Method for producing a pharmaceutical composition comprising immunogenic chikungunya virus CHIKV-DELTA 5NSP3

Abstract

To provide a method for producing an immunogenic live attenuated Chikungunya virus (CHIKV), as well as pharmaceutical compositions comprising the same.SOLUTION: The present invention provides a method including the step of growing CHIKV-Δ5nsP3 in a host cell so as to minimize the existence of mutations that reduce Immunogenicity of CHIKV-Δ5nsP3 virus including a deletion mutation in the non-structural protein 3.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for producing an immunogenic live attenuated chikungunya virus, as well as to pharmaceutical compositions containing the same. [Background technology]

[0002] Chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus of the Alphavirus genus in the Togaviridae family. Chikungunya virus disease is primarily an epidemic disease with a high incidence rate. The virus is transmitted to humans via mosquito vectors and causes fever, rash, fatigue, and severe polyarthralgia. Except for rare cases involving CNS infection, which have a mortality rate of 10% to 30%, CHIKV infections generally resolve spontaneously and are not usually fatal. Infants under 1 year of age and adults over 65 years of age are particularly at risk for CHIKV CNS disease, with infection rates 25 and 6 times higher than those of the general population, respectively. The rate of permanent disability in children after CHIKV encephalitis is estimated to be 30 to 45 percent (Gerardin P, et al. Chikungunya virus-associated encephalitis: A cohort study on La Reunion Island, 2005-2009 (2016) Neurology 86:1-9). Additionally, approximately 30 percent of all chikungunya patients experience joint pain for months or years after recovery. In some cases, neurological, renal, cardiac, respiratory, or liver complications may occur.

[0003] Currently, no vaccines or medications are available to prevent or treat chikungunya virus disease. While past outbreaks have occurred primarily in Africa, the geographic range of the East-Central-Southern African (ECSA) genotype has recently expanded, resulting in outbreaks in India, Asia, and even temperate Europe (Weaver, S., Arrival of Chikungunya Virus in the New World: Prospects for Spread and Impact on Public Health (2014) PLOS Neglected Tropical Diseases 8(6):e2921). CHIKV has been repeatedly introduced into the Americas since 1995, but natural transmission was not reported in the Caribbean until 2013. By 2015, the epidemic had spread to the mainland, resulting in over 1 million suspected cases in 27 countries in the Americas (Pan-American Health Organization (2015) Number of Cumulative Cases of Chikungunya Fever in the Americas). Further epidemics may be partially fueled by the spread of CHIKV mosquito vectors to non-endemic areas, along with the ability of CHIKV to adapt to local mosquito species (Vega-Rua A, et al., Chikungunya Virus Transmission Potential by Local Aedes Mosquitoes in the Americas and Europe (2015) PLOS Neglected Tropical Diseases DOI:10.1371 / journal.pntd.0003780). The high infectivity of Chikungunya virus, its geographical spread, and the potential for long-term complications highlight the need to develop preventative measures, such as a vaccine.

[0004] Vaccines against Chikungunya virus can include live-attenuated CHIKV particles, i.e., live CHIKV particles that have been modified to reduce pathogenicity but still maintain immunogenicity. One example of an attenuated CHIKV contains a deletion mutation in nonstructural protein 3 (CHIKV-Δ5nsP3; see Figure 2). CHIKV-Δ5nsP3 has been shown to confer protective immunity in mice (Hallengard D, et al. Novel attenuated Chikungunya vaccine candidates elicit protective immunity in C57Bl / 6 mice (2014) J. Virol. 88:2858-2866) and non-human primates (Roques P, et al. Attenuated and vectored vaccines protect non-human primates against Chikungunya virus (2017) J. Clin. Invest. Insight 2(6):e83527). These preliminary in vivo studies in mice and non-human primates were performed with CHIKV-Δ5nsP3 virus produced in BHK-21 cells, a cell type generally unfavorable for the production of human vaccines. Therefore, as disclosed herein, it will be necessary to adapt CHIKV-Δ5nsP3 virus production to a more suitable cell culture platform. Such adaptation is not a trivial process; for example, it is known in the art that viral adaptation to specific host cells can result in mutations that alter the surface charge of viral particles. Such acquired mutations serve to attenuate the virus; indeed, serial passaging has been used to develop such attenuated viral particles for use in many vaccines against the virus.With regard to CHIKV, it has been shown that repeated in vitro passage of pathogenic wild-type chikungunya virus can lead to specific point mutations, resulting in partial or complete attenuation of the virus (Gardner CL, et al., Deliberate Attenuation of Chikungunya Virus by Adaptation to Heparan Sulfate-Dependent Infectivity: A Model for Rational Arboviral Vaccine Design (2014) PLOS Neglected Tropical Diseases 8(2):e2719).

[0005] Surprisingly, it has now been found that specific point mutations that result in loss of immunogenicity occur early during the cell substrate adaptation process of attenuated CHIKV-Δ5nsP3, making control or reduction of these point mutations an essential consideration for successful production of vaccine candidates. Accordingly, the present invention provides a process with well-defined parameters for the propagation of CHIKV-Δ5nsP3 vaccine candidates, allowing for the production of highly immunogenic viral particles while simultaneously achieving high production titers in cell culture suitable for industrial applications. Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure relates to pharmaceutical compositions comprising an immunogenic chikungunya virus in an amount sufficient to elicit a neutralizing immune response in a subject, i.e., an immune response that is protective against infection with and / or disease caused by the chikungunya virus. In particular, the present disclosure provides pharmaceutical compositions comprising live attenuated CHIKV-Δ5nsP3 particles, in which the proportion of such viral particles bearing immunogenicity-reducing mutations, particularly in the E2 protein, is minimized. The present disclosure also provides methods for producing pharmaceutical compositions comprising live attenuated CHIKV-Δ5nsP3, which minimize the presence of immunogenicity-reducing mutations in the viral genome, particularly in E168 and / or other E2 residues of the viral E2 protein and / or residues of other structural or non-structural CHIKV proteins. The present disclosure also provides pharmaceutical compositions comprising immunogenic attenuated live chikungunya viruses obtainable by the methods of the present invention. [Means for solving the problem]

[0007] Efforts are currently underway to develop a vaccine against chikungunya virus. One of the most advanced vaccine candidates provides a chimeric construct of the measles virus platform (see http: / / www.themisbio.com / # / news). The vaccine, currently in Phase 2 trials, is delivered in two doses (https: / / clinicaltrials.gov / ct2 / show / NCT02861586?term=themis&recrs=a&rank=2). A one-shot vaccine would be a distinct advantage in the field.

[0008] Thus, in one embodiment, it is an object of the present invention to provide a stable, well-defined, safe and effective pharmaceutical composition, such as a vaccine against Chikungunya virus, preferably an improved vaccine that confers protection with only one vaccination, i.e. a so-called "one-shot" vaccine on an industrial scale using a common cell substrate, a method for producing such a stable, well-defined, safe and effective vaccine, as well as methods and uses of said stable, well-defined, safe and effective vaccine.

[0009] The accompanying drawings are not intended to be drawn to scale. The drawings are merely illustrative and are not necessary to practice the present disclosure. For clarity, not every component is labeled in every drawing. The drawings are as follows: [Brief explanation of the drawings]

[0010] [Figure 1] Map of the pMX plasmid used for the full assembly of the CHIKV-Δ5nsP3 genome from synthesized fragments. For the full assembly of the CHIKV-Δ5nsP3 sequence, pMX plasmids carrying an ampicillin resistance cassette (pMA) were used. The pMX vector series is based on pUC-like cloning vectors but excludes unnecessary promoters (biosafety level 1). [Figure 2] Schematic diagram of the genome structure of CHIKV-Δ5nsP3. The Chikungunya virus genome encodes two polyproteins: nonstructural proteins 1 to 4 (nsP1 to nsP4) and structural proteins (C, E3, E2, 6K, and E1). Compared to the wild-type genome sequence, the CHIKV-Δ5nsP3 sequence contains a 183-bp deletion in the 3' portion of the sequence encoding nsP3 (amino acids 1656 to 1717 of the nsP1-4 polyprotein), resulting in a deletion of 60 amino acids in the nsP3 replicase protein (denoted Δ60aa). SP, subgenomic promoter; UTR, untranslated region. (Figure adapted from Hallengard D, et al., 2014, supra.) [Figure 3-1] Cloning strategy for assembly of the CHIKV-Δ5nsP3 genome in pMA. (A) Schematic design of synthetic polynucleotide fragments covering the entire genome of CHIKV-Δ5nsP3. (B) Cloning strategy for assembly of the CHIKV-Δ5nsP3 genome in pMA plasmid. 1. Cloning of CHIKV-Δ5nsP3 fragment 2 into pMA (pMA fragment 1) containing fragment 1 via EcoRI and PacI. 2. Assembly of fragment 4 and fragment 3 via ClaI and PacI. 3. Preparation of XhoI- and PacI-digested fragment 5 in pMA for final full assembly. 4. Full assembly of the CHIKV-Δ5nsP3 genome in pMA by fusing AgeI / XhoI-digested fragment 3 and fragment 4, and XhoI / PacI-digested fragment 5 with AgeI / PacI-linearized pMA fragment 1 and fragment 2. The correct CHIKV-Δ5nsP3 genome assembly was verified by Sanger sequencing. [Figure 3-2] Cloning strategy for assembly of the CHIKV-Δ5nsP3 genome in pMA. (A) Schematic design of synthetic polynucleotide fragments covering the entire genome of CHIKV-Δ5nsP3. (B) Cloning strategy for assembly of the CHIKV-Δ5nsP3 genome in pMA plasmid. 1. Cloning of CHIKV-Δ5nsP3 fragment 2 into pMA (pMA fragment 1) containing fragment 1 via EcoRI and PacI. 2. Assembly of fragment 4 and fragment 3 via ClaI and PacI. 3. Preparation of XhoI- and PacI-digested fragment 5 in pMA for final full assembly. 4. Full assembly of the CHIKV-Δ5nsP3 genome in pMA by fusing AgeI / XhoI-digested fragment 3 and fragment 4, and XhoI / PacI-digested fragment 5 with AgeI / PacI-linearized pMA fragment 1 and fragment 2. The correct CHIKV-Δ5nsP3 genome assembly was verified by Sanger sequencing. [Figure 4-1]Yield, plaque size, and immunogenicity of CHIKV-Δ5nsP3 after passage in Vero cells. Virus was passaged in three parallel replicates (A, B, and C) starting from a common P0 (rescue) as detailed in Table 2. (A) Viral titers 24 h after infection of Vero cells from passages 0 to 16. The average titers of the three replicates (A, B, and C) are shown. (B) Relative titers of CHIKV-Δ5nsP3 from P0, P5, and P15 assessed by plaque assay. Vero cells were seeded at a density of 4 × 10 cells per well in 6-well plates in MEM supplemented with 5% FBS, 2 mM L-glutamine, and 1% antibiotic-antimycotic (Anti-Anti) and incubated overnight at 35°C and 5% CO2. The next day, culture supernatant was removed from Vero cells, and serial dilutions of CHIKV-Δ5nsP3 were added to the cells. After 1 hour of incubation at 35°C / 5% CO2, a 2% final concentration of methylcellulose overlay was added, and the cells were further incubated at 35°C / 5% CO2 for 3 days. Finally, plaques were counted after crystal violet staining (0.5% crystal violet in 5% formaldehyde) to assess viral titer (pfu / ml) and plaque morphology. (C) Immunogenicity of CHIKV-Δ5nsP3 P0, P5B, P8B, and P15C as assessed by neutralization of CHIKV-Δ5nsP3 (P0) in PRNT in Vero cells. Vero cells were seeded at a density of 3 × 105 in 12-well plates and incubated overnight at 35°C / 5% CO2. Groups of five C57Bl / 6 mice were immunized subcutaneously once with 10 TCID of each CHIKV-Δ5nsP3 passage. 10 TCID of P0 CHIKV-Δ5nsP3 (virus rescue) was used as a positive control. Four-fold serial dilutions of the day 21 serum pool, ranging from 1:20 to 1:327680, were mixed with 560 pfu / ml of CHIKV-Δ5nsP3 (P0) and incubated for 1 hour. The CHIKV-Δ5nsP3 / neutralization mixture was then added to Vero cells, and the plates were incubated for 2 hours at 35°C / 5% CO2. This step was followed by a 2% methylcellulose overlay, and the plates were incubated for approximately 60 hours at 35°C / 5% CO2.After removing the overlay, cells were stained with crystal violet / 5% formaldehyde and plaques were counted. [Figure 4-2]Yield, plaque size, and immunogenicity of CHIKV-Δ5nsP3 after passage in Vero cells. Virus was passaged in three parallel replicates (A, B, and C) starting from a common P0 (rescue) as detailed in Table 2. (A) Viral titers 24 h after infection of Vero cells from passages 0 to 16. The average titers of the three replicates (A, B, and C) are shown. (B) Relative titers of CHIKV-Δ5nsP3 from P0, P5, and P15 assessed by plaque assay. Vero cells were seeded at a density of 4 × 10 cells per well in 6-well plates in MEM supplemented with 5% FBS, 2 mM L-glutamine, and 1% antibiotic-antimycotic (Anti-Anti) and incubated overnight at 35°C and 5% CO2. The next day, culture supernatant was removed from Vero cells, and serial dilutions of CHIKV-Δ5nsP3 were added to the cells. After 1 hour of incubation at 35°C / 5% CO2, a 2% final concentration of methylcellulose overlay was added, and the cells were further incubated at 35°C / 5% CO2 for 3 days. Finally, plaques were counted after crystal violet staining (0.5% crystal violet in 5% formaldehyde) to assess viral titer (pfu / ml) and plaque morphology. (C) Immunogenicity of CHIKV-Δ5nsP3 P0, P5B, P8B, and P15C as assessed by neutralization of CHIKV-Δ5nsP3 (P0) in PRNT in Vero cells. Vero cells were seeded at a density of 3 × 105 in 12-well plates and incubated overnight at 35°C / 5% CO2. Groups of five C57Bl / 6 mice were immunized subcutaneously once with 10 TCID of each CHIKV-Δ5nsP3 passage. 10 TCID of P0 CHIKV-Δ5nsP3 (virus rescue) was used as a positive control. Four-fold serial dilutions of the day 21 serum pool, ranging from 1:20 to 1:327680, were mixed with 560 pfu / ml of CHIKV-Δ5nsP3 (P0) and incubated for 1 hour. The CHIKV-Δ5nsP3 / neutralization mixture was then added to Vero cells, and the plates were incubated for 2 hours at 35°C / 5% CO2. This step was followed by a 2% methylcellulose overlay, and the plates were incubated for approximately 60 hours at 35°C / 5% CO2.After removing the overlay, cells were stained with crystal violet / 5% formaldehyde and plaques were counted. [Figure 5] Effect of a controlled MOI (0.01) during CHIKV-Δ5nsP3 passaging on immunogenicity. (A) Schematic diagram of CHIKV-Δ5nsP3 passaging in Vero cells under uncontrolled and controlled conditions. CHIKV-Δ5nsP3 P0 was passaged in Vero cells under uncontrolled conditions at various MOIs up to P3B (outlined in Table 2; replicate B). P3B was used as the starting material, and a controlled infection process was performed with all subsequent infections at a defined MOI of 0.01 to generate one P4 passage, two P5 passages, and one P6 passage for analysis of immunogenicity in mice. (B) P0 (○), P2B (□), and P5#1 (●) were assessed by neutralization of CHIKV-Δ5nsP3 (P2) in PRNT in Vero cells.

number

[0011] During the industrialization of the CHIKV-Δ5nsP3 attenuated virus vaccine candidate, we observed that virus passaging in Vero cells resulted in higher viral titers with increasing passage numbers, but also a concomitant increase in sequence heterogeneity of the CHIKV-Δ5nsP3 viral genome. Specific point mutations arising during passage in Vero cells were found to be reproducible from batch to batch and appeared already in early passages on new cell substrates. Surprisingly, some of these mutations were observed to correlate with a significant loss or reduction in the neutralizing immunogenicity conferred by the CHIKV-Δ5nsP3 virus. Other reproducible mutations did not reduce immunogenicity and / or acted as "rescue" mutations for immunogenicity-reducing mutations. Although a correlation between low multiplicity of infection ("MOI") and the generation of increased sequence heterogeneity of CHIKV-Δ5nsP3 was identified, a high MOI is generally not suitable for industrial use due to the need for a single virus source over many years of production. Therefore, it was initially unclear whether culture conditions could be found that would allow the generation of immunogenic CHIKV-Δ5nsP3 particles with sufficient production yield for reproducible and reliable manufacturing (the objective of the present invention).

[0012] Provided herein are methods that control and minimize the immunogenicity-reducing mutations observed herein while still allowing for high production yields. Also provided herein are pharmaceutical compositions comprising an effective amount of an immunogenic Chikungunya virus together with a residual amount of a non-immunogenic mutant of the Chikungunya virus. In a preferred embodiment, the pharmaceutical composition is produced using a low MOI, such as an MOI of less than 0.1, e.g., 0.1 or 0.001, under controlled conditions (e.g., reduced passage number after rescue, optimized temperature and host cell confluency) to minimize the amount of the non-immunogenic mutant(s) of the Chikungunya virus described herein. In some embodiments, the viral particle is a live virus, a chimeric virus, an attenuated live virus, a modified live virus, or a recombinant live virus. In one embodiment, the viral particles of the present invention may optionally be inactivated. In some embodiments, the viral particle is an attenuated form of the viral particle. For example, the virus may have reduced infectivity, virulence, and / or replication in a host compared to a wild-type virus. In some embodiments, the virus is a mutant or modified virus, e.g., the viral nucleic acid may contain at least one mutation, such as a substitution or deletion, compared to the wild-type virus. In some embodiments, the virus is a live recombinant virus, meaning a virus that has been produced recombinantly and may contain nucleic acid sequences from different sources. In some aspects, the wild-type chikungunya virus is inactivated. In a preferred embodiment, the virus is inactivated with formaldehyde.

[0013] In one embodiment, the immunogenic chikungunya virus is a live-attenuated virus. In a preferred embodiment, the live-attenuated chikungunya virus is the protective CHIKV-Δ5nsP3 described by Hallengard D, et al. (supra), herein referred to as CHIKV-Δ5nsP3, and defined by the nucleic acid sequence of SEQ ID NO: 1. Briefly, the wild-type CHIKV genome possesses an 11-kb positive-sense, single-stranded RNA genome containing two open reading frames encoding nonstructural proteins (nsP1-nsP4) and structural proteins (C, E3, E2, 6K, and E1), respectively. The attenuated CHIK virus CHIKV-Δ5nsP3, based on the La Reunion CHIKV strain LR2006-OPY1, was constructed by replacing amino acid residues 1656 to 1717 of the P1234 polyprotein with a small linker (AA sequence AYRAAAG) in the hypervariable region of the nsP3 protein (see Figure 2). CHIKV-Δ5nsP3 has been shown to be infectious, highly immunogenic, and protective against challenge with wild-type CHIKV (Hallengard D, et al., supra, and Hallengard D, et al., Prime-Boost Immunization Strategies against Chikungunya Virus (2014) J. Virology, 88(22):13333-13343, supra, and Roques P, et al. 2017, supra). In one embodiment, the live attenuated chikungunya virus may be a variant of the CHIKV-Δ5nsP3 attenuated mutant virus. In a preferred embodiment, the live attenuated chikungunya virus provided herein comprises CHIKV-Δ5nsP3 encoded by the nucleic acid sequence defined by SEQ ID NO:1. As used herein, the term "CHIKV-Δ5nsP3" is used interchangeably with "CHIKV-Δ5nsP3 virus," "CHIKV-Δ5nsP3 particle," "CHIKV-Δ5nsP3 viral particle," or multiple versions thereof.

[0014] Provided herein is a pharmaceutical composition comprising an effective amount of CHIKV-Δ5nsP3. In one embodiment, an effective amount of immunogenic CHIKV-Δ5nsP3 virus is defined as an amount sufficient to induce neutralizing antibodies against Chikungunya virus. In a further embodiment, an effective amount of immunogenic CHIKV-Δ5nsP3 virus is defined as an amount that induces an immune response in a vaccinated subject that confers protective immunity against Chikungunya virus infection. In a preferred embodiment, an effective amount of CHIKV-Δ5nsP3 is at least 10 2 Pieces, at least 10 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 pcs, preferably at least 10 3 Immunogenic CHIKV-Δ5nsP3 particles are defined as CHIKV-Δ5nsP3 particles expressing an E2 structural protein defined by the polypeptide sequence of SEQ ID NO:2. In one aspect, the E2 structural protein contains one or more point mutations that do not affect, i.e., reduce, the immunogenicity of the virus. In one embodiment, the point mutations that do not affect the immunogenicity of the virus may be at amino acids 232 and / or 247 of the E2 protein, such as H232Y and / or E247K. In one embodiment, the E2 structural protein of CHIKV-Δ5nsP3 contains about 10 or fewer point mutations. In one embodiment, the E2 structural protein of CHIKV-Δ5nsP3 contains no more than 9, 8, 7, 6, 5, or 4 point mutations. In a preferred embodiment, the E2 structural protein of CHIKV-Δ5nsP3 contains no more than three point mutations, most preferably only one or two point mutations.

[0015] As defined herein, immunogenic CHIKV-Δ5nsP3 may be, for example, about 3×10 4 TCID 50When delivered at a dose of 0.05 mg / mL, the immunogenic CHIKV-Δ5nsP3 is capable of eliciting an effective immune response in vivo, i.e., an immune response resulting in the production of neutralizing antibodies sufficient to alleviate or prevent signs or symptoms of Chikungunya virus disease. In a preferred embodiment, the immunogenic CHIKV-Δ5nsP3 defined herein is a CHIKV-Δ5nsP3 that expresses an E2 structural protein according to the amino acid sequence provided by SEQ ID NO:2. In a further preferred embodiment, the immunogenic CHIKV-Δ5nsP3 defined herein is defined by the polynucleotide sequence of SEQ ID NO:1. Alternatively or additionally, the immunogenic CHIKV-Δ5nsP3 of the present invention has a neutralizing capacity in an immunized subject, i.e., elicits the production of antibodies with neutralization of Chikungunya virus in an in vitro assay of at least 50%, preferably at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% at a serum dilution of 1:80 or greater.

[0016] As defined herein, a non-immunogenic CHIKV-Δ5nsP3 is a CHIKV-Δ5nsP3 that elicits a level of neutralizing antibodies in a vaccinated subject that is insufficient to prevent signs or symptoms of Chikungunya virus disease. In a preferred embodiment, the non-immunogenic CHIKV-Δ5nsP3 is a CHIKV-Δ5nsP3 that expresses an E2 structural protein having at least one amino acid substitution, particularly an amino acid substitution in the E2 structural protein, particularly an E168K and / or G55R substitution, particularly an E168K substitution. A non-immunogenic CHIKV-Δ5nsP3 is further defined as one that elicits antibodies in an immunized subject that exhibit a reduced ability to neutralize infection of cells by Chikungunya virus (wild-type or attenuated) in in vitro assays. In particular, non-immunogenic CHIKV-Δ5nsP3 is defined as one that elicits a level of neutralizing antibodies in immunized subjects that results in neutralization of Chikungunya virus in an in vitro neutralization assay of less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, and especially less than 10% at serum dilutions of 1:80 or greater.

[0017] In a further embodiment, an effective amount of CHIKV-Δ5nsP3 is defined as an amount sufficient to induce neutralizing antibodies against wild-type Chikungunya virus. In one embodiment, the pharmaceutical composition is a two-shot pharmaceutical composition. In a preferred embodiment, the pharmaceutical composition is a one-shot pharmaceutical composition. In a preferred embodiment, the pharmaceutical composition comprises at least 10 nucleotides, with or without point mutations, particularly point mutations that reduce immunogenicity, contained in the total pool of particles. 2 Pieces, at least 10 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 pieces, preferably about 10 3 ~10 pieces 5 total CHIKV-Δ5nsP3 viral particles, specifically approximately 10 3 Pieces or 10 4 In a preferred embodiment, the pharmaceutical composition comprises a detectable amount of non-immunogenic CHIKV-Δ5nsP3 as defined herein, preferably non-immunogenic CHIKV-Δ5nsP3 having at least one point mutation compared to the wild-type E2 protein defined by SEQ ID NO:2.

[0018] In a preferred embodiment, the pharmaceutical composition comprises CHIKV-Δ5nsP3 and contains an increased amount of non-immunogenic mutant(s) of CHIKV-Δ5nsP3 compared to a vaccine composition comprising CHIKV-Δ5nsP3 produced in BHK-21 cells, for example in the mouse study described above in Hallengard D, et al. 2014, but still contains sufficient immunogenic particles of CHIKV-Δ5nsP3 to confer protective immunity in the vaccinated subject. For example, the pharmaceutical composition may comprise: (i) CHIKV-Δ5nsP3 expressing an E2 structural protein defined by the polypeptide sequence of SEQ ID NO: 2 in an amount sufficient to confer protective immunity in a vaccinated subject; (ii) CHIKV-Δ5nsP3 having an increased amount of at least one mutation in the E2 structural protein compared to a vaccine composition containing CHIKV-Δ5nsP3 produced in BHK-21 used in the mouse study described above in Hallengard D, et al. 2014; and (iii) optionally, a pharmaceutically acceptable excipient.

[0019] It has been demonstrated herein that production of CHIKV-Δ5nsP3 by five or more serial passages in Vero cells results in high levels of sequence heterogeneity, particularly in the E2 structural protein (see, e.g., Example 2, below). For example, E168K and / or G55R mutations in the E2 protein frequently appear by passage 5 (see, e.g., Table 3, below), both of which correlate with reduced immunogenicity. Thus, as described in Hallengard D, et al. 2014 supra, production of CHIKV-Δ5nsP3 using five or more passages in Vero cells may disadvantageously result in high levels of non-immunogenic mutants of CHIKV-Δ5nsP3 (e.g., E168K[E2]) in vaccine compositions. In contrast, it is demonstrated below that the sequence heterogeneity of the E2 structural protein after fewer than five passages was much lower (see, e.g., Example 3 - the E168K mutation was present after three passages, but at a frequency of only 18%).

[0020] Thus, in one aspect, a pharmaceutical composition comprises (i) CHIKV-Δ5nsP3 and (ii) optionally, a pharmaceutically acceptable excipient, wherein at least 30% of the CHIKV-Δ5nsP3 particles present in the composition express an E2 structural protein defined by the polypeptide sequence of SEQ ID NO: 2. In this embodiment, at least 30% of the CHIKV-Δ5nsP3 particles are non-mutant with respect to the E2 structural protein, i.e., express the E2 structural protein of SEQ ID NO: 2. In other words, the frequency of sequence heterogeneity (i.e., mutant CHIKV-Δ5nsP3 particles expressing at least one mutation in the E2 structural protein of SEQ ID NO: 2) is 70% or less. Unless otherwise specified, references to "CHIKV-Δ5nsP3" or "CHIKV-Δ5nsP3 particles" in general are intended to encompass both non-mutant and mutant forms of CHIKV-Δ5nsP3, i.e., CHIKV-Δ5nsP3 expressing the E2 structural protein of SEQ ID NO:2, and CHIKV-Δ5nsP3 expressing an E2 structural protein with one or more mutations in SEQ ID NO:2. In one embodiment, the E2 structural protein of CHIKV-Δ5nsP3 contains no more than about 10 point mutations. In one embodiment, the E2 structural protein of CHIKV-Δ5nsP3 contains no more than 9, 8, 7, 6, 5, or 4 point mutations. In a preferred embodiment, the E2 structural protein of CHIKV-Δ5nsP3 contains no more than 3 point mutations, most preferably only 1 or 2 point mutations.

[0021] In a preferred embodiment, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the CHIKV-Δ5nsP3 particles present in the composition are non-mutant, i.e., express the E2 structural protein defined by the polypeptide sequence of SEQ ID NO:2.

[0022] In one aspect, a pharmaceutical composition comprises (i) CHIKV-Δ5nsP3 and (ii) optionally a pharmaceutically acceptable excipient, wherein less than 70% of the CHIKV-Δ5nsP3 particles present in the composition express an E2 structural protein having one or more mutations with respect to the polypeptide sequence of SEQ ID NO: 2.

[0023] In one aspect, a pharmaceutical composition comprises (i) CHIKV-Δ5nsP3 and (ii) optionally a pharmaceutically acceptable excipient, wherein less than 70% of the CHIKV-Δ5nsP3 particles present in the composition express an E2 structural protein having the mutation E168K in the polypeptide sequence of SEQ ID NO: 2.

[0024] In a preferred embodiment, the mutation in the E2 structural protein (e.g., the E168K mutation) is present at a frequency of 70% or less, e.g., less than 70% of all CHIKV-Δ5nsP3 particles contain one or more mutations (or the E168K mutation), and 30% or more of all CHIKV-Δ5nsP3 particles express a non-mutated E2 structural protein or an E2 structural protein that does not contain the E168K mutation.

[0025] In a preferred embodiment, less than 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the CHIKV-Δ5nsP3 particles present in the composition express an E2 structural protein having one or more mutations (e.g., E168K) with respect to the polypeptide sequence of SEQ ID NO:2. For example, the composition may contain 1% to 70%, 1% to 50%, 1% to 30%, 1% to 20%, 5% to 70%, 5% to 50%, 5% to 30%, 5% to 20%, 10% to 70%, 10% to 50%, 10% to 30%, or 10% to 20% mutant particles (i.e., CHIKV-Δ5nsP3 particles expressing an E2 structural protein having one or more mutations (e.g., E168K) with respect to the polypeptide sequence of SEQ ID NO: 2) relative to the total number of CHIKV-Δ5nsP3 particles (mutant and non-mutant) present in the composition. In one embodiment, CHIKV-Δ5nsP3 particles expressing an E2 structural protein having an E168K mutation further comprise a mutation that mitigates the loss of immunogenicity conferred by the E168K mutation. In one embodiment, the mutation is in the nsP1 protein, specifically at residue A38. In a preferred embodiment, CHIKV-Δ5nsP3 particles expressing an E2 structural protein with an E168K mutation also express an nsP1 with an A38S mutation.

[0026] Also provided herein is a method for producing a pharmaceutical composition of the present invention, comprising: 1) propagating a CHIKV-Δ5nsP3 virus in a cell line; and 2) minimizing the presence of mutations that reduce the immunogenicity of the CHIKV-Δ5nsP3 virus. In one embodiment, the immunogenic CHIKV-Δ5nsP3 virus is propagated in a cell line selected from the group consisting of EB66, Vero, Vero-αHis, HeLa, HeLa-S3, 293, PC12, CHO, 3T3, PerC6, MDSK, chicken embryo fibroblast, duck, and diploid avian cell lines. In some embodiments, the cell line is a duck cell line. In some embodiments, the cell line is a diploid avian cell line. In some embodiments, the cell line is an EB66 cell line. In a preferred embodiment, the cell line is a Vero cell line.

[0027] In one embodiment, the presence of immunogenicity-reducing mutations is minimized by passage of CHIKV-Δ5nsP3 less than five times, preferably less than four times, preferably less than three times, preferably less than two times, more preferably only once, and most preferably at most three times. As used herein, passage number refers to the number of in vitro passages after virus rescue (P0). In a preferred embodiment, the virus is passaged in Vero cells. In one aspect, the virus is grown at an optimal temperature. In a preferred embodiment, the optimal temperature is about 28°C to 37°C, preferably about 35°C.

[0028] In one embodiment, host cell cultures are infected with CHIKV-Δ5nsP3 at an optimal MOI. In one aspect, the optimal MOI is defined as an MOI low enough to avoid the need for excessive amounts of working virus seed bank culture, yet high enough to minimize the immunogenicity-reducing mutations described herein. In a preferred embodiment, the optimized MOI is less than 0.1, preferably about 0.1-0.001, more preferably about 0.09-0.0011, even more preferably about 0.05-0.005, and most preferably about 0.01. In one embodiment, the confluency of host cells is assessed prior to infection. In one aspect, the confluency of host cells is about 20%-90%, preferably about 30%-75%, more preferably about 40%-60%, and particularly about 50%-60%. In one embodiment, the cell culture is infected at an optimal time point after seeding the host cells, i.e., between 2 and 5 days after seeding the host cells, preferably at about day 4 after seeding the host cells. In one embodiment, the viral particles are harvested between 1 and 6 days after infection of the host cells, preferably between 1 and 4 days, preferably 1 or 2 days after infection of the host cells, preferably both 1 and 2 days after infection of the host cells.

[0029] In one aspect, the immunogenicity-reducing mutation is a point mutation at any position within the genome of CHIKV-Δ5nsP3 defined by the polynucleotide sequence of SEQ ID NO: 1. In one embodiment, the immunogenicity-reducing mutation is present in the genome at a position other than the E2 protein. In a preferred embodiment, the immunogenicity-reducing mutation is located in the E2 protein, preferably at amino acid residues 55 and / or 168, e.g., the G55R and / or E168K mutations, particularly E168K. In some embodiments, the immunogenicity-reducing mutations described herein are mitigated or "rescued" by other mutations within the genome. In one embodiment, the E168K mitigating mutation is the A38S mutation in nonstructural protein 1 (nsP1).

[0030] In one embodiment, the frequency of the E168K mutation in the E2 protein of CHIKV-Δ5nsP3 is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, preferably less than 50% in the entire pool of CHIKV-Δ5nsP3 collected.

[0031] In one aspect, the present invention provides an immunogenic CHIKV-Δ5nsP3 obtainable by the process provided herein. In another aspect, the present invention provides a pharmaceutical composition comprising an immunogenic CHIKV-Δ5nsP3 obtainable by the process provided herein.

[0032] An aspect of the present invention provides for the use of the methods described herein to manufacture a composition for immunization against Chikungunya virus infection. In a preferred embodiment, the composition is a vaccine. In one embodiment, the vaccine is administered to a subject once, twice, or more than twice. In one aspect, CHIKV-Δ5nsP3 viral particles isolated from an immunized subject have a similar point mutation profile as the administered vaccine composition, particularly with respect to point mutations in the E2 structural protein. In one embodiment, the vaccine is administered once or twice. In a preferred embodiment, the vaccine is administered only once (e.g., a one-shot vaccine). In one aspect, booster vaccinations are optionally applied. In certain preferred aspects, the pharmaceutical composition is provided in lyophilized form.

[0033] Another aspect provides a composition comprising viral particles obtained by the methods described herein for treating and / or preventing Chikungunya virus infection. In one aspect, the composition is for use in a method of stimulating an immune response in a subject and / or a method of treating or preventing Chikungunya virus infection. The term "preventing" as used herein also means "protecting from." In one aspect, Chikungunya virus infection may be caused by a West African, East / Central / South African (ECSA) and / or Asian Chikungunya virus genotype.

[0034] Viral preparations produced using any of the methods described herein may be further subjected to additional processing steps, including additional filtration and / or lyophilization. Viral preparations may also be subjected to analysis of the purity of the preparation. For example, viral preparations may be evaluated for the presence of impurities and contaminants, such as host cell genomic DNA and / or host cell proteins. The purity of a viral preparation may be evaluated using any method known in the art, such as size exclusion chromatography (SEC), optical density at various wavelengths, protein gel electrophoresis (such as SDS-PAGE), Western blotting, ELISA, PCR, and / or qPCR.

[0035] In some embodiments, the virus preparation is evaluated for remaining impurities or contaminants. In some embodiments, the amount of remaining impurities or contaminants is compared to the amount of impurities or contaminants at an earlier stage of the purification process, such as immediately after virus harvest. In some embodiments, the relative reduction of impurities in the final virus preparation is between 60% and 95% compared to the impurities present at the earlier stage of the purification process. In some embodiments, the relative reduction of impurities in the final virus preparation is approximately 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. In some embodiments, the final virus preparation contains less than 5% impurities or contaminants. In some embodiments, the final virus preparation contains less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than 0.1% impurities. In preferred embodiments, the final virus preparation contains less than 1% impurities.

[0036] Any of the methods described herein can be used in the manufacture of a composition comprising a purified virus for administration to a subject. In some embodiments, the subject is a mammalian subject, such as a human or non-human animal, including livestock, pets, or companion animals. In some embodiments, the composition is administered to a subject in need of immunization against a virus or a virus similar to the virus preparation. In some embodiments, a virus preparation or composition comprising a virus purified using the methods described herein treats or prevents infection by a virus or a virus similar to the virus preparation. In a preferred embodiment, a virus preparation or composition comprising a virus purified using the methods described herein treats or prevents chikungunya virus infection, particularly chikungunya virus infection caused by West African, East / Central / South African (ECSA), and / or Asian chikungunya virus genotypes.

[0037] A CHIKV-Δ5nsP3 pharmaceutical composition or CHIKV-Δ5nsP3 virus purified using the methods described herein can be administered to a subject by any route known in the art. In some embodiments, the preparation or composition can be administered via a conventional route, such as parenterally or orally. As used herein, "parenteral" administration includes, but is not limited to, subcutaneous, intracutaneous, intradermal, intravenous, intramuscular, intraarticular, intraperitoneal, intrathecal, or by injection.

[0038] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural 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, the nomenclatures and techniques used in connection with biochemistry, enzymology, molecular and cell 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, unless otherwise indicated, are described in the various general and more specific references cited and discussed throughout this specification. [Table 1]

[0039] Example Example 1. Initial testing of CHIKV-Δ5nsP3 drug substance (DS) production Assembly of synthetic CHIKV-Δ5nsP3 genome The CHIKV-Δ5nsP3 viral genome was synthesized in five fragments at MWG Eurofins (Germany) and fully assembled in the standard cloning vector pMA plasmid (pMX vector with ampicillin resistance). The pMX vector backbone is shown in Figure 1. All cloning and plasmid preparation procedures were performed under TSE-free conditions using electrocompetent NEB10β E. coli cells. An overview of the cloning strategy used for the full assembly of the CHIKV-Δ5nsP3 genome in pMA is shown in Figure 3B. Briefly, the pMA plasmid (shown in Figure 3A) containing fragment 1 encompassing nsP1 and part of nsP2 was linearized by EcoRI / PacI restriction digestion, and fragment 2 encompassing parts of nsP2 and nsP3 was fused to fragment 1. In parallel, fragment 3 (encompassing nsP4 and C) was fused to fragment 4 (encompassing C and E2) via ClaI / PacI cloning. In the third cloning step, fragments 3 and 4 were cloned via AgeI / XhoI, and fragment 5 was cloned via XhoI / PacI into AgeI / PacI-linearized pMA, which already contained fragments 1 and 2. The cloning resulted in the pMA_CHIKV-Δ5nsP3 vector encoding CHIKV-Δ5nsP3, as verified by sequencing.

[0040] CHIKV-Δ5nsP3 rescue from Vero cells ("virus rescue") To produce CHIKV-Δ5nsP3 viral particles from the modified pMA_CHIKV-Δ5nsP3 vector on Vero cells (virus rescue), the pMA_CHIKV-Δ5nsP3 plasmid was linearized by NotI restriction digestion and subjected to in vitro transcription using Ambion's mMessage mMachine SP6 Kit (AM130). RNA integrity was confirmed by gel electrophoresis (not shown). In parallel, Vero cells were prepared for electroporation with viral RNA. Briefly, Vero cells were detached from cell culture flasks using TrypLESelect (Gibco) and washed twice with PBS. All centrifugation steps were performed at 300 g at room temperature. Viral RNA was collected at 8 × 10 ribosomal RNA in 800 μl PBS. 6 The RNA was mixed with 1000 Vero cells, and the Vero cell / RNA mixture was transferred to a 0.4 cm electroporation cuvette. Two pulses were performed at 850 V, 25 μF, and 200 ohms. After electroporation, the Vero cells were kept at room temperature for 10 minutes and finally resuspended in MEM / 5% FCS / 1% antibiotic-antimycotic (anti-anti) / 2 mM L-glutamine and incubated in a T75 flask at 35°C / 5% CO2 for 48 hours. The cell culture supernatant containing the rescued CHIKV-Δ5nsP3 (passage 0; P0) was collected and centrifuged at 3000 g for 10 minutes at 4°C. Virus titers were determined by plaque assay on Vero cells and TCID 50 The genomic structure of the resulting CHIKV-Δ5nsP3 viral vaccine candidate, also designated VLA1553, is shown in Figure 2.

[0041] CHIKV-Δ5nsP3 sequence validation To verify the viral genome sequence, viral nucleic acids were extracted from the harvested cell culture supernatant using the QIAamp MinElute Virus Spin Kit (QIAGEN #57704), and cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System (Life Technologies, catalog no. 18080-051) with random hexamers. PCR using Phusion High Fidelity Polymerase was performed with primers amplifying overlapping regions of the CHIKV-Δ5nsP3 genome, and the PCR products were subjected to Sanger sequencing at MWG Eurofins, Germany. The sequences of the primer pairs used for PCR and sequencing are listed in Table 1. [Table 2]

[0042] Passaging of CHIKV-Δ5nsP3 in Vero cells Following rescue of CHIKV-Δ5nsP3, Vero cells were infected and the virus was repeatedly passaged in triplicate (see Table 2). For passage, Vero cells were seeded into T150 flasks and grown to confluence (1–3 days). They were washed twice with 1x DPBS and then 20 mL of infection medium (serum-free EMEM) was added. The indicated amount of inoculum was added directly to the flask, and the cells were incubated at 35°C and 5% CO2 for 24 hours. For the first passage from virus rescue (P0), Vero cells were infected and passaged in triplicate (A, B, and C) at an MOI of 0.01. A 20 mL harvest was transferred to a 50 mL PP tube at 24 hours postinfection (pi), cell debris was removed by centrifugation (3000 g, 10 minutes), and the supernatant was transferred to a new 50 mL PP tube. A 49% (w / w) sucrose solution was added to a final concentration of 10% (w / w) and 1 mL aliquots of the stabilized harvest were stored at -70°C or below.

[0043] Subsequent infections were performed with harvests without sucrose. Infections were performed using various volumes of harvest, roughly calculated based on the cytopathic effect observed in previous passages and parallel replicates. The volume of harvest used for infection varied from 5 μL to 1 mL. One harvest was performed 24 hours post-infection. The MOI used for production of passages 2 through 16 was 100 TCID 50 Note that the MOI was determined retrospectively after the results of the previous study became available, resulting in a broad ("uncontrolled") range of MOIs throughout the experiment (see Table 2). This procedure was performed up to passage 16 in three parallel replicates (replicates A, B, and C) to allow systematic observation of various parameters during adaptation of CHIKV-Δ5nsP3 to Vero cells. During this experiment, the yield (TCID 50 / mL), infection dose, number of Vero cells per flask, Vero cell passage number, and cytopathic effect (CPE) were recorded. Multiplicity of infection (MOI) was calculated as the TCID 50 CHIKV-Δ5nsP3 passages were simultaneously assessed for plaque size in all three replicates. [Table 3] TIFF0007738032000005.tif92160

[0044] Trends observed during serial passaging under "uncontrolled" MOI conditions During adaptation of CHIKV-Δ5nsP3 to Vero cell passage, we observed a significant increase in the total CHIKV-Δ5nsP3 virus yield with increasing passage numbers in Vero cells, as shown in Table 2. As shown in Figure 4A, passages 6 and above resulted in an approximately 100-fold increase in titer compared to virus rescue (P0) and initial passages. A concomitant decrease in CHIKV-Δ5nsP3 plaque size was also observed (Figure 4B). The effect of in vitro passaging of wild-type chikungunya virus on plaque size in other cell lines has previously been described by Gardner CL, et al. (Gardner CL, et al., 2014, supra). Although high virus yields would be highly desirable for industrial production of inactivated viruses, we also observed herein that CHIKV-Δ5nsP3 decreased in immunogenicity with increasing passage numbers, as shown in Figure 4C. Briefly, to determine the immunogenicity of the different passages of CHIKV-Δ5nsP3 generated in Table 2 above, groups of five C57Bl / 6 mice were cultured for 10 min. 5 TCID 50 Each mouse was subcutaneously immunized once with CHIKV-Δ5nsP3 passage 5 (P5B), passage 8 (P8B), or passage P15 (P15C) at a dose of 10. 5 TCID 50 P0 (virus rescue) CHIKV-Δ5nsP3 was used as a positive control. Day 21 serum pools were evaluated for their ability to neutralize CHIKV-Δ5nsP3 (P0) in PRNT assays in Vero cells by testing four-fold serial serum dilutions ranging from 1:20 to 1:327680. As shown in Figure 4C, the immunogenicity of P5B CHIKV-Δ5nsP3 in mice showed a slightly shifted immunogenicity compared to unpassaged CHIKV-Δ5nsP3 (P0), whereas the P8B virus showed a significant decrease in immunogenicity comparable to the P15C virus. (The P15C virus was non-immunogenic and therefore served as a negative control in subsequent PRNT assays.)

[0045] Finally, the genetic stability of selected passages of CHIKV-Δ5nsP3 was tested by Sanger sequencing. Passaging CHIKV-Δ5nsP3 up to 16 times in Vero cells confirmed that the 60 amino acid deletion in the nsP3 gene, responsible for viral attenuation, was genetically stable and did not revert to wild-type virus, an important consideration for the safety of live-attenuated vaccines.

[0046] Trends observed during serial passage under controlled MOI conditions The use of a high MOI (e.g., greater than 0.1) is not useful for industrial-scale processes because it requires too much starting material. Therefore, we tested the use of a lower MOI (0.01) for three passages to determine the immunogenicity of the resulting passages. As shown in Figure 5A, three passages from replicate B in Table 2 were passaged under uncontrolled conditions with MOIs ranging from 0.01 to 2.23. From the third passage thus obtained (P3B), further passages were performed up to P6 using an MOI of 0.01 for each passage, shown in Figure 5A as "Control Condition with MOI 0.01." Under conditions using an MOI of 0.01, the resulting virus lost its immunogenicity very rapidly, as shown in Figure 5B. By the fifth passage, CHIKV-Δ5nsP3 became non-immunogenic. This result was in contrast to that seen with P5B in Figure 4C, which was obtained at a much higher MOI (see Table 2, replicate B) and remained immunogenic. This observation likely indicates that a lower MOI leads to more rapid selection during Vero cell passaging for mutations that affect the immunogenicity of CHIKV-Δ5nsP3.

[0047] Example 2. Defining sequence heterogeneity of CHIKV-Δ5nsP3 that influences immunogenicity Given the observed decrease / loss of immunogenicity (neutralizing antibody titers) and plaque size at higher passages of CHIKV-Δ5nsP3, it was interesting to analyze the possible sequence heterogeneity within the virus population at different passage numbers. Furthermore, it was interesting to analyze the sequences of individual plaques from the virus population. Unpassaged CHIKV-Δ5nsP3 (P0) did not show sequence heterogeneity based on Sanger sequencing. In general, with increasing passage number, an increase in sequence heterogeneity was observed for all three replicates (replicates A, B, and C, Table 3). When replicate C was passaged at the 8th passage (P8C), the virus population was still heterogeneous (sequence heterogeneity shown in Table 3), whereas the P15C passage showed a defined point mutation ( * The immunogenicity data shown in Figure 4C (Example 1 above) focused on P5B and P8B and demonstrated sequence heterogeneity of the CHIKV-Δ5nsP3 nonstructural proteins (nsPs) and envelope protein E2, as shown in Table 3 below. [Table 4]

[0048] Growth and sequencing of single CHIKV-Δ5nsP3 plaques To understand the effect of individual mutations on immunogenicity and consequently develop a controlled and reproducible production process for a highly immunogenic CHIKV-Δ5nsP3 vaccine, individual plaques of CHIKV-Δ5nsP3 isolates P5B and P8B were selected. Briefly, serial dilutions of P5B and P8B CHIKV-Δ5nsP3 were used to infect Vero cells in a plaque assay (described in Figure 4). After 72 h of incubation, single plaques of various morphologies were selected (small plaques were preferentially selected, as they began to appear after several passages in Vero cells) and then cultured on Vero cells (5 × 10 5) by reinfection. Different CHIKV-Δ5nsP3 samples derived from a single plaque were selected based on mutations in the E2 gene sequence before being propagated again in Vero cells. Clones P5B-02, P5B-03, P5B-04, P5B-07, P5B-11, P8B-01, and P8B-05 were propagated and purified for in vivo immunogenicity experiments. Each isolate was propagated in a single 850cm tube equipped with CellBIND surface. 2 Individual plaques were propagated in roller bottle-grown Vero cells. Upon reaching confluence, cells were washed twice with 100 mL D-PBS+Ca+Mg, followed by the addition of 100 mL infection medium (serum-free EMEM) containing an inoculum at an MOI of 0.01. The cells were then incubated at 35°C and 5% CO for 24 h. At 24 h postinfection (pi), 100 mL of the harvest was transferred to a 50 mL PP tube, and cell debris was removed by centrifugation followed by 0.2 μm filtration using a Steriflip® vacuum filtration device (Merck). Individual clarified virus harvests were first concentrated using an Amicon® Centrifugal Filter device, diafiltered with TBS buffer, and purified by protamine sulfate and Capto™ Core 700 treatment. The purified CHIKV-Δ5nsP3 clone was then used for subsequent studies.

[0049] The whole genome sequences of the propagated CHIKV-Δ5nsP3 samples, P5B+1 and P8B+1; i.e., P5B-02, P5B-03, P5B-04, P5B-07, P5B-11, P8B-01, and P8B-05, obtained from single plaques P5B and P8B, respectively, were evaluated by Sanger sequencing. The observed point mutations of individual plaques are summarized in Table 4, and the schematic genome sequences are shown in Figures 6B and 6D.

[0050] To assess the impact of specific point mutations on the immunogenicity of CHIKV-Δ5nsP3, day 19 mouse sera were generated from mice immunized with virus derived from individual plaques and analyzed by PRNT. 5 TCID 50 A single dose of CHIKV-Δ5nsP3 was administered subcutaneously to C57Bl / 6 mice (10 per treatment group), and pooled sera from day 19 were analyzed for virus neutralization capacity in four-fold serial dilutions ranging from 1:20 to 1:327680 by PRNT assay. Virus neutralized by PRNT corresponded to second-passage CHIKV-Δ5nsP3 (P2, 560 pfu / ml) and showed no sequence heterogeneity, making it identical to unpassaged CHIKV-Δ5nsP3 (P0). The neutralization mix (560 pfu / ml CHIKV-Δ5nsP3P2 and serial serum dilutions) was incubated at room temperature for 1 h, added to Vero cells, and then incubated for 2 h. Finally, a methylcellulose overlay (0.8%) was added, followed by 72 h of incubation. Plaque reading was performed after crystal violet staining (0.5% crystal violet in 5% formaldehyde). [Table 5]

[0051] As can be seen in Figure 6A, the immunogenicity of CHIKV-Δ5nsP3 P5B-11 and P5B-03, both of which contain single point mutations, E247K and H232Y, respectively, in the E2 protein (Figure 6B), is unaffected compared to that of CHIKV-Δ5nsP3 P0. The neutralizing ability of P0 serum was demonstrated in two independent mouse experiments (4415; P0#2 and 4399; P0#1), demonstrating an acceptable range of immunogenicity. On the other hand, P8B-05, which features three point mutations, G577W, G55R, and H232Y, in nsP2 and E2, respectively, is non-immunogenic in mice. The G55R mutation has been previously described by Gardner CL, et al. as a result of in vitro passaging of CHIKV. Viral particles are affected by an increased dependency on heparan sulfate binding in vitro, which leads to attenuation in vivo (Gardner CL, et al., 2014, supra).

[0052] As can be seen in Figure 6C, only one of the four CHIKV-Δ5nsP3 samples derived from a single plaque (P5B-02) was still immunogenic, characterized by two point mutations in nsP1 and E2, A38S and E168K, respectively (Figure 6D). Furthermore, P8B-01, which contains three point mutations, R470S in nsP3 and H99Y and E168K in the E2 protein, was non-immunogenic in mice and comparable to the negative control P15C. P5B-04 and P5B-07 each contained a single point mutation in E2, i.e., glutamic acid 168 was mutated to lysine (E168K), which directly affected immunogenicity by abolishing their ability to neutralize CHIKV-Δ5nsP3.

[0053] In summary, we observed that most of the mutations occurring during passage in Vero cells were in the E2 protein. Some of the point mutations identified in the E2 protein and / or other parts of the genome, particularly the H232Y[E2] and E247K[E2] mutations, did not substantially affect the immunogenicity of the virus. However, some of the other mutations identified in CHIKV-Δ5nsP3, particularly the frequently occurring E168K[E2] mutation, resulted in a loss of immunogenicity, whether alone or in combination with other mutations. An interesting exception was a mutant carrying both the E168K[E2] and A38S[nsP1] mutations, which maintained immunogenicity. This finding suggests that the A38S[nsP1] mutation has a mitigating effect on the reduced immunogenicity caused by the E168K[E2] mutation. Furthermore, isolates carrying the G55R / H232Y[E2] and G577W[nsP2] mutations also demonstrated poor immunogenicity, likely due primarily to the G55R mutation in E2, as the H232Y mutation alone had little effect (see P5B-03).

[0054] The E168K and G55R mutations in the chikungunya virus E2 protein have previously been described as increasing the positive surface charge, enhancing interactions with heparan sulfate and / or other glycosaminoglycans (GAGs), ultimately enhancing specific infectivity. In a wild-type CHIKV background, the mutations were shown to reduce plaque size due to reduced plate spreading mediated by binding to heparan sulfate. Furthermore, the mutations attenuated CHIKV in a mouse model of musculoskeletal disease (MSD), resulting in reduced organ spread and lower levels of viremia in mice (Gardner CL, et al., 2014, supra; Silva LA, et al., A single-amino-acid polymorphism in Chikungunya virus E2 glycoprotein influences glycosaminoglycan utilization (2014) J Virol.; 88(5):2385-97). The fact that the presence of the E168K and G55R mutations in wild-type CHIKV otherwise resulted in intermediate attenuation is consistent with the present disclosure regarding reduced plaque size or immunogenicity in vivo. However, it was unexpected that these two mutations in the attenuated CHIKV-Δ5nsP3 background resulted in the loss of immunogenicity in mice reported herein.

[0055] In addition, Sindbis virus (SINV; Klimstra WB, et al., Infection of neonatal mice with Sindbis virus results in a systemic inflammatory response syndrome (1999) J. Virol.; 73(12):10387-98; Klimstra WB, et al., The furin protease cleavage recognition sequence of Sindbis virus PE2 can mediate virion attachment to cell surface heparan sulfate (1999) J. Virol.; 73(8):6299-306; Byrnes and Griffin, Binding of Sindbis virus to cell surface heparan sulfate (1998) J. Virol.; 72(9):7349-56) and Ross River virus (RRV; Heil ML, et al., An amino acid substitution in the coding region of the E2 glycoprotein adapts Ross River virus to utilize heparan sulfate as an attachment site. It has been reported that, as a general phenomenon in other cell culture-passaged alphaviruses such as the virus moiety (2001) J. Virol.; 75(14):6303-9) and Semliki Forest virus (SFV; Smit JM, et al., Adaptation of alphaviruses to heparan sulfate: interaction of Sindbis and Semliki forest viruses with liposomes containing lipid-conjugated heparin (2002) J. Virol.; 76(20):10128-37), substitution of positively charged residues in E2 enhances heparan sulfate-dependent infectivity in vitro, and that these mutations can be selected for by several in vitro passages.Furthermore, such mutations have been shown to attenuate the virus in vivo (Byrnes AP and DE Griffin, Large-plaque mutants of Sindbis virus show reduced binding to heparan sulfate, heightened viremia, and slower clearance from the circulation (2000) J. Virol.; 74(2):644-51; Klimstra WB, et al. 1999, supra).

[0056] Since sequence heterogeneity accompanied by reduced immunogenicity was already evident in the P5 and P8 passages of CHIKV-Δ5nsP3, the sequence heterogeneity at early passages as well as its impact on immunogenicity were investigated in more detail as outlined below.

[0057] Example 3. Defining sequence heterogeneity and immunogenicity of CHIKV-Δ5nsP3 in passage P3 The occurrence in later passages of sequence heterogeneity, which adversely affected the immunogenicity of CHIKV-Δ5nsP3 as measured by neutralizing antibody titers, warranted the discovery of an optimal passage characterized by both high immunogenicity and sufficient viral titers for the production of an effective vaccine.

[0058] To determine the genetic stability of CHIKV-Δ5nsP3 during production of MVSB (P1), WVSB (P2), and CHIKV-Δ5nsP3 drug substance ("VLA1553") (P3), independently generated passages 1, 2, and 3 were sequenced. P0 (virus rescue), P1 (MVSB), and P2 (WVSB) showed no obvious sequence heterogeneity as determined by Sanger sequencing. The next step was to use P2 (WVSB) for infection to demonstrate the reproducibility of the genetic stability of the purified drug substance (DS) derived from P3. For infection (MOI 0.01), P2 (WVSB) was used to generate a total of four independent P3 harvests, consisting of the combined day 1 and day 2 harvests, in two T150 T-flasks. For each replicate, the individual harvests from days 1 and 2 were pooled (total volume: approximately 50 mL) and concentrated approximately 10-fold (Amicon 100 kDa ultrafiltration device). Diafiltration was performed against 25 mM Tris / 150 mM NaCl, pH 7.4, followed by protamine sulfate treatment (final concentration: 2 mg / mL) to precipitate host cell DNA. The clear supernatant was then further purified by batch adsorption chromatography using CaptoCore 700 resin (approximately 1 mL of a 50% slurry in Tris / NaCl buffer). The resin was removed by centrifugation, and sucrose was added to a final concentration of 10% to allow for freezing and thawing of CHIKV-Δ5nsP3. The final preparation was then 0.2 μm sterile filtered and stored frozen (<-65°C) until further processing.

[0059] At passage 3 (P3), no heterogeneity was detected by automated base calling (Eurofins—both less than 20%). However, visual inspection revealed that a small proportion of the virus population showed a consistent increase in sequence heterogeneity at E168K and E247K in the CHIKV glycoprotein E2 gene, which was absent in the rescued CHIKV-Δ5nsP3 (P0) and MVSB and WVSB samples. Figure 7 shows sequencing chromatograms of four independently generated P3 DS samples (Examples 1–4) compared with passage 0 (P0) virus. As indicated by the box, G / A heterogeneity at the codon for amino acid 168 (genomic position 8882) was detectable in all four replicates and verified by reverse sequencing, revealing the same heterogeneity. The same position in the P0 sample showed a sharp peak of G in the forward sequencing reaction (and C in the reverse sequencing reaction). On the other hand, although heterogeneity at E247K (genomic nucleic acid position 9119) was present, it was barely detectable in the sequencing chromatogram.

[0060] Furthermore, to quantify the abundance of E168K and E247K in the viral population, we performed next-generation sequencing of P3 and compared it with sequencing of the first passage (P1-MVSB). As can be seen in Figure 8, only very low levels of sequence heterogeneity were detectable at the P1-MVSB stage (both below background; the 15% cutoff indicated by the dotted line). However, two representative P3 samples showed an overall increase in sequence heterogeneity, with two mutations in the E2 gene reaching or exceeding background (E168K at position 8882 in 18% and 15% of the viral population, respectively, and E247K at position 9119 in a lower degree).

[0061] In summary, the presence of the E168K mutation in the E2 protein of CHIKV-Δ5nsP3 was identified by Sanger sequencing and NGS of eight independently generated P3 samples, demonstrating the reproducibility of these results. Representative sequence examples are shown in Figures 7 and 8. These data demonstrate that the appearance of the E168K mutation in the E2 protein of CHIKV-Δ5nsP3 virus was highly reproducible upon passage in Vero cells and attenuated the immunogenicity of the attenuated CHIKV-Δ5nsP3 virus, as shown in Example 2, where viruses derived from P5B plaques (P5B-04 and P5B-07 in Figure 6) were non-immunogenic. Like E168K, two additional mutations, G55R and G82R, observed during passaging of CHIKV-Δ5nsP3 in Vero cells have been reported to affect CHIKV virulence (Gardner CL, et al., 2014, supra; and Silva LA, et al., 2014, supra; and Gorchakov R, et al., "Attenuation of Chikungunya virus vaccine strain 181 / clone 25 is determined by two amino acid substitutions in the E2 envelope glycoprotein (2012) J. Virol.; 86(11):6084-96; Epub 2012 / 03 / 30)), resulting in viral attenuation. These two mutations also negatively impact the immunogenicity of CHIKV-Δ5nsP3, and therefore must be avoided for the production of highly immunogenic CHIKV-Δ5nsP3 vaccine candidates.

[0062] Figure 9 shows the positions of amino acids prone to mutation within the E1 / E2 dimer. Because the three E2 amino acids G55, G82, and E168 are all surface-accessible, mutation of these amino acids may affect the interaction of CHIKV with its cellular receptor. In contrast, E247[E2] is more buried within the protein structure and may not be as surface-exposed. The location of E247 may be one of the reasons why the E247K mutation did not significantly affect the immunogenicity of CHIKV-Δ5nsP3, as shown above.

[0063] Example 4. Determining the threshold of the E168K mutation for loss of immunogenicity of heterogeneous CHIKV-Δ5nsP3 virus populations The above observations indicated that the E168K[E2] mutation appears early and frequently during passaging of CHIKV-Δ5nsP3 in Vero cells and is associated with loss of immunogenicity. To develop a reliable manufacturing process for an effective, immunogenic live-attenuated chikungunya virus vaccine, we tested the tolerance of this mutation in CHIKV-Δ5nsP3 vaccine samples by preparing various ratios of P3 drug substance and virus P5B-07 (E168K single mutant, see Table 3).

[0064] Passage 3 (P3) drug substance, exhibiting approximately 20% E168K heterogeneity (data not shown), was mixed with preparations of CHIKV-Δ5nsP3 from the P5B-07 isolate (an E168K mutant) at ratios of 1:0.1, 1:1, and 1:10. The mixtures were sequenced to confirm the approximate frequency of the E168K mutation in each virus preparation. As shown in Figure 10 (see rows labeled "heterogeneity"), the relative amount of nucleotide A compared to the wild-type nucleotide G increased with increasing levels of P5B-07 isolate (up to 100% A in the E168K control).

[0065] To determine the effect of the E168K mutation on immunogenicity, various CHIKV-Δ5nsP3 samples were used, as shown in Figure 10, to measure the immunogenicity of 3 × 10 4TCID 50 C57Bl / 6 mice were subcutaneously immunized with a planned dose of 1:0.1 (actual doses are shown in Figure 11D). Mouse sera were collected on day 21, and individual sera from the 1:0.1, 1:1, and 1:10 groups were tested and compared with pooled sera from two control groups, P3 (group 6) and P5B-07 (E168K, group 7), using a viral replicon particle (VRP)-based neutralization assay (Figure 11). VRP resembles a replication-deficient wild-type CHIKV that displays the capsid and envelope proteins of the LR2006-OPY1 virus. The method was essentially performed as described by Glasker S, et al. (Virus replicon particle-based Chikungunya virus neutralization assay using Gaussia luciferase as readout (2013) Virol. J.; 10:235). VRPs were generated by cotransfecting BHK-21 cells with a CHIKV replicon expressing Gaussia luciferase (GLuc) and two helper RNAs expressing wild-type CHIKV capsid protein and the remaining structural proteins (E3, E2, 6K, and E1), respectively. The resulting single, circular infectious particles were used in a CHIKV neutralization assay using secreted GLuc as a readout. Neutralization of VRPs in a luciferase assay measured the reduction in GLuc expression by BHK-21 cells. The ability to induce neutralizing immunity was observed to decrease with increasing amounts of the E168K mutant (Figures 11A, 11B, and 11C, respectively). The numbers of individual mice exhibiting positive, low-positive, or negative immune responses to Chikungunya virus are shown in Figure 11D.

[0066] This finding confirms that increasing the ratio of E168K mutant to wild-type virus particles in the viral population attenuates the immunogenicity of CHIKV-Δ5nsP3 in mice. Therefore, careful monitoring of the E168 position in E2 is important to ensure high immunogenicity of the CHIKV-Δ5nsP3 vaccine. Based on the previous passaging process and quantification of E168K in the viral population at passage 8, when mouse serum pools were analyzed in PRNT, we observed that approximately 70% of the E168K mutations in the CHIKV-Δ5nsP3 population lost immunogenicity.

[0067] Example 5. Upstream processes that reduce the E168K mutation in CHIKV-Δ5nsP3 The objective of this example was to characterize an optimized Vero cell culture-based process for producing CHIKV-Δ5nsP3 in roller bottles. The effects of several upstream process parameters (MOI, days of Vero cell infection after inoculation, and incubation temperature) on viral productivity and sequence heterogeneity of the E2 protein were tested using a GMP working virus seed bank (GMP WVSB B3005044; passage 2, also referred to herein as "P2 CHIKV-Δ5nsP3") and an R&D VERO working cell bank to produce the drug substance (DS; passage 3, also referred to as "P3 CHIKV-Δ5nsP3").

[0068] Preparation of GMP WVSB B3005044 A characterized pre-master virus seed bank (PMVSB, pre-master virus seed bank AFR886 / 197579 derived from virus rescue from Vero cells) was established under R&D conditions, and the pre-master virus seed bank was used to generate a master virus seed bank of CHIKV-Δ5nsP3 under GMP conditions. A GMP working virus seed bank, VLA78-1553-WVSB-2016, batch B3005044, was produced at Halix BV using the same production method and GMP conditions as described for VLA78-1553-MVSB-2016, batch number B3005567. Briefly, the VERO working seed bank (internal designation: ICB 2014 / 002) was prepared using the T75cm ELISA kit as shown in Figure 12. 2 Flask (1x), T175cm 2 Flasks (3x), final stage 6x850cm 2 Four roller bottles were grown in a seed row in four stages. Four of the six roller bottles were used to generate the CHIKV-Δ5nsP3 seed bank. The total amount of cells in the four roller bottles was determined. The pre-master virus seed bank was used to generate a CHIKV-Δ5nsP3 master virus seed bank. The CHIKV-Δ5nsP3 master virus seed bank was then used to generate a CHIKV-Δ5nsP3 working virus seed bank. Infections were performed at an MOI of 0.01 in all stages. After 24 hours of infection (35°C; 5% CO2; 0.5 RPM), CHIKV-Δ5nsP3 was harvested. Tris and sucrose stock solutions were added to a final concentration of 5% sucrose and 25 mM Tris in the harvest pool. After compounding, the pool was filtered through a steam-sterilized 0.22 μm filter using a peristaltic pump and placed into a sterile 500 mL bioprocess vessel. Vials were filled one at a time with 0.7 mL of dispensed, filtered harvest using a peristaltic filling pump. 2D Matrix cryovials were opened and closed using a ThermoFisher capper / decapper device. GMP seed bank filled vials were stored below -65°C.

[0069] Vero cell culture Vero cells were cultured at 35°C, 5% CO2, and 75cm 2 (T75), T175cm 2 (T175) T-flask and 850cm 2 Experiments were performed in roller bottles (850RB). Vero cells used in various experiments were derived from the GMP Master Cell Bank MCB ICB / 2014 / 001. The internal name of this research working cell bank is Bk5685. The GMP Master Cell Bank originated from the Institut Merieux (Aventis Pasteur) P129 bank and ultimately from the WHO Vero Cell Bank 10-87 P134, which was derived from the original ATCC CCL 81 P113 bank. Further details regarding the cell culture sequence are shown in Figure 12. Cells were maintained in MEM medium supplemented with 10% FBS and 2 mM L-glutamine.

[0070] 850cm 2 Virus production in roller bottles After 2, 4, or 5 days of cell growth at 35°C in 850RB, cells were washed with PBS and incubated with 0.1, 0.01, or 0.001 TCID 50 The cells were infected with CHIKV-Δ5nsP3 (WVSB B3005044) at an MOI of 100 / cell. For virus production, the infected cells were incubated in 100 mL of MEM medium supplemented with 2 mM glutamine at 37°C, 35°C, or 28°C.

[0071] Virus titration TCID 50The assay was used to determine virus titers in Vero cells. 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 1 week of incubation at 35°C / 5% CO2, virus-induced cytopathic effects were monitored, and virus titers were calculated according to the Reed and Muench method (Reed, LJ; Muench, HA. A simple method of estimating fifty percent endpoints (1938) The American Journal of Hygiene 27:493-497).

[0072] Viral genome extraction and sequencing Viral nucleic acids were extracted and purified from Vero cell culture supernatants at the indicated time points using the QIAamp MinElute Virus Spin Kit (Qiagen), and cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System (ThermoFischer) with random hexamers. For sequencing of the E2 gene region, PCR was first performed using Phusion High Fidelity Polymerase (ThermoFischer) to amplify overlapping regions of the CHIKV E2 gene with primers 16F, 16R, 17F, 17R, 18F, and 18R (see Table 1 for primer sequences). After purification of the PCR amplicons, Sanger sequencing was performed at MWG Eurofins in Germany. In addition to analyzing sequence heterogeneity detected by automated base calling (>20%), all sequencing chromatograms were manually read to detect heterogeneity below the detection limit (<20%).

[0073] Optimization of the production process for immunogenic P3CHIKV-Δ5nsP3 drug substance To optimize the process for producing third-passage CHIKV-Δ5nsP3 in Vero cells, various combinations of MOI, time of Vero cell infection after inoculation, and incubation temperature were tested, as shown in Table 5. Furthermore, the yield was analyzed at different days post-infection. Three characteristics of the harvested viruses were monitored: viral productivity, titer stability, and the level of sequence heterogeneity of the E2 structural protein. [Table 6]

[0074] Virus production The kinetics of CHIKV-Δ5nsP3 production achieved under all conditions tested are shown in Figure 13. The currently used optimal process for producing CHIKV-Δ5nsP3 (35°C, MOI 0.01, infection on day 4 after Vero cell seeding) was also included in the experiment, resulting in 10 nsP3 at 24 hours post-infection. 8.0~8.5 The results showed viral productivity at the expected level of 100 / mL (Figure 13B).

[0075] Compared with the MOI and infection time, temperature had the greatest effect on virus production kinetics. At 37°C and 35°C (Figures 13A and 13B, respectively), maximum productivity was achieved on day 1 postinfection. CHIKV-Δ5nsP3 infectivity significantly decreased over the next 24 hours, with a slight decrease in virus titer loss at 35°C. When the temperature was lowered to 28°C, the virus kinetics significantly changed. Depending on the MOI used, maximum virus productivity was delayed by 1 to 4 days, and a significant improvement in virus titer stability was observed (Figure 13C). Figure 14 shows the total virus yields produced from all harvests. Under the 28°C condition (Figure 13A), the yields of two harvests were combined (days 1 and 2 postinfection). Under the 35°C condition (Figure 13B), the yields of all three harvests were combined (days 1 to 3). Under the 37°C condition (Figure 13C), the yields of five harvests (days 1 to 5) were combined.

[0076] To complete the initial observations, virus productivity and titer stability data were analyzed using a quadratic response surface model (Figures 15 and 16). The maximum virus titer was used for virus productivity. The virus titer stability was calculated by subtracting the maximum titer from the titer measured after one day of the production process, and the delta value was used. The individual titers at each time point were added to determine total virus productivity.

[0077] ANOVA analysis of both models revealed statistically significant influencing factors (Figures 15A and 16A). Confirming previous observations, temperature was the strongest factor affecting virus productivity and stability. In particular, lower temperatures (28°C) resulted in higher virus titers while keeping the virus titers reasonably stable over time. However, at this temperature, overall total virus productivity was not significantly higher compared to 35°C (see, e.g., Figure 14).

[0078] The time of infection after seeding Vero cells also affected the response, but to a lesser extent. The MOI did not have a significant effect. For both models, infection 72 hours after cell seeding was sufficient time for cell infection. Conversely, the highest virus yield was observed at 35°C, and the most stable titer was observed at 28°C, suggesting that no single temperature could combine optimal virus production and titer stability (Figures 15B and 16B). The highest total virus productivity within the shortest time post-infection was achieved at 35°C (see Figure 14).

[0079] Analysis of the E2 protein gene sequence Virus samples collected on either day 2 or day 5 (infection on day 4 after seeding) postinfection of Vero cells were selected for analysis of the genomic RNA sequence of the viral E2 structural protein. These samples were most representative of Vero cell confluence in roller bottles. Tables 6 and 7 below summarize the estimated percentage of heterogeneity for four amino acid (AA) positions based on the nucleic acid sequences determined by Sanger sequencing. Table 6 shows data for CHIKV-Δ5nsP3 grown at three different temperatures and harvested on day 2 postinfection, while Table 7 shows data for CHIKV-Δ5nsP3 grown at 28°C and harvested on day 5 postinfection. [Table 7] [Table 8]

[0080] MOI, temperature, infection date after Vero cell inoculation, and sample collection date all affected the productivity and quality of CHIKV-Δ5nsP3 when produced in Vero cells. However, the magnitude of each parameter varied in importance. For example, the results suggested a correlation between MOI and heterogeneity level; that is, the lower the virus input at infection, the higher the level of heterogeneity at collection. Except for position 9119 (E247K), where a higher level of heterogeneity was observed at 37°C (Table 6), incubation temperature did not appear to affect the stability of nucleotide sequences. Additionally, samples collected later in the viral kinetics resulted in slightly higher levels of heterogeneity for the same AA position.

[0081] To complete this initial analysis, mathematical modeling of the raw data was also performed (Figures 17 and 18). Because no significant model was calculated for position 8543, only three nucleic acid positions (8882, 9119, and 9649) could be analyzed. As shown in Figure 17, temperature and MOI had different effects depending on the nucleic acid position examined. MOI significantly affected the heterogeneity observed at nucleic acid positions 8882 and 9649, but not at 9112. Temperature affected positions 9112 and 9649, but not at 8822 (Figure 17). Comparison of D2 and D5 postinfection harvest samples from viruses produced at 28°C showed that MOI consistently affected the level of heterogeneity, with cells receiving a high dose of virus (MOI 0.1 TCID 50 We observed minimal levels of AA heterogeneity when inoculated at 0.1 MOI (1 / cell). This observation may prompt the establishment of new process parameter settings with higher MOIs as a means of achieving maximum virus production / titer stability while ensuring low levels of heterogeneity within the E2 protein. However, an MOI of 0.1 significantly increases the consumption of GMP working virus seed banks, limiting its practical industrial applicability.

[0082] The harvest date after infection only affected the variation at nucleic acid position 9119 (Figure 18).

[0083] array SEQ ID NO: 1 Nucleotide sequence of CHIKV-△5nsP3 SEQ ID NO: 2 Amino acid sequence of the E2 protein from the LR2006_OPY1 chikungunya virus strain - amino acids 339 to 742 derived from the structural protein. GenBank accession number: ABD95938.1 (amino acids 1 to 1248). STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAGLFVRTsAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLT TTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEEPNYQEEWVMHKKEV VLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA Some E2 variants identified herein SEQ ID NO: 3 E168K mutant of the E2 protein from chikungunya virus STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAGLFVRTsAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEI KVHMPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLL SYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA SEQ ID NO:4 G55R mutant of the E2 protein from chikungunya virus STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQI R IKTDDSHDWTKLRYMDNHMPADAERAGLFVRTsAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNS PLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA SEQ ID NO:5 E247K mutant of the E2 protein from chikungunya virus STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAGLFVRTsAPCTITGTMGHFILARCPKGETLTVGFTDSRKISH SCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNA K LGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA SEQ ID NO:6 G82R mutant of the E2 protein from chikungunya virus STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERA R LFVRTsAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAELGDRK GKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA SEQ ID NO:7 H232Y mutant of the E2 protein from chikungunya virus STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAGLFVRTsAPCTITGTMGHFILARCPKGETLTVGF TDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTN Y KKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA SEQ ID NO:8 Primer 1F for CHIKV-Δ5nsP3 sequencing ttaggatccGATGGCTGCGTGAGACAC SEQ ID NO:9 Primer 1R for CHIKV-Δ5nsP3 sequencing taactcgagCCGTCAGGTCTGTTGAACAT SEQ ID NO: 10 Primer 2F for CHIKV-Δ5nsP3 sequencing ttaggatccTACCACCAGGCGATTAAAG SEQ ID NO: 11 Primer 2R for CHIKV-Δ5nsP3 sequencing taactcgagCTTTGCCCACTTACTGAAGG SEQ ID NO: 12 Primer 3F for CHIKV-Δ5nsP3 sequencing ttaggatccTGCTACAGAAGTCACGCC SEQ ID NO: 13 Primer 3R for CHIKV-Δ5nsP3 sequencing taactcgagGCCAAAGCGTGAATCAG SEQ ID NO: 14 Primer 4F for CHIKV-Δ5nsP3 sequencing ttaggatccAACAGCTTGAGGACAGAGCG SEQ ID NO: 15 Primer 4R for CHIKV-Δ5nsP3 sequencing taactcgagCTCTGTCTCATCACGTCGG SEQ ID NO: 16 Primer 5F for CHIKV-Δ5nsP3 sequencing ttaggatccAAATTGCAGTCATAGGAGTCTTC SEQ ID NO: 17 Primer 5R for CHIKV-Δ5nsP3 sequencing taactcgagAGTACGTTGACGTGCTCTGA SEQ ID NO: 18 Primer 6F for CHIKV-Δ5nsP3 sequencing ttaggatccGTGGGTTAAACAACTGCAAA SEQ ID NO: 19 Primer 6R for CHIKV-Δ5nsP3 sequencing taactcgagGGTTAAAGTCTTCTATCCTCCTGG SEQ ID NO: 20 Primer 7F for CHIKV-Δ5nsP3 sequencing ttaggatccGGATAACCACTGGGATAATAGG SEQ ID NO: 21 Primer 7R for CHIKV-Δ5nsP3 sequencing taactcgagAGTTGTGAAATTCCTTCTGCC SEQ ID NO: 22 Primer 8F for CHIKV-Δ5nsP3 sequencing ttaggatccCGCAGATAGAACCAGTGAAC SEQ ID NO: 23 Primer 8R for CHIKV-Δ5nsP3 sequencing taactcgagCAGCAGCTCTACTTGGGTC SEQ ID NO: 24 Primer 9F for CHIKV-Δ5nsP3 sequencing ttaggatccAGGAGGGAAAGACAGGCT SEQ ID NO: 25 Primer 9R for CHIKV-Δ5nsP3 sequencing taactcgagCCCTCGCCTTCTTCTG SEQ ID NO: 26 Primer 10F for CHIKV-Δ5nsP3 sequencing ttaggatccCAAAATAGAAGGAGTGCAAAAAG SEQ ID NO: 27 Primer 10R for CHIKV-Δ5nsP3 sequencing taactcgagCCTGGAGTTTCTTAAGTAATAGTTGC SEQ ID NO: 28 Primer 11F for CHIKV-Δ5nsP3 sequencing ttaggatccACCGGTCCAGGTCATTTA SEQ ID NO: 29 Primer 11R for CHIKV-Δ5nsP3 sequencing taactcgagGCAGCAAATTCTTCCCAG SEQ ID NO: 30 Primer 12F for CHIKV-Δ5nsP3 sequencing ttaggatccCCATTCCAGAACACACTACAG SEQ ID NO: 31 Primer 12R for CHIKV-Δ5nsP3 sequencing taactcgagATACCTGATTTCATCATGGC SEQ ID NO: 32 Primer 13F for CHIKV-Δ5nsP3 sequencing ttaggatccCCTTTGATAAGAGCCAAGATG SEQ ID NO: 33 Primer 13R for CHIKV-Δ5nsP3 sequencing taactcgagTACAAAGTTATGACGGGTCCT SEQ ID NO: 34 Primer 14F for CHIKV-Δ5nsP3 sequencing ttaggatccCAACGAACAGGGCTAATTG SEQ ID NO: 35 Primer 14R for CHIKV-Δ5nsP3 sequencing taactcgagGACCGCTTAAAGGCCAG SEQ ID NO: 36 Primer 15F for CHIKV-Δ5nsP3 sequencing ttaggatccGTGCATGAAAATCGAAAATG SEQ ID NO: 37 Primer 15R for CHIKV-Δ5nsP3 sequencing taactcgagTGGTCTTGTGGCTTTATAGACA SEQ ID NO: 38 Primer 16F for CHIKV-Δ5nsP3 sequencing ttaggatccAACCGGAGGAAACCCTAC SEQ ID NO: 39 Primer 16R for CHIKV-Δ5nsP3 sequencing taactcgagGTACCGCACCGTCTGG SEQ ID NO: 40 Primer 17F for CHIKV-Δ5nsP3 sequencing ttaggatccAGCTACCTTGCAGCACGT SEQ ID NO: 41 Primer 17R for CHIKV-Δ5nsP3 sequencing taactcgagCCCACCATCGACAGG SEQ ID NO: 42 Primer 18F for CHIKV-Δ5nsP3 sequencing ttaggatccCGAGCCGTATAAGTATTGGC SEQ ID NO: 43 Primer 18R for CHIKV-Δ5nsP3 sequencing taactcgagCGCCGGTGAAGACCTTAC SEQ ID NO: 44 Primer 19F for CHIKV-Δ5nsP3 sequencing ttaggatccACTACTGTCAGTCACTTTGGAGC SEQ ID NO: 45 Primer 19R for CHIKV-Δ5nsP3 sequencing taactcgagTACCGGGTTTGTTGCTATTT SEQ ID NO: 46 Primer 20F for CHIKV-Δ5nsP3 sequencing ttaggatccCACAACTGGTACTGCAGAGAC SEQ ID NO: 47 Primer 20R for CHIKV-Δ5nsP3 sequencing taactcgagGCGTAGCCCTTTGATCTATAG SEQ ID NO: 48 Primer 21F for CHIKV-Δ5nsP3 sequencing ttaggatccGGTGCTATGCGTGTCGT SEQ ID NO: 49 Primer 21R for CHIKV-Δ5nsP3 sequencing taactcgagATCTCCTACGTCCCTGTGGG

Claims

1. 1. A pharmaceutical composition comprising CHIKV-Δ5nsP3 particles, said composition comprising: (i) at least 10 strains expressing the E2 structural protein defined by the amino acid sequence of SEQ ID NO:2; ３ CHIKV-Δ5nsP3 particles, and (ii) A pharmaceutical composition comprising 1 to 70% of CHIKV-Δ5nsP3 particles expressing an E2 structural protein having at least one mutation compared to the amino acid sequence of SEQ ID NO:

2.

2. The pharmaceutical composition of claim 1, wherein the CHIKV-Δ5nsP3 particles of (ii) express an E2 structural protein having 1 to 10 mutations compared to the amino acid sequence of SEQ ID NO:

2.

3. The pharmaceutical composition of claim 1, wherein the CHIKV-Δ5nsP3 particles of (ii) express an E2 structural protein having 1 to 10 mutations compared to the amino acid sequence of SEQ ID NO:

2.

4. The pharmaceutical composition of claim 1, wherein the CHIKV-Δ5nsP3 particles of (ii) express an E2 structural protein having no more than 9, 8, 7, 6, 5, or 4 mutations, no more than 3 mutations, or 1 or 2 mutations compared to the amino acid sequence of SEQ ID NO:

2.

5. The pharmaceutical composition of claim 1, wherein the particles of (ii) have 1 to 10 mutations, and the mutations are selected from mutations that affect the immunogenicity of the CHIKV-Δ5nsP3 particles and mutations that do not affect the immunogenicity of the CHIKV-Δ5nsP3 particles.

6. The pharmaceutical composition of claim 1, wherein the CHIKV-Δ5nsP3 particles having at least one mutation in the E2 structural protein (ii) further comprise an A38S mutation in nonstructural protein 1, and preferably the mutation in the E2 structural protein is an E168K mutation.

7. The pharmaceutical composition may comprise up to 10 ３ pieces ~ 10 ５ 2. The pharmaceutical composition of claim 1, comprising CHIKV-Δ5nsP3 particles.

8. 2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises 1 to 50% or 5 to 30% CHIKV-Δ5nsP3 particles expressing an E2 structural protein having at least one mutation with respect to the amino acid sequence of SEQ ID NO:

2.

9. The pharmaceutical composition of claim 1, wherein the CHIKV-Δ5nsP3 particles of (i) are encoded by the DNA sequence of SEQ ID NO:

1.

10. 2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition induces neutralizing antibodies against CHIKV-Δ5nsP3 in mice immunized with the pharmaceutical composition, resulting in serum containing the neutralizing antibodies, and the serum neutralizes Chikungunya virus (CHIKV) infection of Vero cells by at least 80%, at least 90%, or at least 95% at a serum dilution of 1:80 in an in vitro neutralization assay.

11. 2. The pharmaceutical composition of claim 1, which is obtained or obtainable by production of CHIKV-Δ5nsP3 particles in Vero cells, wherein said production comprises passage of CHIKV-Δ5nsP3 particles comprising an RNA genome corresponding to the DNA sequence of SEQ ID NO: 1 less than five times or at most three times in Vero cells in culture.

12. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is a one-shot pharmaceutical composition.

13. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is a vaccine and is in lyophilized form and / or formulated for subcutaneous administration.

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