阅读说明：本技术 用于将寨卡病毒灭活和用于确定灭活完全性的方法 (Method for inactivating Zika virus and for determining completeness of inactivation ) 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 2018-11-05 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。(The present disclosure relates to methods for inactivating Zika virus, which may be used in vaccines and immunogenic compositions. The present disclosure also relates to a method for determining the completeness of inactivation of an arbovirus preparation.)

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; and

(b) the Zika virus preparation was treated with 0.005% to 0.02% w/v formaldehyde.

2. The method of claim 1, wherein the cell is a non-human cell.

3. The method of claim 1 or 2, wherein said Zika virus preparation is treated with 0.01% formaldehyde.

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

5. The method of claim 4, wherein said Zika virus preparation is treated for ten days.

6. The method of any one of the preceding claims, wherein the Zika virus formulation is treated at a temperature of 15 ℃ to 30 ℃.

7. The method of claim 6, wherein said Zika virus preparation is treated at a temperature of 22 ℃.

8. The method of any one of the preceding claims, further comprising step (c) determining the completeness of inactivation.

9. The method of claim 8, 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.

10. The method of claim 9, wherein the insect cell is selected from the group consisting of CC L-125 cells, Aag-2 cells, RM L-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, L SB-AA695BB cells, NIID-CTR cells, and TRA-171 cells, e.g., C6/36 cells.

11. The method of claim 9 or 10, wherein the first period of time is 3 to 7 days.

12. The method of any one of claims 9 to 11, wherein the mammalian cells are selected from VERO cells, LL C-MK2 cells, MDBK cells, MDCK cells, ATCC CC L34 MDCK (NB L2) cells, MDCK33016 (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.

13. The method of any one of claims 9 to 12, wherein the second period of time is 3 to 14 days.

14. The method of any one of the preceding claims, further comprising step (d) neutralizing the formaldehyde-treated Zika virus preparation with sodium metabisulfite.

15. The method of claim 14, 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.

16. The method of any one of the preceding claims, further comprising the step of (e) preparing a pharmaceutical composition comprising an inactivated Zika virus preparation.

17. The method of claim 16, wherein said Zika virus preparation is mixed with an adjuvant.

18. The method of claim 17, wherein the adjuvant is selected from the group consisting of aluminum salts, toll-like receptor (T L R) agonists, monophosphoryl lipid A (M L A), synthetic lipid A, lipid A mimetics or analogs, M L A derivatives, cytokines, saponins, Muramyl Dipeptide (MDP) derivatives, CpG oligonucleotides, lipopolysaccharide of gram-negative bacteria (L PS), polyphosphazenes, emulsions, viral particles, cochleates, poly (lactide-co-glycolide) (P L G) microparticles, poloxamer particles, microparticles, liposomes, Complete Freund's Adjuvant (CFA), and Incomplete Freund's Adjuvant (IFA).

19. The method of claim 17, wherein the adjuvant is an aluminum salt, such as aluminum phosphate, aluminum hydroxide, aluminum potassium sulfate, and Alhydrogel 85.

20. The method of any one of claims 17-19, 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.

21. 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.

22. The method of claim 21, wherein the mutation is a Trp98Gly mutation in SEQ ID No. 1.

23. The method of claim 21 or 22, wherein said Zika virus does not comprise a mutation in the envelope protein (E).

24. The method of claim 23, wherein the sequence encoding the envelope protein is identical to the corresponding sequence in SEQ ID No. 2.

25. A pharmaceutical composition comprising inactivated zika virus obtainable by the method of any one of claims 1 to 24.

26. A pharmaceutical composition comprising inactivated zika virus and having a residual formalin content of less than 0.5 μ g/ml.

27. The pharmaceutical composition of claim 26, obtainable by the method of any one of claims 1 to 24.

28. 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.

29. The method of claim 28, wherein the arbovirus is a flavivirus or alphavirus.

30. The method of claim 28 or 29, 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 Maya virus.

31. The method of any one of claims 28 to 30, wherein the arbovirus preparation is subjected to a detergent, formalin, hydrogen peroxide, β -propiolactone (BP L), diethylamine (BEI), acetylethyleneimine, methylene blue or psoralen treatment.

32. The method of any one of claims 28 to 31, wherein the insect cell is selected from the group consisting of CC L-125 cells, Aag-2 cells, RM L-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, L SB-AA695BB cells, NIID-CTR cells, and TRA-171 cells, e.g., C6/36 cells.

33. The method of any one of claims 28-32, wherein the first period of time is 3 to 7 days.

34. The method of any one of claims 28 to 33, wherein the mammalian cells are selected from VERO cells, LL C-MK2 cells, MDBK cells, MDCK cells, ATCC CC L34 MDCK (NB L2) cells, MDCK33016 (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.

35. The method of any one of claims 28-34, wherein the second period of time is 3 to 14 days.

36. The method of any one of claims 28 to 35, wherein the method is capable of detecting less than 1.0TCID₅₀The arbovirus of (1).

Technical Field

The present disclosure relates to methods for inactivating Zika virus (Zika virus) useful in vaccines and immunogenic compositions. The present disclosure also relates to a method for determining the completeness of inactivation of an arbovirus preparation.

Background

Flaviviridae (Flaviviridae) flaviviruses classified with other mosquito-borne viruses (e.g., yellow fever virus, dengue fever virus (dengue), West Nile virus (West Nile), and Japanese encephalitis virus), which have spread rapidly since their introduction into Brazil in 2013, becoming a hemispheric epidemic, have reached Central and North America, including the United states territory, and are therefore now threatening the American continent, indeed, the genomes of the Zika virus strain PRVABC59 have been sequenced at least three times from the sera of a person who had traveled to Puerto Rico in 2015, (see L and, et al, Emerg. Infect. month.2016; 22 (933-5) and Anfeo No. 501215.1; GenBank accession No. GenBank 087101.3; Genun K18, et al; Genunox accession No. 364; and No. 3618).

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 2015, fetal abnormalities (e.g., microcephaly) and Guillain-Barre syndrome (GBS) have increased significantly in widespread areas of infection with zika virus, alerting that zika virus may be more lethal than originally thought, which prompted the World Health Organization (WHO) to announce it as an emergent Public Health event of International interest (Public Health of International conference, PHEIC) (Heymann et al (2016) L anchor 387(10020): 719-21.) although zika virus poses a considerable threat to Public Health, no FDA-approved vaccine or treatment is currently available, and the only preventative measures for controlling zika virus involve 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-L ucarelli 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, and developed and applied a two-plasmid system.

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.

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.

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; and

(b) the Zika virus preparation was treated with 0.005% to 0.02% w/v formaldehyde.

In some embodiments, the cell is a non-human cell. In some embodiments, the zika virus preparation is treated with 0.01% formaldehyde. In some embodiments, the zika virus formulation is treated for eight to twelve days, e.g., ten days. In some embodiments, the Zika virus formulation is treated at a temperature of 15 ℃ to 30 ℃, for example at a temperature of 22 ℃.

The method may further comprise the step of (c) determining the completeness of inactivation. In some embodiments, step (c) comprises:

(i) inoculating cultured insect cells with a Zika virus preparation treated with 0.005% to 0.02% w/v formaldehyde 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 CC L-125 cell, an Aag-2 cell, a RM L-12 cell, a C6/36 cell, a C7-10 cell, an AP-61 cell, a.t.GRIP-1 cell, a.t.GRIP-2 cell, a.t.GRIP-3 cell, a UM-AVE1 cell, a Mos.55 cell, a Sua1B cell, a 4a-3B cell, a mos.42 cell, a MSQ43 cell, a L SB-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, LL C-MK2 cell, MDBK cell, MDCK cell, ATCC CC L34 MDCK (NB L2) cell, MDCK33016 (as described in WO97/37001 under accession number DSM ACC2219) cell, BHK21-F cell, HKCC cell, and 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 can further comprise the step of (d) neutralizing the formaldehyde-treated Zika virus preparation with sodium metabisulfite, for example, neutralizing the formaldehyde-treated Zika virus preparation at least five days, at least seven days, at least nine days, at least 11 days, or at least 14 days after the formaldehyde treatment.

The method may further comprise the step of (e) preparing a pharmaceutical composition comprising the inactivated Zika virus.

The Adjuvant may be selected from the group consisting of aluminum salts, toll-like receptor (T L R) agonists, monophosphoryl lipid A (M L A), synthetic lipid A, lipid A mimetics or analogs, M L A derivatives, cytokines, saponins, Muramyl Dipeptide (MDP) derivatives, CpG oligonucleotides, lipopolysaccharide of gram-negative bacteria (L PS), polyphosphazenes, emulsions, viral particles, cochleates, poly (lactide-co-glycolide) (P L G) 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, and 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 treated viral formulation 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 SEQ ID NO 2.

Some aspects of the present disclosure relate to a pharmaceutical composition comprising an inactivated zika virus obtainable by any one of the methods disclosed herein.

Some aspects of the present disclosure relate to a pharmaceutical composition comprising inactivated Zika virus and having a residual formalin (formalin) content of less than 0.5 μ g/ml. In some embodiments, the pharmaceutical composition is obtainable by any one of the methods disclosed herein.

Some aspects of the present disclosure relate to a method for determining residual formalin content in a pharmaceutical composition comprising 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 pharmaceutical 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 formalin.

In some embodiments, the pharmaceutical composition contains an adjuvant. In some embodiments, the pharmaceutical composition contains aluminum hydroxide as an adjuvant. In some embodiments, the pharmaceutical composition contains 0.1mg/ml to 1.0mg/ml aluminum hydroxide as an adjuvant. In some embodiments, the pharmaceutical composition contains 0.4mg/ml aluminum hydroxide as an adjuvant.

In some embodiments, a volume of 1ml of the pharmaceutical composition of (a) is mixed with 20. mu.l of 15 to 25% (v/v) phosphoric acid and 50. mu.l of 0.9 to 1.1mg/ml DNPH. In some embodiments, a 1ml volume of the pharmaceutical composition of (a) is mixed with 20. mu.l of 20% (v/v) phosphoric acid and 50. mu.l of 1.0mg/ml DNPH.

In some embodiments, the pharmaceutical composition of (a) is incubated with a mixture of phosphoric acid and 2, 4-Dinitrophenylhydrazine (DNPH) at room temperature. In some embodiments, the pharmaceutical composition of (a) is incubated with a mixture of phosphoric acid and 2, 4-Dinitrophenylhydrazine (DNPH) for 10 to 30 minutes. In some embodiments, the pharmaceutical composition of (a) is incubated with a mixture of phosphoric acid and 2, 4-Dinitrophenylhydrazine (DNPH) at room temperature for 20 minutes.

In some embodiments, the pharmaceutical composition of (a) is analyzed by HP L C for mixtures with phosphoric acid and 2, 4-dinitrophenylhydrazine (DNPH.) in some embodiments, HP L C is reversed phase HP L C in some embodiments, a mixture of water and acetonitrile (1:1, v/v) is used as the mobile phase in HP L C in some embodiments, the detection wavelength is 360 nm.

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.

Some aspects of the present disclosure 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 Zika virus, West Nile virus, yellow fever virus, Japanese Encephalitis virus (Japanese Encephalitis virus), tick-borne Encephalitis virus, dengue virus (dengue virus), St.Louis Encephalitis virus (St. L ouis Encephalitisvirus), Chikungunya virus (Chikungunya virus), Arsennion virus (O 'nyong' nyong virus), or Mayara virus (Mayarovirus).

In some embodiments, the arbovirus preparation is subjected to inactivation with detergent, formalin, hydrogen peroxide, β -propiolactone (BP L), diethylamine (BEI), acetylethyleneimine, methylene blue, or psoralen.

In some embodiments, the insect cell is selected from the group consisting of a CC L-125 cell, an Aag-2 cell, a RM L-12 cell, a C6/36 cell, a C7-10 cell, an AP-61 cell, a.t.GRIP-1 cell, a.t.GRIP-2 cell, a.t.GRIP-3 cell, a UM-AVE1 cell, a Mos.55 cell, a Sua1B cell, a 4a-3B cell, a mos.42 cell, a MSQ43 cell, a L SB-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, LL C-MK2 cell, MDBK cell, MDCK cell, ATCC CC L34 MDCK (NB L2) cell, MDCK33016 (as described in WO97/37001 under accession number DSM ACC2219) cell, BHK21-F cell, HKCC cell, and 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 arbovirus less than 1.0TCID 50.

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 ZIKAV PRVABC59P 1 on Vero cell monolayers.

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); S L EV (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); S L EV (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 ZIKAV PRVABC59P 1 virus.

FIG. 9 shows the average spot size of ZIKAV PRVABC59P6 virus clones compared to ZIKAV PRVABC59P 1 virus.

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) By dotted line tablesShown in the figure.

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.

FIG. 15 shows serum viremia of individual AG129 mice on two days post challenge, reported as PFU/m L, solid line represents mean value of one group, limit of detection (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/m L.

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 the serum viremia of individual AG129 mice three days after MVS stocks prior to infection with P6a and P6e Zika virus, reported as PFU/m L.

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. mu.g/m L (b), 0.098. mu.g/m L (c), 0.196. mu.g/m L (d), 0.491. mu.g/m L (e), 0.982. mu.g/m L (f) and 1.964. mu.g/m L (g) formaldehyde.

Detailed Description

General technique

Examples of such Methods include those described in The following, those skilled in The art generally better understand and commonly use The techniques and programs described or mentioned herein, such as library et al, Molecular Cloning: A L laboratory Manual 3 (2001) Cold Spring Harbor L laboratory Press, Cold Spring Harbor N.Y., Current Protocols in Molecular Biology (F.M.Audio. editor, (2003), Methods in enzyme engineering series (Academic Press), PCR 2: A Practical application, P.P.D. Hayes and G.R.Taylor editor (Harlow and L ane editor, 1988) edit, Culture Press, Cell L, 671997, 1987, 7, Cell J.1987, edited by soil et al, Culture Press, 7, Cell J.1987, edited by soil et al, Culture, 7, Cell 1987, edited by U.S. A.A.7, Cell, 7, Cell 1987, 7, edited by U.A.A.A.A.A.7, Cell 1988, 7, edited by Molecular research.

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, Arthron 3/Tatari III, Arthro III, Ar III, Ard III, Ar III, E III, E, R103451, 103344, 8375, JMB-185, ZIKV/H, homo sapiens/Brazil (Brazil)/Natta (Natal)/2015, SPH2015, ZIKV/Hu/thousand leaves (Chiba)/S36/2016 and/or Cuba (Cuba) 2017. In some embodiments, the virus strain PRVABC59 is used in the present disclosure.

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.

The Zika virus used 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, e.g., insect cells (e.g., mosquito cells such as CC L-125 cells, Aag-2 cells, RM L-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, 46B cells, 4a-3B cells, Mos.42 cells, MSQ 45 cells, L SB-AA695BB cells, NIID-CTR cells, TRA-171 cells, and cells derived from e.g. Aedes and Aedes mosquitoes (Aedes aeypypaedes), white Aedes (Aedes aegyptis), pseudolepideus (MDC) cells, or mammalian strains such as isolated from Aedes sinensis cells (insect cells) such as the insect cells isolated from Aedes sinensis strains (insect cells, insect cells (e.g., Tokyo), the insect cells isolated from the insect cells of the insect strains, the insect strains such as the insect strains (e.g., the insect strains of Aedes sinensis mosquito fasciola sinensis strains, the insect strains (e.g., insect strains).

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 NS 5.

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:

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, null bits are ignored in the final sequence identity calculation. The difference between the two, i.e., a gap at a single position is counted as a non-identical position versus ignoring 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 the final sequence identity calculation, e.g., the program needle (emboss) that executes the algorithm of needle and Wunsch (needle and Wunsch,1970, j.mol.biol.48: 443-.

Sequence identity can be calculated from a pairwise alignment showing the two sequences over their full length, thus showing the full length of the first and second sequences ("overall sequence identity"). for example, the program needle (embosss) generates such alignments,% sequence identity (number of identical residues/alignment length) × 100) ].

Sequence identity may be calculated from pairwise alignments of local regions showing only the first or second sequence ("local identity"). for example, the program blast (ncbi) generates such alignments,% sequence identity (number of identical residues/length of alignment) × 100) ].

Sequence alignments are preferably generated by using algorithms of Needleman and Wunsch (j.mol. biol. (1979)48, page 443-453.) preferably, The program "NEED L E" (The european molecular Biology Open Software Suite (embos)) is used together with program default parameters (gap opening 10.0, gap extension 0.5 and matrix of proteins EB L OSUM62, and matrix of nucleotides EDNAFU LL.) sequence identity can then be calculated from an alignment displaying both sequences over The full length, thus displaying The full length of The first and second sequences ("overall sequence identity"). for example,% sequence identity (number of identical residues/alignment length) × 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 NS-1 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 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 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 that uses default parameters (e.g., gap opening penalty of 10.0, gap extension penalty of 0.5, using EB L OSUM62 scoring matrix).

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 are preferably of mammalian origin and include, but are not limited to, insect cells (e.g., mosquito cells, VERO cells (from monkey kidney), horses, cows (e.g., MDBK cells), sheep, dogs (e.g., MDCK cells from canine kidney, ATCC CC L MDCK (NB L) or MDCK33016 as described herein, accession No. DSM 2219 as described in WO 97/37001), cats and rodents (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.

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 either Iscove's Medium, Ultra-CHO Medium (BioWhittaker), or EX-CE LL (JRH Bioscience). commonly used serum-containing media include Eagle's Basal Medium (BME) or Minimal Essential Medium (MEM) (Eagle, Science,130,432(1959)) or Darber's Modified Eagle Medium (Dulbecco's Modified Eagle Medium, DMEM or EDM) which are typically used with up to 10% fetal calf serum or similar additives optionally, Minimal Essential Medium (MEM) (Eagle, Science,130,432(1959)) or Darber's Modified Eagle Medium (DMEM or EDM) can be used without any serum-containing additives.

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 a strain of 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. The cultured cells are inoculated with the virus (measured by PFU or TCID50) to a cell ratio of 1:500 to 1:1, preferably 1:100 to 1: 5. The virus is added to the cell suspension or applied to the cell monolayer and is pipetted 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, supernatants from infected cells are harvested 3 to 7 days post infection. More preferably, the supernatant of infected cells is harvested 3 to 5 days post 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, parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus, reovirus, polyomavirus, birnaviruses, circovirus and/or parvovirus (WO 2006/027698).

In the case where the virus has been grown on cell lines, it is standard practice to minimize the amount of residual cell line DNA in the final vaccine to minimize any oncogenic activity of the host cell DNA, removal of contaminating DNA during vaccine preparation may be enhanced by nuclease treatment, e.g. by use of dnase, a convenient Method for reducing host cell DNA contamination disclosed in the reference (L undibulad (2001) Biotechnology and Applied Biochemistry 34:195-197, guidelines for Industry: biochemical Method variation, u.s.department of health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) for viral expression and Research (cver) treatment, and removal of contaminating DNA during this procedure may be carried out by use of a two-step nuclease, e.g. a cellulase-linked nuclease, β, during growth, a two-step nuclease treatment may be used to remove the viral DNA.

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 a cell line suitable for producing the zika virus of the present disclosure may also be a cell 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, ATCCCC L (mdc25) or MDCK33016, DSM 2219 as described in WO 97/37001), cats, and rodents (e.g., hamster cells, e.g., BHK21-F, HKCC cells or hamster ovary cells (CHO cells)), and may be obtained from a number of stages including, e.g., adult, neonatal, fetal, and embryonic, and in certain embodiments, the cells are immortalized (e.g., perc.6 cells, as described in WO 01/38362 and WO02/40665, and as deposited under eck 2, as endothelial cells in endothelial cells, endothelial 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.

The term "purified inactivated whole zika virus" thus refers to a zika virus obtainable/obtained from a process wherein the purified zika virus is treated with formaldehyde in an amount of about 0.01% w/v for 10 days at a temperature of 20 ℃ to 24 ℃ and provides a main peak representing at least 85% 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 for inactivating the virus include, without limitation, 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, β -propiolactone (BP L), diethylamine (BEI), acetylethyleneimine, heat, electromagnetic radiation, x-ray radiation, gamma radiation, ultraviolet (UV radiation), UV-A radiation, UV-B radiation, UV-C radiation, methylene blue, psoralen, carboxyfullerene (C60), hydrogen peroxide, and any combination of any of these.

In certain embodiments of the present disclosure, at least one virus is chemically inactivated, in certain embodiments, at least one virus is chemically inactivated with one or more of BP L, hydrogen peroxide, formalin, or BEI, in certain embodiments, at least one virus is chemically inactivated with BP L, in certain embodiments, the virus may contain one or more modifications.

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 where 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 for producing the zika virus preparation; and (b) treating the Zika virus preparation with from about 0.005% to about 0.02% v/v formaldehyde.

Suitable non-human mammalian cells include, but are not limited to, VERO cells, LL C-MK2 cells, MDBK cells, MDCK cells, ATCC CC L34 MDCK (NB L2) cells, MDCK33016 (accession number DSM ACC2219 as described in WO 97/37001), BHK21-F cells, HKCC cells, and chinese hamster ovary cells (CHO cells).

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, the Zika virus preparation 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 in the range 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 for eight to twelve days at a temperature of 15 ℃ to 30 ℃. In some embodiments, the Zika virus formulation 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 0.01% (w/v) formalin for ten days at a temperature of 22 ℃.

Inactivated whole zika virus preparations are believed to be obtainable/obtained from a process wherein zika virus is treated with formaldehyde in an amount in the range of about 0.02% w/v for 14 days at a temperature of 22 ℃. In some embodiments, it is believed that the inactivated Zika virus preparation can be obtained/obtained from a method wherein the Zika virus is treated with formaldehyde in an amount of about 0.01% w/v for 10 days at a temperature of 22 ℃.

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 viral agent 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 strain PRVABC59 in 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.

Determination of completeness of inactivation

In contrast to assays using only one cell type and in contrast to other methods, such as the TCID50 method, the method has an unexpectedly low limit of detection (L OD) — furthermore, the method avoids the use of animals to determine the infectivity of an inactivated virus.

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. Including 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 Maya 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 village card that differs from the virus strain PRVABC59 in that it has 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.

Suitable insect cells include, but are not limited to, CC L-125 cells, Aag-2 cells, RM L-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, L SB-AA695BB cells, NIID-CTR cells, and TRA-171 cells.

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.

Suitable mammalian cells include, but are not limited to, VERO cells, LL C-MK2 cells, MDBK cells, MDCK cells, ATCC CC L34 MDCK (NB L2) cells, MDCK33016 (as described in WO97/37001 under accession number DSM 2219), BHK21-F cells, HKCC cells, and Chinese hamster ovary cells (CHO cells). in some embodiments, the mammalian cells are Vero cells.

In some embodiments, a method for determining the completeness of inactivation of an arbovirus preparation comprises the steps of:

(i) inoculating C6/36 cells with the arbovirus preparation that has undergone the inactivation step and incubating the insect 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 the cultured Vero 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.

In some embodiments, a method for determining the completeness of inactivation of an arbovirus preparation comprises the steps of:

(i) inoculating C6/36 cells with the arbovirus preparation that has undergone the inactivation step and incubating the insect 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 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 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 the insect 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 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 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 the insect 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 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 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 the insect 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 mammalian cells for 3 to 14 days; 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 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 the insect 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 mammalian cells for 3 to 14 days; 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 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 the insect 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 mammalian cells for eight days; and

(iii) determining whether the virus preparation contains residual replication virus that produces cytopathic effects on mammalian 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 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 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.

Suitable non-human mammalian cells include, but are not limited to, VERO cells, LL C-MK2 cells, MDBK cells, MDCK cells, ATCC CC L34 MDCK (NB L2) cells, MDCK33016 (accession number DSM ACC2219 as described in WO 97/37001), BHK21-F cells, HKCC cells, and chinese hamster ovary cells (CHO cells).

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 adjuvanting 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 fashion and practice application of Adovants", 1995, Duncan E.S. Stewart-Tull (eds.), John Wiley & Sons L td, ISBN 0-471-.

Exemplary adjuvants may include, but are not limited to, aluminum salts, calcium phosphate, toll-like receptor (T L R) agonists, monophosphoryl lipid a (M L a), M L a derivatives, synthetic lipid a, lipid a mimetics or analogs, cytokines, saponins, Muramyl Dipeptide (MDP) derivatives, CpG oligonucleotides, lipopolysaccharide (L PS) of gram-negative bacteria, polyphosphazenes, emulsions (oil emulsions), chitosan, vitamin D, stearoyl or octadecyl tyrosine, viral particles, cochleates, poly (lactide-co-glycolide) (P L G) microparticles, poloxamer particles, microparticles, liposomes, Complete Freund's Adjuvant (CFA), and Incomplete Freund's Adjuvant (IFA).

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 of the zika viruses is a zika virus comprising a non-human cell-adaptive mutation (e.g., a non-human cell-adaptive mutation in protein NS1, such as a Trp98Gly mutation). In some embodiments, at least one Zika virus is a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from 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 population of zika viruses is heterogeneous (e.g., comprises two or more genotypes). 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. 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 virus 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 of additional plaque purifications of zika virus clone isolates (e.g., one or more, two or more, three or more, four or more, five or more, etc.). 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 (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).

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, subcutaneously or intramuscularly, as is customary. 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 dosage of the vaccine or immunogenic composition will depend on the route of administration and may vary depending on the age of the person to be vaccinated and the formulation of the antigen.

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 presented initially as 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 administering step comprises one or more administrations. 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 any one of the immunological classes from, for example, 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.