Chikungunya virus vaccine multidose formulation and use thereof in outbreaks

Abstract

The present invention relates to a multidose lyophilized vaccine composition for the prevention or treatment of a chikungunya virus infection and / or a chikungunya virus associated clinical illness of any severity. Further provided are methods for manufacturing lyophilized multidose compositions comprising an attenuated chikungunya virus and kits comprising such compositions.

Description

[0001] CHIKUNGUNYA VIRUS VACCINE MULTIDOSE FORMULATION AND USE THEREOF IN OUTBREAKS

[0002] FIELD OF THE INVENTION

[0003] The present invention is related to multidose lyophilized formulations of Chikungunya virus vaccine useful for the purpose of vaccination of individuals in endemic areas or during outbreaks.

[0004] BACKGROUND OF THE INVENTION

[0005] The Chikungunya virus (CHIKV) is currently regarded as one of the most-likely viruses to spread globally or at least regionally, and morbidity due to this virus is considered a serious threat to global and regional public health, raising an urgent demand for efficient prophylaxis. Eminently, due to climate change, the threat posed by Chikungunya could be amplified increasing the size of the human population at risk of infection. However, at present there is no treatment or vaccine available against this CHIKV-induced debilitating disease and its various symptoms. CHIKV has been reported in over 100 countries with more than 2.2 million suspected cases in the Americas alone (World Health Organization. Number of reported cases of chikungunya fever in the Americas by country or territory 2013-2016: http: / / www.paho.org / hq / index.php?option=com topics) .

[0006] CHIKV is a small spherical RNA virus and a member of the Alphavirus genus in the family Togaviridae. The arthropod-borne virus is closely related to other viruses in Africa, South America and Australia that cause similar symptoms such as Ross River Virus, Mayaro virus (MAYV) or O’nyong’nyong virus (ONNV). The chikungunya virus is vectored by the daytime-biting Aedes aegypti mosquito, which also transmits yellow fever, Zika and dengue viruses. CHIKV can also be transmitted by Aedes albopictus mosquitoes, a more cold-tolerant mosquito that could easily facilitate the spread of Chikungunya to more temperate areas of the world. An 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. Individuals who are at higher risk of more serious complications include infants, the elderly and individuals with chronic medical conditions. 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 mosquitoes.

[0007] Recently, a live-attenuated Chikungunya virus vaccine candidate designed for active immunization for the prevention of disease caused by CHIKV has been developed (Hallengard et al., Novel Attenuated Chikungunya Vaccine Candidates Elicit Protective Immunity in C57BL / 6 mice, Journal of Virology, 2014, Vol. 88(5) p. 2858-2866). The candidate vaccine was further developed with the aim to prevent CHIKV disease in the general population living in endemic regions, as well as to serve as a Valneva Austria GmbH PAT071-PCT prophylactic measure for travelers to epidemic areas or areas at risk for an upcoming outbreak (see also WO2019057793, WO2017109223, WO2017109224). The replicating CHIKV vaccine comprises a large deletion of 61 amino acids in the nsP3 gene encoding the non-structural replicase complex protein nsP3, which results in attenuation of the virus in vivo. The candidate vaccine is based on the La Reunion strain of the East Central South African genotype and is produced in Vero cells and purified by centrifugation, ultrafiltration, batch-chromatography and sucrose gradient centrifugation. In C57BL / 6 mice, the vaccine elicited high titers of binding and neutralizing antibodies after a single immunization and mice were subsequently protected from a high dose CHIKV challenge (Hallengard D. et al. Novel Attenuated Chikungunya Vaccine Candidates Elicit Protective Immunity in C57BL / 6 mice. 2014 J Virol 88:2858-66. doi: 10.1128 / JVI.03453-1311). Essentially, a single immunization in non-human primates protected against a wild-type CHIKV infection (Roques P, et al. Attenuated and vectored vaccines protect nonhuman primates against Chikungunya virus. 2017 JCI Insight 2:e83527. doi: 10.1172 / jci.insight.83527). The novel vaccine is designed to protect against all circulating genotypes of CHIKV throughout the world.

[0008] For worldwide distribution of CHIKV vaccines, it is necessary to formulate vaccines such that they are stable under a variety of environmental conditions and over an extended period of time. Components used to stabilize vaccines are known. However, particular formulations of components useful to stabilize CHIKV vaccines must be determined and might not achieve suitable stability of the vaccine, e.g. of the attenuated vaccine candidate described above. Unfortunately, the live-attenuated forms of CHIKV proposed for vaccine use may be rather unstable in various environments. Thus, one object of the present invention is to provide formulations which stabilize CHIKV vaccines, and in particular which stabilize the CHIKV vaccine at a particular dose.

[0009] Further, the development of a multidose formulation for efficient vaccination of populations at risk of chikungunya infection, such as those in endemic or outbreak areas, is of particular interest.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention provides novel multidose formulations of attenuated chikungunya virus useful as vaccines for subjects in endemic areas and during outbreaks.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings are not intended to be drawn to scale. The Figures are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings: Valneva Austria GmbH PAT071-PCT

[0014] Figure 1 Graph of Infectious virus concentration (logic TCIDso / mL) over time for twelve lyophilized CHIKV- A5nsP3 formulations tested in an accelerated stability study (values shown in Table 3). The lyophilized samples were stored at 37°C for 14 days and tested for virus titer at the indicated timepoints.

[0015] Figure 2 Stability of a multidose (3X) lyophilized formulation following reconstitution. A) Stability of virus titer over six hours at room temperature (RT) of lyophilized 3X formulation reconstituted with 1.8 mb sWFI (“Vial 1” and “Vial 2”) compared with three PPQ samples (“PPQ 1-3”). B) Stability of virus titer over six hours at 2 to 8°C of lyophilized 3X formulation reconstituted with 1.8 mb sWFI (“Vial 1” and “Vial 2”) or 0.5 mb sWFI (“0.5 mb”). The virus titer (logic TCIDso / OA mb) was assessed at the indicated timepoints. The resulting values were plotted and a slope calculated to compare the rates of degradation.

[0016] DETAILED DESCRIPTION OF THE INVENTION

[0017] Disclosed herein are multidose attenuated chikungunya virus compositions in lyophilized format as well as liquid formulations reconstituted from such lyophilized compositions, containers comprising such compositions, methods for producing such compositions and kits comprising such compositions. The compositions, methods and kits are particularly useful for vaccination of subjects in chikungunya endemic regions or in an outbreak situation.

[0018] Therefore, in a first aspect, provided is a lyophilized attenuated CHIKV-A5nsP3 chikungunya virus pharmaceutical composition for multidose administration, wherein the composition is lyophilized from a liquid formulation, preferably with the same volume and excipient composition as for a single dose formulation, and wherein the formulation allows the withdrawal of more than one effective dose after reconstitution.

[0019] For instance, in one embodiment there is provided a lyophilized attenuated CHIKV-A5nsP3 chikungunya virus pharmaceutical composition for multidose administration, wherein the composition is lyophilized from a liquid formulation comprising a) two or more effective doses of a CHIKV- A5nsP3 attenuated chikungunya virus; b) sucrose at a concentration of 3-10% (w / v); c) potassium phosphate at a concentration of 3-10 mM; d) sodium citrate at a concentration of 15-50 mM; e) MgCL at a concentration of 3-10 mM; f) D-sorbitol at a concentration of 0.3-1.0% (w / v), g) L-methionine at a concentration of 3-20 mM; and h) recombinant human albumin (rHA) at a concentration of 0.003- 0.02 % (w / v), and wherein the pH of the formulation is 7.3±0.2; optionally wherein the multidose pharmaceutical composition is present in an individual vial that allows the withdrawal of two or more effective doses after reconstitution. Human albumin (HA) is often alternatively referred to as human serum albumin (HSA), as albumin is an abundant protein in human sera. Recombinant human (serum) Valneva Austria GmbH PAT071-PCT albumin is produced using gene expression systems and therefore is safer to use than albumin isolated from the serum of human beings. The human albumin used in the studies described herein is exclusively a recombinant protein.

[0020] A particular advantage of the multidose formulation of the first aspect is that the same formulation buffer and volume as for a single dose formulation is used, meaning that processes, manufacturing equipment and packaging materials do not require modification. The main changes are to add a higher CHIKV-A5nsP3 titer to the drug product (DP) to be lyophilized and to use a higher volume of aqueous solution for reconstitution of the lyophilized cake before administration of the composition. This results in a reduced cost of goods per vaccine composition dose.

[0021] In a second aspect is provided a multidose pharmaceutical composition, wherein the composition is lyophilized from a liquid formulation comprising a) more than one effective dose (e.g. two or more effective doses) of a CHIKV-A5nsP3 attenuated chikungunya virus; b) sucrose at a concentration of 5% (w / v); c) potassium phosphate at a concentration of 5 mM; d) sodium citrate at a concentration of 25 mM; e) MgCb at a concentration of 5 mM; f) D-sorbitol at a concentration of 0.5% (w / v), g) L- methionine at a concentration of 10 mM; and h) recombinant human albumin at a concentration of 0.01% (w / v), and wherein the pH of the formulation is 7.3±0.2. Preferably the total volume of the liquid formulation per individual vial before lyophilization is 0.3 to 1.0 mb, more preferably 0.4 to 0.6 mb, most preferably about 0.5 mb. This liquid formulation for preparation of a lyophilized composition has been demonstrated to provide stability to the single-dose CHIKV-A5nsP3 vaccine (see, e.g., WO 2021028406 Al). Provided in Table Al is a recipe for preparing 16 L of buffer for formulation of a preferred liquid formulation for the preparation of a multidose pharmaceutical composition of the invention. Any other volumes may be prepared using the same proportions.

[0022] Table Al. Recipe for the preparation of 16 L formulation buffer (DS buffer) with a final pH of 7.3 (range 7.1 - 7.5). Valneva Austria GmbH PAT071-PCT

[0023] *WFI may also be added up to a final weight of 16 kg, in which case the concentrations would be indicated as w / w or mmol / kg. This recipe may be scaled up or down according to need.

[0024] In a third aspect, the multidose pharmaceutical composition of the current invention is lyophilized from a liquid formulation comprising i) more than one effective dose (e.g. two or more effective doses) of a CHIKV-A5nsP3 attenuated chikungunya virus, ii) 1.02 mg trisodium citrate dihydrate, iii) 0.09 mg di-potassium hydrogen phosphate, iv) 6.94 mg sucrose, v) 0.14 mg magnesium chloride hexahydrate, vi) 0.69 mg D-sorbitol, vii) 0.21 mg L-methionine, and viii) 0.014 mg recombinant human albumin (rHA), wherein the pH of the formulation is 7.3±0.2, and wherein the total volume of the formulation per individual vial is 0.5 mL. In case of a higher or lower total volume of the formulation per individual vial, the amount of excipients may be increased or reduced accordingly.

[0025] In a fourth aspect, the multidose pharmaceutical composition according to the current invention is defined as comprising between two and twenty-five effective doses, preferably between three and ten effective doses, preferably three or more effective doses, four or more effective doses, five or more effective doses, six or more effective doses, seven or more effective doses, eight or more effective doses, nine or more effective doses, preferably five or ten effective doses, most preferably three effective doses. In a particularly preferred embodiment, the multidose pharmaceutical composition is formulated to deliver three effective doses up to two years after manufacture. In general, an effective dose is >103CHIKV-A5nsP3, particularly >103CHIKV-A5nsP3 particles which do not comprise mutations which abolish immunogenicity of the CHIKV-A5nsP3 particle (for further information, see, e.g., WO 2019057793 Al).

[0026] In a fifth aspect, the multidose pharmaceutical composition according to the current invention comprises an attenuated CHIKV-A5nsP3 virus which comprises essentially an attenuated chikungunya virus of SEQ ID NO: 1, a variant with 99% sequence identity to SEQ ID NO: 1 also containing the 61aa deletion in the nsP3 protein as SEQ ID NO: 1 and / or combinations thereof. In the course of producing a virus on host cells, some variation in the genomic nucleotide sequence is expected to arise. Mutations occurring in the nucleotide sequence according to SEQ ID NO: 1 may not result in changes in the amino acid sequence of the encoded polyprotein (silent mutations). Further, some mutations may result in changes to the amino acid sequence, but not affect the immunogenicity of the viral particle in a significant way. In some cases, the mutations may lead to loss of immunogenicity. The production of the CHIKV-A5nsP3 particle for vaccine manufacture, special measures are taken to ensure the presence of sufficient numbers of immunogenic particles even in the presence of variants (see WO 2019057793 Al). Valneva Austria GmbH PAT071-PCT

[0027] In a sixth aspect, the multidose pharmaceutical composition according to the current invention comprises an attenuated CHIKV-A5nsP3 virus which is selected from a population that comprises substantially two variants, said two variants both expressing the El wild type amino acid sequence as encoded in the relevant part of nucleic acid sequence SEQ ID NO: 1 and wherein one variant expresses the wild type E2 structural protein as defined in SEQ ID NO: 2 and wherein the other variant expresses the E168K mutation in the E2 structural protein as defined in SEQ ID NO: 3. The E168K mutation is an immunogenicity-destroying mutation which has been observed to arise during production of the CHIKV-A5nsP3 virus for vaccine manufacture. As mentioned above, the effects of the E168K mutants can be mitigated (see WO 2019057793 Al).

[0028] In a seventh aspect, the multidose pharmaceutical composition according to the current invention comprises an attenuated CHIKV-A5nsP3 virus which is selected from a population that comprises substantially two variants, said two variants expressing E2 structural proteins as defined by the amino acid sequences of SEQ ID NO: 2 (wild-type E2 protein) and SEQ ID NO: 3 (E2 with E168K), respectively.

[0029] In an eighth aspect, the multidose pharmaceutical composition according to the current invention comprises an attenuated CHIKV-A5nsP3 virus which is selected from a population that comprises one or more variants and wherein the variant has one or more mutations in E2 which mutations are shown in the group of variants encoding for an E2 amino acid sequence with E168K (SEQ ID NO: 3), G55R (SEQ ID NO: 4), E247K (SEQ ID NO: 5), G82R (SEQ ID NO: 6) and / or H232Y (SEQ ID NO: 7).

[0030] In a ninth aspect, the multidose pharmaceutical composition according to the current invention comprises an effective dose of CHIKV-A5nsP3 particles. An effective dose is defined as a number of immunogenic CHIKV-A5nsP3 which, when administered to a subject, is able to induce a protective immune response. As used herein, an effective dose of CHIKV-A5nsP3 virus may be defined as at least 102, at least 103, at least 104, at least 105, at least 106, preferably at least 103CHIKV-A5nsP3 particles which express an E2 structural protein as defined by the amino acid sequence of SEQ ID NO: 2, preferably between 103and 105, especially between about 103and 2xl04CHIKV-A5nsP3 particles which express an E2 structural protein as defined by the amino acid sequence of SEQ ID NO: 2. In a preferred aspect the dose is 5xl03.

[0031] In particularly preferred embodiments, the total number of CHIKV-A5nsP3 particles in an effective dose comprises at least 6.0 logic particles / dose, e.g. 6.0 to 9.0, 6.0 to 8.0 or 6.0 to 7.0 logic CHIKV- A5nsP3 particles. In some embodiments, an effective dose comprises 6.1 to 6.7, 6.2 to 6.6, 6.3 to 6.5, or about 6.4 logic total CHIKV-A5nsP3 particles. Valneva Austria GmbH PAT071-PCT

[0032] The liquid formulation from which the multidose pharmaceutical composition according to the current invention is lyophilized may comprise e.g. at least 6.0 logic total CHIKV-A5nsP3 particles / ml, e.g. at least 6.1, at least 6.3 or at least 6.5 logic total CHIKV-A5nsP3 particles / ml. For instance, the liquid formulation may comprise 6.0 to 8.0, 6.2 to 7.0, or 6.3 to 6.9, 6.5 to 6.8 or about 6.7 logw CHIKV- A5nsP3 particles / ml.

[0033] In a tenth aspect, said liquid formulation from which the multidose pharmaceutical composition according to the current invention is lyophilized comprises between 4.5 and 6.0 logic TCID50 CHIKV- A5nsP3 / mL, e.g. between 4.5 and 6.0 logic infectious CHIKV-A5nsP3 particles / mL. Preferably the liquid formulation comprises at least 5.0, preferably between 5.0 and 5.9, preferably between 5.0 and 5.8, preferably between 5.0 and 5.7, preferably between 5.0 and 5.6, preferably between 5.0 and 5.5, preferably between 5.1 and 5.4, most preferably between 5.2 to 5.3 logic TCID50 CHIKV-A5nsP3 / mL.

[0034] The total number of CHIKV-A5nsP3 particles in the formulation may be determined using standard techniques, e.g. reverse transcription quantitative polymerase chain reaction (RT-qPCR). Amplification of viral sequences may be performed e.g. using the following primers, which target the nsP3 gene in CHIKV-A5nsP3:

[0035] Forward primer - 5 ’-GCTTATAGAGGTGCCGCTGC-3’ (SEQ ID NO: 8)

[0036] Reverse primer - 5 -CGACAGGTACGGTGCTCATT-3’ (SEQ ID NO: 9)

[0037] TCID50 is a measure used to indicate the infectious viral titer, i.e. a level of replication competent infectious virus. TCID50 may be determined using an assay as described in the examples below. TCID50 may be determined on Vero cells, e.g. using an endpoint dilution assay. TCID50 may be defined as an amount of virus required to infect 50% of tissue culture cells, e.g. Vero cells. Infection may be determined by the detection of a cytopathic effect by microscopic evaluation, or by use of a cell dye to detect cell health.

[0038] In an eleventh aspect, the multidose pharmaceutical composition according to the current invention is reconstituted with a sterile aqueous solution. In one aspect, the sterile aqueous solution is sterile water for injection (sWFI) or Phosphate Buffered Saline (PBS).

[0039] In a twelfth aspect, after reconstitution in a volume of 1.5 to 2.0 mL, preferably 1.6 to 1.9 mb, preferably 1.7 to 1.9 mL, preferably about 1.8 mL, the multidose pharmaceutical composition according to the current invention comprises between 3.5 and 6.0 logic TCID50 CHIKV-A5nsP3 / mL, preferably less than 5.0, preferably between 4.0 and 4.9, preferably between 4.2 and 4.9, preferably Valneva Austria GmbH PAT071-PCT between 4.5 and 4.8, most preferably between 4.7 to 4.8 or about 4.75 logic TCID50 CHIKV- A5nsP3 / mL.

[0040] In a thirteenth aspect, the multidose pharmaceutical composition according to the current invention does not exceed potency losses of logic = [0.5] when stored at 5 ± 3°C for up to 2 years. In one aspect, the potency loss at a storage temperature of 5 ± 3°C is less than 0.5 logic, preferably less than 0.4 logic especially less than 0.3 logic, most preferably no more than 0.5 logic.

[0041] In a fourteenth aspect, the multidose pharmaceutical composition of the current invention is stable for at least about 6 hours at room temperature after reconstitution of the composition. Furthermore, in a fifteenth aspect, the multidose pharmaceutical composition of the current invention is stable for at least about 6 hours at room temperature after a first dose withdrawal. As used herein, stability is defined as maintaining a CHIKV-A5nsP3 virus titer which is at or above the level of an effective dose.

[0042] In a sixteenth aspect, the multidose pharmaceutical composition as disclosed herein is suitable for use during an outbreak. In particular, it is considered that recently manufactured lyophilized multidose formulations might be used to deliver more doses directly after manufacture as compared to the end of shelf life, when the titer has dropped. Recently manufactured formulations will contain more effective doses than formulations which have been stored for 2 years.

[0043] In a seventeenth aspect, there is provided a pharmaceutical composition for multidose administration, comprising a liquid formulation reconstituted from a lyophilized pharmaceutical composition, the reconstituted liquid formulation comprising a) two or more effective doses of a CHIKV-A5nsP3 attenuated chikungunya virus; b) sucrose at a concentration of 1-3% (w / v); c) potassium phosphate at a concentration of 1-3 mM; d) sodium citrate at a concentration of 5-15 mM; e) MgCF at a concentration of 1-3 mM; f) D-sorbitol at a concentration of 0.1-0.3% (w / v), g) L-methionine at a concentration of 1-7 mM; and h) recombinant human albumin at a concentration of 0.001-0.007% (w / v), and wherein the pH of the formulation is 7.3±0.2.

[0044] In an eighteenth aspect, there is provided a vial, syringe or container comprising a lyophilized pharmaceutical composition or a reconstituted liquid formulation according to the invention. Preferably the vial, syringe or container is suitable for administration of two or more effective doses of the CHIKV-A5nsP3 attenuated chikungunya virus. Valneva Austria GmbH PAT071-PCT

[0045] In a nineteenth aspect, the vial, syringe or container according to the invention comprises a volume of 1.5 to 2.0 mL, preferably 1.6 to 1.9 mL, 1.7 to 1.9 mL, or about 1.8 mb of the reconstituted liquid formulation.

[0046] In a twentieth aspect, the reconstituted liquid formulation according to the invention comprises between 3.5 and 6.0 logic TCID50 CHIKV-A5nsP3 / mL, preferably less than 5.0, preferably between 4.0 and 4.9, preferably between 4.2 and 4.9, preferably between 4.5 and 4.8, most preferably between 4.7 to 4.8 or about 4.75 logw TCID50 CHIKV-A5nsP3 / mL.

[0047] In a twenty-first aspect is provided a method for preparing a lyophilized attenuated CHIKV-A5nsP3 virus pharmaceutical composition for multidose administration, comprising the following steps:

[0048] (i) cultivating a CHIKV-A5nsP3 virus on host cells and mixing the CHIKV-A5nsP3 virus with a concentrated stabilizing solution to form a virus bulk (i); and, optionally,

[0049] (ii) dialyzing the virus bulk (i) to form a composition (ii); wherein said composition (ii) comprises a) more than one effective dose (e.g. two or more effective doses) of a CHIKV- A5nsP3 attenuated chikungunya virus; b) sucrose at a concentration of 3-10%, preferably 5% (w / v); c) potassium phosphate at a concentration of 3-10 mM, preferably 5 mM; d) sodium citrate at a concentration of 15-50 mM, preferably 25 mM; e) MgCL at a concentration of 3-10 mM, preferably 5 mM; f) D-sorbitol at a concentration of 0.3-1.0 % (w / v), preferably 0.5% (w / v), g) L-methionine at a concentration of 0.003-0.02 % (w / v), preferably 10 mM; and h) recombinant human albumin at a concentration of 0.01% (w / v), and wherein the pH of the formulation is 7.3±0.2; and

[0050] (iii) lyophilizing said virus bulk (i) or composition (ii), wherein said virus bulk (i) or composition (ii) comprise the more than one effective dose (e.g. two or more effective doses) of CHIKV-A5nsP3 virus, preferably in the same volume as for a single dose; e.g. wherein said virus bulk (i) or composition (ii) comprises two or more effective doses of CHIKV-A5nsP3 virus per mL, more preferably two or more effective doses of CHIKV-A5nsP3 virus per 0.4 to 0.6 mL, more preferably two or more effective doses of CHIKV-A5nsP3 virus per 0.5 mL, most preferably four, five or six or more effective doses of CHIKV-A5nsP3 virus per mL.

[0051] In a twenty-second aspect, the CHIKV-A5nsP3 cultivated in step (i) of the method according to the current invention is defined by the polynucleotide sequence of SEQ ID NO: 1. In a twenty-third aspect, lyophilizing step (iii) of the method provided herein results in the multidose pharmaceutical composition according to the current invention. Valneva Austria GmbH PAT071-PCT

[0052] In an twenty-fourth aspect, for the pharmaceutical compositions herein as well as for the methods for producing multidose pharmaceutical compositions, multidose is defined as between two and twenty- five doses, preferably between three and ten doses, preferably three or more doses, four or more doses, five or more doses, six or more doses, seven or more doses, eight or more doses, nine or more doses, preferably five or ten doses, most preferably three doses.

[0053] In a twenty-fifth aspect, for the pharmaceutical compositions herein as well as for the methods for producing multidose pharmaceutical compositions, a dose is defined as a number of immunogenic CHIKV-A5nsP3 particles which, when administered to a human subject, is able to induce a protective immune response against chikungunya virus infection, preferably at least 102, at least 103, at least 104, at least 105, at least 106CHIKV-A5nsP3 particles, preferably at least 103CHIKV-A5nsP3 particles which express an E2 structural protein as defined by the amino acid sequence of SEQ ID NO: 2, preferably between 103and 105, most preferably between about 103and 2xl04CHIKV-A5nsP3 particles, especially about 5xl03particles, which express an E2 structural protein as defined by the amino acid sequence of SEQ ID NO: 2.

[0054] In a twenty-sixth aspect is provided a method for producing multidose pharmaceutical compositions wherein the virus bulk (i) or composition (ii) as produced in the method is aliquoted into vials in a volume of less than or equal to 1.0 mb, preferably 0.3 to 1.0 mb, more preferably 0.4 to 0.6 mb, most preferably about 0.5 mb before lyophilization step (iii). Alternatively, if more doses per vial are desired, the virus bulk (i) or composition (ii) as produced in the method of the current invention may be aliquoted in volumes higher than 0.5 mb, such as 0.6 mb, 0.7 mb, 0.8 mb, 0.9 mb, 1.0 mb, up to 2.0 mb

[0055] In a twenty-seventh aspect, the pharmaceutical composition produced by the method according to the current invention is reconstituted in a volume of 1.5 to 2.0 mb, preferably 1.6 to 1.9 mb, 1.7 to 1.9 mb, or about 1.8 mb before administration. Alternatively, a higher volume for reconstitution may be used, particularly if more than three doses are formulated in the composition. Higher volumes for reconstitution may be, for example, 1.9 mb, 2.0 mb, 2.5 mb, 3.0 mb or 4.0 mb.

[0056] In a twenty-eighth aspect, the pharmaceutical composition of the current invention is administered to a subject in a volume of between 0.1 and 0.5 mb, preferably between 0.2 and 0.5 mb, such as 0.3 or 0.4 mb, most preferably about 0.5 mb. The volume of administration will depend on the virus concentration in the reconstituted composition. This value is dependent on the titer added at the time of formulation as well as the time in storage; i.e., the amount of titer loss as a function of time. Valneva Austria GmbH PAT071-PCT

[0057] In a twenty-ninth aspect is provided a method for producing multidose pharmaceutical compositions wherein the virus bulk (i) or composition (ii) as produced in the method comprises between 4.5 and 6.0 logic TCID50 CHIKV-A5nsP3 / mL, preferably between 4.6 and 5.9, preferably between 4.7 and 5.8, preferably between 4.8 and 5.7 , preferably between 4.9 and 5.6 TCID50 CHIKV-A5nsP3 / mL. More preferably the virus bulk comprises at least 5.0 logic TCID50 CHIKV-A5nsP3 / mL, e.g. between 5.0 and 5.9, preferably between 5.0 and 5.8, preferably between 5.0 and 5.7, preferably between 5.0 and 5.6, preferably between 5.0 and 5.5, preferably between 5.1 and 5.4, most preferably between 5.2 to 5.3 logio TCID50 CHIKV-A5nsP3 / mL.

[0058] In a thirtieth aspect is provided a multidose CHIKV-A5nsP3 pharmaceutical composition for use in a method of vaccinating two or more human subjects to stimulate a protective immune response against chikungunya virus in said subjects. For example, multiple subjects may be vaccinated within a short time period using the multidose composition according to the current invention. This aspect is useful, e.g., for application in chikungunya endemic areas, or in the case of a chikungunya outbreak. In a thirty-first aspect, the multidose pharmaceutical composition of the current invention is a vaccine.

[0059] In a thirty-second aspect is provided a kit for preparing and / or administering a multidose composition or vaccine of the current invention, wherein the kit comprises the following components: (a) a lyophilized multidose composition as described herein; and (b) at least one sterile aqueous component (b) for reconstituting component (a). In a thirty-third aspect, the sterile aqueous component (b) comprises phosphate buffered saline (PBS) or sterile water for injection (sWFI). In a thirty-fourth aspect, the kit comprises at least one means for combining component (a) and component (b) to obtain the multidose composition or vaccine. In one aspect, the means for combining component (a) and component (b) comprises a syringe and a needle. In a thirty-fifth aspect, the kit comprises at least one means for administering the multidose composition or vaccine to a subject or subjects in need thereof. In one aspect, the at least one means for administering the multidose composition or vaccine to a subject or subjects comprises at least one syringe and at least one needle or, alternatively, multiple syringes and / or needles.

[0060] In a thirty-sixth aspect, the pharmaceutical composition for multidose administration and the kit according to any of the embodiments of the current invention are suitable for use as a medicament. Further, in a thirty-seventh aspect, the pharmaceutical composition for multidose administration or the kit according to the current invention are suitable for use in the treatment or prevention of an infection with chikungunya virus or of a disease related to such an infection.

[0061] In a thirty-eighth aspect, is provided a method for the treatment or prevention of infection and disease caused by chikungunya virus in a subject in need thereof, such method comprising administering to Valneva Austria GmbH PAT071-PCT the subject a therapeutically effective amount of the pharmaceutical composition according to the current disclosure. In a thirty-third aspect is provided the use of the pharmaceutical composition or the kit according to the current invention, for the treatment or prevention of an infection or disease caused by chikungunya virus.

[0062] Any of the pharmaceutical compositions or vaccines provided in the current invention may further comprise an adjuvant and / or a pharmaceutically acceptable excipient. Excipients include substances such as buffers, stabilizers or further active ingredients, especially ingredients known in connection with pharmaceutical compositions and / or vaccine production. In particular, the compositions or vaccines described herein may be administered to a subject with, prior to, or after administration of one or more adjuvants. An adjuvant is a molecule or formulation that enhances a response in a subject, such as an immune response, to an antigen or other molecule. In some embodiments, an adjuvant may stabilize an antigen or other molecule. Determining whether a virus vaccine or pharmaceutical composition is administered with an adjuvant depends on various factors (e.g., type and extent of response desired) and will be evident to one of skill in the art.

[0063] The term “preventing” can also be interpreted herein as “protecting from”. Any of the methods or uses described herein may be used for the prevention or treatment of a chikungunya virus infection in a subject. As used herein, the terms “prevent,” “preventing” and “protecting from” include the administration of a virus vaccine or composition to a subject to reduce or delay the onset of the manifestation of clinical or subclinical symptoms, complications, pathologies or biochemical indicia of a disease or infection, or to reduce or inhibit the spread / transmission of a virus; i.e., to treat or prevent a chikungunya virus infection and / or a chikungunya virus associated clinical illness of any severity. As used herein, antigen(s), such as an inactivated, live attenuated or vector virus provided by the processes described herein, that is administered to a subject prophylactically (e.g., prior to infection) may be referred to as a vaccine.

[0064] The current invention is not limited in its application to the details of construction and the arrangement of components set forth in the description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including”, “comprising”, or “having”, “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0065] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Valneva Austria GmbH PAT071-PCT

[0066] Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, virology, cell or tissue culture, genetics and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0067] The present invention is further illustrated by the following examples, which in no way should be construed as further limiting. The entire contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove. However, the citation of any reference is not intended to be an admission that the reference is prior art.

[0068] EXAMPLES

[0069] Example 1. Testing of different formulation buffer compositions for preparation of a lyophilized chikungunya vaccine and effects on stability

[0070] The attenuated chikungunya vaccine of the current invention comprises the attenuated CHIKV- A5nsP3 virus in a lyophilized Drug Product (DP), which is a powder for reconstitution, yielding a solution for injection. The release specification for infectious virus concentration of 3.6-4.6 logic TCIDso / OAmL, a titer demonstrated to ensure an effective dose (>3 logic TCID50) up to at least 24 months of storage at 5±3°C. For the single dose lyophilized formulation, 0.5 mb of the composition is aliquoted into vials and lyophilized. At the time of vaccine administration, the lyophilized cake is reconstituted and delivered in a volume of 0.5 mb sWFL At constant process parameters, the stability of the drug product is driven mainly by the composition of the formulation buffer. The standard composition of the formulation buffer is as below:

[0071] -Sucrose (5% w / w)

[0072] -Trisodium citrate dihydrate (25 mM)

[0073] -di-Potassium hydrogen phosphate and Potassium di-hydrogen phosphate (5 mM, combined)

[0074] -Magnesium chloride hexahydrate (5 mM)

[0075] -D-Sorbitol (0.5% w / w)

[0076] -L-Methionine (10 mM)

[0077] -Recombinant human albumin (rHA; 0.1 mg / mL)

[0078] -Water for Injection (quantity sufficient)

[0079] -Final pH 7.3 Valneva Austria GmbH PAT071-PCT

[0080] Sucrose and D-sorbitol are bulking agents in the formulation which determine the cake quality and also function as lyoprotectants for antigen during lyophilization. L-methionine acts as an antioxidant and rHA prevents unspecific surface adsorption of the antigen during manufacturing. To assess the stability of CHIKV-A5nsP3 DP at lower concentrations of these four excipients, the current study was performed using formulation buffers with reduced concentrations of sucrose, D-Sorbitol, L- Methionine and rHA as shown in Table 1.

[0081] Procedure Twelve formulation buffers, 250 g each were prepared using a similar procedure to that of the large-scale production process followed for manufacturing. Table 1 provides an overview of the compositions of the twelve formulation buffers prepared.

[0082] Table 1. Twelve formulation buffer compositions (FB) tested in the experiment. The first 4 components listed in the table were tested at different concentrations as indicated.

[0083] *FB-1 represents the standard formulation.

[0084] Using sterile filtered formulation buffer, liquid CHIKV Drug Product was prepared at a target infectious virus concentration of 5.5 logic TCIDso / O.S mL dose. This higher concentration of virus particles was used in case degradation of virus particles under accelerated conditions might potentially sink below the limit of detection. DS thawing and DP formulation were performed following the GMP process. Thirty vials per formulation were manually filled using an auto-pipette (0.5 mL / vial) and all 360 vials were lyophilized in a small-scale lyophilizer (SP Scientific VirTis Advantage Pro).

[0085] Lyophilized DP vials were stored at 37° C for 2 weeks and samples were analysed in triplicate at TO, day3 (d3), d7, dlO and dl4 time points for infectious virus concentration (TCID50). Additionally, the percent residual moisture was analyzed at TO and dl4 and the drug products were visually inspected for their cake quality. Valneva Austria GmbH PAT071-PCT

[0086] TCID50 Assay Virus titers were determined on vero cells using the TCID50 assay. TCID50 is the measure to indicate the level of replication competent infectious virus from culture supernatant. This endpoint dilution assay quantifies the amount of virus required to infect 50% of tissue culture cells through the detection of cytopathic effect by microscopic evaluation or by use of a cell dye to detect cell health. Briefly, cells were seeded in microplates and infected with 10-fold serially diluted virus samples in EMEM supplemented with 0.5% FBS and 2 mM glutamine. After a one-week incubation at 35°C / 5% CO2, virus-induced cytopathic effects were assessed and viral titers were calculated according to the Reed and Muench method (Reed, L.J. and Muench, H. A simple method of estimating fifty percent endpoints (1938) The American Journal of Hygiene 27:493-497). Assay control samples were included in each analysis. The assay variability between individual runs was estimated as ± 0.3 logic TCID50.

[0087] Results The pH values of the formulations were in line with the standard CHIKV-A5nsP3 formulation buffer, which is approximately 7.3 (see Table 2). In addition, visual inspection of the lyophilized DP derived from all 12 formulations indicated no observable defects in the cake quality.

[0088] Table 2. pH values of the formulation buffers immediately after preparation.

[0089] TCID50 analysis data obtained at various time points for the lyophilized drug products (DP) formulated using the respective experimental formulation buffers confirmed a similar trend in the degradation profile of all 12 drug products (see Table 3 and Figure 1).

[0090] Table 3. TCID50 analysis (values in logic TCIDso / mL) Valneva Austria GmbH PAT071-PCT

[0091] Considering the assay variability of the TCID50 analysis method (±0.3 logic TCID50), linear fitting of the data indicates no observable difference in the degradation profile of the 12 formulations (see Figure 1). Formulation buffer 7 contained the least amount of rHA, D-Sorbitol, Sucrose and L- Methionine compared to the standard DP, yet the slope values indicating losses in infectious virus concentration for Formulation 1 and Formulation 7 were -0.0586 and -0.0523 logic TCIDso / day, respectively, confirming a similar degradation profile.

[0092] As expected, the residual moisture content was observed to increase in correspondence with a decrease in sucrose / sorbitol concentration and with increasing storage time (see Table 4).

[0093] Table 4. Percent residual moisture of lyophilized DP at TO and Day 14.

[0094] This study confirmed that, even with lower concentrations of the stability determining formulation buffer excipients sucrose, D-sorbitol, L-methionine and rHA in the CHIKV-A5nsP3 DP, the infectious virus concentration remains the same and stable. Under accelerated degradation conditions of 37° C, the formulations with lower concentrations of above four excipients assessed in this experiment have shown a similar degradation profile as the CHIKV-A5nsP3 drug product formulation with standard excipient concentrations. Long term stability studies to confirm these observations are currently ongoing. Valneva Austria GmbH PAT071-PCT

[0095] Notably, results from this study suggest that DP prepared using higher concentrations of the four tested excipients may also remain stable, as stability is generally adversely affected when the concentrations of stability determining excipients are reduced. However, this possibility needs to be confirmed experimentally.

[0096] Example 2. Preparation of an exemplary stable multidose lyophilized chikungunya vaccine

[0097] The objective was to test the feasibility of a multidose drug product for lyophilized CHIKV-A5nsP3. The exemplary study described herein tested the same formulation buffer, pre-lyophilized volume, and primary packaging materials as used for the single dose Drug Product (DP) but with a 3 -fold increase in the infectious virus concentration achieved by adding more Drug Substance (DS=purified CHIKV-A5nsP3) in the Final Bulk Formulation prior to lyophilization.

[0098] Procedure A recently manufactured lab-scale batch of lyophilized CHIKV-A5nsP3 vaccine with an infectious virus concentration of 5.40 Logic TCIDso / mL (5.1 logic TCIDso / OA mL) was used. To ensure the withdrawal of three full doses of 0.5 mL each, an excess volume as per USP of 0.1 mL / dose was used, i.e., reconstitution of the multidose vial with 1.8 mL of sWFI, resulting in an infectious virus concentration of 4.84 logic TCID mL. For feasibility of the format, the in-use stability profde of the multidose preparation would need to match the stability profde of the single dose preparation which is already validated.

[0099] In-use stability studies were performed by reconstituting the multidose vials with 1.8 mL sWFI and assessing the infectious virus concentration after 3h and 6h at room temperature storage and comparing the value with the titer at time zero (TO). Two vials were tested at the 3h and 6h timepoints, while one vial was tested at TO. All samples were collected and frozen at -80°C after each respective time point and were thawed on the day of TCID50 analysis. Control samples in the form of 1.8 mL reconstitution volume and storage at 2-8°C were included. Moreover, another set of control samples (one vial each, using the regular (0.5 mL) reconstitution volume) was included as part of the in-use stability testing.

[0100] Results As shown in Table 5 below, the observed titers confirm that reconstitution with 1.8 mL sWFI of the CHIKV-A5nsP3 lyophilized cake, which was formed with 0.5 mL Final bulk, fulfill the criteria of expected outcome; i.e., an infectious virus concentration after reconstitution with 1.8 mL at T0= 4.84 loglO TCIDso / mL.

[0101] Table 5. In-use stability at RT and 2-8°C. Stability comparison of single dose vs. 3X dose formulation. Valneva Austria GmbH PAT071-PCT

[0102] A graphical comparison of three PPQ single dose preparations and duplicate samples of a 3X dose formulation held at RT for up to six hours, along with the resulting slope values, indicates a similar stability profile (i.e., a loss of ~ 0.05 log per hour; see Fig. 2A). As per ANVISA guidelines for lyophilized multidose product, in-use stability should also be assessed at the long-term storage conditions (i.e., 2-8°C). In this regard, virus titers of a single dose and duplicate samples of a 3X dose formulation held for up to 6 hours at 2-8°C are shown in Fig. 2B. The losses observed were less than for the RT study; i.e., 0.025 log per hour or -0.15 log in 6 hours.

[0103] These small-scale results support the feasibility of developing a scaled-up production of a multidose formulation using the same formulation buffer, liquid volume, and primary container closure system as for the single dose. The current lab-scale batch is not expected to fully correlate to process losses at large scale; however, the current data combined with experience from process performance qualification (PPQ) campaigns may be used for selection of a suitable target infectious CHIKV- A5nsP3 virus concentration after dilution with formulation buffer, needs to be confirmed with the help of engineering runs.

[0104] Process performance qualification (PPQ) campaigns have previously indicated a 61.1-82.2% (min- max) deviation from the expected target. Based on these values and the current observations, possible stability scenarios are provided in Table 6 below. The values include the titer present at the time of release (TO) and at the end of 24 months at 2-8°C (24M), as well as in-use stability up to 6h at RT following reconstitution at TO and 24M. The expected losses are based on observed losses in actual formulations (Actual 1 and 2) and on stability studies such as shown in Example 1. The projected CHIKV / 0.5 m dose after 24M storage and use at RT as shown indicate that optimal values of CHIKV at TO are between 5.2 and 5.3 log 10.

[0105] Table 6. Possible scenarios considering best and worst possible acceptable values per dose at the time of release and at the end of 24M shelf-life at 2-8°C along with in-use stability up to 6h at RT. For an effective vaccine, the value must not go below 3.0 logw / dose. The values obtained from actual single dose preparations reconstituted with 0.5 m (“Actual 1 and 2”), as well as losses observed in stability studies, were used to project expected values for proposed multidose (3X) formulations (“Proposal 1- Valneva Austria GmbH PAT071-PCT

[0106] *values may reach or go below the specification of >3.0 log / dose.

[0107] The possibility of errors in the amount of diluent used for reconstitution was also considered, as a difference in dilution volume would impact the infectious virus concentration per dose. The recommended sWFI fdl volume in the diluent vial provided with the 3X lyophilized formulation is 2 mL and the recommended sWFI volume for reconstitution of CHIKV-A5nsP3 lyo cake is 1.8 mL. A practical study of volume transfer by using 2 mL syringe and V / 25G needle indicated a volume transfer of 1.77 ± 0.01 mL (n=3). Nonetheless, the impact on the actual dose delivered in the case of a dilution variation of 1.8 ± 0.1 mL (i.e add 1.7 or 1.9 mL) during reconstitution of CHIKV-A5nsP3 lyo cake was also projected (see Table 7). As observed, a variability of 0. 1 mL during reconstitution with 1.8 mL does not significantly change the infectious virus amount per dose. As such, a 2 mL fill volume for sWFI in the vial and 1.8 mL sWFI for reconstitution of the CHIK lyo-cake is appropriate for delivery of three doses in a clinical set-up.

[0108] Table 7. Projected impact on the infectious virus concentration per dose with different dilution volumes (in case of reconstitution volume inaccuracy).

[0109] The current process for single dose formulation involves 4.75 logic TCIDso / mL as target infectious virus concentration after dilution. Based on the observations provided herein, the target infectious virus concentration for a 3X dose formulation batch is 5.2-5.3 logic TCIDso / mL. Valneva Austria GmbH PAT071-PCT

[0110] SEQUENCES

[0111] SEQ I D NO: 1

[0112] Nucleotide sequence of CHIKV-A5nsP3 GATGGCTGCGTGAGACACACGTAGCCTACCAGTTTCTTACTGCTCTACTCTGCAAAGCAAGAGATTAATAACCCATCATGGATCCTGTGT

[0113] ACGTGGACATAGACGCTGACAGCGCCTTTTTGAAGGCCCTGCAACGTGCGTACCCCATGTTTGAGGTGGAACCAAGGCAGGTCACACC GAATGACCATGCTAATGCTAGAGCGTTCTCGCATCTAGCTATAAAACTAATAGAGCAGGAAATTGACCCCGACTCAACCATCCTGGATAT CGGCAGTGCGCCAGCAAGGAGGATGATGTCGGACAGGAAGTACCACTGCGTCTGCCCGATGCGCAGTGCGGAAGATCCCGAGAGACT CGCCAATTATGCGAGAAAGCTAGCATCTGCCGCAGGAAAAGTCCTGGACAGAAACATCTCTGGAAAGATCGGGGACTTACAAGCAGTA ATGGCCGTGCCAGACACGGAGACGCCAACATTCTGCTTACACACAGACGTCTCATGTAGACAGAGAGCAGACGTCGCTATATACCAAGA CGTCTATGCTGTACACGCACCCACGTCGCTATACCACCAGGCGATTAAAGGGGTCCGAGTGGCGTACTGGGTTGGGTTCGACACAACCC CGTTCATGTACAATGCCATGGCGGGTGCCTACCCCTCATACTCGACAAACTGGGCAGATGAGCAGGTACTGAAGGCTAAGAACATAGG ATTATGTTCAACAGACCTGACGGAAGGTAGACGAGGCAAGTTGTCTATTATGAGAGGGAAAAAGCTAAAACCGTGCGACCGTGTGCTG TTCTCAGTAGGGTCAACGCTCTACCCGGAAAGCCGCAAGCTACTTAAGAGCTGGCACCTGCCATCGGTGTTCCATTTAAAGGGCAAACT CAGCTTCACATGCCGCTGTGATACAGTGGTTTCGTGTGAGGGCTACGTCGTTAAGAGAATAACGATGAGCCCAGGCCTTTATGGAAAAA CCACAGGGTATGCGGTAACCCACCACGCAGACGGATTCCTGATGTGCAAGACTACCGACACGGTTGACGGCGAAAGAATGTCATTCTC GGTGTGCACATACGTGCCGGCGACCATTTGTGATCAAATGACCGGCATCCTTGCTACAGAAGTCACGCCGGAGGATGCACAGAAGCTG TTGGTGGGGCTGAACCAGAGAATAGTGGTTAACGGCAGAACGCAACGGAATACGAACACCATGAAAAATTATCTGCTTCCCGTGGTCG CCCAAGCCTTCAGTAAGTGGGCAAAGGAGTGCCGGAAAGACATGGAAGATGAAAAACTCCTGGGGGTCAGAGAAAGAACACTGACCT GCTGCTGTCTATGGGCATTCAAGAAGCAGAAAACACACACGGTCTACAAGAGGCCTGATACCCAGTCAATTCAGAAGGTTCAGGCCGA GTTTG ACAGCTTTGTG GTACCG AGTCTGTG GTCGTCCG GGTTGTCAATCCCTTTG AG G ACTAG AATCAAATG GTTGTTAAG CAAG GTG C CAAAAACCGACCTGATCCCATACAGCGGAGACGCCCGAGAAGCCCGGGACGCAGAAAAAGAAGCAGAGGAAGAACGAGAAGCAGAA CTGACTCGCGAAGCCCTACCACCTCTACAGGCAGCACAGGAAGATGTTCAGGTCGAAATCGACGTGGAACAGCTTGAGGACAGAGCGG GCGCAGGAATAATAGAGACTCCGAGAGGAGCTATCAAAGTTACTGCCCAACCAACAGACCACGTCGTGGGAGAGTACCTGGTACTCTC CCCGCAGACCGTACTACGTAGCCAGAAGCTCAGTCTGATTCACGCTTTGGCGGAGCAAGTGAAGACGTGCACGCACAACGGACGAGCA GGGAGGTATGCGGTCGAAGCGTACGACGGCCGAGTCCTAGTGCCCTCAGGCTATGCAATCTCGCCTGAAGACTTCCAGAGTCTAAGCG AAAGCGCAACGATGGTGTATAACGAAAGAGAGTTCGTAAACAGAAAGCTACACCATATTGCGATGCACGGACCAGCCCTGAACACCGA CGAAGAGTCGTATGAGCTGGTGAGGGCAGAGAGGACAGAACACGAGTACGTCTACGACGTGGATCAGAGAAGATGCTGTAAGAAGG AAGAAGCCGCAGGACTGGTACTGGTGGGCGACTTGACTAATCCGCCCTACCACGAATTCGCATATGAAGGGCTAAAAATCCGCCCTGCC TGCCCATACAAAATTGCAGTCATAGGAGTCTTCGGAGTACCGGGATCTGGCAAGTCAGCTATTATCAAGAACCTAGTTACCAGGCAGGA CCTGGTGACTAGCGGAAAGAAAGAAAACTGCCAAGAAATCACCACCGACGTGATGAGACAGAGAGGTCTAGAGATATCTGCACGTACG GTTGACTCGCTGCTCTTGAATGGATGCAACAGACCAGTCGACGTGTTGTACGTAGACGAGGCGTTTGCGTGCCACTCTGGAACGCTACT TGCTTTGATCGCCTTGGTGAGACCAAGGCAGAAAGTTGTACTTTGTGGTGACCCGAAGCAGTGCGGCTTCTTCAATATGATGCAGATGA AAGTCAACTATAATCACAACATCTG CACCCAAGTGTACCACAAAAGTATCTCCAGG CG GTGTACACTG CCTGTG ACCG CCATTGTGTCAT CGTTGCATTACGAAGGCAAAATGCGCACTACGAATGAGTACAACAAGCCGATTGTAGTGGACACTACAGGCTCAACAAAACCTGACCCT GGAGACCTCGTGTTAACGTGCTTCAGAGGGTGGGTTAAACAACTGCAAATTGACTATCGTGGATACGAGGTCATGACAGCAGCCGCAT CCCAAGGGTTAACCAGAAAAGGAGTTTACGCAGTTAGACAAAAAGTTAATGAAAACCCGCTCTATGCATCAACGTCAGAGCACGTCAAC GTACTCCTAACGCGTACGGAAGGTAAACTGGTATGGAAGACACTTTCCGGCGACCCGTGGATAAAGACGCTGCAGAACCCACCGAAAG GAAACTTCAAAGCAACTATTAAGGAGTGGGAGGTGGAGCATGCATCAATAATGGCGGGCATCTGCAGTCACCAAATGACCTTCGATAC ATTCCAAAATAAAG CCAACGTTTGTTG GG CTAAG AGCTTG GTCCCTATCCTCG AAACAG CGG G G ATAAAACTAAATG ATAG GCAGTG GT CTCAGATAATTCAAGCCTTCAAAGAAGACAAAGCATACTCACCTGAAGTAGCCCTGAATGAAATATGTACGCGCATGTATGGGGTGGAT CTAGACAGCGGGCTATTTTCTAAACCGTTGGTGTCTGTGTATTACGCGGATAACCACTGGGATAATAGGCCTGGAGGGAAAATGTTCGG ATTTAACCCCGAGGCAGCATCCATTCTAGAAAGAAAGTATCCATTCACAAAAGGGAAGTGGAACATCAACAAGCAGATCTGCGTGACTA CCAGGAGGATAGAAGACTTTAACCCTACCACCAACATCATACCGGCCAACAGGAGACTACCACACTCATTAGTGGCCGAACACCGCCCA

[0114] GTAAAAGGGGAAAGAATGGAATGGCTGGTTAACAAGATAAACGGCCACCACGTGCTCCTGGTCAGTGGCTATAACCTTGCACTGCCTA CTAAGAGAGTCACTTGGGTAGCGCCGTTAGGTGTCCGCGGAGCGGACTACACATACAACCTAGAGTTGGGTCTGCCAGCAACGCTTGG TAGGTATGACCTAGTGGTCATAAACATCCACACACCTTTTCGCATACACCATTACCAACAGTGCGTCGACCACGCAATGAAACTGCAAAT GCTCGGGGGTGACTCATTGAGACTGCTCAAACCGGGCGGCTCTCTATTGATCAGAGCATATGGTTACGCAGATAGAACCAGTGAACGA GTCATCTGCGTATTGGGACGCAAGTTTAGATCGTCTAGAGCGTTGAAACCACCATGTGTCACCAGCAACACTGAGATGTTTTTCCTATTC AGCAACTTTGACAATGGCAGAAGGAATTTCACAACTCATGTCATGAACAATCAACTGAATGCAGCCTTCGTAGGACAGGTCACCCGAGC AGGATGTGCACCGTCGTACCGGGTAAAACGCATGGACATCGCGAAGAACGATGAAGAGTGCGTAGTCAACGCCGCTAACCCTCGCGG GTTACCGGGTGGCGGTGTTTGCAAGGCAGTATACAAAAAATGGCCGGAGTCCTTTAAGAACAGTGCAACACCAGTGGGAACCGCAAAA ACAGTTATGTGCGGTACGTATCCAGTAATCCACGCTGTTGGACCAAACTTCTCTAATTATTCGGAGTCTGAAGGGGACCGGGAATTGGC AGCTGCCTATCGAGAAGTCGCAAAGGAAGTAACTAGGCTGGGAGTAAATAGTGTAGCTATACCTCTCCTCTCCACAGGTGTATACTCAG GAGGGAAAGACAGGCTGACCCAGTCACTGAACCACCTCTTTACAGCCATGGACTCGACGGATGCAGACGTGGTCATCTACTGCCGCGA CAAAGAATGGGAGAAGAAAATATCTGAGGCCATACAGATGCGGACCCAAGTAGAGCTGCTGGATGAGCACATCTCCATAGACTGCGAT ATTGTTCGCGTGCACCCTGACAGCAGCTTGGCAGGCAGAAAAGGATACAGCACCACGGAAGGCGCACTGTACTCATATCTAGAAGGGA CCCGTTTTCATCAGACGGCTGTGGATATGGCGGAGATACATACTATGTGGCCAAAGCAAACAGAGGCCAATGAGCAAGTCTGCCTATAT Valneva Austria GmbH PAT071-PCT

[0115] GCCCTGGGGGAAAGTATTGAATCGATCAGGCAGAAATGCCCGGTGGATGATGCAGACGCATCATCTCCCCCCAAAACTGTCCCGTGCCT TTGCCGTTACGCTATGACTCCAGAACGCGTCACCCGGCTTCGCATGAACCACGTCACAAGCATAATTGTGTGTTCTTCGTTTCCCCTCCCA AAGTACAAAATAG AAG G AGTG CAAAAAGTCAAATGCTCTAAG GTAATG CTATTTG ACCACAACGTG CCATCGCG CGTAAGTCCAAGG G CTTATAGAGGTGCCGCTGCCGGTAACCTTGCGGCCGTGTCTGATTGGGTAATGAGCACCGTACCTGTCGCGCCGCCCAGAAGAAGGCG AGGGAGAAACCTGACTGTGACATGTGACGAGAGAGAAGGGAATATAACACCCATGGCTAGCGTCCGATTCTTTAGGGCAGAGCTGTGT CCGGTCGTACAAGAAACAGCGGAGACGCGTGACACAGCAATGTCTCTTCAGGCACCACCGAGTACCGCCACGGAACCGAATCATCCGC CGATCTCCTTCGGAGCATCAAGCGAGACGTTCCCCATTACATTTGGGGACTTCAACGAAGGAGAAATCGAAAGCTTGTCTTCTGAGCTAC TAACTTTCGGAGACTTCTTACCAGGAGAAGTGGATGACTTGACAGACAGCGACTGGTCCACGTGCTCAGACACGGACGACGAGTTAAG ACTAGACAGGGCAGGTGGGTATATATTCTCGTCGGACACCGGTCCAGGTCATTTACAACAGAAGTCAGTACGCCAGTCAGTGCTGCCGG TGAACACCCTGGAGGAAGTCCACGAGGAGAAGTGTTACCCACCTAAGCTGGATGAAGCAAAGGAGCAACTATTACTTAAGAAACTCCA GGAGAGTGCATCCATGGCCAACAGAAGCAGGTATCAGTCGCGCAAAGTAGAAAACATGAAAGCAGCAATCATCCAGAGACTAAAGAG AGGCTGTAGACTATACTTAATGTCAGAGACCCCAAAAGTCCCTACTTACCGGACTACATATCCGGCGCCTGTGTACTCGCCTCCGATCAA CGTCCGATTGTCCAATCCCGAGTCCGCAGTGGCAGCATGCAATGAGTTCTTAGCTAGAAACTATCCAACTGTCTCATCATACCAAATTAC CGACGAGTATGATGCATATCTAGACATGGTGGACGGGTCGGAGAGTTGCCTGGACCGAGCGACATTCAATCCGTCAAAACTCAGGAGC TACCCGAAACAGCACGCTTACCACGCGCCCTCCATCAGAAGCGCTGTACCGTCCCCATTCCAGAACACACTACAGAATGTACTGGCAGCA GCCACGAAAAGAAACTGCAACGTCACACAGATGAGGGAATTACCCACTTTGGACTCAGCAGTATTCAACGTGGAGTGTTTCAAAAAATT CGCATGCAACCAAGAATACTGGGAAGAATTTGCTGCCAGCCCTATTAGGATAACAACTGAGAATTTAGCAACCTATGTTACTAAACTAAA AGGGCCAAAAGCAGCAGCGCTATTCGCAAAAACCCATAATCTACTGCCACTACAGGAAGTACCAATGGATAGGTTCACAGTAGATATGA AAAGGGACGTAAAGGTGACTCCTGGTACAAAGCATACAGAGGAAAGACCTAAGGTGCAGGTTATACAGGCGGCTGAACCCTTGGCGA CAGCATACCTATGTGGGATTCACAGAGAGCTGGTTAGGAGGCTGAACGCCGTCCTCCTACCCAATGTACATACACTATTTGACATGTCTG CCGAGGATTTCGATGCCATCATAGCCGCACACTTTAAGCCAGGAGACACTGTTTTGGAAACGGACATAGCCTCCTTTGATAAGAGCCAA GATGATTCACTTGCGCTTACTGCTTTGATGCTGTTAGAGGATTTAGGGGTGGATCACTCCCTGCTGGACTTGATAGAGGCTGCTTTCGGA GAGATTTCCAGCTGTCACCTACCGACAGGTACGCGCTTCAAGTTCGGCGCCATGATGAAATCAGGTATGTTCCTAACTCTGTTCGTCAAC ACATTGTTAAACATCACCATCGCCAGCCGAGTGCTGGAAGATCGTCTGACAAAATCCGCGTGCGCGGCCTTCATCGGCGACGACAACAT AATACATGGAGTCGTCTCCGATGAATTGATGGCAGCCAGATGTGCCACTTGGATGAACATGGAAGTGAAGATCATAGATGCAGTTGTAT CCTTGAAAGCCCCTTACTTTTGTGGAGGGTTTATACTGCACGATACTGTGACAGGAACAGCTTGCAGAGTGGCAGACCCGCTAAAAAGG CTTTTTAAACTGGGCAAACCGCTAGCGGCAGGTGACGAACAAGATGAAGATAGAAGACGAGCGCTGGCTGACGAAGTGATCAGATGG CAACGAACAGGGCTAATTGATGAGCTGGAGAAAGCGGTATACTCTAGGTACGAAGTGCAGGGTATATCAGTTGTGGTAATGTCCATGG CCACCTTTG CAAGCTCCAG ATCCAACTTCG AG AAG CTCAG AG G ACCCGTCATAACTTTGTACGG CG GTCCTAAATAG GTACG CACTACAG CTACCTATTTTG CAG AAG CCG ACAG CAAGTATCTAAACACTAATCAG CTACAATG G AGTTCATCCCAACCCAAACTTTTTACAATAG GAG GTACCAGCCTCGACCCTGGACTCCGCGCCCTACTATCCAAGTCATCAGGCCCAGACCGCGCCCTCAGAGGCAAGCTGGGCAACTTGCCC AGCTGATCTCAGCAGTTAATAAACTGACAATGCGCGCGGTACCACAACAGAAGCCACGCAGGAATCGGAAGAATAAGAAGCAAAAGCA AAAACAACAGGCGCCACAAAACAACACAAATCAAAAGAAGCAGCCACCTAAAAAGAAACCGGCTCAAAAGAAAAAGAAGCCGGGCCG CAGAGAGAGGATGTGCATGAAAATCGAAAATGATTGTATTTTCGAAGTCAAGCACGAAGGTAAGGTAACAGGTTACGCGTGCCTGGTG GGGGACAAAGTAATGAAACCAGCACACGTAAAGGGGACCATCGATAACGCGGACCTGGCCAAACTGGCCTTTAAGCGGTCATCTAAGT ATGACCTTGAATGCGCGCAGATACCCGTGCACATGAAGTCCGACGCTTCGAAGTTCACCCATGAGAAACCGGAGGGGTACTACAACTG GCACCACGGAGCAGTACAGTACTCAGGAGGCCGGTTCACCATCCCTACAGGTGCTGGCAAACCAGGGGACAGCGGCAGACCGATCTTC GACAACAAGGGACGCGTGGTGGCCATAGTCTTAGGAGGAGCTAATGAAGGAGCCCGTACAGCCCTCTCGGTGGTGACCTGGAATAAA GACATTGTCACTAAAATCACCCCCGAGGGGGCCGAAGAGTGGAGTCTTGCCATCCCAGTTATGTGCCTGTTGGCAAACACCACGTTCCC CTG CTCCCAGCCCCCTTG CACG CCCTG CTG CTACG AAAAG GAACCGGAG G AAACCCTACG CATG CTTG AG G ACAACGTCATG AG ACCTG G GTACTATCAG CTG CTACAAG CATCCTTAACATGTTCTCCCCACCGCCAG CGACG CAG CACCAAG G ACAACTTCAATGTCTATAAAG CCA CAAGACCATACTTAGCTCACTGTCCCGACTGTGGAGAAGGGCACTCGTGCCATAGTCCCGTAGCACTAGAACGCATCAGAAATGAAGCG ACAGACGGGACGCTGAAAATCCAGGTCTCCTTGCAAATCGGAATAAAGACGGATGACAGCCACGATTGGACCAAGCTGCGTTATATGG ACAACCACATGCCAGCAGACGCAGAGAGGGCGGGGCTATTTGTAAGAACATCAGCACCGTGTACGATTACTGGAACAATGGGACACTT CATCCTGGCCCGATGTCCAAAAGGGGAAACTCTGACGGTGGGATTCACTGACAGTAGGAAGATTAGTCACTCATGTACGCACCCATTTC ACCACGACCCTCCTGTGATAGGTCGGGAAAAATTCCATTCCCGACCGCAGCACGGTAAAGAGCTACCTTGCAGCACGTACGTGCAGAGC ACCGCCGCAACTACCGAGGAGATAGAGGTACACATGCCCCCAGACACCCCTGATCGCACATTAATGTCACAACAGTCCGGCAACGTAAA GATCACAGTCAATGGCCAGACGGTGCGGTACAAGTGTAATTGCGGTGGCTCAAATGAAGGACTAACAACTACAGACAAAGTGATTAAT AACTGCAAGGTTGATCAATGTCATGCCGCGGTCACCAATCACAAAAAGTGGCAGTATAACTCCCCTCTGGTCCCGCGTAATGCTGAACTT GGGGACCGAAAAGGAAAAATTCACATCCCGTTTCCGCTGGCAAATGTAACATGCAGGGTGCCTAAAGCAAGGAACCCCACCGTGACGT ACGGGAAAAACCAAGTCATCATGCTACTGTATCCTGACCACCCAACACTCCTGTCCTACCGGAATATGGGAGAAGAACCAAACTATCAA GAAGAGTGGGTGATGCATAAGAAGGAAGTCGTGCTAACCGTGCCGACTGAAGGGCTCGAGGTCACGTGGGGCAACAACGAGCCGTAT AAGTATTGGCCGCAGTTATCTACAAACGGTACAGCCCATGGCCACCCGCATGAGATAATTCTGTATTATTATGAGCTGTACCCCACTATG ACTGTAGTAGTTGTGTCAGTGGCCACGTTCATACTCCTGTCGATGGTGGGTATGGCAGCGGGGATGTGCATGTGTGCACGACGCAGAT GCATCACACCGTATGAACTGACACCAGGAGCTACCGTCCCTTTCCTGCTTAGCCTAATATGCTGCATCAGAACAGCTAAAGCGGCCACAT ACCAAGAGGCTGCGATATACCTGTGGAACGAGCAGCAACCTTTGTTTTGGCTACAAGCCCTTATTCCGCTGGCAGCCCTGATTGTTCTAT GCAACTGTCTGAGACTCTTACCATGCTGCTGTAAAACGTTGGCTTTTTTAGCCGTAATGAGCGTCGGTGCCCACACTGTGAGCGCGTACG

[0116] AACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAAGACTCTAGTCAATAGACCTGGCTACAGCCCCATGGTATTGGAGATG Valneva Austria GmbH PAT071-PCT

[0117] GAACTACTGTCAGTCACTTTGGAGCCAACACTATCGCTTGATTACATCACGTGCGAGTACAAAACCGTCATCCCGTCTCCGTACGTGAAG TGCTGCGGTACAGCAGAGTGCAAGGACAAAAACCTACCTGACTACAGCTGTAAGGTCTTCACCGGCGTCTACCCATTTATGTGGGGCGG CGCCTACTGCTTCTGCGACGCTGAAAACACGCAGTTGAGCGAAGCACACGTGGAGAAGTCCGAATCATGCAAAACAGAATTTGCATCAG CATACAGGGCTCATACCGCATCTGCATCAGCTAAGCTCCGCGTCCTTTACCAAGGAAATAACATCACTGTAACTGCCTATGCAAACGGCG ACCATGCCGTCACAGTTAAGGACGCCAAATTCATTGTGGGGCCAATGTCTTCAGCCTGGACACCTTTCGACAACAAAATTGTGGTGTACA AAGGTGACGTCTATAACATGGACTACCCGCCCTTTGGCGCAGGAAGACCAGGACAATTTGGCGATATCCAAAGTCGCACACCTGAGAGT AAAGACGTCTATGCTAATACACAACTGGTACTGCAGAGACCGGCTGTGGGTACGGTACACGTGCCATACTCTCAGGCACCATCTGGCTT TAAGTATTGGCTAAAAGAACGCGGGGCGTCGCTGCAGCACACAGCACCATTTGGCTGCCAAATAGCAACAAACCCGGTAAGAGCGGTG AACTGCGCCGTAGGGAACATGCCCATCTCCATCGACATACCGGAAGCGGCCTTCACTAGGGTCGTCGACGCGCCCTCTTTAACGGACAT GTCGTGCGAGGTACCAGCCTGCACCCATTCCTCAGACTTTGGGGGCGTCGCCATTATTAAATATGCAGCCAGCAAGAAAGGCAAGTGTG CGGTGCATTCGATGACTAACGCCGTCACTATTCGGGAAGCTGAGATAGAAGTTGAAGGGAATTCTCAGCTGCAAATCTCTTTCTCGACG G CCTTAG CCAG CG CCG AATTCCG CGTACAAGTCTGTTCTACACAAGTACACTGTGCAG CCG AGTG CCACCCCCCG AAG G ACCACATAGT CAACTACCCGGCGTCACATACCACCCTCGGGGTCCAGGACATCTCCGCTACGGCGATGTCATGGGTGCAGAAGATCACGGGAGGTGTG G G ACTG GTTGTTG CTGTTG CCG CACTG ATTCTAATCGTG GTG CTATGCGTGTCGTTCAG CAGG CACTAACTTG ACAATTAAGTATG AAG G TATATGTGTCCCCTAAGAGACACACTGTACATAGCAAATAATCTATAGATCAAAGGGCTACGCAACCCCTGAATAGTAACAAAATACAAA ATCACTAAAAATTATAAAAACAGAAAAATACATAAATAGGTATACGTGTCCCCTAAGAGACACATTGTATGTAGGTGATAAGTATAGAT CAAAGGGCCGAATAACCCCTGAATAGTAACAAAATATGAAAATCAATAAAAATCATAAAATAGAAAAACCATAAACAGAAGTAGTTCAA AGGGCTATAAAACCCCTGAATAGTAACAAAACATAAAATTAATAAAAATCAAATGAATACCATAATTGGCAAACGGAAGAGATGTAGGT ACTTAAGCTTCCTAAAAGCAGCCGAACTCACTTTGAGAAGTAGGCATAGCATACCGAACTCTTCCACGATTCTCCGAACCCACAGGGACG TAGGAGATGTTATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAA

[0118] SEQ I D NO: 2

[0119] Amino acid sequence of E2 protein from LR2006_OPY1 Chikungunya virus strain— amino acids 339-742 from structural polyprotein GenBank Accession: ABD95938.1 (1-1248 aa) STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERI RNEATDGTLKIQVSLQIGI KTDDSHDWTKLRYMDNHMPADAERAGLFVRTSAPCTI TGTMGHFI LARCPKGETLTVGFTDSRKISHSCTHPFHH DPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSG NVKITVNGQTVRYKCNCGGSN EGLTTTDKVINNCKVDQCHAAVTNH KKWQYNSPLVPRNAELGDRKGKIH IPFPLANVTCRVPKARNPTVTY GKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGN NEPYKYWPQLSTNGTAHGHPH EII LYYYELYPTMTVV VVSVATFI LLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA

[0120] SEQ I D NO: 3

[0121] E168K variant of E2 protein from Chikungunya virus

[0122] STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERI RNEATDGTLKIQVSLQIGI KTDDSHDWTKLRYMDNHMPADAERAGLFVRTSAPCTI TGTMGHFI LARCPKGETLTVGFTDSRKISHSCTHPFHH DPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIKVHMPPDTPDHTLMSQQSG NVKITVNGQTVRYKCNCGGSN EGLTTTDKVINNCKVDQCHAAVTNH KKWQYNSPLVPRNAELGDRKGKIH IPFPLANVTCRVPKARNPTVTY GKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGN NEPYKYWPQLSTNGTAHGHPH EII LYYYELYPTMTVV VVSVATFI LLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA

[0123] SEQ I D NO: 4

[0124] G55R variant of E2 protein from Chikungunya virus

[0125] STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERI RNEATDGTLKIQVSLQI RIKTDDSH DWTKLRYMDN HMPADAERAGLFVRTSAPCTI TGTMGHFI LARCPKGETLTVGFTDSRKISHSCTHPFHH DPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSG NVKITVNGQTVRYKCNCGGSN EGLTTTDKVINNCKVDQCHAAVTNH KKWQYNSPLVPRNAELGDRKGKIH IPFPLANVTCRVPKARNPTVTY GKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGN NEPYKYWPQLSTNGTAHGHPH EII LYYYELYPTMTVV VVSVATFI LLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA

[0126] SEQ I D NO: 5

[0127] E247K variant of E2 protein from Chikungunya virus

[0128] STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERI RNEATDGTLKIQVSLQIGI KTDDSHDWTKLRYMDNHMPADAERAGLFVRTSAPCTI TGTMGHFI LARCPKGETLTVGFTDSRKISHSCTHPFHH DPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSG NVKITVNGQTVRYKCNCGGSN EGLTTTDKVINNCKVDQCHAAVTNH KKWQYNSPLVPRNAKLGDRKGKIH IPFPLANVTCRVPKARNPTVT YGKNQVI MLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEI ILYYYELYPTMTV VVVSVATFI LLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA

[0129] SEQ I D NO: 6

[0130] G82R variant of E2 protein from Chikungunya virus

[0131] STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERI RNEATDGTLKIQVSLQIGI KTDDSHDWTKLRYMDNHMPADAERARLFVRTSAPCTI TGTMGHFI LARCPKGETLTVGFTDSRKISHSCTHPFHH DPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSG NVKITVNGQTVRYKCNCGGSN EGLTTTDKVINNCKVDQCHAAVTNH KKWQYNSPLVPRNAELGDRKGKIH IPFPLANVTCRVPKARNPTVTY GKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGN NEPYKYWPQLSTNGTAHGHPH EII LYYYELYPTMTVV VVSVATFI LLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA Valneva Austria GmbH PAT071-PCT

[0132] SEQ ID NO: 7

[0133] H232Y variant of E2 protein from Chikungunya virus

[0134] STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHIVIPADAERAGLFVRTSAPCTI TGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLIVISQQSG NVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTNYKKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTY GKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVV VVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA

[0135] SEQ ID NO: 8

[0136] CHIKV A5nsP3 forward primer GCTTATAGAGGTGCCGCTGC

[0137] SEQ ID NO: 9

[0138] CHIKV A5nsP3 reverse primer CG ACAG GTACG GTG CTCATT

Claims

1. Valneva Austria GmbH PAT071-PCTCLAIMSWhat is claimed is:

1. A lyophilized attenuated CHIKV-A5nsP3 chikungunya virus pharmaceutical composition for multidose administration, wherein the composition is lyophilized from a liquid formulation comprising a) two or more effective doses of a CHIKV-A5nsP3 attenuated chikungunya virus; b) sucrose at a concentration of 3-10% (w / v); c) potassium phosphate at a concentration of 3-10 mM; d) sodium citrate at a concentration of 15-50 mM; e) MgCL at a concentration of 3-10 mM; f) D-sorbitol at a concentration of 0.3- 1.0% (w / v), g) L-methionine at a concentration of 3-20 mM; and h) recombinant human albumin (rHA) at a concentration of 0.003-0.02% (w / v), and wherein the pH of the formulation is 7.3±0.2; optionally wherein the multidose pharmaceutical composition is present in an individual vial that allows the withdrawal of two or more effective doses after reconstitution.

2. The multidose pharmaceutical composition according to claim 1, wherein said liquid formulation comprises a) two or more effective doses of a CHIKV-A5nsP3 attenuated chikungunya virus; b) sucrose at a concentration of 5% (w / v); c) potassium phosphate at a concentration of 5 mM; d) sodium citrate at a concentration of 25 mM; e) MgCL at a concentration of 5 mM; f) D-sorbitol at a concentration of 0.5% (w / v), g) L-methionine at a concentration of 10 mM; and h) recombinant human albumin at a concentration of 0.01% (w / v), and wherein the pH of the formulation is 7.3±0.2; preferably wherein the total volume of the liquid formulation per individual vial before lyophilization is 0.3 to 1.0 mL, more preferably 0.4 to 0.6 mL, most preferably about 0.5 mL.

3. The multidose pharmaceutical composition according to claim 1, wherein said liquid formulation comprises: a) two or more effective doses of a CHIKV-A5nsP3 attenuated chikungunya virus, b) 1.02 mg trisodium citrate dihydrate, c) 0.09 mg di-potassium hydrogen phosphate, d) 6.94 mg sucrose, e) 0.14 mg magnesium chloride hexahydrate, f) 0.69 mg D- sorbitol, g) 0.21 mg L-methionine, and h) 0.014 mg recombinant human albumin (rHA), wherein the pH of the formulation is 7.3±0.2, and wherein the total volume of the formulation per individual vial is 0.5 mL.

4. The multidose pharmaceutical composition according to any one of the preceding claims, wherein multidose is defined as comprising between two and twenty-five effective doses, preferably between three and ten effective doses, preferably three or more effective doses, four or more effective doses, five or more effective doses, six or more effective doses, seven orValneva Austria GmbH PAT071-PCT more effective doses, eight or more effective doses, nine or more effective doses, preferably five or ten effective doses, most preferably three effective doses.

5. The multidose pharmaceutical composition according to any one of the preceding claims, wherein said CHIKV-A5nsP3 virus comprises essentially an attenuated chikungunya virus of SEQ ID NO: 1, a variant with 99% sequence identity to SEQ ID NO: 1 also containing the 61aa deletion in the nsP3 protein as SEQ ID NO: 1 and / or combinations thereof.

6. The multidose pharmaceutical composition according to any one of the preceding claims, wherein said CHIKV-A5nsP3 virus is selected from a population that comprises substantially two variants, said two variants both expressing the El wild type amino acid sequence as encoded in the relevant part of nucleic acid sequence SEQ ID NO: 1 and wherein one variant expresses the wild type E2 structural protein as defined in SEQ ID NO: 2 and wherein the other variant expresses the E168K mutation in the E2 structural protein as defined in SEQ ID NO: 3.

7. The multidose pharmaceutical composition according to any one of the preceding claims, wherein said CHIKV-A5nsP3 virus is selected from a population that comprises substantially two variants, said two variants expressing E2 structural proteins as defined by the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 (with E168K), respectively.

8. The multidose pharmaceutical composition according to any one of the preceding claims, wherein said CHIKV-A5nsP3 virus is selected from a population that comprises one or more variants and wherein the variant has one or more mutations in E2 which mutations are shown in the group of variants encoding for an E2 amino acid sequence with E168K (SEQ ID NO: 3), G55R (SEQ ID NO: 4), E247K (SEQ ID NO: 5), G82R (SEQ ID NO: 6) and / or H232Y (SEQ ID NO: 7).

9. The multidose pharmaceutical composition according to any one of the preceding claims, wherein an effective dose of CHIKV-A5nsP3 virus is defined as a number of immunogenic CHIKV-A5nsP3 particles which, when administered to a human subject, is able to induce a protective immune response against chikungunya virus infection, preferably at least 102, at least 103, at least 104, at least 105, or at least 106CHIKV-A5nsP3 particles, preferably at least 103CHIKV-A5nsP3 particles which express an E2 structural protein as defined by the amino acid sequence of SEQ ID NO: 2, preferably between 103and 105, most preferably between about 103and 2xl04CHIKV-A5nsP3 particles which express an E2 structural protein as defined by the amino acid sequence of SEQ ID NO: 2.Valneva Austria GmbH PAT071-PCT10. The multidose pharmaceutical composition according to any one of the preceding claims, wherein said liquid formulation comprises between 4.5 and 6.0 logic TCID50 CHIKV- A5nsP3 / mL, preferably at least 5.0, preferably between 5.0 and 5.9, preferably between 5.0 and 5.8, preferably between 5.0 and 5.7, preferably between 5.0 and 5.6, preferably between 5.0 and 5.5, preferably between 5.1 and 5.4, most preferably between 5.2 to 5.3 logic TCID50 CHIKV- A5nsP3 / mL.

11. The multidose pharmaceutical composition according to any one of the preceding claims, wherein said composition is reconstituted with sterile water for injection (sWFI) or Phosphate Buffered Saline (PBS).

12. The multidose pharmaceutical composition according to any one of the preceding claims, wherein after reconstitution in a volume of 1.5 to 2.0 mL, preferably 1.6 to 1.9 mb, preferably1.7 to 1.9 mL, preferably about 1.8 mL, the formulation comprises between 3.5 and 6.0 logic TCID50 CHIKV-A5nsP3 / mL, preferably less than 5.0, preferably between 4.0 and 4.9, preferably between 4.2 and 4.9, preferably between 4.5 and 4.8, most preferably between 4.7 to4.8 or about 4.75 logw TCID50 CHIKV-A5nsP3 / mL.

13. The multidose pharmaceutical composition according to any one of the preceding claims, wherein said composition does not exceed potency losses of logic = [0.5] when stored at 5 ± 3 °C for up to 2 years.

14. The multidose pharmaceutical composition according to any one of the preceding claims, wherein the composition is stable for at least about 6 hours at room temperature after reconstitution of the composition.

15. The multidose pharmaceutical composition according to any one of the preceding claims, wherein the composition is stable for at least about 6 hours at room temperature after a first dose withdrawal.

16. The multidose pharmaceutical composition according to any one of claims 1 to 15, wherein the composition is for use during an outbreak.

17. A pharmaceutical composition for multidose administration, comprising a liquid formulation reconstituted from a lyophilized pharmaceutical composition, the reconstituted liquid formulation comprising a) two or more effective doses of a CHIKV-A5nsP3 attenuated chikungunya virus; b) sucrose at a concentration of 1-3% (w / v); c) potassium phosphate at aValneva Austria GmbH PAT071-PCT concentration of 1-3 mM; d) sodium citrate at a concentration of 5-15 mM; e) MgCL at a concentration of 1-3 mM; f) D-sorbitol at a concentration of 0.1-0.3% (w / v), g) L-methionine at a concentration of 1-7 mM; and h) recombinant human albumin at a concentration of 0.001- 0.007% (w / v), and wherein the pH of the formulation is 7.3±0.2.

18. A vial, syringe or container comprising the lyophilized pharmaceutical composition as defined in any one of claims 1 to 16, or the reconstituted liquid formulation as defined in claim 17; preferably wherein the vial, syringe or container is suitable for administration of two or more effective doses of the CHIKV-A5nsP3 attenuated chikungunya virus.

19. The vial, syringe or container according to claim 18, comprising a volume of 1.5 to 2.0 mb, preferably 1.6 to 1.9 mb, 1.7 to 1.9 mb, or about 1.8 mb of the reconstituted liquid formulation.

20. The vial, syringe or container according to claim 18 or 19, wherein the reconstituted liquid formulation comprises between 3.5 and 6.0 logic TCID50 CHIKV-A5nsP3 / mL, preferably less than 5.0, preferably between 4.0 and 4.9, preferably between 4.2 and 4.9, preferably between 4.5 and 4.8, most preferably between 4.7 to 4.8 or about 4.75 logic TCID50 CHIKV- A5nsP3 / mL.

21. A method for preparing a lyophilized attenuated CHIKV-A5nsP3 virus pharmaceutical composition for multidose administration, comprising:(i) cultivating a CHIKV-A5nsP3 virus on host cells and mixing the CHIKV-A5nsP3 virus with a concentrated stabilizing solution to form a virus bulk (i); and, optionally,(ii) dialyzing the virus bulk (i) to form a composition (ii); wherein said composition (ii) comprises a) two or more effective doses of a CHIKV-A5nsP3 attenuated chikungunya virus; b) sucrose at a concentration of 3-10%, preferably 5% (w / v); c) potassium phosphate at a concentration of 3-10 mM, preferably 5 mM; d) sodium citrate at a concentration of 15-50 mM, preferably 25 mM; e) MgCL at a concentration of 3-10 mM, preferably 5 mM; f) D-sorbitol at a concentration of 0.3- 1.0% (w / v), preferably 0.5% (w / v), g) L-methionine at a concentration of 3-20 mM, preferably 10 mM; and h) recombinant human albumin at a concentration of 0.003-0.02% (w / v), preferably 0.01% (w / v), and wherein the pH of the formulation is 7.3±0.2; and(iii) lyophilizing said virus bulk (i) or composition (ii), wherein said virus bulk (i) or composition (ii) comprises the two or more effective doses of CHIKV-A5nsP3 virus; preferably wherein said virus bulk (i) or composition (ii) comprises two or more effective doses of CHIKV-A5nsP3 virus per mL, more preferably two or more effective doses of CHIKV-A5nsP3 virus per 0.4 to 0.6 mL, more preferably 1Valneva Austria GmbH PAT071-PCT two or more effective doses of CHIKV-A5nsP3 virus per 0.5 mL, most preferably four, five or six or more effective doses of CHIKV-A5nsP3 virus per mL.

22. The method according to claim 21, wherein said CHIKV-A5nsP3 virus cultivated in step (i) is defined by the polynucleotide sequence of SEQ ID NO: 1.

23. The method according to claim 21 or claim 22, wherein said lyophilizing step (iii) results in the multidose pharmaceutical composition according to any one of claims 1 to 16.

24. The method according to any one of claims 21 to 23, wherein multidose is defined as between two and twenty-five doses, preferably between three and ten doses, preferably three or more doses, four or more doses, five or more doses, six or more doses, seven or more doses, eight or more doses, nine or more doses, preferably five or ten doses, most preferably three doses.

25. The method according to any one of claims 21 to 24, wherein an effective dose of CHIKV- A5nsP3 virus is defined as a number of immunogenic CHIKV-A5nsP3 particles which, when administered to a human subject, is able to induce a protective immune response against chikungunya virus infection, preferably at least 102, at least 103, at least 104, at least 105, or at least 106CHIKV-A5nsP3 particles, preferably at least 103CHIKV-A5nsP3 particles which express an E2 structural protein as defined by the amino acid sequence of SEQ ID NO: 2, preferably between 103and 105, most preferably between about 103and 2xl04CHIKV-A5nsP3 particles which express an E2 structural protein as defined by the amino acid sequence of SEQ ID NO: 2.

26. The method according to any one of claims 21 to 25, wherein the virus bulk (i) or composition (ii) is aliquoted into vials in a volume of less than or equal to 1.0 mL, preferably 0.3 to 1.0 mL, more preferably 0.4 to 0.6 mL, most preferably about 0.5 mL, before lyophilization step (iii).

27. The method according to any one of claims 21 to 26, wherein the reconstitution volume before administration is 1.5 to 2.0 mL, preferably 1.6 to 1.9 mL, 1.7 to 1.9 mL, or about 1.8 mL.

28. The method according to any one of claims 21 to 27, wherein the volume for administration to a subject is between 0.1 and 0.5 mL, preferably between 0.2 and 0.5 mL, most preferably about 0.5 mL.

29. The method according to any one of claims 21 to 28, wherein the virus bulk (i) or composition (ii) comprises between 4.5 and 6.0 logic TCID50 CHIKV-A5nsP3 / mL, preferably at least 5.0,Valneva Austria GmbH PAT071-PCT preferably between 5.0 and 5.9, preferably between 5.0 and 5.8, preferably between 5.0 and 5.7, preferably between 5.0 and 5.6, preferably between 5.0 and 5.5, preferably between 5.1 and 5.4, most preferably between 5.2 to 5.3 logic TCID50 CHIKV-A5nsP3 / mL.

30. The multidose pharmaceutical composition according to any one of claims 1 to 17 for use in a method of vaccinating two or more human subjects to stimulate a protective immune response against chikungunya virus in said subjects.

31. The multidose pharmaceutical composition according to any one of claims 1 to 17, wherein said composition is a vaccine.

32. A kit for preparing and / or administering a multidose composition or vaccine wherein the kit comprises the following components: (a) the lyophilized multidose composition according to any one of claims 1 to 16; and (b) at least one sterile aqueous component (b) for reconstituting component (a).

33. The kit according to claim 32, wherein the sterile aqueous component (b) comprises a phosphate buffered saline (PBS) or sterile water for injection (sWFI).

34. The kit according to claim 32 or 33, wherein the kit comprises at least one means for combining component (a) and component (b) to obtain the multidose composition or vaccine.

35. The kit according to any one of claims 32 to 34, wherein the kit comprises at least one means for administering the multidose composition or vaccine to a subject or subjects in need thereof.

36. The pharmaceutical composition for multidose administration according to any one of claims 1 to 17, or the kit according to any one of claims 32 to 35, for use as a medicament.

37. The pharmaceutical composition for multidose administration according to any one of claims 1 to 17, or the kit according to any one of claims 32 to 35, for use in the treatment or prevention of an infection with chikungunya virus or of a disease related to such an infection.

38. A method for the treatment or prevention of infection and disease caused by chikungunya virus in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 17.Valneva Austria GmbH PAT071-PCT39. Use of the pharmaceutical composition according to any one of claims 1 to 17 or the kit according to any one of claims 32 to 35, for the treatment or prevention of an infection or disease caused by chikungunya virus.

Images