Chikungunya fever vaccine composition comprising chikungunya virus envelope recombinant protein

Abstract

The present invention relates to a chikungunya fever vaccine composition comprising a chikungunya virus envelope recombinant protein.

Description

Chikungunya fever vaccine composition containing recombinant Chikungunya virus envelope protein

[0001] The present invention relates to a chikungunya fever vaccine composition comprising a recombinant protein of the chikungunya virus envelope.

[0002] Chikungunya fever is transmitted by the bite of an infected female Aedes mosquito and causes fever, severe joint pain, muscle pain, headache, nausea, fatigue, and a rash. While Chikungunya fever is generally not a fatal disease, the joint pain can be debilitating and last for weeks to years.

[0003] To date, only one vaccine (Valneva) has been approved and is scheduled for commercialization in 2024, and there is currently no specific treatment available. Since this virus is primarily transmitted by mosquitoes in densely populated areas such as cities, reliance is placed on methods that simply eradicate mosquitoes, despite their limited effectiveness. As global temperatures rise, mosquito vectors are now able to thrive in more regions worldwide, and this climate change could amplify the risk of Chikungunya fever.

[0004] The Chikungunya virus has an RNA gene of about 12kb and consists of four types of non-structural proteins (nsP1, nsP2, nsP3, nsP4) and five types of structural proteins (three types of glycosylated envelope glycoproteins (E1, E2, E3), nonglycosylated nucleocapsid (C), and cleavage product 6 (6K / TF)). Among the Chikungunya virus structural proteins (E1, E2, E3, C, 6K / TF), E1 performs attachment between the virus and the host cell, E2 performs virus penetration into the host cell, E3 performs interaction with the E2 glycoprotein and spike protein assembly, 6K / TF performs virus assembly and release, and C performs various functions such as nucleocapsid formation as well as genome encapsulation by interacting with RNA. According to previous studies, the Chikungunya Envelope protein (E protein) is a major antigen recognized by the host immune system and serves as a binding site for host cell receptors, making it a primary target for neutralizing antibodies.

[0005] The objective of the present invention is to provide a Chikungunya fever vaccine composition comprising a recombinant Chikungunya virus envelope protein in order to solve the above-mentioned problems.

[0006] To achieve the objective of the present invention, the present invention provides a Chikungunya virus envelope recombinant protein comprising SEQ ID NO. 4, a Chikungunya virus envelope recombinant protein comprising SEQ ID NO. 5, a Chikungunya virus envelope recombinant protein comprising SEQ ID NO. 6, or a Chikungunya virus envelope recombinant protein comprising SEQ ID NO. 7.

[0007] As used herein, the term "protein" is defined as a chain of amino acid residues having a generally defined sequence.

[0008] As used herein, the term protein includes the terms "peptide" and "protein." The term also includes modified amino acid polymers.

[0009] In one example of the present invention, the present invention provides a vaccine composition comprising the recombinant protein.

[0010] The above vaccine composition may further comprise one or more selected from the group consisting of preservatives, diluents, adjuvants, and carriers, and the adjuvants may be squalene or lipids derived therefrom, preferably squalene-based oil-in-water emulsion, and more preferably Addavax, but are not limited thereto.

[0011] Carriers suitable for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, etc. Such carriers may be aqueous solutions, non-aqueous solutions, suspensions, or emulsions. Typical or amorphous organic or inorganic polymers, etc., may be used as adjuvants to increase immunogenicity. Adjuvants are generally known to play a role in promoting immune responses through chemical or physical binding to antigens. Amorphous aluminum gels, oil emulsions, double oil emulsions, and immunosols may be used as adjuvants. In addition, various plant-derived saponins, levamisole, CpG dinucleotides, RNA, DNA, LPS, and various types of cytokines may be used to promote immune responses. Such immune compositions may be used as compositions for inducing an optimal immune response by combining various adjuvants and immune response-promoting additives. Furthermore, stabilizers, inactivators, antibiotics, preservatives, etc., may be used as compositions to be added to the vaccine. Depending on the route of vaccine administration, vaccine antigens may also be mixed with distilled water, buffer solutions, etc.

[0012] The above vaccine composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or unit dosing ampoules or multi-dose injectables, according to conventional methods. When formulating the above vaccine composition, it may be prepared by adding diluents or excipients such as commonly used fillers, volume expanders, binders, wetting agents, disintegrants, or surfactants.

[0013] When the above vaccine composition is prepared as a parenteral formulation, it may be formulated in the form of an injectable, transdermal, nasal inhalant, and suppository according to methods known in the art with a suitable carrier. When formulated as an injectable, suitable carriers may include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof; preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 5% dextrose, etc., may be used. When formulated as a transdermal formulation, it may be formulated in the form of an ointment, cream, lotion, gel, topical solution, paste, liniment, aerosol, etc. In the case of nasal inhalers, they can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide, and when formulated as suppositories, the base may be Witepsol, Tween 61, polyethylene glycols, cocoa starch, laurin starch, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, and sorbitan fatty acid esters.

[0014] The administration route of the above vaccine composition may be administered through any general route as long as it can reach the target tissue, and specifically, the vaccine composition may be selected from the group consisting of compositions for intramuscular administration, subcutaneous administration, intraperitoneal administration, intravenous administration, oral administration, dermal administration, ocular administration, nasal administration, and intracerebral administration.

[0015] The above vaccine composition may be administered in a pharmaceutically effective amount, wherein the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dose level may be determined based on factors including the severity of the disease, drug activity, patient's age, weight, health, gender, patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and elimination rate, the duration of treatment, drugs combined or used concurrently with the composition of the present invention used, and other factors well known in the medical field. The above vaccine composition may be administered alone or in combination with components known to exhibit known preventive or therapeutic effects. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration.

[0016] The dosage of the above vaccine composition may be determined by a person skilled in the art by taking into consideration the purpose of use, the degree of toxicity of the disease, the patient's age, weight, gender, medical history, or the type of substance used as an active ingredient. For example, the vaccine composition of the present invention may be administered at a dose of about 0.1 ng to about 1,000 mg / kg, preferably 1 ng to about 100 mg / kg per adult, and the frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose. The above dosage or frequency of administration does not limit the scope of the present invention in any way.

[0017] The above vaccine composition may be for preventing Chikungunya virus infection.

[0018] The above vaccine composition may be one or more selected from the group consisting of priming vaccine compositions and boosting vaccine compositions, but is not limited thereto.

[0019] In another example of the present invention, the present invention provides a method for inducing an immune response to the Chikungunya virus in non-human mammals, comprising the step of administering the vaccine composition.

[0020] In another example of the present invention, the present invention provides a method for preventing Chikungunya virus infection in non-human mammals, comprising the step of administering the vaccine composition.

[0021] The above-mentioned administering step may include a step of inducing a priming immune response; and a step of inducing a boosting immune response.

[0022] The present invention relates to a Chikungunya fever vaccine composition comprising a recombinant Chikungunya virus envelope protein. A vaccine composition against Chikungunya virus using domestic isolates can secure basic data necessary for research and development of vaccines for future response to potential outbreaks and can help strengthen research capabilities in this regard.

[0023] Figure 1 shows a schematic diagram of a Chikungunya virus E2 protein vaccine candidate.

[0024] Figure 2 shows the sequence of a Chikungunya virus E2-based recombinant protein vaccine candidate.

[0025] Figure 3 shows the evaluation of the expression of a Chikungunya virus E2-based recombinant protein vaccine candidate.

[0026] Figure 4 shows a schematic diagram of a Chikungunya virus E3-E2 protein vaccine candidate.

[0027] Figure 5 shows the sequence of a Chikungunya virus E3-E2-based recombinant protein vaccine candidate.

[0028] Figure 6 shows the evaluation of the expression of a Chikungunya virus E3-E2-based recombinant protein vaccine candidate.

[0029] Figure 7 shows a schematic diagram of a Chikungunya virus E3-E2-E1 protein vaccine candidate.

[0030] Figure 8 shows the sequence of a Chikungunya virus E3-E2-E1-based recombinant protein vaccine candidate.

[0031] Figure 9 shows the evaluation of the expression of a Chikungunya virus E3-E2-E1-based recombinant protein vaccine candidate.

[0032] Figure 10 shows the mouse vaccination schedule for the Chikungunya virus recombinant protein vaccine candidate.

[0033] Figure 11 shows the ELISA results of a Chikungunya virus recombinant protein vaccine candidate.

[0034] Figure 12 shows the PRNT results of the Chikungunya virus recombinant protein vaccine candidate.

[0035] Figure 13 shows the results of the Chikungunya virus recombinant protein vaccine candidate ELISpot.

[0036] Preferred embodiments of the present invention will be described in detail below. In addition, many specific details, such as specific components, are illustrated in the following description; however, these are provided merely to aid in a more comprehensive understanding of the present invention, and it will be obvious to those skilled in the art that the present invention can be practiced without such specific details. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations are omitted if it is determined that such detailed descriptions may unnecessarily obscure the essence of the present invention.

[0037]

[0038] Example 1. Animal experiment

[0039] 100 µg of the vaccine candidate was administered as a first dose to 5-week-old C57BL / 6 mice, and a second dose was administered 3 weeks after the first dose, for a total of 2 immunizations. Blood was collected 2 weeks after vaccination, and a total of 2 blood samples were collected at weeks 2 and 5.

[0040]

[0041] Example 2. Establishment of the T-cell secreted cytokine measurement (ELISpot) method

[0042] The experiment was conducted for two days, with the first day dedicated to cell preparation and the second day to ELISpot analysis. The kit used for ELISpot analysis was the Mouse IFN-γ Kit (#EL485) from R&D Systems, and for the peptides used to stimulate splenocytes, parts corresponding to the E1, E2, E3, and 6K proteins were custom-made and used.

[0043]

[0044] (Day 1) The spleen was extracted from mice vaccinated with the Chikungunya virus, a cell strainer was placed on a 60mm dish, the spleen was released into RPMI medium, and the spleen was ground using a syringe plunger. Subsequently, the ground spleen was transferred to a 15ml conical tube containing 5ml of RPMI medium and centrifuged at a speed of 1800–2000 rpm for 5 minutes. To secure cell pellets from the conical tube, the supernatant was removed, and 5ml of ACK lysis buffer was added and reacted at room temperature for 5 minutes. The mixture was then neutralized using the original RPMI medium. After neutralization, centrifugation was performed at 1800–2000 rpm to remove the supernatant for cell washing, and 10ml of RPMI medium was added to repeat the centrifugation process. The final supernatant was removed, 11 ml of RPMI medium was added, and vortexing was performed to thoroughly loosen the cell pellets in the medium. Subsequently, the mixture was left to stand at room temperature for 1 minute to remove debris, and 10 ml of the cell suspension was collected and transferred to a new 15 ml conical tube. At this stage, only the supernatant was collected to prevent debris contamination. Following the collection of the cell suspension, 200 µl of RPMI medium was dispensed into every well of the ELISpot plate included in the kit, and blocking was performed for 30 minutes. During the 30 minutes, cells were counted from the previously collected suspension to ensure a count of 1 x 10^6 cells per 100 µl. Once the 30-minute blocking process was complete, the medium was removed from the plate, and cells were dispensed at 100 µl per well according to the experimental design. A custom-made peptide was added to stimulate the splenocytes, and the cells were cultured in a 37°C CO2 incubator for 18 hours.

[0045]

[0046] (Day 2) The cell culture plates were washed four times using the wash buffer provided in the kit, and 100 µl of detection antibody was dispensed into each well and incubated at room temperature for approximately 2 hours. Afterward, four washes were performed, 100 µl of Streptavidin-AP solution was dispensed into each well, incubated at room temperature for 2 hours, and four washes were performed. Once the glycolysis was complete, a color reaction was induced using BCIP / NBT reagents. Since BCIP / NBT reagents are sensitive to light, the reaction must be carried out in a light-shielded environment and incubated for 1 hour. After the reaction was complete, the plates were washed with distilled water, dried at room temperature, and then analyzed using an Immunospot instrument.

[0047]

[0048] Example 3. ELISA

[0049] Envelope proteins, which are antigens of the Chikungunya virus, were dispensed into a 96-well plate and coated at 4°C for one day. Subsequently, the plates were washed with PBS-T, and a blocking process was carried out using 5% skim milk at 37°C for approximately one hour. Once blocking was complete, the plates were washed with PBS-T, and the prepared serum was added to the 96-well plate at a 2-fold serial dilution starting from 1:100, followed by an incubation at 37°C for two hours. After the reaction was finished, the plates were washed with PBS-T, HRP conjugate anti-mouse IgG antibody (1:50,000) was added, and the reaction was incubated at 37°C for two hours. Following a PBS-T wash, TMB solution was added, and the color reaction was carried out at room temperature. Once the color reaction occurred, the reaction was stopped with TMB stop solution, and the results were analyzed by measuring the absorbance at an OD of 450 nm.

[0050]

[0051] Example 4. PRNT

[0052] (Day 0) Vero E6 cells were suspended at 2.5 x 10^5 cells / ml, then 1 ml was dispensed into each well of a 12-well plate and cultured for one day in a 37℃ CO2 incubator.

[0053]

[0054] (Day 1) The serum samples to be used for the experiment were incubated at 56°C for 30 minutes, followed by 2-fold serial dilution using culture medium (2% FBS + 1% Pen / Strep + DMEM). Once the serum and cells were prepared, 6 x 10 CHIKV were collected from the stock solution in the BL3 facility. 3The solution was diluted to PFU / ml, and the diluted serum was dispensed at a concentration of 50 PFU and reacted at 37°C for 1 hour. After the reaction was complete, the pre-prepared Vero E6 cell supernatant was completely removed, 200 µl of the serum and virus mixture was dispensed into each well, and the plates were incubated in a 37°C CO2 incubator for 1 hour. After the reaction was complete, the mixture in the plates was removed, and 1 ml of Overlay media (4% FBS MEM (2X) : 1.5% agar = 1:1 ratio) was dispensed into each well, followed by incubation in a CO2 incubator for 3 days.

[0055]

[0056] (Day 3) Once incubation was complete, 1 ml of Crystal violet stain (Crystal violet solution + Formaldehyde solution + EtOH + DW) was dispensed into each well, and the overlay media was stained at room temperature. After a staining process of at least 2 hours, the staining reagent and agar were removed, and the plates were transferred to the BL2 facility. After sufficient drying at room temperature, the number of plaques was counted, and the ND was calculated using the Karber formula. 50 Calculated.

[0057]

[0058] Experimental Example 1. Design of Chikungunya Virus Recombinant Protein Vaccine Antigen

[0059] (Candidate substance G1) Among Chikungunya E proteins, the E2 glycoprotein is known as a promising target for vaccine neutralizing antibodies because the fusion loop of domain B is the site where it binds to host cell receptors and has high antigenicity.

[0060] E2 glycoprotein has a lipidation site, so when the protein is expressed, it is not secreted outside the cell. Therefore, two sections, 346 and 367, were cut to remove the lipidation site, and an E2 protein expression vector was designed.

[0061] It was confirmed that the E2 glycoprotein has a lipidation site that induces cell surface fixation, and an E2 protein expression vector was constructed by removing the corresponding sequences (346, 367) to enable extracellular secretion in an animal cell expression system.

[0062] When protein expression was performed using vectors with E2 glycoprotein sequences 346 and 367 removed, respectively, it was confirmed that the protein was expressed at a concentration of 25 mg / L with vector 346 and no protein was secreted with vector 367, so E2 glycoprotein sequence 346 was selected as the final vaccine candidate.

[0063]

[0064] (Candidate substances G2, G3) Chikungunya E3 glycoprotein interacts with E2 glycoprotein to facilitate binding to host cell receptors. A furin cleavage site (sequence 60 RQRR) was present between the E3 and E2 proteins.

[0065] To prevent cleavage of Chikungunya E3 and E2 proteins, an E3-E2 protein vector was designed by replacing the furin cleavage site sequence (RQRR) with other amino acid sequences (AQRR, RQRE, AQRA).

[0066] When protein expression was performed using each E3-E2 protein vaccine candidate sequence vector, the furin portion of the AQRR vector was cleaved and expressed, while the RQRE vector and AQRA vector were expressed at concentrations of 17 mg / L and 24 mg / L, respectively, so two were selected as the final vaccine candidates.

[0067]

[0068] (Candidate substances G4-G7) It was confirmed that the 6K protein is anchored to the cell membrane between Chikungunya E1 proteins, and structural analysis confirmed that the distance between the E2 and E1 proteins is approximately 39.5 Å. Based on this, the 6K protein was removed and a suitable linker was applied to that site. * By introducing [the element], we designed an E3-E2-E1 protein expression vector capable of extracellular secretion in an animal cell expression system.

[0069] *(G4S)4Linker - ~35Å / (G4S)2(EAAAK)2(G4S)2Linker - ~42Å

[0070] When protein expression was performed using each E3-E2-E1 glycoprotein sequence vector, the E3-E2-(G4S)2-(EAAAK)2-(G4S)2-E1(402), E3-E2-(G4S)2-(EAAAK)2-(G4S)2-E1(415), E3-E2-(G4S)4-E1(402), and E3-E2-(G4S)4-E1(415) vectors were expressed at concentrations of 155 mg / L, 120 mg / L, 98 mg / L, and 100 mg / L, respectively, and thus four were selected as final vaccine candidates.

[0071] Based on the analysis of Chikungunya glycoprotein sequences and structures and the evaluation of expression, a total of seven vaccine candidates were selected.

[0072]

[0073] Experimental Example 2. Evaluation of Immunogenicity of Chikungunya Virus Recombinant Protein Vaccine Candidate

[0074] 1) Evaluation of immunogenicity in mice

[0075] (Animal experiment) After introducing 5-week-old female C57BL / 6 mice, they are placed in mouse cages of 4 for 1 week to undergo an acclimatization process.

[0076] After the ingestion period ended, four animals each were immunized twice over three weeks with a recombinant protein vaccine candidate at a predetermined concentration for each group in the hind leg muscles.

[0077] Group AntigenImmunoadjuvantVaccination methodMarie SuduzAddaVaxPBSAddaVax1:1IM4-G1CHIKV E2-346AddaVax1:1IM4100ugG2CHIKV E3-E2(346) RQREAddaVax1:1IM4100ugG3CHIKV E3-E2(346) AQRAAddaVax1:1IM4100ugG4CHIKV E3-E2-(G4S)2-(EAAAK)2-(G4S)2-E1(402)AddaVax1:1IM4100ugG5CHIKV E3-E2-(G4S)2-(EAAAK)2-(G4S)2-E1(415)AddaVax1:1IM4100ugG6CHIKV E3-E2-(G4S)4-E1(402)AddaVax1:1IM4100ugG7CHIKV E3-E2-(G4S)4-E1(415)AddaVax1:1IM4100ug

[0078] Facial venous blood collection was performed 2 weeks after primary and final immunization, respectively. Serum was separated from the collected blood, and binding antibodies and neutralizing antibodies were measured according to established efficacy evaluation methods (ELISA, PRNT). 2 weeks after final immunization, mouse splenocytes were isolated, and cytokines such as IFN-γ were measured by performing ELISPOT according to established cell-mediated immune response methods.

[0079]

[0080] (Measurement of binding antibody) Recombinant protein E2 (Cat No. 40440-V08B) and VLP (Cat No. CBSV065), which have high antigenicity as host cell receptor binding sites, were coated onto a 96-well plate as antigens. After reacting with diluted isolated mouse serum, an HRP conjugate anti-mouse IgG antibody was conjugated. Subsequently, a color reaction was induced using TMB solution as a substrate, and the absorbance was measured at an OD of 450 nm.

[0081] After administering two doses of the Chikungunya recombinant protein vaccine candidate, mouse serum was collected to confirm the binding antibody titer. The G2 experimental group of vaccine candidates was euthanized at the humane end point after the second dose.

[0082] In the ELISA results coated with Chikungunya VLP proteins (E1, E2, Capsid), it was confirmed that G4, G5, and G7 increased in the serum after the first vaccination compared to the control group (AddaVax). In the serum after the second vaccination, G3 and G6 also increased along with the aforementioned G4, G5, and G7.

[0083] In the ELISA results coated with Chikungunya E2 protein, G1, G2, and G3 increased in the serum after the first vaccination compared to the control group (AddaVax), and G4, G5, G6, and G7 increased in the serum after the second vaccination along with the aforementioned G3.

[0084] In the case of VLP protein, since it is a substance that is a mixture of E1, E2, and Capsid proteins among Chikungunya structural proteins, it is presumed that G4, G5, G6, and G7, which contain E1 and E2 among vaccine candidates, appeared high in the ELISA results using serum after the first vaccination.

[0085] It was confirmed that immunity was well achieved, as the ELISA results using serum after the second vaccination were higher than the ELISA results using serum after the first vaccination.

[0086]

[0087] (Measurement of Neutralizing Antibodies) Vero E6 cells were cultured in a 12-well plate for one day. Non-inactivated mouse serum was serially diluted twofold from the stock solution, reacted with Chikungunya virus, and then inoculated into the Vero E6 cells. After incubating for one hour, 1 ml of Overlay media (4% FBS MEM (2X) : 2% agar = 1:1) was added to each well, and the cells were incubated for three days. Staining was performed using Crystal Violet staining reagent, after which the agar was removed and the cells were thoroughly dried at room temperature. The number of plaques was counted, and the Nd value was calculated using the Karber formula. 50 Calculated.

[0088] After administering two doses of the Chikungunya recombinant protein vaccine candidate, mouse serum was collected to check neutralizing antibody titers. The G2 experimental group of vaccine candidates was euthanized at the humane end point after the second dose.

[0089] As a result of confirming neutralizing antibody titers using the Chikungunya virus, it was confirmed that after the first vaccination, the neutralizing antibody titers in the serum of vaccine candidates G4, G5, G6, and G7 increased compared to the control group (AddaVax). In experiments using serum after the second vaccination, it was confirmed that the neutralizing antibody titers increased in the same vaccine candidate groups, similar to the previous experiments.

[0090] Among the G4, G5, G6, and G7 vaccine candidates, the G4 vaccine candidate showed the highest neutralizing antibody titer in the serum after the first dose and also showed the highest neutralizing antibody titer in the serum after the second dose.

[0091]

[0092] (Analysis of cell-mediated immune response) One week after final immunization with the vaccine candidate, mouse spleens were isolated to obtain splenocytes, which were then seeded into a 96-well plate coated with IFN-γ and stimulated using a pre-prepared peptide pool. The peptide-stimulated cells were reacted with a detection antibody, and further reactions were carried out by adding Streptavidin AP. After the final reaction using a BCIP / NBT substrate, the cells were dried and the spots were counted.

[0093] Cell-mediated immune response analysis was performed on four vaccine candidates, G4, G5, G6, and G7, which showed significant neutralizing antibody titers among the seven vaccine candidates.

[0094] Analysis of cell-mediated immune responses confirmed that the highest IFN-γ T cell immune response was observed in vaccine candidate G6.

Claims

Chikungunya virus envelope recombinant protein consisting of SEQ ID NO. 4 Chikungunya virus envelope recombinant protein consisting of SEQ ID NO. 5 Chikungunya virus envelope recombinant protein consisting of SEQ ID NO. 6 Chikungunya virus envelope recombinant protein consisting of SEQ ID NO. 7 A vaccine composition comprising a recombinant protein according to any one of claims 1 to 4. In claim 5, the vaccine composition is characterized by further comprising one or more selected from the group consisting of preservatives, diluents, adjuvants, and carriers. A vaccine composition according to claim 6, characterized in that the ajuvants are squalene-based oil-in-water emulsions. In claim 5, the vaccine composition is characterized as being for the prevention of Chikungunya virus infection. A method for inducing an immune response to Chikungunya virus in non-human mammals, comprising the step of administering the vaccine composition of claim 5 A method for inducing an immune response against Chikungunya virus, characterized in that, in claim 9, the administering step comprises: a step of inducing a priming immune response; and a step of inducing a boosting immune response. A method for preventing Chikungunya virus infection in non-human mammals comprising the step of administering the vaccine composition of claim 5 A method for preventing Chikungunya virus infection according to claim 11, characterized in that the administering step comprises: a step of inducing a priming immune response; and a step of inducing a boosting immune response.

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