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 a live-attenuated vaccine composition for chikungunya virus infection. The vaccine composition is immunogenic and elicits protective immune response.
BACKGROUND OF THE INVENTION
[002] Chikungunya virus (CHIKV or CHIK virus) is a positive-sense, single-stranded RNA virus from the genus Alphavirus and family Togaviridae. The CHIK virus is vectored by the daytime- biting Aedes aegypti mosquito, which also transmits yellow fever, Zika and dengue viruses. It can also be transmitted by Aedes albopictus mosquitoes, a more cold-tolerant mosquito that could readily promote the spread of Chikungunya to more temperate areas of the world (Vega-Rua A, et al., 2015). Outbreaks in the past have occurred mainly in Africa, but the East-Central South African (ECSA) genotype has recently expanded its geographical range, resulting in outbreaks in India, Asia, and even temperate Europe (Weaver, 2014).

[003] Infection with CHIKV results in chronic and incapacitating arthralgia affecting all gender and age groups, accompanied by an acute febrile disease with headache, muscle pain, and skin rashes. The severe, often debilitating joint pain in infected patients can persist for years, especially in adults. In some cases, neurological, renal, cardiac, respiratory or hepatic complications can also occur. Individuals who are at higher risk of more serious complications include infants, the elderly and individuals with chronic medical conditions. Although infections with CHIKV usually resolve spontaneously, higher risk groups can develop CNS infection (CHIKV encephalitis), which had an overall mortality rate of about 16% in the La Reunion Outbreak of 2005-2006 and a rate of persistent disabilities in children following CHIKV encephalitis of between 30 and 45 percent (Gerardin et al., 2016).

[004] CHIKV is currently regarded as one of the most- likely re-emerged viruses to spread globally, and morbidity to this virus is considered a serious threat to global public health raising an urgent demand for efficient prophylaxis. Further, since neither a specific antiviral treatment nor a vaccine is available to prevent CHIKV infection, prevention against CHIKV infection is therefore limited to non-treatment interventions such as the employment of insecticides, wearing long sleeves and pants, and other means to restrict exposure to vector mosquitos. Preclinical studies in mice and non-human primates (NHPs) have demonstrated the important role of antibodies in protection against CHIKV infection. For example, B cell deficient mice were unable to clear CHIKV viremia, while wild-type mice were competent (Lum F-M. et al., 2013). Specifically, passive transfer of immune sera conferred protection against disease in recipient mice, in contrast to adoptive transfer of primed CD8+ T cells, which had no impact on viremia (Couderc T. et al., 2009; Chu H. et al., 2013; Kam Y-W. et al.2012). A combination of neutralizing monoclonal antibodies protected against a lethal challenge with CHIKV in a mouse model (Pal P. et al. 2013). Additionally, the administration of a human neutralizing monoclonal antibody blocked CHIKV spread and inflammation in NHPs (Broeckel R. et al., 2017). Importantly, the findings of a prospective longitudinal cohort study in the Philippines further supports the surrogate endpoint for development of the CHIKV-D5nsP3 attenuated vaccine. Briefly, acute febrile illnesses were investigated via community-based active surveillance over a period of 12 months in 853 subjects. PRNT assays were performed from blood samples obtained at enrolment and at 12 months. In addition, symptomatic CHIKV infections were identified by positive CHIKV PCR in acute blood samples and/or CHIKV IgM/IgG ELISA seroconversion in paired acute/convalescent samples. The authors reported that a baseline CHIKV PRNT80 titer ³10 was associated with 100% (95% CI: 46.1, 100.0) protection from symptomatic CHIKV infection. The aforementioned studies in humans as well as in animals suggest that the induction of CHIKV neutralizing antibodies is likely to predict clinical benefit (Yoon et al., 2015). Adding further support to the importance of neutralizing antibodies are studies with several vaccine candidates in development, which have demonstrated that vaccines which induced neutralizing antibodies protected against infection, whereas those inducing mainly CD8-specific T-cells did not (Ahola T. et al, 2015). These preclinical and clinical observations are strongly supported by findings from natural CHIKV infections in humans, such as the Yoon et al. study cited above. Robust IgM/IgG antibody responses are elicited following CHIKV infection in humans that primarily target E1/E2 structural proteins. In addition, it is known that natural CHIKV infection induces a durable antibody response that is believed to confer life-long immunity (Galatas B. et al., 2016; Nitatpattana N. et al.; 2014). Several report and studies are available, however, at present there is no treatment or vaccine available against this CHIKV-induced debilitating disease and its various symptoms. Therefore, there is a need in the art to develop a vaccine for the prevention of Chikungunya infection.

[005] Accordingly, 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 of the present invention has developed a vaccine composition which is safe, immunogenic and stimulates a quick protective immune response.

SUMMARY OF THE INVENTION
[006] 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 vaccine composition comprising a live-attenuated CHIKV virus. The vaccine composition developed for active immunization is for the prevention of disease caused by CHIKV in populations living in endemic regions, as well as for travelers to endemic areas or areas at risk for outbreak. The replicating CHIKV vaccine candidate comprises a deletion of specific amino acids in the non-structural protein gene, leading to attenuation of the virus in vivo. The vaccine composition, based on the attenuated strain isolated from the clinical sample of the patient, is produced in vero cells and purified by centrifugation.
[007] In an aspect of the present invention, there is provided a vaccine composition comprising a live-attenuated CHIKV particles along with a pharmaceutically acceptable excipient/carrier. The vaccine composition of the present invention comprises a sufficient amount of immunogenic Chikungunya virus to elicit a neutralizing immune response in a subject i.e., an immune response that is protective against infection with and/or disease caused by Chikungunya virus. In particular, the invention provides a vaccine composition comprising live-attenuated CHIKV particles wherein the percentage of said viral particles with immunogenicity-reducing mutations, particularly immunogenicity-reducing mutations in the E2 protein, are minimal.
[008] In another aspect, the present invention also relates to a process for preparation of a live-attenuated vaccine composition against chikungunya virus infection.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[009] Figure 1 illustrates:
A- Control uninfected Vero cells;
B- Vero cells after 24 hours of CHIKV infection; and
C- Vero cells after 48 hours of CHIKV infection.

[0010] Figure 2 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.

DESCRIPTION OF THE INVENTION
[0011] 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.
[0012] 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.
[0013] 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.
[0014] The present invention relates to providing an efficient & effective vaccine composition for Chikungunya virus infection. More particularly, the invention provides a live-attenuated virus-based vaccine composition which is safe and immunogenic.
[0015] The Chikungunya (CHIK) virus vaccine according to the present invention refers to an active ingredient (immunogen) of attenuated chikungunya virus particle that is produced by infecting in a monolayer of Vero cells and passaged for multiple time 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.
[0016] 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 composition of the present invention is provided in a sealed vial in a lyophilized format that can either be administrated to a subject about 0.2 ml /mouse intraperitoneally/ subcutaneously /intramuscularly /intradermally/orally/ intranasally.
[0017] In an aspect of the present invention, there is provided a vaccine composition comprising a live-attenuated chikungunya virus particle which can be delivered to a subject in need thereof, providing protection against infection with chikungunya virus and/or the development of chikungunya fever after vaccination. The methods described in the present invention are also applicable to other genotype/strains of Chikungunya virus. The virus particles obtained from the clinical isolates of patients' serum have been adapted and propagated in vitro in cell monolayers for several passages.
[0018] The attenuated virus particle of the present invention is 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 and gelatin like substances. Such a stable composition of the immunogen in a liquid frozen form in a pharmaceutically acceptable buffer is suitable for administration intraperitoneally /intradermally /subcutaneously /intramuscularly in human host.
[0019] Thus, in accordance with the present invention, there is provided a vaccine composition for chikungunya virus infection comprising:
(a) a live attenuated chikungunya virus antigen; and
(b)one or more pharmaceutically acceptable excipients,
wherein the composition induces a sustained protective immune response against chikungunya virus infection.
[0020] In an embodiment of the present invention, the pharmaceutically acceptable excipients is selected from sucrose, potassium phosphate, glutamate and gelatin.
[0021] In another embodiment of the present invention, the composition optionally comprises an adjuvant selected from aluminium hydroxide, aluminium oxy-hydroxide aluminium phosphate or aluminium hydroxide or oxy-hydroxide in combination with fructans and derivatives.
[0022] In still another embodiment of the present invention, the fructans such as inulin is selected from oligosaccharides and polysaccharides.
[0023] In yet another embodiment of the present invention, the antigen expresses E2 protein as defined by SEQ ID NO: 2.
[0024] In an embodiment of the present invention, the composition stimulates seroconversion in at least 25%, up to 100% of vaccinated subjects within 7 days of vaccination, wherein seroconversion is defined as reaching a neutralizing CHIKV antibody titer of at least 10, preferably at least 20.
[0025] In another embodiment of the present invention, the composition increases serum antibody titers to virus by at least 1 log relative to a control within about 28 days from primary immunization.
[0026] In a preferred embodiment of the present invention, there is provided a stable vaccine composition comprising:
(a) a live attenuated chikungunya virus antigen; and
(b) 200-400 mMol sucrose, 2-4 mMol monobasic potassium phosphate, 6-8 mMol dibasic potassium phosphate, 3-6 mMol potassium glutamate and 0.2%-0.5% gelatin.
[0027] In another aspect of the present invention, there is provided a process for preparing the vaccine composition comprising inoculating an appropriate host cell line such as Vero cells with the virus, and maintaining the infected cells in continuous culture, infecting the host cell monolayer with the virus and harvesting the virus in sufficient quantities from the infected host cells; repeating the procedure multiple times to generate the attenuated CHIKV strain in the process of serial passaging. 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.
[0028] 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, a regulatory qualified cells are selected which allow the virus to grow well. For example Vero (ATCC No. CCL-81), are preferably used. The Vero cell line used in the current invention has been validated for use as a host cell for vaccine production. For maintenance in cell culture of the above-mentioned cell lines, stationary culture in monolayers, perfusion system culture, shake flasks, roller tube, suspension culture, microcarrier culture, cell factories and cell stacks can be adopted. For example, commercially available cell culture tray (cell factories) are used as a microcarrier, and other commercially available animal cell culture devices can be used.
[0029] For potency testing of the vaccine, the vaccine formulations 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 attenuated strain of CHIKV. A booster dose was given 21 days after the first administration of the antigen. The resultant serum was assayed by in vitro neutralization tests Seroconversion was observed in the animals immunized with the vaccine formulation.
[0030] 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.
[0031] Example 1: Propagation/Maintenance of cell line for virus culture:
The Vero cell line was selected as the potential host for cultivating Chikungunya virus (CHIKV), and successful viral propagation was achieved. Vero cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, #11965084), supplemented with 10% fetal bovine serum (FBS), following the manufacturer's guidelines. Incubation was carried out at 37°C until the cell monolayer reached 80-100% confluence. Subsequently, cell counting was performed. In an alternative approach, Vero cells were cultured in a serum-free medium. To scale up cell production for virus infection, one cryovial containing 2-5 x 10^6 viable Vero cells from the working cell bank was utilized to seed a T175 cell culture-grade flask. Revival and cell replenishment were achieved using DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Upon reaching 90% confluence in T175 flasks, the cells were trypsinized and further propagated in cell factories or cell stacks.
[0032] 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.
[0033] Example 3: Production of the attenuated 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. The process was repeated upto 40-45 passages and virus titers were determined on Vero cells using the TCID50 assay.
[0034] Example 4: CHIKV Freeze Dried (Lyo) Product Formulation Development:
The attenuated CHIKV antigen stabilized in 200-400 mMol sucrose, 2-4 mMol monobasic potassium phosphate, 6-8 mMol dibasic potassium phosphate, 3-6 mMol potassium glutamate and 0.2%-0.5% Gelatin. The Aluminium hydroxide or aluminum oxy-hydroxide alone/ CHIK V antigen were prepared by adding adjuvant stock drop wise to a 1X PBS solution containing 30 ug formulated dose in a 1:9 ratio. Alternatively, polysaccharide like inulin was used to formulate the attenuated CHIK V antigen. The vaccine vials containing the above composition were then freeze dried in Scan Vac freeze drier at -96°C and pressure of about 0.1 hPa for 16 hours. Frozen vials were lyophilized in Scan Vac freeze drier at -96°C and pressure of about 0.1 hPa for 16 hours. Upon completion, vacuum was released and the lyophilized vials were sealed and stored at 4°C.
[0035] Example 5: 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 lyophilized formulations contained the attenuated viral antigen along with Aluminium oxy-hydroxide adjuvant for one group and Fructan (a polysaccharide) were first resuspended in DMEM. No mortality and morbidity were observed in any of the treated animal throughout the study period.
[0036] Blood samples were collected at 0, 14, 28, 42, 56, 72 and 90 days after the first immunization. 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 2A-2B). 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
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2. Broeckel R, Fox JM, Haese N, Kreklywich CN, Sukulpovi-Petty S, Legasse A, Smith PP, Denton M, Corvey C, Krishnan S, Colgin LM. Therapeutic administration of a recombinant human monoclonal antibody reduces the severity of chikungunya virus disease in rhesus macaques. PLoS neglected tropical diseases. 2017 Jun 19;11(6):e0005637.

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We Claim:
1. A vaccine composition for chikungunya virus infection comprising:
(a) a live attenuated chikungunya virus antigen; and
(b) one or more pharmaceutically acceptable excipients,
wherein the composition induces a sustained protective immune response against chikungunya virus infection.

2. The vaccine composition as claimed in claim 1, wherein the pharmaceutically acceptable excipients is selected from sucrose, potassium phosphate, glutamate and gelatin.

3. The vaccine composition as claimed in claim 1, wherein the composition optionally comprises an adjuvant selected from aluminium hydroxide, aluminium oxy-hydroxide aluminium phosphate or aluminium hydroxide or oxy-hydroxide in combination with fructans and derivatives.

4. The vaccine composition as claimed in claim 3, wherein the fructans such as inulin is selected from oligosaccharides and polysaccharides.

5. The vaccine composition as claimed in claim 1, wherein the antigen express E2 protein as defined by SEQ ID NO: 2.
6. The vaccine composition as claimed in claim 1, wherein the composition stimulates seroconversion in at least 25%, up to 100% of vaccinated subjects within 7 days of vaccination, wherein seroconversion is defined as reaching a neutralizing CHIKV antibody titer of at least 10, preferably at least 20.
7. The vaccine composition as claimed in any one of claims 1 to 6, wherein the composition increases serum antibody titers to virus by at least 1 log relative to a control within about 28 days from primary immunization.
8. A stable vaccine composition comprising:
(a) a live attenuated chikungunya virus antigen; and
(b) 200-400 mMol sucrose, 2-4 mMol monobasic potassium phosphate, 6-8 mMol dibasic potassium phosphate, 3-6 mMol potassium glutamate and 0.2%-0.5% gelatin.
9. 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) repeating step (i) multiple times by serial passaging to obtain attenuated CHIKV infected cells;
(iii) centrifuging suspension of cells of step (ii) and harvesting the infected cells;
(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.
10. The process as claimed in claim 9, wherein the composition is freeze dried at a temperature of -96°C and pressure of 0.1 hPa for a duration of 16 hours to obtain in lyophilized form.
, Claims: