We claim:
1. A stable vaccine composition comprising one or more arbovirus antigens selected from Zika virus, Chikungunya virus and Japanese encephalitis virus, said antigens being formulated with or without an adjuvant in pharmaceutically acceptable buffer and optionally a preservative, wherein the vaccine composition elicits protective immune response to each of the viruses in mammals.
2. The vaccine composition as claimed in claim 1, wherein the said antigens are inactivated whole virion (virus) antigens.
3. The vaccine composition as claimed in claim 1, wherein the said antigens are purified recombinant antigens.
4. The vaccine composition as claimed in claim 1, wherein the said antigens are prepared using Vero cells as cell substrate by adapting the viruses to Vero cells.
5. The vaccine composition as claimed in claim 1, wherein said antigens are purified and concentrated antigens obtained from one or more methods selected from:
a. ultracentrifugation;
b. density gradient centrifugation;
c. clarification of the viral harvest using membrane filtration, followed by
purification by column chromatography; and
d. tangential flow filtration using membranes with cut off from 100 kDa to 300 kDa,
wherein tangential filtration is carried out either before or after virus inactivation.
6. The vaccine composition as claimed in claim 5, wherein said purification by column chromatography comprises gel filtration, mixed mode resin column chromatography, ion exchange column chromatography, affinity matrix chromatography and hydrophobic interaction chromatography.
7. The vaccine composition as claimed in claim 6, wherein the column chromatography elutes majority of the virus antigen in the flow through using mixed mode size exclusion matrix.
8. The vaccine composition as claimed in claim 2, wherein the said whole viruses are inactivated by at least one or more of a chemical inactivating agent, a physical inactivating agent, an irradiating agent.
9. The vaccine composition as claimed in claim 8, wherein the inactivation is carried out before or after purification of the viruses.
10. The vaccine composition as claimed in claim 9, wherein the viruses are inactivated by chemical inactivating agent selected from formalin (formaldehyde), beta propiolactone (BPL) and hydrogen peroxide.
11. The vaccine composition as claimed in claim 10, wherein the viruses are inactivated by any one of the following methods selected from:
a. Formalin treatment at any concentration ranging from 1: 500 up to 1: 4000 v/v of formalin: virus, at 80C to 370C, preferably 25±30C, for at least 1 to 7 days;
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b. Formalin treatment at any concentration ranging from 1:500 up to 1: 4000 v/v
of formalin: virus, at 20C to 80C for at least 10 to 30 days;
c. Beta propiolactone (henceforth BPL) at any concentration ranging from 1:500
up to 1: 4000 v/v of BPL: virus, for at least 24 to 48 hrs at temperatures
ranging from 80C to 300C, preferably 25±30C, for 48 hours;
d. Beta propiolactone at any concentration ranging from 1: 500 up to 1:4000
(BPL: virus, v/v), at 20C to 80C for at least 3-7 days;
e. A combination of BPL and formalin at any of the aforementioned conditions,
preferably BPL inactivation at 1:3000 (BPL: virus, v/v) for 24 hours followed
by formalin inactivation at 1: 3000 (formalin: virus, v/v) for 24to 48 hours at
150C to 300C, preferably 25±30C;
f. Hydrogen peroxide at any concentration from 0.1 to 3%, preferably 0.1 to 1%
at any temperature from 20 - 300C for 5 minutes to 120 minutes.
12. The vaccine composition as claimed in Claim 8, wherein the inactivation of the viruses by irradiating agent comprises inactivation by gamma irradiation by exposure from 10 kGy (Kilo Gray) up to 35 kGy, preferably 25 kGy to 30 kGy from a 60Co source.
13. The vaccine composition as claimed in claim 8, wherein inactivation of the viruses by irradiating agent comprises inactivation by UV irradiation by exposure to 254 nm for 30 – 60 minutes.
14. The vaccine composition as claimed in claim 8, wherein the virus is inactivated by heat treatment at a temperature between 50°C to 65°C for 30 min up to 2 hrs.
15. The vaccine composition as claimed in claim 1, wherein the buffer is selected from the list comprising of phosphate buffer, citrate buffer, phosphate citrate buffer, borate buffer, tris(hydroxymethyl)aminomethane (Tris) containing buffer, succinate buffer, buffers containing glycine or histidine as one of the buffering agents.
16. The vaccine composition as claimed in claim 15, wherein phosphate buffer is sodium phosphate buffer at concentration of 5 mM up to 200 mM of phosphate ions of any pH between 6.50 to pH 9, and optionally containing sodium chloride at a concentration of 50 to 200 mM.
17. The vaccine composition as claimed in claim 1, wherein the buffer maintains the pH in a liquid composition above pH 6.5, preferably above pH 7.0 throughout the bioprocess from viral culture up to preparation of purified inactivated virus bulk.
18. The vaccine composition as claimed in claim 8, wherein the inactivation is carried out in the presence of a stabilizing agent selected from lactose, sucrose, trehalose, maltose, mannose, iso-maltose, raffinose, stachyose, lactobiose, sorbitol, mannitol, lactobionic acid, dextran, L-glycine, L-histidine, L-glutamic acid, L-aspartic acid and human serum albumin or combinations thereof.
19. The vaccine composition as claimed in claim 18, wherein the stabilizing agent is selected from:
a. 2% sorbitol and 1% L-glycine;
b. 1% sorbitol and 0.5 % L-glycine;
c. 1% mannitol and 0.5% L-glycine;
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d. 1% mannitol and 0.5% L-glutamic acid; and
e. 1% sorbitol and 0.5% L-glycine, 1% human serum albumin.
20. The vaccine composition as claimed in 8, wherein the inactivation of Zika virus comprises inactivation of any genotype/strain, live attenuated Zika virus, deactivated virus, virus like particles, chimeric virus particles that carry any Zika virus antigens particularly the E protein in any heterologous virus backbone, in vectored vaccines and infectious synthetic virus particles derived in vitro or in vivo using the sequence of any Zika virus genome.
21. The vaccine composition as claimed in claim 3, wherein the purified recombinant Zika virus comprises antigens of Zika virus comprising the envelope (E) protein, membrane (M) protein and optionally the non-structural 1 (NS1) protein as vaccine antigens for eliciting immune response for prophylaxis of Zika virus infections.
22. The vaccine composition as claimed in claim 21, wherein the Zika virus has the structural protein sequences as disclosed in SEQ. ID No. 3 and SEQ. ID No.4 corresponding to nucleotide sequences of SEQ ID. No. 1 and SEQ ID No.2 respectively, for use as vaccine antigens against Zika virus infections caused by genotypes or variants thereof.
23. The vaccine composition as claimed in claim 3, wherein the Recombinant DNA constructs comprises a (i) vector (ii) at least one nucleic acid fragment corresponding to SEQ ID NO.1 or SEQ ID NO. 2 encoding the amino acid sequence of the proteins of SEQ ID NO.3, SEQ ID NO.4 respectively which is applicable to any Zika virus protein sequences that share at least 70% amino acid identity to the aforementioned SEQ ID NO. 3 and SEQ ID NO.4.
24. The vaccine composition as claimed in claim 23 comprising a recombinant DNA construct, wherein the vector is an eukaryotic plasmid vector being cloned in a eukaryotic host such as baculovirus for expression in insect cells as virus like particles (VLPs).
25. The vaccine composition as claimed in claim 22, wherein the recombinant proteins of the viruses are obtained by the process comprising the steps of:
a. transfecting the recombinant plasmid DNA in insect cells;
b. harvesting the cells and isolating the recombinant protein therefrom;
c. purifying the protein by a method selected from ion exchange
chromatography, gel filtration, affinity chromatography, hydrophobic column
chromatography, mixed mode resin chromatography, diafiltration,
ultracentrifugation, density gradient centrifugation and fractionation with salt.
26. The vaccine composition as claimed in claim 1, wherein the structural antigens of the viruses are expressed in prokaryotic and eukaryotic expression system including baculovirus mediated expression in insect cells.
27. The vaccine composition as claimed in claim 1, wherein the composition is obtained by a process wherein neutralizing antibodies are largely elicited against the Envelope protein such as in optimally inactivated virus, live attenuated virus, deactivated virus, DNA vaccine, virus like particles, chimeric virus particles that display the Zika virus
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E protein in any heterologous virus backbone such as in vectored vaccines and synthetic virus particles derived from any Zika virus genomic RNA sequence.
28. The vaccine composition as claimed in claim 1, further comprising an adjuvant.
29. The vaccine composition as claimed in claim 28, wherein the adjuvant is selected from the group consisting of a) aluminum salts comprising aluminum hydroxide, aluminum phosphate, aluminum sulphate phosphate; b) inulin; c) algammulin which is a combination of inulin and aluminium hydroxide; d) monophosphoryl lipid A (MPL); e) resiquimod; f) muramyl dipeptide (MDP); g) N-glycolyl dipeptide (GMDP); h) polyIC; i) CpG oligonucleotide; j) aluminum hydroxide with MPL; k) water in oil emulsion; l) oil in water emulsion that contains one or more of the following constituents: squalene or its analogues or any pharmaceutically acceptable oil, tween-80, sorbitan trioleate, alpha-tocopherol, cholecalciferol and aqueous buffer, or analogues and derivatives of the molecules thereof, used individually or in combinations.
30. The vaccine composition as claimed in claim 29, wherein the composition comprises aluminum hydroxide in a concentration range of 0.1 mg to 1.5 mg of aluminum per vaccine dose, preferably 0.25 mg to 0.5 mg aluminum per vaccine dose.
31. The vaccine composition as claimed in claim 29, wherein the adjuvant confers mucosal immunity and systemic immunity when administered in mammals.
32. The vaccine composition as claimed in claim 1, wherein the composition with the virus antigens is administered at any dose ranging from 0.125 µg to 100 µg per dose with or without an adjuvant, either as a single dose or in two or more doses to elicit an immune response in a mammal.
33. The vaccine composition as claimed in claim 1 comprising Zika virus and Japanese encephalitis virus antigens in a combination vaccine that elicits protective immune response in mammals against each of the viruses.
34. The vaccine composition as claimed in claim 33, wherein the virus inactivated antigens are present in the combination vaccine at concentrations ranging 5 µg to 50 µg of each antigen in a pharmaceutically acceptable formulation without an adjuvant, or with an adjuvant.
35. The vaccine composition as claimed in claim 34, wherein the adjuvant is selected from the group consisting of a) aluminum salts comprising aluminum hydroxide, aluminum phosphate, aluminum sulphate phosphate; b) inulin; c) algammulin which is a combination of inulin and aluminium hydroxide; d) monophosphoryl lipid A (MPL); e) resiquimod; f) muramyl dipeptide (MDP); g) N-glycolyl dipeptide (GMDP); h) polyIC; i) CpG oligonucleotide; j) aluminum hydroxide with MPL; k) water in oil emulsion; l) oil in water emulsion that contains one or more of the following constituents: squalene or its analogues or any pharmaceutically acceptable oil, tween-80, sorbitantrioleate, alpha-tocopherol, cholecalciferol and aqueous buffer, or any of the analogues and derivatives of the molecules thereof, used individually or in combinations.
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36. The vaccine composition as claimed in claim 35, wherein the adjuvant is aluminium hydroxide with 0.25 mg to 1.0 mg of aluminium content per vaccine dose.
37. The vaccine composition as claimed in claim 36, wherein the virus antigens are present in a combination vaccine at concentrations ranging from 5 µg to 50 µg of each antigen in a pharmaceutically acceptable formulation without an adjuvant, or with an adjuvant.
38. The vaccine composition as claimed in claim 37, wherein the adjuvant is selected from the group consisting of a) aluminum salts comprising aluminum hydroxide, aluminum phosphate, aluminum sulphate phosphate; b) inulin; c) algammulin which is a combination of inulin and aluminium hydroxide; d) monophosphoryl lipid A (MPL); e) resiquimod; f) muramyl dipeptide (MDP); g) N-glycolyl dipeptide (GMDP); h) polyIC; i) CpG oligonucleotide; j) aluminum hydroxide with MPL; k) any water in oil emulsion; l) any oil in water emulsion that contains one or more of the following constituents: squalene or its analogues or any pharmaceutically acceptable oil, tween-80, sorbitantrioleate, alpha-tocopherol, cholecalciferol and aqueous buffer, or any of the analogues and derivatives of the molecules thereof, used individually or in combinations.
39. The vaccine composition as claimed in claim 38, wherein the adjuvant is aluminium hydroxide at 0.25 mg to 1.5 mg of aluminium content per vaccine dose.
40. The vaccine composition as claimed in claim 1 comprising Zika virus, Chikungunya virus and Japanese encephalitis virus antigens in a combination vaccine that elicits protective immune response in mammals against each of the viruses.
41. The vaccine composition as claimed in claim 40, wherein the virus antigens are present in a combination vaccine at concentrations ranging from 5 µg to 50 µg of each antigen in a pharmaceutically acceptable formulation without an adjuvant, or with an adjuvant.
42. The vaccine composition as claimed in claim 41, wherein the adjuvant is selected from the group consisting of a) aluminum salts comprising aluminum hydroxide, aluminum phosphate, aluminum sulphate phosphate; b) inulin; c) algammulin which is a combination of inulin and aluminium hydroxide; d) monophosphoryl lipid A (MPL); e) resiquimod; f) muramyl dipeptide (MDP); g) N-glycolyl dipeptide (GMDP); h) polyIC; i) CpG oligonucleotide; j) aluminum hydroxide with MPL; k) any water in oil emulsion; l) any oil in water emulsion that contains one or more of the following constituents: squalene or its analogues or any pharmaceutically acceptable oil, tween-80, sorbitantrioleate, alpha-tocopherol, cholecalciferol and aqueous buffer, or any of the analogues and derivatives of the molecules thereof, used individually or in combinations.
43. The vaccine composition as claimed in claim 42, wherein the adjuvant is aluminium hydroxide at 0.25 mg to 1.0 mg of aluminium content per vaccine dose.
44. The vaccine composition as claimed in claim 1, wherein the preservative is 2-phenoxyethanol in a concentration ranging from 2.5 to 5 mg/mL.
45. The vaccine composition as claimed in claim 1, when administered in a single dose or in two or more doses in mammals elicits both Th1 and Th2 immune response
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against any of the arbovirus antigens comprising Zika Virus, Chikungunya virus and Japanese Encephalitis virus and is suitable for administration to humans.
46. A method for preparation of a vaccine composition comprising one or more arbovirus
antigens selected from Zika virus, Chikungunya virus and Japanese encephalitis
virus, the method comprising one or more steps of inactivation, producing
recombinant protein, expressing structural antigens, purification and concentration of
the virus antigen wherein said purification and concentration of Zika, Chikungunya
and Japanese Encephalitis virus comprises one or more steps selected from:
a. ultracentrifugation;
b. density gradient centrifugation;
c. clarification of the viral harvest using membrane filtration;
d. purification by column chromatography;
e. tangential flow filtration using membranes with cut off from 100 kDa to 300
kDa, wherein tangential filtration is carried out either before or after virus
inactivation.
47. The method as claimed in claim 46, wherein the column chromatography method comprises gel filtration, mixed mode resin column chromatography, any ion exchange column chromatography, affinity matrix chromatography and hydrophobic interaction chromatography.
48. The method as claimed in claim 46, wherein the column chromatography elutes majority of the virus antigen in the flow through using mixed mode size exclusion matrix.
49. The method as claimed in claim 46, wherein the viruses are inactivated by one or more inactivating agents selected from a chemical inactivating agent, a physical inactivating agent and an irradiating agent.
50. The method as claimed in claim 46, wherein inactivation of the viruses is carried out before or after purification of the viruses.
51. The method as claimed in claim 50, wherein the viruses are inactivated by chemical inactivating agent selected from formalin (formaldehyde), beta propiolactone (BPL) and hydrogen peroxide.
52. The method as claimed in claim 49, wherein the virus bulk are inactivated by any one of the following methods selected from:
a. Formalin treatment at any concentration ranging from 1: 500 up to 1: 4000 v/v
of formalin: virus, at 80C to 370C, preferably 25±30C, for at least 1 to 7 days;
b. Formalin treatment at any concentration ranging from 1:500 up to 1: 4000 v/v
of formalin: virus, at 20C to 80C for at least 10 to 30 days;
c. Beta propiolactone (henceforth BPL) at any concentration ranging from 1:500
up to 1: 4000 v/v of BPL: virus, for at least 24 to 48 hrs, if not more, at
temperatures ranging from 80C to 300C, preferably 25±30C, for 48 hours;
d. Beta propiolactone at any concentration ranging from 1: 500 up to 1:4000
(BPL: virus, v/v), at 20C to 80C for at least 3-7 days;
e. a combination of BPL and formalin at any of the aforementioned conditions,
preferably BPL inactivation at 1:3000 (BPL:virus, v/v) for 24 hours followed by
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formalin inactivation at 1: 3000 (formalin: virus, v/v) for 24to 48 hours at 15 C to 30°C, preferably 25±3°C ;
f Hydrogen peroxide at any concentration from 0.1 to 3%, preferably 0.1 to 1% at any temperature from 20 - 30°C for 5 minutes to 120 minutes.
53. The method as claimed in claim 49, wherein the viruses are inactivated by gamma irradiation by exposure from 20 kGy (Kilo Gray) up to 35 kGy, preferably 25 kGy to 30 kGy from a 60Co source.
54. The method as claimed in claim 49, wherein the viruses are inactivated by UV irradiation by exposure to 254 nm for 30-60 minutes.
55. The method as claimed in claim 49, wherein the viruses are inactivated by heat treatment from 50°C to 65°C for 30 min up to 2 hrs, preferably, 65°C for 1 hr.
56. The method as claimed in claim 49, wherein the inactivation is carried out in the presence of stabilizing agent selected from lactose, sucrose, trehalose, maltose, mannose, iso-maltose, raffinose, stachyose, lactobiose, sorbitol, mannitol, lactobionic acid, dextran, L-glycine, L-histidine, L-glutamic acid, L-aspartic acid and human serum albumin or combinations thereof.
57. The method as claimed in claim 56, wherein the stabilizing agent is selected from:
a. 2% sorbitol and 1% L-glycine;
b. 1% sorbitol and 0.5 % L-glycine;
c. 1% mannitol and 0.5% L-glycine;
d. 1% mannitol and 0.5% L-glutamic acid; and
e. 1% sorbitol and 0.5% L-glycine, 1% human serum albumin.
58. The method as claimed in claim 46, wherein the method of producing the
recombinant protein comprises the steps of:
a. transfecting recombinant plasmid DNA in insect cells;
b. harvesting the cells and isolating the recombinant protein therefrom;
c. purifying the protein by at least one of the methods comprising of ion exchange
chromatography, gel filtration, affinity chromatography, hydrophobic column
chromatography, mixed mode resin chromatography, diafiltration,
ultracentrifugation, density gradient centrifugation, fractionation with salt.
59. The method as claimed in claim 46, wherein the method of expressing the structural
antigens of the virus comprising expression system is prokaryotic or eukaryotic
expression system including baculovirus mediated expression in insect cells.