the low concentration of formaldehyde applied to the virus at room temperature, relatively briefly, effectively inactivated the Zika virus. In addition, an assay was developed which allows to determine with high sensitivity whether infectious virions are still present after inactivation. Finally, a method was developed which allows the detection of low levels of residual formaldehyde in the final drug product or pharmaceutical composition.
Accordingly, certain aspects of the present disclosure relate to a method for inactivating a Zika virus preparation, the method comprising:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, 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 group consisting of:
(i) the filter paper is subjected to deep filtration,
(ii) buffer exchange and/or dilution;
(iii) ion exchange chromatography; and
(b) the Zika virus preparation was treated with formaldehyde, wherein the value of the concentration of formaldehyde as measured in% (w/v) multiplied by the incubation time under formaldehyde as measured in days resulted in 0.025 to 0.5.
In some embodiments, the cell is a non-human cell or a Vero cell.
In some embodiments, the Zika virus preparation is obtained from an inoculum containing a heterogeneous population of Zika viruses.
In some embodiments, the zika virus preparation is obtained from a clinical isolate.
In some embodiments, the Zika virus preparation is obtained from a Zika virus clone isolate. Clones isolated from Zika virus were obtained by plaque purification. A plurality of cells can be inoculated with an inoculum containing a heterogeneous population of zika virus prior to plaque purification.
Some aspects of the present disclosure relate to a method for inactivating a Zika virus preparation, the method comprising:
(a) obtaining a Zika virus preparation from a clinical isolate; and
(b) the Zika virus preparation was treated with formaldehyde, wherein the value of the concentration of formaldehyde as measured in% (w/v) multiplied by the incubation time under formaldehyde as measured in days resulted in 0.025 to 0.5.
Some aspects of the present disclosure relate to 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; and
(b) the Zika virus preparation was treated with formaldehyde, wherein the value of the concentration of formaldehyde as measured in% (w/v) multiplied by the incubation time under formaldehyde as measured in days resulted in 0.025 to 0.5.
In some embodiments, the Zika virus preparation is treated with formaldehyde at a concentration of 0.005% (w/v) to 0.02% (w/v).
In some embodiments, the zika virus formulation is treated for eight to twelve or ten days.
In some embodiments, the Zika virus formulation is treated at a temperature of 15 ℃ to 30 ℃ or 22 ℃.
The method may further comprise step (c): the completeness of inactivation was determined.
In some embodiments, step (c) 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) (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
(iii) determining whether said Zika virus preparation contains residual replication virus that produces a cytopathic effect on said mammalian cells.
In some embodiments, the insect cell is selected from the group consisting of a CCL-125 cell, an Aag-2 cell, an RML-12 cell, a C6/36 cell, a C7-10 cell, an AP-61 cell, an A.t.GRIP-1 cell, an A.t.GRIP-2 cell, an A.t.GRIP-3 cell, a UM-AVE1 cell, a mos.55 cell, a Sua1B cell, a 4a-3B cell, a mos.42 cell, an MSQ43 cell, a LSB-AA695BB cell, a NIID-CTR cell, and a TRA-171 cell, e.g., a C6/36 cell.
In some embodiments, the first period of time is 3 to 7 days.
In some embodiments, the mammalian cell is selected from the group consisting of a VERO cell, an LLC-MK2 cell, an MDBK cell, an MDCK cell, an ATCC CCL34MDCK (NBL2) cell, an MDCK33016 (deposited under number DSM ACC2219 as described in WO 97/37001) cell, a BHK21-F cell, an HKCC cell, and a Chinese hamster ovary cell (CHO cell), e.g., a VERO cell.
In some embodiments, the second period of time is 3 to 14 days.
The method may further comprise step (d): the formaldehyde-treated Zika virus preparation is neutralized with sodium metabisulfite, for example, at least five days, at least seven days, at least nine days, at least 11 days, or at least 14 days after formaldehyde treatment.
The method may further comprise step (e): preparing a pharmaceutical composition comprising an inactivated Zika virus preparation.
In some embodiments, the zika virus preparation is mixed with an adjuvant. The adjuvant may be selected from the group consisting of: aluminum salts, toll-like receptor (TLR) agonists, Monophosphoryl 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 (poloxamer particles), microparticles, liposomes, Complete Freund's adjuvant, CFA, and Incomplete Freund's Adjuvant (IFA).
In some embodiments, the adjuvant is an aluminum salt, such as aluminum phosphate, aluminum hydroxide, aluminum potassium sulfate, or Alhydrogel 85.
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 the one or more antigens in the zika virus preparation are adsorbed to the adjuvant.
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.
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
Some aspects of the present disclosure also relate to a pharmaceutical composition comprising an inactivated zika virus obtainable by any one of the methods described herein.
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 μ g/ml. In some embodiments, the pharmaceutical composition is obtainable by the method of any one of
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:
(i) inoculating the cultured insect cells with the arbovirus preparation that has undergone the inactivation step and incubating the insect cells for a first period of time, thereby producing an insect cell supernatant;
(ii) (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
(iii) determining whether said arbovirus preparation contains residual replication virus that produces a cytopathic effect on said mammalian cells.
In some embodiments, the arbovirus is a flavivirus or alphavirus. In some embodiments, the arbovirus is a zaka virus, a west nile virus, a yellow fever virus, a Japanese Encephalitis virus (Japanese Encephalitis virus), a tick-borne Encephalitis virus, a dengue virus (dengue virus), a st.
In some embodiments, the arbovirus preparation is subjected to detergent, formalin, hydrogen peroxide, beta-propiolactone (BPL), Binary Ethylamine (BEI), acetyl ethyleneimine, methylene blue, or psoralen treatment.
In some embodiments, the insect cell is selected from the group consisting of a CCL-125 cell, an Aag-2 cell, an RML-12 cell, a C6/36 cell, a C7-10 cell, an AP-61 cell, an A.t.GRIP-1 cell, an A.t.GRIP-2 cell, an A.t.GRIP-3 cell, a UM-AVE1 cell, a mos.55 cell, a Sua1B cell, a 4a-3B cell, a mos.42 cell, an MSQ43 cell, a LSB-AA695BB cell, a NIID-CTR cell, and a TRA-171 cell, e.g., a C6/36 cell.
In some embodiments, the first period of time is 3 to 7 days.
In some embodiments, the mammalian cell is selected from the group consisting of a VERO cell, an LLC-MK2 cell, an MDBK cell, an MDCK cell, an ATCC CCL34MDCK (NBL2) cell, an MDCK33016 (deposited under number DSM ACC2219 as described in WO 97/37001) cell, a BHK21-F cell, an HKCC cell, and a Chinese hamster ovary cell (CHO cell), e.g., a VERO cell.
In some embodiments, the second period of time is 3 to 14 days.
In some embodiments, the method is capable of detecting less than 1.0TCID50The arbovirus of (1).
Some aspects of the present disclosure also relate to a method for determining residual formaldehyde content in a pharmaceutical composition comprising an inactivated virus, the method comprising the steps of:
(a) providing a pharmaceutical composition comprising a virus that has been treated with formaldehyde;
(b) mixing the composition of (a) with phosphoric acid and 2, 4-Dinitrophenylhydrazine (DNPH), thereby providing a mixture;
(c) incubating the mixture of (b) under suitable conditions; and
(d) the mixture was analyzed for the presence of residual formaldehyde.
In some embodiments, the composition of (a) contains an adjuvant, which may be aluminum hydroxide. In some embodiments, the composition of (a) contains 0.1mg/ml to 1.0mg/ml aluminum hydroxide as an adjuvant.
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 1.1mg/ml DNPH.
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-dinitrophenyl hydrazine (DNPH) for 10 to 30 minutes.
In some embodiments, the composition of (a) is analyzed by HPLC, which may be reverse phase HPLC, with a mixture of phosphoric acid and 2, 4-Dinitrophenylhydrazine (DNPH). In some embodiments, a mixture of water and acetonitrile (1:1, v/v) is used as the mobile phase in HPLC.
In some embodiments, the virus is an inactivated Zika virus. In some embodiments, inactivated Zika virus has been treated with 0.01% (w/v) formaldehyde at 22 ℃ 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.
Drawings
Figure 1 shows bright field microscopic images of Vero cell monolayers mimicking infection (top) or infection with the ZIKAV strain PRVABC59 (bottom).
FIG. 2 illustrates, for example, passing TCID50Growth kinetics determined for
FIG. 3 shows the potency assay Test (TCID) for clones a-f of Zika virus PRVABC59P 550)。
Figure 4 shows bright field microscopy images depicting cytopathic effect (CPE) of growth of zaka virus PRVABC59P6 clone a-f on Vero cell monolayers.
FIG. 5 shows the potency assay Test (TCID) for clones a-f of Zika virus PRVABC59P650)。
FIG. 6 shows an amino acid sequence alignment comparing Zika virus envelope glycoprotein sequences from Zika virus strains PRVABC59P 6e (SEQ ID NO:8) and PRVABC59(SEQ ID NO:9) near residue 330 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 116007 (SEQ ID NO: 14); DENV 216681 (SEQ ID NO: 15); DENV 316562 (SEQ ID NO: 16); and DENV 41036 (SEQ ID NO: 17)).
FIG. 7 shows an amino acid sequence alignment comparing the Zika virus NS1 protein sequence near residue 98 from Zika virus strains PRVABC59P 6e (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 116007 (SEQ ID NO: 24); DENV 216681 (SEQ ID NO: 25); DENV 316562 (SEQ ID NO: 26); and DENV 41036 (SEQ ID NO: 27)).
FIG. 8 shows the plaque phenotype of ZIKAV PRVABC59P6 virus clone a-f compared to
FIG. 9 shows the average spot size of ZIKAV PRVABC59P6 virus clones compared to
FIG. 10 shows the growth kinetics of ZIKAV PRVABC59P6 clone a-f in Vero cells under serum-free growth conditions.
Figure 11 shows a schematic of the steps taken to prepare PRVABC59P6b and P6e formulated drugs for immunization experiments.
FIG. 12A shows a schedule for administering vaccine formulations derived from ZIKAV PRVABC59P6b and P6e clones to CD-1 mice. PBS was used as placebo.
Figure 12B shows serum ZIKAV neutralizing antibody titers of CD-1 mice immunized with vaccine formulations derived from ZIKAV PRVABC59P6B and P6e clones as described in figure 12A. ZIKAV neutralizing antibody titers were determined by Reporter Virus Particle (RVP) neutralization assay. The solid line represents the geometric mean of a group. Detection limit (1.93 log)10) Indicated by the dashed line.
Fig. 13A shows the schedule of administration of vaccine formulations derived from ZIKAV PRVABC59P6b and P6e clones to AG129 mice. PBS was used as placebo.
Figure 13B shows serum ZIKAV neutralizing antibody titers of AG129 mice immunized with vaccine formulations derived from ZIKAV PRVABC59P6B and P6e clones as described in figure 13A. The solid line represents the geometric mean of a group. Detection limit (1.30 log)10) Indicated by the dashed line. Partition with no detectable titer: (<1.30) of animals 0.5.
Fig. 14 shows the average weight of the AG129 test group after challenge, expressed as a percentage of the starting weight. Error bars indicate standard deviation.
Figure 15 shows serum viremia of individual AG129 mice two days after challenge, reported as PFU/mL. The solid line represents the mean of one group. Detection limit (2.0 log)10) Indicated by the dashed line.
Fig. 16 shows survival analysis of AG129 test group after challenge.
Figure 17 shows pre-challenge serum circulating ZIKAV neutralizing antibody (Nab) titers after passive transfer of pooled sera from vaccinated and challenged AG129 mice.
Figure 18 shows the average body weight of passively transferred mice and control mice challenged with zika virus.
Figure 19 shows serum viremia of individual AG129 mice three days after challenge, reported as PFU/mL.
Figure 20 shows survival analysis of passively transferred mice and control mice challenged with zika virus.
Figure 21 shows the correlation between ZIKAV neutralizing antibody titers and viremia observed in passively transferred mice.
FIG. 22 shows the survival analysis of AG129 mice using Kaplan Meier survival curves (Kaplan Meier surviv curve) after MVS stocks prior to infection with P6a and P6e Zika virus.
FIG. 23 shows the average body weight, expressed as a percentage of the initial weight, of MVS stock solution prior to infection with Zika virus P6a and P6e, at the time of the invention. The dotted line represents 100% of the starting weight for reference.
FIG. 24 shows serum viremia of individual AG129 mice three days after MVS stocks prior to infection with P6a and P6e Zika virus, reported as PFU/mL. The dotted line indicates the detection limit of the assay.
Figure 25 shows compiled inactivation kinetics data. Data compare infection efficacy (TCID50) to RNA copies, and inactivation Completeness (COI) of four toxicology lot samples. These data indicate that the sensitivity of the COI assay exceeds TCID 50.
FIG. 26 shows a comparison of C6/36 and Vero sensitivity in assays as evidenced by input virus titers of 0.31TCID 50.
FIG. 27 shows a log regression analysis of CPE versus log TCID50 using C6/36 cells, sites including 99% confidence intervals around the target value of 0.01TCID 50/well (-2log TCID 50/well); the model predicts that 0.85% of the wells will be positive.
FIG. 28 shows chromatograms of PBS (a) and PBS solutions containing 0.049 μ g/mL (b), 0.098 μ g/mL (c), 0.196 μ g/mL (d), 0.491 μ g/mL (e), 0.982 μ g/mL (f), and 1.964 μ g/mL (g) formaldehyde.
Detailed Description
General technique
The techniques and procedures described or referenced herein are generally better understood and commonly employed by those skilled in the art using conventional methods, such as the widely utilized methods described in the following: sambrook et al, Molecular Cloning, A
Zika virus
Certain aspects of the present disclosure relate to a purified inactivated Zika virus that can be used in a vaccine and/or immunogenic composition.
Zika virus (ZIKV) is a mosquito-borne flavivirus that was first isolated in 1947 from sentinel rhesus monkeys in Wugan Dazhai forest (Zikaforest). Since then, the virus has been isolated from humans in africa and asia, and in recent years in america. ZIKV was found to have two (possibly three) lineages: african lineages (perhaps independent east africa and west african lineages) and asian lineages. Thus, examples of suitable zika viruses of the present disclosure include, without limitation, 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 of Zika virus have been previously identified within African and Asian lineages. 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, 6330656, 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 985-99, ArA982-99, ArA 20199, ArA27101, ArA 27290, ArA 27118, ArA 27153, ArB (Arkuraney III) III, Arthro III,
In some embodiments, an example of a Zika virus genomic sequence is set forth as SEQ ID NO:2 below:
in some embodiments, the zika virus may comprise the genomic sequence of GenBank accession No. KU 501215.1. In some embodiments, zika virus is from the strain PRVABC 59. In some embodiments, the genomic sequence of GenBank accession number KU501215.1 comprises the sequence of SEQ ID NO. 2. In some embodiments, zika virus may comprise a genomic 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.
In some embodiments, the Zika virus may comprise at least one polypeptide encoded by the sequence of SEQ ID NO. 2. In some embodiments, the zika virus may comprise 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.
Thus, in some embodiments, the inactivated zika virus of the present disclosure may be used in any of the vaccines and/or immunogenic compositions disclosed herein. For example, the inactivated zika virus of the present disclosure may be used to provide one or more antigens that may be used to treat or prevent infection by zika virus 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.
Zika virus for use in the present disclosure may be obtained from one or more cells in cell culture (e.g., purified via plaque). Any suitable cell known in the art for producing Zika virus may be used, for example 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, A.t.GRIP-1 cells, A.t.GRIP-2 cells, A.t.GRIP-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 derived from, for example, Aedes aegypti (Aedes aegypti), Aedes albopictus (Aedes albopictus), Aedes pseudolepiperis (Aedes pseudolepiperis), Anedes triphylla (Anedes), Aedes triandrus (Aedes aegypti), Cuaedes aee (Culex), Cultis quine (Culex), Cultis fatigus (Culex), Cultis fatigus (Culeya) may be used in the art, Additional cells or cell lines of the mosquito species Culex hilgardii (Culex theileri), Culex tritaeniorhynchus (Culex bitaeniorhynchus), and/or aren megamosquito (toxorhynchies), 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, MDCK33016 (accession number ACC2219 as described in WO 97/37001), BHK21-F cells, HKCC cells, or chinese hamster ovary cells (CHO cells). In some embodiments, the Zika virus (e.g., a Zika virus clone isolate) is produced by a non-human cell. In some embodiments, the Zika virus (e.g., a Zika virus clone isolate) is produced by an insect cell. In some embodiments, the zika virus (e.g., a zika virus clone isolate) is produced by mosquito cells. In some embodiments, the Zika virus (e.g., Zika virus clone isolate) is produced by a mammalian cell. In some embodiments, the Zika virus (e.g., Zika virus clone isolate) is produced by VERO cells.
Zika virus has a positive-sense single-stranded RNA genome encoding structural and non-structural polypeptides. The genome also contains non-coding sequences at the 5 'and 3' end regions that are functional in viral replication. Structural polypeptides encoded by these viruses include, but are not limited to, capsid (C), precursor membrane (prM), and envelope (E). Non-structural (NS) polypeptides encoded by these viruses include, but are not limited to, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and
In certain embodiments, the Zika virus comprises a mutation in Zika virus non-structural protein 1(NS 1). 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 mutation is within the NS1 polypeptide. The amino acid sequence of the wild-type NS1 polypeptide from an exemplary zika virus strain is shown below:
DVGCSVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEDGICGISSVSRMENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNELPHGWKAWGKSYFVRAAKTNNSFVVDGDTLKECPLKHRAWNSFLVEDHGFGVFHTSVWLKVREDYSLECDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMKTCEWPKSHTLWTDGIEESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRKEPESNLVRSMVT(SEQ ID NO:1)。
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 can 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 can be amino acid positions 795 to 1145 of an amino acid sequence encoded by the sequence of GenBank accession No. KU 501215.1. In some embodiments, the amino acid sequence of the NS1 polypeptide can be from zika virus strain PRVABC 59.
"sequence identity", "percent identical" or "sequence alignment" means a comparison of a first amino acid sequence to a second amino acid sequence, or a 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 the "same percentage" or "ID percentage".
In general, sequence alignments can be used to calculate sequence identity by one of two different methods. In the first approach, both mismatches at a single position and gaps at a single position are counted as non-identical positions in the final sequence identity calculation. In the second approach, mismatches at a single position in the final sequence identity calculation are counted as non-identical positions; however, gaps at a single position in the final sequence identity calculation are not counted as non-identical positions (neglected). In other words, in the second method, the null bits are ignored in the final sequence identity calculation. The difference between the two approaches, i.e., gaps at a single position are counted as non-identical position contrasts neglecting gaps, may cause variability in sequence identity values between the two sequences.
In some embodiments, sequence identity is determined by a program that generates an alignment and calculates identity by calculating mismatches at a single position and gaps at a single position as non-identical positions in a final sequence identity calculation. For example, the program needle (EMBOSs) which implements the algorithm of needle man and Wunsch (needle man and Wunsch,1970, J.mol.biol.48: 443. sup. 453) and calculates the sequence identity at each default setting by first creating an alignment between a first sequence and a second sequence, then counting the number of identical positions over the length of the alignment, then dividing the number of identical residues by the length of the alignment, and then multiplying this number by 100, yielding the% sequence identity [ (% identical residues number/length of alignment) × 100) ].
Sequence identity can be calculated by pairwise alignments showing both sequences over their full length, thus showing the full length of the first and second sequences ("overall sequence identity"). For example, the program needle (emboss) generates such alignments; percent sequence identity ═ (number of identical residues/length aligned) × 100) ].
Sequence identity may be calculated from pairwise alignments of local regions displaying only the first or second sequence ("local identity"). For example, the program blast (ncbi) generates such alignments; percent sequence identity ═ (number of identical residues/length aligned) × 100) ].
Sequence alignments are preferably generated using the algorithms of Needleman and Wunsch (J.mol.biol. (1979)48, p. 443-453). Preferably, The program "needlet" (The european molecular Biology Open Software Suite, EMBOSS)) is used together with program default parameters (gap opening 10.0, gap elongation 0.5 and matrix of proteins EBLOSUM62, and matrix of nucleotides EDNAFULL). Sequence identity can then be calculated from an alignment that displays both sequences over their full length, thus displaying the full length of the first and second sequences ("overall sequence identity"). For example: percent sequence identity ═ (number of identical residues/length aligned) × 100) ].
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.
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 in viruses other than Zika virus that correspond to the tryptophan residue at position 98 of SEQ ID NO:1 are shown in FIG. 7 of the present application, where 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.
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 envelope protein E. In some embodiments, the whole inactivated virus contains at least one mutation in Zika virus non-structural protein 1(NS1) and does not include a mutation in 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 within envelope protein E. In some embodiments, the fully inactivated 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 and does not include a mutation in zika virus envelope protein e (env). In some embodiments, the whole inactivated virus contains at least one mutation in the non-structural protein 1(NS1) of zika virus 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 fully 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 fully 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.
In some embodiments, the Zika virus contains at least one mutation that increases genetic stability compared to the 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 the Zika virus lacking the at least one mutation. In some embodiments, zika virus contains at least one mutation that reduces or otherwise inhibits the occurrence of an undesirable mutation, e.g., within envelope protein e (env) of zika virus.
In the above embodiments of the present disclosure, one exemplary pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm, which uses default parameters (e.g., gap opening penalty of 10.0, gap extension penalty of 0.5, using EBLOSUM62 scoring matrix). The algorithm is preferably implemented in the needle tool in the EMBOSS package.
In some embodiments, inactivated Zika virus may be used in vaccines and immunogenic compositions. For example, inactivated zika virus may be used to treat or prevent infection by zika virus 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.
Production of vaccines and immunogenic compositions
Other aspects of the present disclosure relate to zika virus vaccines and immunogenic compositions comprising a purified inactivated whole 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. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated 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 PRVABC 59. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated 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 one embodiment, the vaccine and immunogenic composition comprises a plaque-purified cloned Zika virus isolate. Such vaccines and immunogenic compositions are useful, for example, in treating or preventing Zika virus infection in a subject in need thereof and/or inducing an immune response, e.g., a protective immune response, against Zika virus in a subject in need thereof.
Production of the vaccines and/or immunogenic compositions of the present disclosure includes growth of zika virus. Growth in cell culture is one method for preparing the vaccine and/or immunogenic compositions of the present disclosure. Cells for virus growth may be cultured in suspension or under adherent conditions.
Cell lines suitable for growth of 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), horses, cows (e.g., MDBK cells), sheep, dogs (e.g., MDCK cells from canine kidney, ATCC CCL34MDCK (NBL2), or MDCK33016, as described in WO97/37001, accession number DSM ACC2219), cats, and rodents (e.g., hamster cells, such as 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, neonatal, fetal, and embryonic Lung), endothelial cells (e.g., aorta, coronary artery, lung, blood vessels, cutaneous microvasculature, umbilicus), hepatocytes, keratinocytes, immune cells (e.g., T cells, B cells, macrophages, NK, dendrites), breast cells (e.g., epithelium), smooth muscle cells (e.g., blood vessels, aorta, coronary artery, uterus, bronchi, cervix, periretinal cells), melanocytes, nerve cells (e.g., astrocytes), prostate cells (e.g., epithelium, smooth muscle), kidney cells (e.g., epithelium, mesentery, proximal tubule), skeletal cells (e.g., chondrocytes, osteoclasts, osteoblasts), muscle cells (e.g., myoblasts, skeletal muscle cells, smooth muscle cells, bronchi), hepatocytes, retinoblasts, and stromal cells. WO 97/37000 and WO97/37001 describe the generation of animal cells and cell lines that are capable of growth in suspension and in serum-free media and that can be used to produce and replicate viruses. In one embodiment, the cells used for growth of the at least one virus are Vero cells.
Culture conditions for the above cell types are known and described in various publications. Alternatively, media, supplements, and conditions are commercially available, such as described in catalogues and additional literature of Cambrex Bioproducts (East Rutherford, n.j.).
In certain embodiments, the cells used in the methods described herein are cultured in serum-free and/or protein-free media. If the culture medium does not contain any additives from human or animal derived serum, it is referred to as serum-free medium in the context of the present disclosure. Protein-free is understood to mean a culture in which cell proliferation occurs with removal of 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.
Known serum-free media include Iscove's medium, Ultra-CHO medium (BioWhittaker) or EX-CELL (JRH bioscience). Commonly used serum-containing media include Eagle's Basic Medium (BME) or Minimal Essential Medium (MEM) (Eagle, Science,130,432(1959)) or Dulbecco's modified Eagle Medium (DMEM or EDM), which is typically used with up to 10% fetal bovine serum or similar additives. Optionally, Minimal Essential Medium (MEM) (Eagle, Science,130,432(1959)) or Darber's modified Eagle's 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, Peptidase or HyPep.TM. (all from Quest International) or lactalbumin hydrolysate (Gibco and other manufacturers) are also well known in the art. Media additives based on plant hydrolysates have the particular advantage that contamination by viruses, mycoplasma or unknown infectious agents can be excluded.
Cell culture conditions (temperature, cell density, pH, etc.) vary over a wide range due to the suitability of the cell lines employed according to the present disclosure, and may be adapted to the requirements of a particular virus strain.
Methods for propagating viruses in cultured cells generally include the steps of: inoculating the cultured cells with the strain of the virus to be cultured; culturing the infected cells for a period of time required for virus propagation, e.g., as determined by virus titer or antigen expression (e.g., between 24 hours and 168 hours post inoculation); and collecting the propagated virus. In some embodiments, the virus is collected via plaque purification. Cultured cells are inoculated with 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 the cell suspension or applied to the cell monolayer and is allowed to wick onto the cells at 25 ℃ to 40 ℃, preferably 28 ℃ to 38 ℃ 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 typically less than 300 minutes. Infected cell cultures (e.g., monolayers) can be removed by harvesting the supernatant (without cells), freeze-thawing, or by enzymatic action to increase the viral content of the harvested culture supernatant. The harvested fluid is then inactivated or stored frozen. The cultured cells may 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, the cells are infected at an MOI of about 0.01. During infection, the ratio of medium to cell culture vessel area may be lower than during cell culture. Keeping this ratio low allows the virus to infect the cells to the maximum possible extent. Supernatants of infected cells can be harvested 30 to 60 hours or 3 to 10 days post infection. In certain preferred embodiments, the supernatant of infected cells is harvested 3 to 7 days post infection. More preferably, the supernatant of infected cells is harvested 3 to 5 days after infection. In some embodiments, a protease (e.g., trypsin) may be added during cell culture to allow for virus release, and the protease may be added at any suitable stage during culture. Alternatively, in certain embodiments, the supernatant of the infected cell culture can be harvested and the virus can be isolated or otherwise purified from the supernatant.
The virus inocula and virus cultures are preferably free of (i.e. tested and given negative results of contamination with) herpes simplex virus, respiratory syncytial virus,
In the case where the virus has been grown on cell lines, it is standard practice to minimise the amount of residual cell line DNA in the final vaccine to minimise any oncogenic activity of the host cell DNA. Standard purification procedures such as chromatography can be used to remove contaminating DNA during vaccine preparation. The removal of residual host cell DNA can be enhanced by nuclease treatment, for example by using dnase. A convenient Method for reducing DNA contamination of host cells disclosed in the reference (Lundblad (2001) Biotechnology and Applied Biochemistry 34:195- & 197, guidelines for Industry: Bioanalytical Method differentiation. U.S. Deparatment of health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Viral Medicine (CVM) 5 months 2001) involves a two-step process using first a deoxyribonuclease (e.g., nuclease (Benzonase)) which can be used during virus growth, followed by a cationic detergent (e.g., CTAB) which can be used during virion disruption. Can also be removed by treatment with beta-propiolactone. In one embodiment, contaminating DNA is removed by totipotent nuclease treatment of the culture supernatant.
Antigen production
Zika virus may 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 purified inactivated Zika virus.
In some embodiments, the inactivated virus may be produced as described in the section entitled "production of vaccines and immunogenic compositions" above.
In certain embodiments, the zika virus of the present disclosure may 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 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 kidney, ATCCCCL34 MDCK (NBL2) or MDCK33016, as described in WO97/37001 under accession number DSM ACC2219), cats and rodents (e.g. hamster cells, e.g. BHK21-F, HKCC cells or chinese hamster ovary cells (CHO cells)), and may be obtained from a variety of developmental stages including, for example, adult, neonatal, 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 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, blood vessels, skin microvasculature, umbilicus), hepatocytes, keratinocytes, immune cells (e.g., T cells, B cells, macrophages, NK, dendrites), breast cells (e.g., epithelium), smooth muscle cells (e.g., blood vessels, aorta, coronary arteries, uterus, bronchi, cervix, periretinal cells), melanocytes, nerve cells (e.g., astrocytes), prostate cells (e.g., epithelium, smooth muscle), kidney cells (e.g., epithelium, mesentery, proximal tubule), skeletal cells (e.g., chondrocytes, osteoclasts, osteoblasts), muscle cells (e.g., myoblasts, skeletal muscle cells, smooth muscle cells, bronchi), hepatocytes, retinoblasts, and stromal cells. WO 97/37000 and WO97/37001 describe the generation of animal cells and cell lines that can be grown in suspension and in serum-free media and can be used to produce viral antigens. In certain embodiments, the non-human cells are cultured in serum-free media. In certain embodiments, the zika virus of the present disclosure may be produced by culturing Vero cells.
Inactivation of viruses
Certain embodiments of the present disclosure relate to Zika virus vaccines and/or immunogenic compositions comprising purified inactivated Zika virus. The term "inactivated Zika virus" as used herein is intended to encompass Zika virus that has been treated with an inactivation method, e.g., treatment with an effective amount of formalin. Specifically, inactivated Zika virus can be obtained/obtained from a method in which Zika virus is treated with formaldehyde in an amount of about 0.01% w/v at a temperature of 20 ℃ to 24 ℃ for 10 days. Inactivated Zika virus is no longer able to infect host cells that may be infected with Zika virus that has not been inactivated. In one embodiment, the inactivated Zika virus is no longer capable of infecting VERO cells and exerting a cytopathic effect on VERO cells.
The term "purified Zika virus" means that 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 compared to unpurified Zika virus. The purity of purified Zika virus was determined by size exclusion chromatography. The major peak of purified Zika virus in size exclusion chromatography may 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.
Thus, the term "purified inactivated whole zika virus" refers to a zika virus obtainable/obtained from a process 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 ℃ to 24 ℃ and provides a main peak of 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 ℃ to 24 ℃ and provides a main peak that is 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 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 ℃ to 24 ℃ and provides a main peak that is at least 95% of the total area under the curve in size exclusion chromatography. In certain embodiments, the purified inactivated Zika virus is a clonal isolate obtained/obtainable by plaque purification.
Methods of inactivating or killing viruses to destroy their ability to infect mammalian cells without destroying the secondary, tertiary or quaternary structure and immunogenic epitopes of the virus are known in the art. Such methods include both chemical and physical means. Suitable means for inactivating the virus include, but are not limited to, treatment with an effective amount of one or more agents selected from the group consisting of: detergents, formalin (also referred to herein as "formaldehyde"), hydrogen peroxide, beta-propiolactone (BPL), Binary Ethylamine (BEI), acetyl ethylene imine, heat, electromagnetic radiation, x-ray radiation, gamma radiation, ultraviolet light (UV 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 purposes of this application, the terms "formalin" and "formaldehyde" are used interchangeably. When referring to formaldehyde concentration herein, it refers to formaldehyde concentration rather than formalin concentration. Thus, a "formaldehyde concentration of 0.01% (w/v)" refers to 0.01% (w/v) formaldehyde, and it is not necessary to further correct this concentration for the concentration of formaldehyde in the formalin stock solution (which typically contains 37% formaldehyde by mass). For example, such formaldehyde concentrations in a virus 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 desired concentration when mixed with a virus preparation such as a zika virus preparation.
In certain embodiments of the present disclosure, at least one virus is chemically inactivated. Agents for chemical inactivation and methods of 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 where 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 can include modifying a nucleic acid. In some embodiments, the modified nucleic acid is an alkylated nucleic acid. In other embodiments, the one or more modifications may comprise modifying the polypeptide. In some embodiments, the modified polypeptide contains modified amino acid residues, including modifications of one or more of cysteine, methionine, histidine, aspartic acid, glutamic acid, tyrosine, lysine, serine, and threonine.
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 comprise modifying the polypeptide. In some embodiments, the one or more modifications may comprise cross-linking the polypeptide. In certain embodiments, wherein the at least one virus is chemically inactivated with formalin, the vaccine or immunogenic composition further comprises 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 can include modifying a nucleic acid. In some embodiments, the modified nucleic acid is an alkylated nucleic acid.
In certain embodiments in which at least one virus is chemically inactivated with formalin, any residual unreacted formalin may be neutralized with sodium metabisulfite, may be leached, and/or may be buffer exchanged to remove residual unreacted formalin. In some embodiments, sodium metabisulfite is added in excess. In some embodiments, the solution may be mixed using a mixer, such as a static mixer in series, followed by filtration or further purification (e.g., using a cross-flow filtration system).
Certain embodiments of the present disclosure relate to a method for inactivating a Zika virus preparation. In some embodiments, the method comprises:
(a) isolating a Zika virus preparation from one or more cells cultured in vitro, 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 group consisting of:
(i) the filter paper is subjected to deep filtration,
(ii) buffer exchange and/or dilution;
(iii) ion exchange chromatography; and
(b) the Zika virus preparation was treated with formaldehyde, wherein the value of the concentration of formaldehyde as measured in% (w/v) multiplied by the incubation time under formaldehyde as measured in days resulted in 0.025 to 0.5.
For example, when the formaldehyde concentration is 0.01% (w/v) and the incubation time under formaldehyde is 10 days, the value obtained by multiplying the formaldehyde concentration by the incubation time under formaldehyde is 0.01 × 10 ═ 0.1.
In some embodiments, the formaldehyde concentration as measured in% (w/v) multiplied by the incubation time under formaldehyde measured in days results in a value of 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 results in a value of 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 results in a value of 0.1.
In some embodiments, the formaldehyde concentration is from 0.005% (w/v) to 0.02% (w/v). In some embodiments, the formaldehyde concentration is from 0.0075% (w/v) to 0.015% (w/v). In some embodiments, the formaldehyde concentration is 0.01% (w/v).
In some embodiments, the formaldehyde concentration as measured in% (w/v) multiplied by the incubation time under formaldehyde measured in days results in a value of 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 results in a value of 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 results in a value of 0.025 to 0.5 and the formaldehyde concentration is 0.01% (w/v).
In some embodiments, the formaldehyde concentration as measured in% (w/v) multiplied by the incubation time under formaldehyde as measured in days results in a value of 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 results in a value of 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 results in a value of 0.05 to 0.25 and a formaldehyde concentration of 0.01% (w/v).
In some embodiments, the formaldehyde concentration as measured in% (w/v) multiplied by the incubation time under formaldehyde measured in days results in a value of 0.075 to 0.15 and a formaldehyde concentration of 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 results in a value of 0.075 to 0.15 and a formaldehyde concentration of 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 results in a value of 0.075 to 0.15 and a formaldehyde concentration of 0.01% (w/v).
In some embodiments, the value of the formaldehyde concentration as measured in% (w/v) multiplied by the incubation time under formaldehyde as measured in days results in 0.1 and the formaldehyde concentration is 0.005% (w/v) to 0.02% (w/v). In some embodiments, the value of the formaldehyde concentration as measured in% (w/v) multiplied by the incubation time under formaldehyde as measured in days results in 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 as measured in days results in a value of 0.1 and the formaldehyde concentration is 0.01% (w/v).
In some embodiments, the formaldehyde concentration as measured in% (w/v) multiplied by the incubation time under formaldehyde measured in days results in a value 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 results in a value 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 results in a value of 0.025 to 0.5 and the incubation time under formaldehyde is ten days.
In some embodiments, the numerical result of multiplying the formaldehyde concentration as measured in% (w/v) by the incubation time under formaldehyde is from 0.05 to 0.25 and the incubation time under formaldehyde is from 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 results in a value of 0.05 to 0.25 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 results in a value of 0.05 to 0.25 and the incubation time under formaldehyde is ten days.
In some embodiments, the formaldehyde concentration as measured in% (w/v) multiplied by the incubation time under formaldehyde measured in days results in a value 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 results in a value 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 results in a value of 0.075 to 0.15 and the incubation time under formaldehyde is ten days.
In some embodiments, the formaldehyde concentration as measured in% (w/v) multiplied by the incubation time under formaldehyde measured in days results in a value of 0.1 and the incubation time under formaldehyde is eight to twelve days. In some embodiments, the value of the formaldehyde concentration as measured in% (w/v) multiplied by the incubation time under formaldehyde measured in days results in 0.1 and the incubation time under formaldehyde is nine to eleven days. In some embodiments, the value of the formaldehyde concentration as measured in% (w/v) multiplied by the incubation time under formaldehyde measured in days results in 0.1 and the incubation time under formaldehyde is ten days.
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 CCL34MDCK (NBL2) cells, MDCK33016 (deposited under number DSM ACC2219 as described in WO 97/37001) cells, BHK21-F cells, HKCC cells, and Chinese hamster ovary cells (CHO cells). In some embodiments, the mammalian cell is a Vero cell.
In certain embodiments of the method, the Zika virus preparation is treated with formalin at a temperature in the range of about 2 ℃ to about 42 ℃. For example, Zika virus preparations may be treated with formalin at a temperature in the range of about 2 ℃ to about 42 ℃, about 2 ℃ to about 8 ℃, about 15 ℃ to about 37 ℃, about 17 ℃ to about 27 ℃, about 20 ℃ to about 25 ℃, or at a temperature of about 2 ℃, about 4 ℃, about 8 ℃, about 10 ℃, about 15 ℃, about 17 ℃, about 18 ℃, about 19 ℃, about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27 ℃, about 28 ℃, about 29 ℃, about 30 ℃, about 37 ℃ or about 42 ℃. In some embodiments, the Zika virus preparation is treated with formalin at a temperature of 15 ℃ to 30 ℃. In some embodiments, the Zika virus preparation is treated with formalin at a temperature of 18 ℃ to 25 ℃. 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 ℃.
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.
In the middle of the inactivation treatment period, the mixture of the virus preparation and formalin may be filtered to remove aggregates. After filtration, the mixture of the virus preparation and formalin is transferred to a new vessel and further treated with formalin until the end of the inactivation treatment period. In some embodiments, if the entire formalin treatment period is eight to twelve days, the mixture of the virus preparation and formalin is filtered after four to six days of formalin treatment. In some embodiments, if the entire formalin treatment period is nine to eleven days, the mixture of the virus preparation and formalin is filtered after five to six days of formalin treatment. In some embodiments, if the entire formalin treatment period is ten days, the mixture of the virus preparation and formalin is filtered five days after formalin treatment. One suitable filter for this step is a 0.2 μm filter.
In some embodiments, the Zika virus formulation is treated with 0.005 to 0.02% (w/v) formalin at a temperature of 15 ℃ to 30 ℃ for eight to twelve days. In some embodiments, the Zika virus formulation is treated with 0.005 to 0.02% (w/v) formalin at a temperature of 15 ℃ to 30 ℃ for nine to eleven days. In some embodiments, the Zika virus formulation is treated with 0.005 to 0.02% (w/v) formalin for ten days at a temperature of 15 ℃ to 30 ℃. In some embodiments, the zika virus preparation is treated with 0.008 to 0.015% (w/v) formalin at a temperature of 15 ℃ to 30 ℃ 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 ℃ to 30 ℃ 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 ℃ to 30 ℃ for ten days. In some embodiments, the Zika virus formulation is treated with 0.01% (w/v) formalin at a temperature of 15 ℃ to 30 ℃ 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 ℃ to 30 ℃ 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 ℃ to 30 ℃ for ten days.
In some embodiments, the Zika virus formulation is treated with 0.005 to 0.02% (w/v) formalin at a temperature of 18 ℃ to 25 ℃ for eight to twelve days. In some embodiments, the Zika virus formulation is treated with 0.005 to 0.02% (w/v) formalin at a temperature of 18 ℃ to 25 ℃ for nine to eleven days. In some embodiments, the Zika virus formulation is treated with 0.005 to 0.02% (w/v) formalin for ten days at a temperature of 18 ℃ to 25 ℃. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w/v) formalin at a temperature of 18 ℃ to 25 ℃ 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 ℃ to 25 ℃ 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 ℃ to 25 ℃ for ten days. In some embodiments, the Zika virus formulation is treated with 0.01% (w/v) formalin at a temperature of 18 ℃ to 25 ℃ 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 ℃ to 25 ℃ 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 ℃ to 25 ℃ for ten days.
In some embodiments, the Zika virus formulation is treated with 0.005 to 0.02% (w/v) formalin at a temperature of 22 ℃ for eight to twelve days. In some embodiments, the Zika virus formulation is treated with 0.005 to 0.02% (w/v) formalin at a temperature of 22 ℃ for nine to eleven days. In some embodiments, the Zika virus formulation is treated with 0.005 to 0.02% (w/v) formalin for ten days at a temperature of 22 ℃. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w/v) formalin at a temperature of 22 ℃ 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 ℃ 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 ℃ for ten days. In some embodiments, the Zika virus formulation is treated with 0.01% (w/v) formalin at a temperature of 22 ℃ 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 ℃ 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 ℃ for ten days.
It is believed that the inactivated Zika virus preparation can be obtained/obtained from a process wherein Zika virus is treated with formaldehyde in an amount in the range of about 0.02% w/v at a temperature of 22 ℃ for 14 days. In some embodiments, it is believed that the inactivated Zika virus preparation can be obtained/obtained from a method wherein Zika virus is treated with formaldehyde in an amount of about 0.01% w/v at a temperature of 22 ℃ for 10 days.
In some embodiments, the method further comprises neutralizing unreacted formalin with an effective amount of sodium metabisulfite. In some embodiments, an effective amount of sodium metabisulfite ranges from about 0.01mM to about 100 mM. For example, sodium metabisulfite may be added at an effective concentration of about 0.01mM to about 100mM, about 0.1mM to about 50mM, about 0.5mM to about 20mM, or about 1mM to about 10mM, or at a concentration of about 0.01mM, about 0.05mM, about 0.1mM, about 0.25mM, about 0.5mM, about 0.75mM, about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 20mM, about 30mM, about 40mM, about 50mM, about 75mM, or about 100 mM. In some embodiments, the formalin is neutralized with about 2mM sodium metabisulfite.
In some embodiments, the zika virus preparation is treated with hydrogen peroxide. In some embodiments, the zika virus formulation is treated with hydrogen peroxide at a concentration in the range of 0.1 to 3% or 0.1 to 1% for 5 to 120 minutes at any temperature from 20 ℃ to 30 ℃. In some embodiments, the zika virus preparation is treated with hydrogen peroxide at a final concentration of 0.01% for 60 minutes or less.
In some embodiments, the method comprises (a) isolating a zika virus preparation from one or more cells cultured in vitro for producing the 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 known in the art for purifying a virus preparation can be used to isolate zika virus, including without limitation the use of cross-flow filtration (CFF), multimodal chromatography, size exclusion chromatography, cation exchange chromatography, and/or anion exchange chromatography. In some embodiments, the virus preparation is isolated by cross-flow filtration (CFF). In some embodiments, the viral 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.
In some embodiments, the zika virus may 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 20159, 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 975-99, Ara982-99, ArA 986-99, ArA 1368, ArB 1362, Nile Hizia 3668, Are Guiya 10366, Are Guinea, ArB 10372, ArB 27433, Hai/Hai, Sp 27433, Are/K3680, Are/K27433, ArK 41519, ArA 368, ArK V3668, Ark III, Ara 41519, Ara III, ArA 36982-III, ArA, Arb 3668, Arb 3699, Ark III, K V III, Thailand (Thailand) SVO127/14, Philippine (Philippine) COC 0740, Brazilian Futalza (Brazil Fortalaza) 2015, and cuba 2017.
In certain embodiments, the Zika virus comprises a mutation in Zika virus non-structural protein 1(NS 1). 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 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 PRVABC 59. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated 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 a purified inactivated Zika virus that differs from the virus strain PRVABC59 by having a Trp98Gly mutation at position 98 of SEQ ID NO: 1.
The vaccines and/or immunogenic compositions of the present disclosure comprising one or more antigens from at least one inactivated zika virus may be used to treat or prevent infection with zika virus 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.
The method for inactivating a zika virus preparation may further comprise step (c) as described below: the completeness of inactivation was determined.
Determination of completeness of inactivation
Other aspects of the 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. Furthermore, the method avoids the use of animals to determine the infectivity of inactivated viruses.
A method for determining the completeness of inactivation of an arbovirus preparation comprises the steps of:
(i) inoculating the cultured insect cells with the arbovirus preparation that has undergone the inactivation step and incubating the insect cells for a first period of time, thereby producing an insect cell supernatant;
(ii) (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
(iii) determining whether the virus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
Arboviruses are viruses that are transmitted by arthropods to humans. It includes viruses from the genera flavivirus, togavirus (togavirus) and bunyavirus (bunyavirus). Arbovirus preparations examined by the methods disclosed herein contain arboviruses that are capable of infecting mammalian cells, particularly Vero cells, and causing cytopathic effects on these cells. In some embodiments, the arbovirus is selected from the group consisting of Zika virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, dengue virus, St.Louis encephalitis virus, tick-borne encephalitis virus, chikungunya virus, Arnien-Nion virus, and Mayaro virus. In some embodiments, the arbovirus is Zika virus.
In some embodiments, the zika virus may 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 20159, 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 975-99, Ara982-99, ArA 986-99, ArA 1368, ArB 1362, Nile Hizia 3668, Are Guiya 10366, Are Guinea, ArB 10372, ArB 27433, Hai/Hai, Sp 27433, Are/K3680, Are/K27433, ArK 41519, ArA 368, ArK V3668, Ark III, Ara 41519, Ara III, ArA 36982-III, ArA, Arb 3668, Arb 3699, Ark III, K V III, Thailand SVO127/14, Philippine COC 0740, Brazilian Futalisa 2015 and cuba 2017.
In certain embodiments, the Zika virus comprises a mutation in Zika virus non-structural protein 1(NS 1). 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 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 PRVABC 59. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated 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 a purified inactivated whole zika virus that differs from the virus strain PRVABC59 by having a Trp98Gly mutation at position 98 of SEQ ID No. 1.
The cultured insect cells are inoculated with the arbovirus preparation by adding the arbovirus preparation to a culture of insect cells comprising 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 incubation, any live virus will be secreted into the insect cell supernatant.
The insect cell used may be any insect cell which is capable of being infected by the arbovirus in question and whose viral infection does not alter viability. 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, A.t.GRIP-1 cells, A.t.GRIP-2 cells, A.t.GRIP-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.
The insect cell supernatant produced by incubating the insect cells with the arbovirus preparation is then used to inoculate the cultured mammalian cells. For inoculation, the insect cell supernatant is transferred to mammalian cells and incubated with the mammalian cells for 60 to 120 minutes or 80 to 100 minutes or 90 minutes. After inoculation, cell culture medium is added and the mammalian cells are incubated with the insect cell supernatant for a second period of time under suitable conditions. 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. Thus, 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 incubation, any live virus will exert a cytopathic effect on mammalian cells. During incubation, any residual replication virus will exert a cytopathic effect on mammalian cells, such as Vero cells.
The mammalian cell used may be any mammalian cell which is capable of being infected by the arbovirus in question 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 CCL34MDCK (NBL2) cells, MDCK33016 (deposited under number DSM ACC2219 as described in WO 97/37001) cells, BHK21-F cells, HKCC cells, and Chinese hamster ovary cells (CHO cells). In some embodiments, the mammalian cell is a Vero cell.
In some embodiments, a method for determining the completeness of inactivation of an arbovirus preparation comprises the steps of:
(i) inoculating the C6/36 cells with the arbovirus preparation that has undergone the inactivation step and incubating the C6/36 cells for a first period of time, thereby producing a C6/36 cell supernatant;
(ii) (ii) inoculating cultured mammalian cells with the supernatant of the C6/36 cells produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the virus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In some embodiments, a method for determining the completeness of inactivation of an arbovirus preparation comprises the steps of:
(i) inoculating the cultured insect cells with the arbovirus preparation that has undergone the inactivation step and incubating the insect cells for a first period of time, thereby producing an insect cell supernatant;
(ii) (ii) inoculating Vero cells with the insect cell supernatant produced in (i) and incubating the Vero cells for a second period of time; and
(iii) determining whether the virus preparation contains residual replication virus which produces cytopathic effects on Vero cells.
In some embodiments, a method for determining the completeness of inactivation of an arbovirus preparation comprises the steps of:
(i) inoculating the C6/36 cells with the arbovirus preparation that has undergone the inactivation step and incubating the C6/36 cells for a first period of time, thereby producing a C6/36 cell supernatant;
(ii) (ii) inoculating Vero cells with the supernatant of C6/36 cells produced in (i) and incubating the Vero cells for a second period of time; and
(iii) determining whether the virus preparation contains residual replication virus which produces cytopathic effects on Vero cells.
In some embodiments, a method for determining the completeness of inactivation of a zika virus preparation comprises the steps of:
(i) inoculating the C6/36 cells with the arbovirus preparation that has undergone the inactivation step and incubating the C6/36 cells for a first period of time, thereby producing a C6/36 cell supernatant;
(ii) (ii) inoculating Vero cells with the supernatant of C6/36 cells produced in (i) and incubating the Vero cells for a second period of time; and
(iii) determining whether the virus preparation contains residual replication virus which produces cytopathic effects on Vero cells.
In some embodiments, a method for determining the completeness of inactivation of a zika virus preparation comprises the steps of:
(i) inoculating C6/36 cells with the arbovirus preparation that has undergone the inactivation step and incubating C6/36 cells for three to seven days, thereby producing a C6/36 cell supernatant;
(ii) (ii) inoculating Vero cells with the supernatant of C6/36 cells produced in (i) and incubating the Vero cells for a second period of time; and
(iii) determining whether the virus preparation contains residual replication virus which produces cytopathic effects on Vero cells.
In some embodiments, a method for determining the completeness of inactivation of a zika virus preparation comprises the steps of:
(i) inoculating the C6/36 cells with the arbovirus preparation that has undergone the inactivation step and incubating the C6/36 cells for a first period of time, thereby producing a C6/36 cell supernatant;
(ii) (ii) inoculating Vero cells with the supernatant of C6/36 cells produced in (i) and incubating the Vero cells for 3 to 14 days; and
(iii) determining whether the virus preparation contains residual replication virus which produces cytopathic effects on Vero cells.
In some embodiments, a method for determining the completeness of inactivation of a zika virus preparation comprises the steps of:
(i) inoculating C6/36 cells with the arbovirus preparation that has undergone the inactivation step and incubating C6/36 cells for three to seven days, thereby producing a C6/36 cell supernatant;
(ii) (ii) inoculating Vero cells with the supernatant of C6/36 cells produced in (i) and incubating the Vero cells for 3 to 14 days; and
(iii) determining whether the virus preparation contains residual replication virus which produces cytopathic effects on Vero cells.
In some embodiments, a method for determining the completeness of inactivation of a zika virus preparation comprises the steps of:
(i) inoculating C6/36 cells with the arbovirus preparation that has undergone the inactivation step and incubating C6/36 cells for six days, thereby producing a C6/36 cell supernatant;
(ii) (ii) inoculating Vero cells with the supernatant of C6/36 cells produced in (i) and incubating the Vero cells for eight days; and
(iii) determining whether the virus preparation contains residual replication virus which produces cytopathic effects on Vero cells.
At the end of the second period, it is determined whether the viral preparation has a cytopathic effect on mammalian cells. Cytopathic effects are any changes in cell structure caused by virus invasion, infection and germination from cells during virus replication. In the methods of the present disclosure, if the cells are cultured in media containing phenol red, the cytopathic effect is determined by the color of the media changing from pink to orange or yellow, or by microscopic examination of the mammalian cells. If microscopic examination of mammalian cells reveals that the cells are rounded, begin to leave the tissue culture vessel (plate, well, or flask) or are cleared from the tissue culture plate/flask, then a cytopathic effect is considered to be present. Other hallmarks of cytopathic effects include fusion of adjacent cells to form syncytia and the presence of nuclear or cytoplasmic inclusion bodies.
As discussed above, the methods disclosed herein have extremely low detection limits. Using this method, less than 1.0TCID can be detected50The viral content of (a). In some embodiments, less than 0.8TCID can be detected50The viral content of (a). In some embodiments, less than 0.5TCID can be detected50The viral content of (a). In some embodiments, less than 0.2TCID can be detected50The viral content of (a). In some embodiments, less than 0.1TCID can be detected50The viral content of (a).
The above method for determining the completeness of inactivation may be used in any method of inactivating an arbovirus. In one embodiment, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.005% to 0.02% w/v formaldehyde;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.0075% to 0.015% w/v formaldehyde;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.01% w/v formaldehyde;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.005% to 0.02% w/v formaldehyde for a period of eight to twelve days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.0075% to 0.015% w/v formaldehyde for a period of eight to twelve days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.01% w/v formaldehyde for a period of eight to twelve days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.005% to 0.02% w/v formaldehyde for a period of nine to eleven days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.0075% to 0.015% w/v formaldehyde for a period of nine to eleven days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.01% w/v formaldehyde for a period of nine to eleven days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.005% to 0.02% w/v formaldehyde for a period of ten days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.0075% to 0.015% w/v formaldehyde for a period of ten days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.01% w/v formaldehyde for a period of ten days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In some embodiments, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.1% to 3% hydrogen peroxide at a temperature of 20 ℃ to 30 ℃ for 5 to 120 minutes;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
In some embodiments, a method for inactivating an arbovirus preparation comprises:
(a) isolating an arbovirus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the arbovirus preparation;
(b) treating the arbovirus preparation with 0.01% hydrogen peroxide at a temperature of 20 ℃ to 30 ℃ for 60 minutes;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the arbovirus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
The above method for determining the completeness of inactivation can be used for any method for inactivating Zika virus. In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.005% to 0.02% w/v formaldehyde;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.005% to 0.02% w/v formaldehyde;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.0075% to 0.015% w/v formaldehyde;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.01% w/v formaldehyde;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.005% to 0.02% w/v formaldehyde for a period of eight to twelve days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.0075% to 0.015% w/v formaldehyde for a period of eight to twelve days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.01% w/v formaldehyde for a period of eight to twelve days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.005% to 0.02% w/v formaldehyde for a period of nine to eleven days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.0075% to 0.015% w/v formaldehyde for a period of nine to eleven days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.01% w/v formaldehyde for a period of nine to eleven days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.005% to 0.02% w/v formaldehyde for a period of ten days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.0075% to 0.015% w/v formaldehyde for a period of ten days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In one embodiment, a method for inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the arbovirus preparation with 0.01% w/v formaldehyde for a period of ten days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In some embodiments, a method of inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.1% to 3% hydrogen peroxide at a temperature of 20 ℃ to 30 ℃ for 5 to 120 minutes;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In some embodiments, a method of inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.01% hydrogen peroxide at a temperature of 20 ℃ to 30 ℃ for 60 minutes;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating the mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) it was determined whether the Zika virus preparation contained residual replication virus that produced cytopathic effects on mammalian cells.
In some embodiments, a method of inactivating a zika virus preparation comprises:
(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation;
(b) treating the Zika virus preparation with 0.05% formalin at a temperature of 20 ℃ to 30 ℃, e.g., 22 ℃, for seven days;
(c) inactivation completeness was determined by:
(i) inoculating the 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;
(ii) (ii) inoculating cultured mammalian cells with the supernatant produced in (i) and incubating the mammalian cells for a second period of time; and
(iii) determining whether the virus preparation contains residual replication virus that produces cytopathic effects on mammalian cells.
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 CCL34MDCK (NBL2) cells, MDCK33016 (deposited under number DSM ACC2219 as described in WO 97/37001) cells, BHK21-F cells, HKCC cells, and Chinese hamster ovary cells (CHO cells). In some embodiments, the mammalian cell is a Vero cell.
Pharmaceutical composition
Other aspects of the present disclosure relate to pharmaceutical compositions comprising inactivated zika virus obtainable by the methods disclosed herein. These pharmaceutical compositions have a particularly low content of residual formaldehyde. Without wishing to be bound by any particular theory, it is hypothesized that the low level of residual formaldehyde reduces the risk of the subject developing side effects.
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 United states Pharmacopeia (US Pharmacopeia), the upper limit of residual formaldehyde in a vaccine comprising inactivated bacteria or viruses is 0.02%, corresponding to 100. mu.g/ml formaldehyde.
The residual formaldehyde content can be determined by any method known to the skilled artisan. One suitable method is described in EMEA, VICH Topic GL25, Biologicals: Testing of residual formaldehyde for the middle 30.4.2002, and involves the use of methylbenzothiazolinone hydrazone hydrochloride (MBTH). Other methods include acetylacetone titration, ferric chloride titration, and basic fuchsin testing. A particularly suitable method is described herein.
The pharmaceutical compositions are in a form that can be administered to a subject and typically contain one or more pharmaceutically acceptable excipients.
The residual formaldehyde content in the pharmaceutical composition is less than 50 μ g/ml. In one embodiment, the residual formaldehyde content in the pharmaceutical composition is less than 45 μ g/ml, less than 40 μ g/ml, less than 35 μ g/ml, less than 30 μ g/ml, less than 25 μ g/ml, less than 20 μ g/ml, less than 15 μ g/ml or less than 10 μ g/ml. In one embodiment, the residual formaldehyde content in the pharmaceutical composition is less than 9.5 μ g/ml, less than 9 μ g/ml, less than 8.5 μ g/ml, less than 8 μ g/ml, less than 7.5 μ g/ml, less than 7 μ g/ml, less than 6.5 μ g/ml, less than 6 μ g/ml, less than 5.5 μ g/ml, less than 5 μ g/ml, less than 4.5 μ g/ml, less than 4 μ g/ml, less than 3.5 μ g/ml, less than 3 μ g/ml, less than 2.5 μ g/ml, less than 2 μ g/ml, less than 1.5 μ g/ml, less than 1 μ g/ml or less than 0.5 μ g/ml. In one embodiment, the residual formaldehyde content of the pharmaceutical composition is less than 0.5 μ g/ml.
Method for determining residual formaldehyde content
Other aspects of the present disclosure relate to a method for determining residual formaldehyde content in a pharmaceutical composition comprising an inactivated virus, the method comprising the steps of:
(a) providing a composition comprising a virus that has been treated with formaldehyde;
(b) mixing the composition of (a) with phosphoric acid and 2, 4-Dinitrophenylhydrazine (DNPH), thereby providing a mixture;
(c) incubating the mixture of (b) under suitable conditions; and
(d) the mixture was analyzed for the presence of residual formaldehyde.
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 invention is particularly suitable for detecting residual formaldehyde in vaccines containing adjuvants such as aluminium hydroxide. The method was validated in terms of specificity, linearity, accuracy, repeatability, robustness and stability according to the International Conference on harmony (ICH) Q2 guidelines.
Compositions comprising viruses that have been treated with formaldehyde may also comprise adjuvants. In one embodiment, the adjuvant is aluminum hydroxide. In one embodiment, the composition comprising the virus which has been treated with formaldehyde comprises 0.1mg/ml to 1.0mg/ml aluminium hydroxide as adjuvant. In one embodiment, the composition comprising the virus which has been treated with formaldehyde comprises 0.2mg/ml to 0.9mg/ml aluminium hydroxide as adjuvant. In one embodiment, the composition comprising the virus which has been treated with formaldehyde comprises 0.3mg/ml to 0.8mg/ml aluminium hydroxide as adjuvant. In one embodiment, the composition comprising the virus which has been treated with formaldehyde comprises 0.3mg/ml to 0.7mg/ml aluminium hydroxide as adjuvant. In one embodiment, the composition comprising the virus which has been treated with formaldehyde comprises 0.3mg/ml to 0.6mg/ml aluminium hydroxide as adjuvant. In one embodiment, the composition comprising the virus which has been treated with formaldehyde comprises 0.3mg/ml to 0.5mg/ml aluminium hydroxide as adjuvant. In one embodiment, a composition comprising a virus that has been treated with formaldehyde comprises 0.4mg/ml aluminum hydroxide as an adjuvant.
In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde is mixed with 1 part of 15 to 25% (v/v) phosphoric acid and 2.5 parts of 0.9 to 1.1mg/ml DNPH. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde is mixed with 1 part of 20% (v/v) phosphoric acid and 2.5 parts of 1.0mg/ml DNPH. In some embodiments, 1ml of a composition comprising a virus that has been treated with formaldehyde is mixed with 20. mu.l of 20% (v/v) phosphoric acid and 50. mu.l of 1.0mg/ml DNPH.
In some embodiments, a 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 ℃ to 30 ℃. In some embodiments, a 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 ℃ to 25 ℃. In some embodiments, a 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 ℃.
In some embodiments, the composition comprising the 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 the 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 the virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH for 20 minutes.
In some embodiments, the composition comprising the virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 18 ℃ to 30 ℃ for 10 to 30 minutes. In some embodiments, the composition comprising the virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 18 ℃ to 30 ℃ for 15 to 25 minutes. In some embodiments, the composition comprising the virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH for 20 minutes at a temperature of 18 ℃ to 30 ℃.
In some embodiments, the composition comprising the virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 20 ℃ to 25 ℃ for 10 to 30 minutes. In some embodiments, the composition comprising the virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 20 ℃ to 25 ℃ for 15 to 25 minutes. In some embodiments, the composition comprising the virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH for 20 minutes at a temperature of 20 ℃ to 25 ℃.
In some embodiments, the composition comprising the virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 22 ℃ for 10 to 30 minutes. In some embodiments, the composition comprising the virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH at a temperature of 22 ℃ for 15 to 25 minutes. In some embodiments, a composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of phosphoric acid and DNPH for 20 minutes at a temperature of 22 ℃.
In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of 1 part of 20% phosphoric acid and 2.5 parts of 1.0mg/ml DNPH for 10 to 30 minutes at a temperature of 18 ℃ to 30 ℃. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of 1 part of 20% phosphoric acid and 2.5 parts of 1.0mg/ml DNPH for 15 to 25 minutes at a temperature of 18 ℃ to 30 ℃. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of 1 part of 20% phosphoric acid and 2.5 parts of 1.0mg/ml DNPH for 20 minutes at a temperature of 18 ℃ to 30 ℃.
In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of 1 part of 20% phosphoric acid and 2.5 parts of 1.0mg/ml DNPH for 10 to 30 minutes at a temperature of 20 ℃ to 25 ℃. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of 1 part of 20% phosphoric acid and 2.5 parts of 1.0mg/ml DNPH for 15 to 25 minutes at a temperature of 20 ℃ to 25 ℃. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of 1 part of 20% phosphoric acid and 2.5 parts of 1.0mg/ml DNPH for 20 minutes at a temperature of 20 ℃ to 25 ℃.
In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of 1 part of 20% phosphoric acid and 2.5 parts of 1.0mg/ml DNPH for 10 to 30 minutes at a temperature of 22 ℃. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of 1 part of 20% phosphoric acid and 2.5 parts of 1.0mg/ml DNPH for 15 to 25 minutes at a temperature of 22 ℃. In some embodiments, 50 parts of a composition comprising a virus that has been treated with formaldehyde is incubated with a mixture of 1 part of 20% phosphoric acid and 2.5 parts of 1.0mg/ml DNPH for 20 minutes at a temperature of 22 ℃.
After incubation, the mixture of the composition comprising the virus that has been 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 that has been 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 that has been treated with formaldehyde and phosphoric acid and DNPH is analyzed by reverse phase HPLC. In one embodiment, the ligand of the reverse phase HPLC column is selected from the group consisting of C18, n-butyl (n-butal), n-octyl, phenyl, and cyanopropyl. In one embodiment, the ligand of the reverse 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 reverse phase HPLC. In one embodiment, the detection wavelength is 360 nm.
In one embodiment, the present disclosure provides a method for determining residual formaldehyde content in a pharmaceutical composition comprising an inactivated virus, the method comprising the steps of:
(a) providing a composition comprising a virus that has been treated with formaldehyde;
(b) mixing 50 parts of the composition of (a) with 1 part of 20% phosphoric acid and 2.5 parts of 1mg/ml2, 4-Dinitrophenylhydrazine (DNPH), thereby providing a mixture;
(c) incubating the mixture of (b) for 20 minutes at room temperature; and
(d) the mixture was analyzed by reverse phase HPLC for the presence of residual formaldehyde using a C18 column and a mixture of water and acetonitrile (1:1, v/v) as the mobile phase.
In one embodiment, the present disclosure provides a method for determining residual formaldehyde content in a pharmaceutical composition comprising inactivated zika virus, the method comprising the steps of:
(a) providing a composition comprising Zika virus that has been treated with formaldehyde;
(b) mixing 50 parts of the composition of (a) with 1 part of 20% phosphoric acid and 2.5 parts of 1mg/ml2, 4-Dinitrophenylhydrazine (DNPH), thereby providing a mixture;
(c) incubating the mixture of (b) for 20 minutes at room temperature; and
(d) the mixture was analyzed by reverse phase HPLC for the presence of residual formaldehyde using a C18 column and a mixture of water and acetonitrile (1:1, v/v) as the mobile phase.
Adjuvant
Other aspects of the present disclosure relate to Zika virus vaccines and/or immunogenic compositions comprising 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, e.g., 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 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, and one or more adjuvants. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated 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, and one or more adjuvants. In one embodiment, the vaccine and immunogenic composition comprises a plaque-purified cloned Zika virus isolate and one or more adjuvants. Such adjuvanted vaccines and/or immunogenic compositions of the present disclosure 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.
Various methods of achieving the adjuvant effect of the vaccine are known and may 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 therapy and practical application of Adjuvants", 1995, Duncan E.S. Stewart-Tull (eds.), John Wiley & SonsLtd, ISBN 0-471-.
Exemplary adjuvants may include, but are not limited to, aluminum salts, calcium phosphate, toll-like receptor (TLR) agonists, Monophosphoryl Lipid A (MLA), MLA derivatives, synthetic lipid a, lipid a mimetics or analogs, cytokines, saponins, Muramyl Dipeptide (MDP) derivatives, CpG oligonucleotides, lipopolysaccharides of gram-negative bacteria (LPS), polyphosphazenes, emulsions (oil emulsions), chitosan, vitamin D, stearoyl or octadecyl tyrosine, viral particles, cochleates, 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.
In some embodiments, the adjuvant comprises at least one of alum, aluminum phosphate, aluminum hydroxide, aluminum potassium sulfate, and Alhydrogel 85. In some embodiments, the aluminum salt adjuvants of the present disclosure have been found to increase antigen adsorption of the zika virus vaccines and/or immunogenic compositions 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.
Certain embodiments of the present disclosure include a process for preparing a zika virus vaccine or immunogenic composition with an adjuvant, the process comprising (a) mixing a vaccine or immunogenic composition with an aluminum salt adjuvant, wherein the vaccine or immunogenic composition comprises 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 in the range of 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 methods, at least one zika virus is a zika virus comprising a non-human cell-adaptive mutation (e.g., a non-human cell-adaptive mutation in protein NS1, e.g., 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 the virus strain PRVABC 59. 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 the virus strain PRVABC59 comprising a genomic sequence according to SEQ ID NO: 2.
Virus purification
Other aspects of the disclosure relate to methods of purifying Zika virus. In some embodiments, the method comprises inoculating a plurality of cells with an inoculum containing a population of zika viruses, and obtaining a zika virus clonal 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.
In some embodiments, the population of zika viruses is heterogeneous, i.e., 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 population of zika viruses comprises a clinical isolate of zika virus (e.g., from the strain PRVABC59) and/or one or more zika viruses that have been previously passaged in cell culture. Clinical isolates of Zika virus were obtained from samples of patients infected with Zika virus. In some embodiments, plaque purification (e.g., as described herein) allows for the substantial and/or complete isolation (genetically homogeneous) of clonal isolates from a heterogeneous viral population. In some embodiments, the monkey cells are from a VERO cell line (e.g., VERO 10-87 cells). In some embodiments, the inoculum comprises human serum. In some embodiments, the inoculum comprises one or more foreign factors (e.g., one or more contaminating viruses). In some embodiments, plaque purification (e.g., as described herein) allows for the substantial and/or complete purification (genetically homogeneous) of a clonal purification strain free of one or more foreign elements.
In some embodiments, methods described for isolating and/or purifying zika virus clones include 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 clone isolate. In some embodiments, methods described for isolating and/or purifying a zika virus clone isolate comprise passaging the zika virus clone isolate one or more times (e.g., one or more times, two or more times, three or more times, four or more times, five or more times, etc.) in cell culture (e.g., in insect cells such as a mosquito cell line and/or in mammalian cells such as a VERO cell line).
Other aspects of the disclosure relate to methods of purifying zika virus for preparation of a vaccine or immunogenic composition. In some embodiments, the method comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, or six or more) of the following steps (in any order, including the following order): subjecting a sample or preparation containing Zika virus to depth filtration; 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 comprising Zika virus to an ion exchange membrane (e.g., anion exchange membrane, cation exchange membrane) to produce a bound portion, wherein the bound portion comprises Zika virus, and eluting the bound portion from the ion exchange membrane; treating a sample containing Zika virus with an effective amount of any of the chemical inactivators described herein; neutralizing a sample containing chemically inactivated Zika virus with sodium metabisulfite; and/or purifying a neutralized sample comprising chemically inactivated Zika virus (e.g., by cross-flow filtration (CFF)). In some embodiments, the method comprises the steps of: (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 comprises zika virus; (c) binding the first retentate to an ion exchange membrane to produce a first bound portion, wherein the first bound portion comprises Zika virus, and eluting the first bound portion from the ion exchange membrane to produce a second eluate, wherein the second eluate comprises Zika virus; (d) passing the second eluate through a second depth filter to produce a second retentate, wherein the second retentate comprises 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).
In the methods of the invention, the zika virus preparation is isolated from one or more cells cultured in vitro, wherein the cells are used to produce the zika virus preparation, by one or more steps selected from the group consisting of depth filtration, buffer exchange and/or dilution and ion exchange chromatography. In one embodiment, the zika virus preparation is isolated from one or more cells cultured in vitro, wherein the cells are used to produce the zika virus preparation, by the steps of depth filtration, buffer exchange and/or dilution and ion exchange chromatography. In one embodiment, the zika virus preparation is isolated from one or more cells cultured in vitro, wherein the cells are used to produce the zika virus preparation, by the steps of depth filtration, buffer exchange and/or dilution and ion exchange chromatography, wherein the steps are performed in the order of depth filtration, buffer exchange and/or dilution and ion exchange chromatography.
Depth filtration means the use of porous filter media in which particles are retained within the media and not just on the media surface.
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 from the filter surface rather than into the filter.
In one embodiment, the step of ion exchange chromatography involves anion exchange chromatography. In one embodiment, anion exchange chromatography uses an anion exchange membrane comprising quaternary ammonium ligands. In some embodiments, the virus is eluted from the anion exchange membrane by a step elution, for example using 250mM NaCl, 500mM NaCl, and 750mM NaCl.
Formulation of vaccines and/or immunogenic compositions
Other aspects of the disclosure relate to formulations of the vaccines and/or immunogenic compositions of the disclosure containing 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 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 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 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 the virus strain PRVABC 59. In some embodiments, the purified inactivated 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 the virus strain PRVABC59 comprising a genomic sequence according to SEQ ID NO. 2.
Such vaccines and/or immunogenic compositions of the present disclosure comprising one or more antigens from zika virus described herein are useful for treating or preventing infection by zika virus in a subject in need thereof and/or inducing an immune response, e.g., a protective immune response, against zika virus in a subject in need thereof.
Typically, the vaccine and/or immunogenic compositions of the present disclosure are prepared as injectable liquids in the form of liquid solutions or suspensions; solid forms suitable for dissolution or suspension in a liquid prior to injection may also be prepared. Such formulations may also be emulsified or produced in dry powder form. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, sucrose, glycerol, ethanol, and the like, and combinations thereof. Furthermore, if necessary, the vaccine or immunogenic composition may contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents or adjuvants which enhance the efficacy of the vaccine or immunogenic composition.
The vaccine or immunogenic composition may be administered parenterally, by injection, for example subcutaneously, transdermally, intradermally, subdermally, or intramuscularly as is conventional. Additional formulations suitable for other modes of administration include suppositories and, in some cases, oral, intranasal, buccal, sublingual, intraperitoneal, intravaginal, anal and intracranial formulations. For suppositories, conventional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10% or even 1-2%. In certain embodiments, a low melting wax, e.g., a mixture of fatty acid glycerides or cocoa butter, is first melted and the zika virus vaccines and/or immunogenic compositions described herein are uniformly dispersed, e.g., by stirring. The molten homogeneous mixture is then poured into a suitably sized mold and allowed to cool and solidify.
The vaccines and/or immunogenic compositions of the present disclosure can be administered in a manner compatible with the administration of the formulation and in an amount that will be therapeutically effective and immunogenic. The amount to be administered will depend 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 desired. Suitable dosage ranges may include, for example, from about 0.1 μ g to about 100 μ g of purified inactivated Zika virus. The amount of purified inactivated Zika virus can be determined by establishing a standard curve using a determined amount of recombinant Zika envelope protein by the Bradford assay (Bradford et al (1976) anal. biochem.72: 248-254).
Suitable regimens for initial administration and booster injections are also variable, but are typically initial administration followed by vaccination or other administration.
The manner of application may vary widely. Any conventional method for administering a vaccine or immunogenic composition is suitable. These methods include oral administration, parenteral administration, by injection, and the like, on a solid physiologically acceptable base or in a physiologically acceptable dispersion. The dosage of the vaccine or immunogenic composition will depend on the route of administration and may vary according to the age of the person to be vaccinated and the formulation of the antigen. The vaccine or immunogenic composition can have a unit dose volume of more than 0.5mL, or less than 0.5mL, as described herein. For example, its administration volume may be 0.25 mL.
For controlling tonicity, physiological salts, such as sodium salts, are preferably included. Sodium chloride (NaCl) is preferred, and may be present in an amount of between 1 and 20 mg/ml. Other salts that may be present include potassium chloride, monopotassium phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, and the like.
The vaccine and/or immunogenic compositions of the present disclosure may include one or more buffering agents. Typical buffers include: phosphate buffer; tris buffer solution; a borate buffer; a succinate buffer; histidine buffer (especially with aluminum hydroxide adjuvant); or citrate buffer. The content of buffer is typically in the range of 5-20 mM.
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 7.5, between 7.0 and 8.5, between 7.0 and 8.0 or between 7.0 and 7.8. Thus, the manufacturing process of the present disclosure may include the step of adjusting the pH of the bulk vaccine prior to packaging.
The vaccine or immunogenic composition is preferably sterile. It is preferably pyrogen-free, e.g. containing <1EU (endotoxin unit, standard measure) per dose, and preferably <0.1EU per dose. It is preferably gluten-free.
In certain embodiments, the vaccine and/or immunogenic compositions of the present disclosure can include an effective concentration of detergent. In some embodiments, an effective amount of detergent may include, without limitation, from about 0.00005% v/v to about 5% v/v or from about 0.0001% v/v to about 1% v/v. In certain embodiments, an 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, the detergent helps maintain the vaccine and/or immunogenic composition of the present disclosure in a dissolved state and helps prevent aggregation of the vaccine and/or immunogenic composition.
Suitable detergents include, for example, polyoxyethylene sorbitan ester surfactants (known as 'Tweens'), octoxynol (e.g., octoxynol-9 (Triton X100) or t-octylphenoxypolyethoxyethanol), cetyltrimethyl ammonium bromide ('CTAB'), and sodium deoxycholate. The detergent may be present in only trace amounts. Trace amounts of other remaining components may be antibiotics (e.g. neomycin, kanamycin, polymyxin b). In some embodiments, the detergent contains a polysorbate. In some embodiments, an effective concentration of detergent comprises a range of about 0.00005% v/v to about 5% v/v.
The vaccine and/or immunogenic composition is preferably stored at between 2 ℃ and 8 ℃. Ideally, it avoids direct light. The antigen and emulsion are typically mixed, but may be initially presented in the form of a kit of separate components, which are mixed at the point of use. The vaccine and/or immunogenic composition will generally be in aqueous form when administered to a subject.
Methods of the present disclosure
Other aspects of the present disclosure relate to methods of treating or preventing zika virus in a subject in need thereof and/or eliciting an immune response to zika virus in a subject in need thereof using the vaccines and/or immunogenic compositions described herein that contain a purified inactivated 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). Other aspects of the present disclosure relate to methods of treating or preventing zika virus in a subject in need thereof and/or eliciting an immune response to zika virus in a subject in need thereof using the vaccines and/or immunogenic compositions described herein comprising 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 present disclosure relate to methods of treating or preventing zika virus in a subject in need thereof and/or eliciting an immune response to zika virus in a subject in need thereof using the vaccines and/or immunogenic compositions described herein comprising 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 the strain PRVABC 59. Other aspects of the present disclosure relate to methods of treating or preventing zika virus in a subject in need thereof and/or eliciting an immune response to zika virus in a subject in need thereof using the vaccines and/or immunogenic compositions described herein comprising 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 the virus strain PRVABC59 comprising a genomic sequence according to SEQ ID NO: 2.
In some embodiments, the disclosure relates to a method 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 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 disclosure relates to a method 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 disclosure containing 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 disclosure relates to a method 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 disclosure 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 the strain PRVABC 59. In some embodiments, the disclosure relates to a method 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 disclosure 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 the virus strain PRVABC59 comprising a genomic sequence according to SEQ ID No. 2.
In some embodiments, the disclosure relates to a method for eliciting 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 disclosure containing a purified inactivated 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 disclosure relates to a method for eliciting 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 disclosure comprising 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 the strain PRVABC 59. In some embodiments, the disclosure relates to a method for eliciting 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 disclosure containing 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 the virus strain PRVABC59 comprising a genomic sequence according to SEQ ID No. 2.
In some embodiments, the administering step elicits a protective immune response in the subject against zika virus. In some embodiments, the subject is a human. In some embodiments, the subject is pregnant or is intended to be pregnant.
In some embodiments, the step of administering comprises administering one or more times. Administration can be via a single dose schedule or a multiple dose (prime-boost) schedule. In a multi-dose schedule, various doses may be administered by the same or different routes, e.g., parenteral priming and mucosal boosting, mucosal priming and parenteral boosting, and the like. Typically, they are administered by the same route. Multiple doses will typically be 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.). It is assumed that it is particularly useful to have two doses 25-30 days apart (e.g., 28 days).
The methods of the present disclosure comprise administering a therapeutically effective or immunogenic amount of a zika virus vaccine and/or immunogenic composition of the present disclosure. A therapeutically effective or immunogenic amount can be an amount of a vaccine and/or immunogenic composition of the present disclosure that will elicit a protective immune response in an uninfected, infected, or unexposed subject to which it is administered. Such a response will generally result in a secreted, cellular, and/or antibody-mediated immune response to the vaccine being manifested in the subject. Typically, such responses include (but are not limited to) one or more of the following effects; producing antibodies from any one of the immunological classes, such as immunoglobulin A, D, E, G or M; b and T lymphocyte proliferation; providing activation, growth and differentiation signals to immune cells; helper T cells, suppressor T cells, and/or cytotoxic T cell expansion.
Preferably, the therapeutically effective amount or immunogenic amount is sufficient to treat or prevent the symptoms of the disease. The exact amount required will vary depending upon, among other things: a subject of treatment; the age and overall condition of the subject to be treated; the ability of the immune system of the subject to synthesize antibodies; the degree of protection required; the severity of the condition being treated; the specific Zika virus antigen selected and its mode of administration. One of ordinary skill in the art can readily determine the appropriate therapeutically effective or immunogenic amount. The therapeutically effective amount or immunogenic amount will be within a relatively wide range as can be determined by routine experimentation.
The present disclosure will be more fully understood by reference to the following examples. The examples, however, should not be construed as limiting any aspect or scope of the disclosure in any way.