Description:FIELD OF THE INVENTION
[001] The present invention relates to the field of vaccines. In particular, the invention relates to providing an efficient & effective vaccine for Chikungunya. More particularly, the invention provides an inactivated vaccine composition for Chikungunya virus infection. The vaccine composition is immunogenic and elicits protective immune response.
BACKGROUND OF THE INVENTION
[002] Chikungunya virus (CHIKV) is an arthritogenic arbovirus which belongs to the genus alphavirus of the Togaviridae family which includes Ross River virus (RRV), Semliki Forest virus (SFV) and Sindbis virus (SINV). CHIKV is responsible for recurring epidemics over the years worldwide (Mason and Haddow, 1957) and is considered an important public health problem because it is endemic in tropical and subtropical regions of the globe. The transmission occurs by the bite of infected mosquitoes from genus Aedes spp., mainly Aedes aegypti and Aedes albopictus, which are highly domesticated and extremely adaptable to environment changes, respectively, thereby resulting in an efficient spread across the countries and continents (Azevedo et al., 2015; World Health Organization, 2017). The chikungunya virus infection results in a disease known as Chikungunya fever (CHIKF), characterized by high fever, rash, myalgia, headache, and a prominent polyarthralgia (Burt et al., 2017). Indeed, the name “Chikungunya”, which means “to become contorted” in the Kimakonde language, reflects the most remarkable characteristic of this disease, which is the intense and persistent joint pain (Arora et al., 2020). The symptom of the disease is present in more than 90% of the symptomatic cases and can last for weeks, months, or even years in some individuals after complete virus clearance, resulting in a notorious economic and social impact (Wahid et al., 2017; Suhrbier, 2019).

[003] Although CHIKF is known as a non-deadly disease, a typical and severe acute manifestation can evolve to multiple organ failure and death. The mortality rates can range from 0.024 up to 0.7% and depends on both the virus genotype/strain and the commitment of neurological system (Jaffar-Bandjee et al., 2010; de Brito, 2017; Dorléans et al., 2018; Freitas et al., 2018; da Silva et al., 2018; Suhrbier, 2019). The disease can be diagnosed by various serological tests, but definitive identification requires verification of the genetic material since many closely related arboviruses cause similar diseases.

[004] Also, not much is known about viral proteins, their function or pathogenicity of CHIKV. The genome consists of 12 kilobases, with a 5' 7mG cap and the 3' poly(A) region, and a base composition of 30% A, 25% C and G, and 20% U. The genome has the sequence of 5'- nsPl-nsP2-nsP3-nsP4-(junction region)-C-E3-E2-6K-El-polyA-3'. The non-structural proteins are translated directly from the 5' two-thirds of the genome, and the structural proteins are produced from the 26S subgenomic RNA which is collinear with the 3' one- third of the genome. The genome contains conserved repeat sequences as well as an internal poly (A) tract within the 3' non-translated region. Based on sequence information, Nonstructural gene sequences consisted of 7,422 nucleotides encoding 2,474 amino acids. The nonstructural polyprotein consists of nsP1 (535 aa), nsP2 (798 aa), nsP3 (530 aa), and nsP4 (611 aa) plays an important role in the viral RNA replication and translation (Badar et al., 2021) while structural proteins C, E3, E2, 6K and El proteins contain 261, 64, 423 and 61 amino acids (Khan et al., 2002; Schuffenecker et al., 2006).

[005] CHIKV infection (whether clinical or silent) is thought to confer life-long immunity. Because of close antigenic relationship, cross-protection between different strains (Casals, 1957; Porterfield, 1961;) as well as reciprocal cross-protection among other alphaviruses (Parks and Price, 1958; Hearn and Rainey, 1963) can be hypothesized, and is demonstrated in animal models. However, there is some available evidence that live attenuated alphavirus vaccines may interfere with a subsequent, related vaccine (McClain et al., 1998). As a prelude to vaccines, initial CHIKV preparations involved either formalin inactivation (Harrison et al., 1967) or tween-ether extraction of virus grown in vitro (Eckels et al., 1970). While formalin kills HA activity, the latter treatment retains the HA activity completely, although both lose infectivity drastically. However, they both elicit similar HA and complement-fixing and neutralization antibodies and also show similar levels of protection in lethal challenge studies (Eckels et al., 1970). US Army Medical Institute of Infectious Diseases in Fort Detrick, Maryland made a candidate vaccine for CHIKV. CHIKV strain 15561 from Thailand (1962 outbreak) was used to develop a small lot of green monkeys passaged, formalin- inactivated preparation that was administered to 16 volunteers who showed high immune responses and no adverse effects (Harrison et al., 1971). The GMK-passaged virus was further serially passaged by plaquing 18 times in MRC-5 cells (Levitt et al., 1986), and found to be safe and immunogenic in phase I trial with 15 alphavirus-naive individuals, viremia occurring on day 2-4 post-inoculation (McClain et al., 1988). In a randomized, double-blind, placebo-controlled, phase II trial, 73 alphavirus-naive volunteers of 18-40 years were injected with 0.5 mL dose containing either ~105 PFU of virus (59 subjects) or placebo (14 subjects) subcutaneously. Serological evaluation involved plaque reduction neutralization titer (PRNT), and a 50% reduction titer of >20 was considered positive. Local and systemic reactions were limited to vaccine take whereas 8% of CHIKV vaccinees (and none of placebo group) showed arthralgia. 98.3% of vaccinates seroconverted by day 28, achieving peak PRNT50 titers of 1:10240 at 28-42 days. Although antibody levels declined somewhat over time, 85% of the vaccines were still seropositive at one year, with titers of 1:1280 at 180-360 days (Edelman et al., 2000).

[006] Accordingly, despite the relevance of CHIKV infection to public health, there is still no vaccine or an effective antiviral drug for either the prevention or treatment of CHIKF. Thus, there is a need in the art to develop a vaccine for the prevention of Chikungunya infection, which is non-infectious, immunogenic and elicits protective immune response.

[007] Therefore, to address the existing problem known in the art, inventors of present invention have devised a vaccine for the prevention of Chikungunya infection. Particularly, the inventors has developed a vaccine composition capable of eliciting protective immune response against Chikungunya virus infection in humans and other mammalian hosts.
SUMMARY OF THE INVENTION
[008] The present invention relates to the field of vaccines. In particular, the invention relates to providing an efficient & effective vaccine for Chikungunya virus infection. More particularly, the invention provides a purified inactivated virus which is suitable for the development of chikungunya vaccine. The method used for the chemical inactivation of the harvested virus uses formalin and the purification of the inactivated virus is performed using single mode ion exchange chromatography.
[009] In an aspect, the present invention provides a vaccine composition capable of eliciting protective immune response against Chikungunya virus (CHIKV) infection in humans and other mammalian hosts. The immunogenic vaccine composition comprises purified inactivated chikungunya virus and a pharmaceutically acceptable excipient/carrier. The inactivated virus preparation is non-infectious, immunogenic and elicits protective immune response in mammalian host. The immunogenic composition is formulated for in vivo administration to humans. Particularly, the vaccine composition comprises a pharmaceutical accepted tris buffered saline in the pH range 7.5-8.0 with added stabilizing agents including non-reducing sugars such as sucrose, inorganic/organic substances like phosphates and amino acids such as glutamate. The stable vaccine composition is formulated in a liquid form suitable for intramuscular /intradermal/ subcutaneous/intravenous administration in a human host.
[0010] In another aspect, the present invention also relates to a method for preparation of an inactivated vaccine composition for chikungunya virus infection.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0011] Figure 1 illustrates:
A- Control uninfected Vero cells
B- Vero cells after 24 hours of CHIKV infection
C- Vero cells after 48 hours of CHIKV infection
[0012] Figure 2 illustrates SDS-PAGE of purified CHIKV preparation depicting the EI/E2 and capsid proteins. The virus sample was run in 10% denaturing SDS-PAGE gel. The two proteins appear to be approximately 46 - 50 kDa in size.
[0013] Figure 3 illustrates viral load dynamics of CHIKV following vaccine administration in mice.
A- Body weight change in mice in comparison to placebo at Day 72. Two way ANOVA has been used to analyze body weights.
B- Assessment of CHIKV vaccine strain by measuring footpad swelling induced by CHIKV of 104 PFU infection.
C- Masson’s Trichrome stained section of footpad showing the thickness, in the images yellow arrow shows the thickness of the inflated area of the footpad.
D- Graphical representation showing thickness of the inflated area.
E- H&E-stained sections of liver.
F- Graphical representation showing percentage cellularity in liver.
G- H&E-stained sections of brain tissue at Day 90
H- Graphical representation showing cell accumulation calculated as percentage cellularity.
[0014] Figure 4 illustrates evaluation of immune responses of the mice following vaccine administration using Plaque Reduction Neutralization Test.
A- Immunogenicity data of inactivated Chikungunya vaccine after 14, 28, 42, 56, 72 and 90 days of immunization.
B- Plaque assay, Plaque morphology of serum samples obtained 28 days after the first immunization in C57B/6 mice.
C- Flow cytometric analysis of T cells in spleen. Single cell suspension was obtained from the spleen at day 90.
D- B cells population in single suspension of spleen in flow cytometry at time point Day 90.
E- Formation of germinal centers in spleen.

DESCRIPTION OF THE INVENTION
[0015] While the invention is susceptible to variations and modifications other than those specifically described herein by specific embodiments and examples. It is to be understood that the present disclosure includes all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims.
[0016] The figures and protocols have been represented for only showing the specific details that are pertinent to understanding of the embodiments of the present invention and not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the disclosure.
[0018] The present invention relates to providing an efficient & effective vaccine for Chikungunya virus infection. The vaccine composition disclosed in the present invention is capable of eliciting protective immune response against Chikungunya virus (CHIKV) infection in humans and other mammalian hosts.
[0019] The immunogenic vaccine composition comprises purified inactivated Chikungunya virus in a stable form. Inactivated virus preparation is non-infectious and immunogenic. The immunogenic vaccine composition is formulated for in vivo administration to humans. The method used to produce inactivated virus particles of Chikungunya virus are also applicable to other genotype/strains of Chikungunya virus. The virus particles obtained from the clinical isolates of patient’s serum from Indian state of Gujarat have been adapted and propagated in vitro in cell monolayers for several passages.
[0020] Particularly, the Chikungunya (CHIK) virus vaccine composition according to the present invention refers to an active ingredient (immunogen) of an inactivated Chikungunya virus that is produced by infecting in a monolayer of Vero cells in continuous cell culture where the cells are used as host cells for culturing the Chikungunya virus. The cell line is qualified through various quality control tests and is approved by FDA/National Regulatory Authority as a vaccine quality cell line. The virus is propagated in large quantities by growing to a high titer (~109) in cell culture and inactivated by chemical agents. The inactivated virus is then purified from infected cells.
[0021] The vaccine composition comprises an inactivated virus particle diluted with any suitable diluent that is pharmaceutically acceptable so as to obtain the desired titer. The buffer used in the formulation is phosphate buffer. A vaccine composition may optionally contain preservative(s), stabilizer(s) etc., including non-reducing sugars such as sucrose, inorganic/organic substances like phosphates and amino acids such as glutamate. Such a stable composition of the immunogen in a liquid form in a pharmaceutically acceptable buffer is suitable foradministrationintraperitoneally/intradermally/subcutaneously/intramuscularly in human host.
[0022] In an aspect of the present invention, there is provided a vaccine composition for chikungunya virus infection comprising:
(a) a chemically inactivated and purified chikungunya virus antigen; and
(b) pharmaceutically acceptable excipient,
wherein the composition elicits a protective immune response against Chikungunya virus infection.
[0023] In an embodiment, there is provided a vaccine composition, wherein the antigen is inactivated using formalin in a concentration ranging from 0.01 to 0.05%.
[0024] In another embodiment, there is provided a vaccine composition, wherein the pharmaceutical acceptable excipient is selected from tris buffered saline, preservatives, stabilizing agents including non-reducing sugars such as sucrose, inorganic/organic substances.
[0025] In still another embodiment, there is provided a vaccine composition, wherein the composition optionally comprises an adjuvant.
[0026] In yet another embodiment, there is provided a vaccine composition, wherein the adjuvant is selected from aluminium hydroxide, aluminium oxy-hydroxide aluminium phosphate or aluminium hydroxide or oxy-hydroxide in combination with fructans and derivatives.
[0027] In an embodiment, there is provided a vaccine composition, wherein the fructans such as inulin is selected from oligosaccharides and polysaccharides.
[0028] In another embodiment, there is provided a vaccine composition, wherein the virus antigen is present in a range of 10 to 50 mcg per dose.
[0029] The immunogen of a Chikungunya virus vaccine is a representative example of an immunogen of a vaccine against infectious diseases caused by Chikungunya viruses of other strain or genotypic variants of the Chikungunya virus. The vaccine is provided in a sealed vial in a liquid format that can either be administrated to a subject an amount 0.2 ml intraperitoneally/ subcutaneously / intramuscularly / intradermally/intravenously orally / intranasally. In an embodiment, there is provided a vaccine composition, wherein the composition is formulated in a liquid form suitable for intramuscular, intradermal, subcutaneous and intravenous administration.
[0030] In another aspect of the present invention, there is provided a process for preparing the vaccine composition comprises inoculating an appropriate host cell line such as vero cells with the virus, maintaining the infected cells in continuous culture, culturing involving infecting the host cell monolayer with the virus and harvesting the virus in sufficient quantities from the infected host cells. The virus grown in cell layers could include a population of the extracellular virus that is obtained in the supernatant of the infected cell culture that can be harvested by centrifugation, and harvesting the virus that is cell associated by centrifugation. Vero cell line propagated in vitro in culture can be used as a host for virus culture. For example, serially passaged Vero cell lines has been used. For propagating Chikungunya virus strains, regulatory qualified cells are selected which allow the virus to grow well.
[0031] The virus replicates fairly rapidly in cell culture. Vero, the African green monkey kidney cell were used for cell culture. Depending on viral dose and on the cell line, the multiplicity of infection, cytopathic effect can be observed in 12-48 hrs. At multiplicities of 1-5, following a 5-6 hr eclipse period, the intracellular virus titer rises sharply and reaches peak by 12 hrs. Extracellular virus can be observed at 8 hrs post-infection and peaks at 12-24hrs depending on the cell system and dose of the inoculum (Chain et al., 1966; Higashi et al., 1967; Hahon and Hankins, 1970; Eckels et al., 1970). Spread of infection through the monolayer differs in different cell types, involving both extracellular and cell-to-cell transmission in BHK21 cells, but only the former in L929 and guinea pig lung cells (Hahon and Zimmerman, 1970). Titers in supernatants can reach as high as that observed with mouse brain preparations (Paul and Singh, 1968; Umrigar and Kadam, 1974). The virus can be concentrated from cell culture supernatant by ultracentrifugation, or precipitation with ammonium sulphate, alum, or polyethylene glycol (Eckels et al., 1970; Baneijee and Ranadive, 1988; Killington et al., 1996). The virus can be further purified by using rate zonal centrifugation, equilibrium density gradient or gel filtration (Eckels et al., 1970; Simizu et al., 1984; Baneijee and Ranadive, 1988). Titration of the virus can be performed by immunofluorescence, ELISA, complement fixation, agar gel immunodiffusion, hemagglutination and inhibition, plaque assays, or neutralization. For example, Vero (ATCC No. CCL-81), are preferably used. The Vero cell line used in the present invention has been validated for use as a host cell for vaccine production.
[0032] For maintenance in cell culture of the above-mentioned cell lines, stationary culture in monolayers, perfusion system culture, shake flasks, roller tube/bottle culture, suspension culture, microcarrier culture, cell factories and cell stacks can be adopted. For example, commercially available cell culture tray factories are used as a microcarrier, and other commercially available animal cell culture devices can be used.
[0033] An inactivating agent such as formalin is added to a virus suspension to inactivate the virus. For example, the amount of formalin to be added is about 0.01% to 0.05% (v/v) and the inactivation temperature is about 2-8°C to about 40°C and the inactivation duration mainly depends upon the inactivation temperature. For example, it could be 6-48 hours at 37°C and about 28-42 days at 2 - 8°C. Inactivation is also effective at intermediate temperatures of around 25 °C for a period of 72-120 hours.
[0034] Further, the purification of the virus is conducted by physical means or chemical means and preferably by a combination of both. Physical methods utilize the physical properties of the virus such as density, size, mass, sedimentation coefficient etc. and includes any of the following techniques but is not limited to membrane filtration (membranes ranging from size of 0.22-0.45 microns), ultrafiltration with membranes with size cut offs ranging from 50 - 200 kDa to remove serum and cellular components. Purification through chemical means employs methods including high performance technology like Ion Exchange chromatography, adsorption/desorption through chemical or physiochemical reactions.
[0035] In an embodiment of the present invention, there is provided a process for preparation of a vaccine composition, the process comprising of:
(i) inoculating a host cell line with virus and maintaining the infected cells in continuous culture;
(ii) harvesting the cells by centrifugation, and chemically inactivating the harvested virus using formalin at a temperature ranging from 25-40 for a duration of 24-96 hours;
(iii) purifying the inactivated virus by filtration using filter membrane ranging from 0.22-0.45 micron and purifying by ion exchange column chromatography; and
(iv) adding tris buffer having a pH ranging from 7.5-8.0, stabilizing agent, sucrose, phosphates and amino acids; and optionally adding an adjuvant to obtain the vaccine composition.
[0036] For potency testing of the vaccine, the vaccine composition were tested in C57/B6 mice. The animals in each group were injected intraperitoneally with about 0.2 ml /mouse with two different formulations of inactivated CHIKV. A booster dose was given 21 days after the first administration of the antigen. A composition of the inactivated virus preparation formulated in aluminium oxy- hydroxide in various combination with fructans like inulin gave a higher immune response. The resultant serum was assayed by in vitro neutralization tests and seroconversion was observed in the animals immunized with the vaccine composition.
[0037] The following examples are included solely to aid in a more complete understanding of the invention described and claimed herein. The examples do not limit the scope of the claimed invention in any fashion it should not construe the scope of the protection of the claims. However, one of the ordinary skilled in the art appreciates the modifications and changes can be made without departing from the scope of the present invention as set forth in the claims below. The benefits, advantages, solutions to problems, and any element(s) that may cause any benefit, advantage, or a solution to occur or become more pronounced are not to be construed as critical, required, or essential features or elements of any or all the claims. The invention is defined solely by the appended claims including any amendments made during the dependency of this application and all equivalents of those claims as issued.
[0038] Example 1: Propagation/Maintenance of cell line for virus culture:
Vero cell line was used as candidate cell line for Chikungunya virus (CHIKV) culture and good viral propagation was observed. Vero cells were prepared in a growth medium consisting of DMEM (Dulbecco's Modified Eagle Medium; Gibco, # 11965084 and used per the manufacturer's instructions) containing 10% fetal bovine serum (FBS). They were statically incubated at 37° C until reaching 80 - 100 % confluence of the monolayer. Thereafter the number of cells was counted. In an alternate procedure, Vero cells were cultured in serum free medium. For scaling the production of cells for virus infection, one cryo vial containing 2-5 x 106 viable Vero cells from working cell bank were used for seeding one T175 Cell culture grade flask. DMEM containing 10 % FBS and 1% penicillin-streptomycin was used for revival and replenishing the cells. After 90% of the confluence of the cell monolayer in T175 flasks, the cells were trypsinized and propagated further in cell factories / cell stacks.
[0039] Example 2: Isolation of the virus:
The CHIKV strain was procured from RCB and the Vero cells with 90% confluency infected with Chikungunya virus at 0.01-0.05 MOI. The virus was harvested 36-48 hours after infection. The cytopathic effect (cpe) of Chikungunya virus isolates of the patient from Gujarat in infected Vero cells was as observed in Figure 1.
[0040] Example 3: Propagation of the CHIKV isolate:
The CHIKV isolate was propagated in continuous cell culture in Vero cells (ATCC No. CCL-81) The medium for the infection was DMEM containing 1% penicillin-streptomycin and 0% FBS. At the end of 48 hours of infection, the cytopathic effect was visible the virus was present largely in the extracellular medium. A TCID50 of the virus upto 109/ml was obtained.
[0041] Example 4: Chemical Inactivation of the virus:
The harvested CHIKV isolate was chemically inactivated by inactivating reagent such as formalin at concentrations ranging from 0.005% to 0.05%, particularly from 0.01%-0.05%. For example: 0.01% formalin inactivate virus in 24 hours and 96 hours at 37°C and 25°C respectively, while incubating at 4°C requires 42 days for inactivation. The detailed kinetics has been listed in Table 1.
The virions inactivated at the various concentrations of formalin were found to be non-infectious when re-infected in Vero cells.

Table 1: VIRUS INACTIVATION KINETICS
Formalin Concentration Temperature Time period (in hours unless stated)
0.01% 37°C 0 5 16 24 48 72 96 120
25°C 0 5 16 24 48 72 96 120
4°C 0 1 day 7 days 14 days 28 days 35 days 42 days 49 days
0.025% 37°C 0 5 16 24 48 72 96 120
25°C 0 5 16 24 48 72 96 120
4°C 0 1 day 7 days 14 days 28 days 35 days 42 days 49 days
0.05% 37°C 0 5 16 24 48 72 96 120
25°C 0 5 16 24 48 72 96 120
4°C 0 1 day 7 days 14 days 28 days 35 days 42 days 49 days

[0042] Example 5: Purification of the virus:
The virus isolate was purified from the infected Vero cell monolayers by filtration using filter membrane ranging from 0.22-0.45 micron The virus was further purified by ion exchange column chromatography using Nuvia HPQ matrix. The desired protein was eluted by passing Elution buffer consisting of 50 mM Tris and 500-1500 mM NaCl. The peaked sample containing the virus particles was collected and the purity of the virus preparation was checked by SDS-PAGE.
[0043] Example 6: Purity of the virus preparation:
The purity of the virus preparation was checked by SDS-PAGE was found to be of good purity. The E1 and E2 envelope glycoproteins could be easily detected. See Fig 2.
[0044] Example 7: Preparation of vaccine composition
CHIKV antigen purified using 50 mM tris buffer with 500-1500 mM NaCl comprises of 200-400 mMol sucrose, 2-4 mMol monobasic potassium phosphate, 6-8 mMol dibasic potassium phosphate, 3-6 mMol potassium glutamate. The virus preparations were formulated in a liquid formulation as adjuvant/antigen ratio. The Aluminium hydroxide or aluminum oxy-hydroxide alone or in combination with fructan like inulin / CHIK V antigen were prepared by adding adjuvant stock dropwise to a 1X PBS solution containing 30 ug formulated dose in a 1:9 ratio.
[0045] Example 8: Animal testing and vaccine immunogenicity:
Fifteen 4 weeks old C57/B6 mice were used for animal testing. Five animals in each of the three groups were taken and injected intraperitoneally with about 0.2 ml /mouse. The animals were administered with booster dose on day 21 after the first immunization. Each formulation contained the viral antigen along with Aluminium oxy-hydroxide adjuvant alone or aluminum oxy-hydroxide in combination with Fructan (a polysaccharide) in 1x PBS, pH 6.8 - 7.2. No mortality and morbidity were observed in any of the treated animal throughout the study period. Increase in body weight was observed at day 72 in mice immunized with both formulated vaccine strains and a booster at Day 21 followed by a challenge at day 42 (Fig 3A). Other findings of the administered vaccines with respect to inflammation and foot pad thickness in mice, no significant adverse effect was observed. Combinatorial formulated vaccine strain showed decreased liver and brain inflammation (Fig 3E-3H).
[0046] Blood samples were collected at 0,14, 28, 42, 56, 72 and 90 days after the first immunization. An equal amount of serum was pooled for each group and used for virus neutralization assay. Remarkable neutralization of CHIKV infection has been observed in both the formulations. Peaked seroconversion rate was found at Day 28 (Fig 4A-4B). A significant increase in CD4+ T helper cells, IFN ?+ CD4 T helper’s cells and memory T cells was found. Also, the population of memory B-cells and plasma blast cells were found to be significantly increased. The mature germinal centers rich with GL7+ B lymphocytes were also observed in the spleen (Fig 4C-4E).
[0047] CHIK V antibody titer was evaluated in PFUs (plaque-forming units/ml) by a plaque-counting method using Vero cells. The method involves two-fold series of sera dilution (ranging from 1:10 to 1:160) against constant CHIK V titer. The plaques could be ready by 36-72 hours and the titers could be determined by 40-72 hours.

References
1. Arora, K., Tomar, P. C., Kumari, P., & Kumari, A. (2020). Medicinal alternative for chikungunya cure: a herbal approach. Journal of microbiology, biotechnology and food sciences, 9(5), 970-978.
2. Azevedo, R., do, S., da, S., Oliveira, C. S., Vasconcelos, P. F., and da, C. (2015). Chikungunya risk for Brazil. Rev. Saude Publica 49:58. doi: 10.1590/S0034-8910.2015049006219
3. Badar N, Ikram A, Salman M, Alam MM, Umair M, Arshad Y, Mushtaq N, Mirza HA, Ahad A, Farooq U, Yasin MT, Qazi J. Chikungunya virus: Molecular epidemiology of nonstructural proteins in Pakistan. PLoS One. 2021 Dec 23;16(12):e0260424. doi: 10.1371/journal.pone.0260424.
4. Baneijee K and Ranadive SN. 1988. Oligonucleotide fingerprinting of Chikungunya virus strains. Ind. J. Med. Res. 87:531-541.
5. Burt, F. J., Chen, W., Miner, J. J., Lenschow, D. J., Merits, A., Schnettler, E., et al. (2017). Chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen. Lancet. Infect. Dis. 17, e107–e117. doi: 10.1016/S1473-3099(16)30385-1
6. Casals J. 1957. The arthropod-borne group of animal viruses. Trans. N. Y. Acad. Sci. 19:219-235.
7. Chain MMT, Doane RW and McLean DM. 1966. Morphological development of Chikungunya virus. Can. . Microbiol. 12:895-899
8. da Silva, G. B. Jr., Pinto, J. R., Mota, R. M. S., da Pires Neto, R. J., and Daher, E. D. F. (2018). Impact of chronic kidney disease on chikungunya virus infection clinical manifestations and outcome: highlights during an outbreak in northeastern Brazil. Am. J. Trop. Med. Hyg. 99, 1327–1330. doi: 10.4269/ajtmh.18-0531
9. de Brito, C. A. A. (2017). Alert: severe cases and deaths associated with Chikungunya in Brazil. Rev. Soc. Bras. Med. Trop. 50, 585–589. doi: 10.1590/0037-8682-0479-2016
10. Dorléans, F., Hoen, B., Najioullah, F., Herrmann-Storck, C., Schepers, K. M., Abel, S., et al. (2018). Outbreak of chikungunya in the french caribbean islands of martinique and guadeloupe: findings from a hospital-based surveillance system (2013-2015). Am. J. Trop. Med. Hyg. 98, 1819–1825. doi: 10.4269/ajtmh.16-0719
11. Eckels KH, Harrison VR and Hetrick FM. 1970. Chikungunya virus vaccine prepared by Tween-ether extraction. Applied Microbiol. 19:321-325.
12. Edelman R, Tacket CO, Wasserman SS, Bodison SA, Perry JG and Magniafico JA. 2000. Phase II safety and immunogenicity study of live chikungunya virus vaccine TSI-GSD-218. Am. J. Trop. Med. Hyg. 62:681-685.
13. Freitas, A. R. R., Alarcón-Elbal, P. M., Paulino-Ramírez, R., and Donalisio, M. R. (2018). Excess mortality profile during the Asian genotype chikungunya epidemic in the dominican republic, 2014. Trans. R. Soc. Trop. Med. Hyg. 112, 443–449. doi: 10.1093/trstmh/try072
14. Gupta S, Vrati S. Molecular cloning and characterization of Chikungunya
virus genes from Indian isolate of 2006 outbreak. J Pharm Res. 2012;55:3860–3863.
15. Hahon N and Hankins WA. 1970. Assay for Chikungunya virus in cell monolayers by immunofluorescence. Applied Microbiol. 19:224-231.
16. Hahon N and Zimmerman WD. 1970. Chikungunya virus infection of cell monolayers by cell-to-cell and extracellular transmission. Applied Microbiol. 19:389-391.
17. Harrison VR, Eckels KH, Bartelloni PJ and Hampton C. 1971. Production and evaluation of a formalin-killed chikungunya vaccine. J. Immunol. 107:643-647.
18. Hearn HJ and Rainey CT. 1963. Cross-protection in aminals infected with group A arboviruses. J. Immunol. 90:720-724.
19. Higashi N, Matsumoto A, Tabata K and Nagatomo Y. 1967. Electron microscope study of development of Chikungunya virus in green monkey kidney stable (Vero) cells. Virology 3:55-69.
20. Jaffar-Bandjee, M. C., Ramful, D., Gauzere, B. A., Hoarau, J. J., Krejbich-Trotot, P., Robin, S., et al. (2010). Emergence and clinical insights into the pathology of Chikungunya virus infection. Exp. Rev. Anti. Infect. Ther. 8, 987–996. doi: 10.1586/eri.10.92
21. Khan A.H., Morita K., Parquet Md Mdel C., Hasebe F., Parquet. Md. E.G., Igarashi A. Complete nucleotide sequence of chikungunya virus and evidence for an internal polyadenylation site. J. Gen. Virol. 2002;83:3075–3084.
22. Killington RA, Stokes A and Hierholzer JC. 1996. Virus purification. In "Virology methods manual," Mahy BWJ and Kangro (Eds). Academic Press, San Diego, CA, pp 71-89.
23. Levitt NH, Ramsburg HH, Hasty SE, Repik PM, Cole FE Jr and Lupton HW. 1986. Development of an attenuated strain of chikungunya virus for use in vaccine production. Vaccine 4:157-162.
24. Liu S-Q, et al. Detection, isolation, and characterization of chikungunya viruses associated with the Pakistan outbreak of 2016–2017. Virologica Sinica. 2017;32(6):511–519. doi: 10.1007/s12250-017-4059-7.
25. Mason, P. J., and Haddow, A. J. (1957). An epidemic of virus disease in southern province, tanganyika territory, in 1952–1953: an additional note on chikungunya virus isolations and serum antibodies. Trans. R. Soc. Trop. Med. Hyg. 51, 238–240. doi: 10.1016/0035-9203(57)90022-6
26. McClain DJ, Pittman PR, Ramsburg HH, Nelson GO, Rossi CA, Mangiafico J A, Schmaljohn AL and Malinoski FJ. 1998. Immunologic interference from sequential administration of live attenuated alphavirus vaccines. J. Infect. Dis. 177:634-641.
27. Melan A, et al. Molecular characterization of chikungunya virus causing the 2017 outbreak in Dhaka Bangladesh. New Microbes New Infect. 2018;24:14–16. doi: 10.1016/j.nmni.2018.03.007.
28. Parks JJ and Price WJ. 1958. Studies on immunologic overlap among certain arthropod-borne viruses. Am. J. Trop. Med. Hyg. 67:187-206.
29. Paul SD and Singh KRP. 1968. Experimental infection of Macaca radiata with Chikungunya virus and transmission of virus by mosquitoes. Ind. J. Med. Res. 56:802-810.
30. Porterfield JS. 1961. Cross-neutralization studies with group A arthropod-borne viruses. Bull WHO 24:735-741
31. Roongaraya, P., & Boonyasuppayakorn, S. (2023). Chikungunya vaccines: An update in 2023. Asian Pacific Journal of Allergy and Immunology, 41(1), 1-11.
32. Schuffenecker I., Iteman I., Michault A., Murri S., Frangeul L., Vaney M.C., Lavenir R., Pardigon N., Reynes J.M., Pettinelli F., Biscornet L., Diancourt L., Michel S., Duquerroy S., Guigon G., Frenkiel M.P., Brehin A.C., Cubito N., Despres P., Kunst F., Rey F.A., Zeller H., Brisse S. Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak. PLoS Med. 2006;3:e263.
33. Simizu B, Yamamoto K, Hashimoto K and Ogata T. 1984. Structural proteins of Chikungunya virus. J. Virol. 51:254-258.
34. Suhrbier, A. (2019). Rheumatic manifestations of chikungunya: emerging concepts and interventions. Nat. Rev. Rheumatol. 15, 597–611. doi: 10.1038/s41584-019-0276-9
35. Umrigar MD and Kadam SS. 1974. Comparative sensitivity of suckling mice and Vero cells for primary isolation of Chikungunya virus. Ind. J. Med. Res. 62:1893- 1895.
36. Wahid, B., Ali, A., Rafique, S., and Idrees, M. (2017). Global expansion of chikungunya virus: mapping the 64-year history. Int. J. Infect. Dis. 58, 69–76. doi: 10.1016/j.ijid.2017.03.006
37. World Health Organization (2017). Global Vector Control Response 2017 – 2030: A Strategic Approach to Tackle Vector-Borne Diseases. Switzerland: WHO.
38. Yap, M. L., Klose, T., Urakami, A., Hasan, S. S., Akahata, W., & Rossmann, M. G. (2017). Structural studies of Chikungunya virus maturation. Proceedings of the National Academy of Sciences, 114(52), 13703-13707.

We Claim:
1. A vaccine composition for chikungunya virus infection comprising:
(a) a chemically inactivated and purified chikungunya virus antigen; and
(c) pharmaceutically acceptable excipient,
wherein the composition elicits a protective immune response against Chikungunya virus infection.
2. The vaccine formulation as claimed in claim 1, wherein the antigen is inactivated using formalin in a concentration ranging from 0.01 to 0.05%.
3. The vaccine composition as claimed in claim 1, wherein the pharmaceutical acceptable excipient is selected from tris buffered saline, preservatives, stabilizing agents including non-reducing sugars such as sucrose, inorganic/organic substances.

4. The vaccine composition as claimed in claim 1, wherein the composition optionally comprises an adjuvant.

5. The vaccine composition as claimed in claim 4, wherein the adjuvant is selected from aluminium hydroxide, aluminium oxy-hydroxide aluminium phosphate or aluminium hydroxide or oxy-hydroxide in combination with fructans and derivatives.

6. The vaccine composition as claimed in claim 5, wherein the fructans such as inulin is selected from oligosaccharides and polysaccharides.

7. The vaccine composition as claimed in claim 1, wherein the virus antigen is present in a range of 10 to 50 mcg per dose.

8. The vaccine composition as claimed in any of preceding claims, wherein the composition is formulated in a liquid form suitable for intramuscular, intradermal, subcutaneous and intravenous administration.

9. A stable liquid vaccine composition comprising of:
(i) a purified and inactivated chikungunya virus antigen;
(ii)50 mM Tris buffer and 500-1500 mM sodium chloride; and
(iii) 1x PBS or phosphate-citrate buffer.

10. A process for preparation of a vaccine composition, the process comprising of:
(i) inoculating a host cell line with virus and maintaining the infected cells in continuous culture;
(ii) harvesting the cells by centrifugation, and chemically inactivating the harvested virus using formalin at a temperature ranging from 25-40 for a duration of 24-96 hours;
(iii) purifying the inactivated virus by filtration using filter membrane ranging from 0.22-0.45 micron and purifying by ion exchange column chromatography; and
(iv) adding tris buffer having a pH ranging from 7.5-8.0, stabilizing agent, sucrose, phosphates and amino acids; and optionally adding an adjuvant to obtain the vaccine composition.
, Claims: