Adaptation of enterovirus to VERO cells and its vaccine preparation

Abstract

The present invention discloses enterovirus D68 adapted to proliferate to high titer in Vero cells and its adaptation method. The present invention also discloses a suitable vaccine composition comprising inactivated enterovirus D68 antigen.

Description

[0001] Related patent applications

[0002] This application claims priority to and the benefit of Indian Patent Application No. 201841049814 filed on December 29, 2018; the disclosure of which is incorporated herein by reference. Field of the Invention

[0003] The present invention relates to the creation of new viral strains by adapting cell substrates recommended for vaccine production and their use for the generation of vaccines and as diagnostic tools. Background of the Invention

[0005] Enterovirus 68 or enterovirus D68 is a newly emerged enterovirus that causes respiratory illness with mild to severe symptoms, often manifested as flu-like symptoms and neurological diseases such as acute flaccid myelitis (AFM). Enterovirus 68 is a positive-sense single-stranded RNA virus belonging to the genus Enterovirus and the family Picornaviridae D. Although the first isolation was in the United States in 1962 (Schieble et al., Am J Epidemiol 1967, 85: 297-310), it was not until the beginning of the 21st century that this pathogen was frequently reported (Oberste et al., J Gen Virol 2004, 85: 2577-2584). Since then, outbreaks caused by enterovirus D68 have been reported worldwide (Eshaghi et al., Front Microbiol 2017, 8: 257). In 2014, a large-scale outbreak of enterovirus D68 spread to various parts of the United States, resulting in 1,153 infections and 14 deaths (CDC. Non-polio enteroviruses: enterovirus D68; Midgley et al., MMWR Morb Vertal Wkly Rep 2014, 63:798-9). During the same period, a surge in enterovirus infections was reported in Canada (Skowronski et al., EuroSurveill, 2015, 20:1-14; Edwin et al., Can CommuniDis Rep 2015, 41 Suppl 1:2-8). Subsequently, respiratory infections caused by enterovirus D68 were reported in different countries (Dyrdak et al., EuroSurveill 2016, 21; Knoester et al., Emerg Infec Dis 2017, 23:140-143; Wang et al., SciRep 2017, 7:1242).

[0006] US20160312314A1 discloses a method for detecting enterovirus D68, by contacting nucleic acid obtained from a sample with an oligonucleotide primer, exposing the contacted sample to a DNA amplification process, which provides for the production of a 98-nucleotide amplification product of the enterovirus D68VP1 gene, and subsequently detecting the 98-nucleotide amplification product, wherein the presence of the amplification product indicates that the sample contains enterovirus D68.

[0007] US20160355897A1 discloses methods and compositions for detecting enterovirus D in a sample, in particular, detecting enterovirus D68. The method includes contacting the sample with at least one primer (e.g., a forward primer and / or a reverse primer) capable of specifically amplifying EV-D68 viral protein 1 (VP1) nucleic acid or a portion thereof and / or a detectably labeled probe capable of specifically hybridizing to EV-D68 VP1 nucleic acid under conditions sufficient to specifically amplify EV-D68 VP1 nucleic acid by at least one primer and / or under conditions sufficient to specifically hybridize the probe to EV-D68 nucleic acid. Detect the amplification of EV-D68 VP1 nucleic acid and / or the hybridization of the probe with EV-D68 VP1 nucleic acid, thereby identifying whether EV-D68 is present in the sample.

[0008] The prior art fails to teach that the known enterovirus D68 is adapted and propagated in Vero cells, a cell substrate recommended for virus propagation for vaccine production. Furthermore, the prior art is limited to methods for detecting enterovirus D strains and does not disclose any composition that can be used as a vaccine / immunogenic composition to provide protection against the virus. A vaccine against this dreaded new emerging virus is necessary but to date none is available. The present invention discloses methods for adapting enterovirus D68 to propagate in Vero cells. The present invention also discloses an enterovirus strain containing a mutated nucleic acid sequence that is translated into a protein that is an antigen suitable for use in a vaccine composition. The present invention also discloses a suitable vaccine composition.

[0009] Purpose of the Invention

[0010] The main object of the present invention is to provide a method for adapting enterovirus D68 strain(s) in a cell matrix, which is a cell matrix recommended for vaccine production (eg Vero cells).

[0011] Another object of the present invention is to provide novel enterovirus D68 strain(s) adapted for propagation on Vero cells and capable of being produced at high titers on Vero cells.

[0012] Another object of the present invention is to provide an inactivated vaccine preparation against enterovirus D68 infection, which comprises the virus strain.

[0013] Another object of the present invention discloses the use of recombinant proteins of Enterovirus D68 adapted to Vero cells or whole Enterovirus D68 virus for producing antibodies against the respective recombinant proteins or whole virus, which antibodies can be used for the diagnosis of the respective antigens. SUMMARY OF THE INVENTION

[0015] One aspect of the present invention is to provide a method for adapting enterovirus D68 in a cell matrix recommended for vaccine production.

[0016] In some embodiments, an enterovirus D68 adapted to be propagated to high titers in Vero cells is provided, which is encoded by a cDNA molecule having a nucleotide sequence of SEQ ID No 1. In some other embodiments, a nucleotide sequence encoding an enterovirus D68 polyprotein adapted to Vero cells disclosed in SEQ ID No 2 is provided. In another embodiment, an amino acid sequence of an enterovirus D68 polyprotein adapted to Vero cells disclosed in SEQ ID No 3 is provided.

[0017] Another aspect of the present invention is to provide changes in amino acid levels (V341L, E647G, M699K, E719K, D1355N, T1406S, H2110Q) in the enterovirus D68 virus adapted to Vero cells.

[0018] In some embodiments, a method for adapting enterovirus D68 to grow to high titers in Vero cells is provided, comprising the steps of:

[0019] (a) infecting the virus by adsorption at 32-35° C., more precisely at 32-33° C., for about 60-120 minutes;

[0020] (b) propagating the virus after adding maintenance medium at 32-35°C, more precisely at 32-33°C;

[0021] (c) diluting the virus stock obtained from each passage at a dilution of 1:3 to 1:10 to infect the next batch of Vero cells for initial passage of the virus at 32-35°C, more precisely at 32-33°C;

[0022] (d) diluting the virus at a dilution of 1:20-1:500 to infect Vero cells at 32-35°C, more precisely at 32-33°C in subsequent / subsequent passages; and

[0023] (e) Viruses were harvested when a cytopathic effect of 90% or greater was achieved during each passage or within 6 days of infection.

[0024] In some other embodiments, a method for adapting Enterovirus D68 to grow to high titers in Vero cells is provided, wherein the Enterovirus D68 virus is subjected to a plaque assay, comprising the following steps:

[0025] (a) Plate 3 to 4 million Vero cells per 12-well or 6-well plate to achieve a confluency suitable for plaque assay;

[0026] (b) adsorbing different dilutions of the virus in different wells in duplicate, triplicate or quadruplicate at 32-35° C., more precisely at 32-33° C., for 1-2 hours;

[0027] (c) covering with ≤0.6% carboxymethyl cellulose or 0.8-1.8% Avicel RC 591 as covering medium;

[0028] (d) Fixing cells with 10% formalin 5-7 days after infection with carboxymethylcellulose as overlay or 3-4 days after infection with Avicel RC 591 as overlay;

[0029] (e) after fixation, removing the fixative and washing the cells with phosphate buffered saline; and

[0030] (f) Add freshly prepared staining solution - crystal violet solution dropwise to the fixed cells and stain at room temperature for 30 minutes to 1 hour or 2 hours.

[0031] In some other embodiments, a method for inactivating enterovirus D68 containing cDNA encoded by SEQ ID No. 1 for immunization is provided, comprising the following steps:

[0032] (a) sterile filtering the harvested enterovirus D68 virus;

[0033] (b) removal of host nucleic acids using nuclease treatment and concentration by tangential flow filtration using a 100 kDa filter;

[0034] (c) inactivation using 1 / 2000-1 / 4000 formalin at temperatures varying from 25-37°C for up to 3 weeks or using BPL at 4-25°C for up to 120 hours or using 0.005% to 3% hydrogen peroxide at 25±5°C for up to 6 hours; and

[0035] (d) Purification of inactivated antigens or purification prior to inactivation of viral antigens is performed by gel filtration using Sepharose CL-4B followed by anion exchange chromatography using DEAE resin, or by two steps / two rounds of size exclusion chromatography, or by cellufine sulphate followed by anion exchange chromatography, or by mixed mode resins such as CHT type II (Biorad) alone or in combination with other resins, or a combination of chromatographic methods.

[0036] In another aspect of the present invention, a formulation for an inactivated enterovirus D68 monovalent vaccine is provided, wherein the inactivating agent for enterovirus D68 includes chemical agents such as formalin, β-propiolactone, hydrogen peroxide (H2O2) or various physical agents such as UV, X-ray and γ-ray radiation.

[0037] In some embodiments, an immunogenic composition is provided, comprising an inactivated Enterovirus D68 viral antigen in a physiologically acceptable vehicle and optionally one or more pharmaceutically acceptable excipients selected from an adjuvant, a stabilizer or a preservative.

[0038] Another aspect of the present invention is to provide a formulation for a combination vaccine comprising inactivated enterovirus D68 and other enteroviruses. In some embodiments, the immunogenic composition according to the present invention further comprises other enteroviruses, including EV71, poliovirus or a combination thereof.

[0039] Another aspect of the present invention relates to an inactivated Enterovirus D68 vaccine capable of inducing an Enterovirus D68-specific humoral immune response, wherein the inactivated vaccine may be adjuvanted or non-adjuvanted.

[0040] Yet another aspect of the present invention relates to an enterovirus D68 vaccine capable of eliciting a significant neutralizing antibody response.

[0041] In some embodiments, an amino acid sequence belonging to Enterovirus D68 is provided, which has amino acids selected from the polyprotein of L341, G647, K699, K719, N1355, S1406, Q2110, or a combination thereof. In some embodiments, an immunogenic composition is provided, which comprises an inactivated Enterovirus D68 viral antigen in a physiologically acceptable vehicle, wherein Enterovirus D68 has amino acids selected from the polyprotein of L341, G647, K699, K719, N1355, S1406, Q2110, or a combination thereof.

[0042] In some embodiments, the immunogenic composition according to the invention is stable for at least 6 months at 2-8°C and for at least 1 month at 37°C.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : Adaptation of enterovirus 68 in Vero cells.

[0045] 1A: Mock-infected Vero cells

[0046] 1B: Vero cells infected with EV68 showed cytopathic effect on day 3 after infection.

[0047] Figure 2 : Titers of enterovirus D68 (US / KY / 14-18953) in Vero cells during different passages.

[0048] Figure 3 : Confirmation of EV68 virus adapted to Vero cells by RT-PCR. RNA was isolated from two different passages of EV68 virus from adapted Vero cells and reverse transcribed and subsequently detected using EV68-specific primer pairs (A) (lane 1: 1 Kb marker (NEB), lanes 2, 3 - Vero-adapted US-KY / 14-18953 (US-KY / 14-18953-Vero) virus and using pan-enteroviral primers), (B) (lanes 1 and 2: Vero-adapted US-KY / 14-18953 / US-KY / 14-18953-Vero, lane 3: negative control, lane 4: 100 bp marker from NEB).

[0049] Figure 4 : Plaque assay of Vero-adapted EVD68 using two different overlay media. (A) Avicel (0.8% and 1.2%) and (B) CMC (0.6%) were tested as overlay media for the progression of the enterovirus D68 plaque assay over different time periods.

[0050] Figure 5 : Expression of Vero-adapted EVD68 proteins. The expression of VP1 and VP2 proteins of EVD68 was examined by Western blotting with VP1 and VP2 antibodies specific for EVD68. The positions and molecular weights of the individual bands of the protein ladder have been mentioned.

[0051] Figure 6 : Stability of EVD68 antigen adapted to Vero cells. The stability of EVD68 antigen adapted to Vero cells is expressed as the percentage of stability after exposure to EVD68 antigen at 2-8°C for 1 month, 3 months and 6 months (A) and at 37°C for 7 days, 15 days and 1 month (B).

[0052] Figure 7:Enterovirus D68 specific IgG titer in Balb / c mouse serum induced by formalin-inactivated enterovirus D68 strain US / KY / 14-18953-Vero.Formalin-inactivated enterovirus D68 strain US / KY / 14-18953-Vero with or without aluminum adjuvant (alum) was used to immunize 6-8 week female Balb / c mice three times subcutaneously at 3 week intervals.Blood was collected and serum was separated before and after each immunization.ELISA was carried out to detect enterovirus D68 specific IgG titer in serum.

[0053] Figure 8 : Enterovirus D68-specific IgG titers in serum of Balb / c mice induced by β-propiolactone-inactivated enterovirus D68 strain US / KY / 14-18953-Vero. Female Balb / c mice of 6-8 weeks were subcutaneously immunized three times at 3-week intervals with β-propiolactone-inactivated enterovirus D68 strain US / KY / 14-18953-Vero formulated with or without aluminum adjuvant. Blood was collected before and after each immunization and serum was separated. ELISA was performed to detect enterovirus D68-specific IgG titers in serum.

[0054] Fig. 9 : The ratio of IgG1 and IgG2a in the serum collected from mice immunized with the enterovirus D68 antigen inactivated by β-propiolactone and formalin prepared with or without aluminum adjuvant. Female Balb / c mice aged 6-8 weeks were subcutaneously immunized three times with formalin and β-propiolactone inactivated enterovirus D68 strain US / KY / 14-18953-Vero prepared with or without aluminum adjuvant at 3-week intervals. 2 weeks after the third immunization, the IgG1 / IgG2a ratio was measured in the serum collected from the immunized mice. DETAILED DESCRIPTION OF THE INVENTION

[0056] Vero cells are the most commonly used cell matrix for vaccine production recommended by the World Health Organization (WHO) in recent years. The main advantage of Vero cells is unlimited life span (continuous cell line), with proven safety and suitability for large-scale production of vaccines (Barrett et al., Expert Rev Vaccines 2009, 8: 607-618). Therefore, Vero cells are valuable for the rapid production of human vaccines. We obtained the clinical enterovirus D68 strain (US / KY / 14-18953) isolated from the USA from ATCC (ATCC, VR-1825) and adapted it in Vero cells.

[0057] In some embodiments, an enterovirus D68 adapted to be propagated to high titers in Vero cells is provided, which is encoded by a cDNA molecule having a nucleotide sequence of SEQ ID No 1. In some other embodiments, a nucleotide sequence encoding an enterovirus D68 polyprotein adapted to Vero cells disclosed in SEQ ID No 2 is provided. In yet another embodiment, an amino acid sequence of an enterovirus D68 polyprotein adapted to Vero cells disclosed in SEQ ID No 3 is provided.

[0058] In some embodiments, a method for adapting enterovirus D68 to grow to high titers in Vero cells is provided, comprising the steps of:

[0059] (a) infecting the virus by adsorption at 32-35° C., more precisely at 32-33° C., for about 60-120 minutes;

[0060] (b) propagating the virus after adding maintenance medium at 32-35°C, more precisely at 32-33°C;

[0061] (c) diluting the virus stock obtained from each passage at a dilution of 1:3 to 1:10 to infect the next batch of Vero cells for initial passage of the virus at 32-35°C, more precisely at 32-33°C;

[0062] (d) diluting the virus at a dilution of 1:20-1:500 to infect Vero cells at 32-35°C, more precisely at 32-33°C in subsequent / subsequent passages; and

[0063] (e) Viruses were harvested during each passage when a cytopathic effect of 90% or greater was achieved or within 6 days of infection.

[0064] Initially, the parent enterovirus D68 strain US / KY / 14-18953 did not reproduce in Vero cells, nor did it show any signs of infection in the form of cytopathic effect or any cell deformity. Therefore, the parent strain did not show any signs of adaptation. Subsequently, a high infection dose of the virus was used for several subsequent passages. The temperature of the entire infection process was maintained at around 32-34°C and was never allowed to exceed 35°C. Through this strategy, the strain began to adapt to Vero cells and showed partial cytopathic effect from the 3rd generation. By the 5th generation, a complete cytopathic effect (CPE) was observed in Vero cells. The enterovirus D68 strain was further passaged using a gradually decreasing infection dose to determine whether the strain is stably adapted in Vero cells. The enterovirus D68 strain disclosed in the present invention has indeed adapted to Vero cells and has been used as a vaccine candidate strain to produce vaccines using Vero cells as a matrix for virus propagation.

[0065] The present invention discloses a method for adapting any enterovirus D68 strain in Vero cells to make it suitable for vaccine production. In particular, one embodiment of the present invention describes a method for adapting enterovirus D68 in new cells such as Vero cells.

[0066] Furthermore, the present invention also discloses one or more polynucleotide sequences of the Vero-adapted enterovirus D68 strain, such as disclosed in SEQ ID No.1 and SEQ ID No.2, and one or more amino acid sequences of the Vero-adapted enterovirus D68 strain, such as disclosed in SEQ ID No.3.

[0067] The present invention further discloses the nucleotide sequences of individual structural proteins of EV68 disclosed as SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10 and the amino acid sequences of individual structural proteins of EV68 disclosed as SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 and SEQ ID No.11, which can be used to prepare recombinant proteins. The nucleotide sequence of SEQ ID No.4 is translated to obtain the amino acid sequence of SEQ ID No.5, the nucleotide sequence of SEQ ID No.6 is translated to obtain the amino acid sequence of SEQ ID No.7, the nucleotide sequence of SEQ ID No.8 is translated to obtain the amino acid sequence of SEQ ID No.9, and the nucleotide sequence of SEQ ID No.10 is translated to obtain the amino acid sequence of SEQ ID No.11. The recombinant protein can be expressed in bacteria, yeast or mammalian systems after codon optimization of the nucleotide sequence suitable for each expression system and cloned into the corresponding expression vector. The antibodies generated after immunization with the expressed recombinant protein can be used to diagnose the complete virus or the corresponding protein antigen.

[0068] In another embodiment, the present invention discloses a standardized method for plaque assay of Vero cell-adapted Enterovirus D68 strain in Vero cells, which can be used to determine the virus titer expressed in plaque forming units, in particular for determining the plaque reduction neutralization titer (PRNT) for vaccine efficacy studies.

[0069] In some other embodiments, a method for adapting Enterovirus D68 to propagate to high titers in Vero cells is provided, wherein the Enterovirus D68 virus is subjected to a plaque assay, comprising the following steps:

[0070] (a) Plate 3 to 4 million Vero cells per 12-well or 6-well plate to achieve a confluency suitable for plaque assay;

[0071] (b) adsorbing different dilutions of virus in different wells in duplicate, triplicate or quadruplicate at 32-35° C., more precisely at 32-33° C., for 1-2 hours;

[0072] (c) covering with ≤0.6% carboxymethyl cellulose or 0.8-1.8% Avicel RC 591 as covering medium;

[0073] (d) Fixing cells with 10% formalin 5-7 days after infection with carboxymethylcellulose as overlay or 3-4 days after infection with Avicel RC 591 as overlay;

[0074] (e) after fixation, removing the fixative and washing the cells with phosphate buffered saline; and

[0075] (f) Add freshly prepared staining solution - crystal violet solution dropwise to the fixed cells and stain at room temperature for 30 minutes to 1 hour or 2 hours.

[0076] The present invention also discloses the preparation of antigens from adapted viruses and their formulation into vaccine compositions to elicit antibodies against enterovirus D68. Vaccine antigens of EV68 adapted to Vero cells can be prepared by inactivation using chemical methods such as formalin, beta-propiolactone (BPL), oxidants such as hydrogen peroxide, physical methods including but not limited to UV or X-radiation or gamma irradiation.

[0077] In some other embodiments, a method for inactivating enterovirus D68 containing cDNA encoded by SEQ ID No. 1 for immunization is provided, comprising the following steps:

[0078] (a) sterile filtering the harvested enterovirus D68 virus;

[0079] (b) removal of host nucleic acids using nuclease treatment and concentration by tangential flow filtration using a 100 kDa filter;

[0080] (c) inactivation using 1 / 2000-1 / 4000 formalin at temperatures varying from 25-37°C for up to 3 weeks or using BPL at 4-25°C for up to 120 hours or using 0.005% to 3% hydrogen peroxide at 25±5°C for up to 6 hours; and

[0081] (d) Purification of the inactivated antigen or purification prior to inactivation of viral antigen is performed by gel filtration using Sepharose CL-4B followed by anion exchange chromatography using DEAE resin, or by two steps / two rounds of size exclusion chromatography, or by cellulose sulfate followed by anion exchange chromatography, or by mixed mode resins such as CHT type II (Biorad) alone or in combination with the above resins, or any combination of the above chromatography steps.

[0082] In yet another embodiment, the inactivated vaccine antigens may be formulated in the presence or absence of an adjuvant. Adjuvants include, but are not limited to, aluminum adjuvants (aluminum hydroxide, aluminum phosphate), emulsions such as oil-in-water adjuvants or water-in-oil adjuvants, Toll-like receptor (TLR) ligands such as monophosphoryl lipid A (MPL), complete or truncated flagellin, adjuvants including bacterial cell components, squalene-based adjuvants such as MF59 or AddaVax, montanide, etc., Ribi adjuvants, CpG and non-CpG oligonucleotides, saponins such as QS-21, immunostimulatory complexes (ISCOM), ISCOMATRIX, etc., vitamins, and immunomodulators including cytokines.

[0083] In some embodiments, an immunogenic composition is provided, comprising an inactivated Enterovirus D68 viral antigen in a physiologically acceptable vehicle and optionally one or more pharmaceutically acceptable excipients selected from an adjuvant, a stabilizer or a preservative.

[0084] In another embodiment, the enterovirus 68 vaccine can be formulated with other enterovirus-based vaccines (one or more) to prepare one or more combination vaccines, wherein the other enteroviruses include but are not limited to enterovirus 71, coxsackieviruses including coxsackievirus A16, coxsackievirus A6, coxsackievirus A10, rhinoviruses and polioviruses. Vaccine formulations may also include enterovirus 68 vaccine in combination with flaviviruses, orthomyxoviruses, or any other virus that can cause respiratory disease, encephalitis or meningitis.

[0085] In some embodiments, an amino acid sequence belonging to Enterovirus D68 is provided, having an amino acid in a polyprotein selected from L341, G647, K699, K719, N1355, S1406, Q2110, or a combination thereof. In some embodiments, an immunogenic composition is provided, comprising an inactivated Enterovirus D68 viral antigen in a physiologically acceptable vehicle, wherein Enterovirus D68 has an amino acid in a polyprotein selected from L341, G647, K699, K719, N1355, S1406, Q2110, or a combination thereof.

[0086] In some embodiments, the immunogenic composition according to the invention is stable at 2-8°C for at least 6 months and at 37°C for at least 1 month. Example

[0087] Example 1: Virus propagation

[0088] Vero cells were infected with a high infectious dose of enterovirus strain D68. Initially, the virus was allowed to adsorb to the T25 cm 2 flask with occasional shaking for about 60 to 120 minutes, and then DMEM is added as maintenance medium. The virus stock obtained from each passage is used to infect the next batch for initial passage at a dilution of about 1:3-1:10. In subsequent / subsequent passages, the virus stock from the previous passage is used for infection at a higher dilution (ranging from 1:20-1:500). The virus is harvested when a cytopathic effect of 90% or more is reached or within 6 days of infection, whichever is earlier. The entire propagation process of enterovirus D68 is carried out at a temperature not higher than 35°C and ideally 32-33°C. The titer of the virus in different passages is determined by TCID50 assay and / or plaque assay and as Figure 2 shown.

[0089] The entire process of selecting lower dilutions of virus for infection (not toxic to Vero cells) during initial passages at a controlled temperature in the range of 32-35°C (preferably 32-33°C) followed by infection with higher dilutions of virus in subsequent passages, as described above, has allowed the successful adaptation of the enterovirus D68 virus in Vero cells. Figure 1 Example illustrates the adaptation of enterovirus 68 in Vero cells, where Figure 1 A shows mock-infected Vero cells, Figure 1 B shows that Vero cells infected with EV68 showed cytopathic effect at day 3 post infection. After successful adaptation, at T75 cm 2 Flask, T175 cm 2 Scale-up of virus production is performed sequentially in flasks and then in cell stacks or in roller bottles.

[0090] Example 2: RNA isolation and RT-PCR

[0091] RNA has been isolated from the cell-free supernatant with infectious virus after harvest by Trizol method according to standard procedures. Briefly, 750 μl Trizol was used for 250 μl supernatant containing virus. If it is necessary to obtain a large amount of viral nucleic acid material, the viral supernatant is concentrated by PEG6000 or PEG8000 (Merck, India) or ultrafiltration using 100 kDa cut-off membrane (Merck, India) before Trizol isolation method. The isolated RNA was reverse transcribed into complementary cDNA (cDNA) using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The presence of viral RNA was confirmed using virus-specific primers, such as Figure 3 The primer sequences are provided as follows:

[0092] Enterovirus D68 specific primers (Calvo et al., Pediatr Infect Dis J 2016, 35:45-49):

[0093] (F)-1:GTTCYTTAATAGGRTTCRTAGCAGC

[0094] (R)-1:CTCTATTRCCAATTATGGCATTRAG

[0095] Pan-enterovirus primers (Thao et al., J. Virol. Methods 2010, 170: 134-139)

[0096] (F):5'-CAAGCACTTCTGTTTCCCCGG-3'

[0097] (R):5'-ATTGTCACCATAAGCAGCCA-3'

[0098] Example 3: Sequencing

[0099] For whole genome sequencing, use an Illumina-compatible UltraTM DirectionalRNALibrary Prep Kit (New England BioLabs, MA, USA) was used to prepare RNA sequencing libraries. The sequencing libraries were initially quantified by Qubit Fluorometer (Thermo Fisher Scientific, MA, USA) and their fragment size distribution was analyzed on Agilent TapeStation. Finally, the sequencing libraries were accurately quantified by quantitative PCR using Kapa Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA). The qPCR-quantified libraries were merged in equimolar amounts to create the final multiplex library set for sequencing on Illumina NextSeq (150x 2 chemistry).

[0100] Table 1 below provides the amino acid differences and corresponding nucleotide level differences between the parental enterovirus D68 strain US / KY / 14-18953 and the Vero cell adapted virus strain US / KY / 14-18953-Vero:

[0101]

[0102]

[0103] Table 1: Sequencing summary

[0104] Example 4: Plaque Assay of Enterovirus D68

[0105] There is currently no comprehensive literature supporting enterovirus D68 plaque assay. The present invention discloses a standardized plaque assay procedure for enterovirus D68. About 3 million to 4 million Vero cells are plated per 12-well plate or 6-well plate (Greiner bio-one) to achieve a confluence suitable for plaque assay. Different dilutions of the virus are adsorbed in different wells for 1-2 hours in duplicate, triplicate or quadruplicate. Subsequently, a 0.8% carboxymethyl cellulose (CMC) (Merck, India) semi-solid overlay is added. 0.6-0.5% concentration of carboxymethyl cellulose is also alternatively used as an overlay medium. Alternatively, Avicel RC591 (FMC Corporation, USA) is used as an overlay at a concentration of 0.8-1.8%. For convenience, Avicel RC 591 will be referred to as Avicel below.

[0106] Cells were fixed with 10% formaldehyde for 1 hour after 4 to 6 days post infection when CMC was used as an overlay or after 2 to 4 days post infection when Avicel was used as an overlay. After fixation, the fixative was removed and the cells were washed with phosphate buffered saline (PBS). After washing, freshly prepared crystal violet solution was added dropwise to the fixed cells and stained for 30 minutes to 1 hour at room temperature. Plaques were visible on the 5th day of infection when CMC was used as an overlay or within 48 hours when Avicel was used. Results are shown in Figure 2. Figure 4 When Avicel was used as overlay medium, plaques were more obvious 3 days after infection, as shown in Figure 4 shown.

[0107] Example 5: Expression of Enterovirus D68

[0108] The expression of enterovirus D68 VP1 and VP2 proteins was examined by Western blotting of enterovirus D68 antigen adapted to Vero cells using enterovirus D68-VP1 and VP2-specific antibodies, as Figure 5 As shown. Specific antigens were separated by 12% SDS-PAGE gel and then transferred to PVDF membrane. The blot was blocked with 1% BSA and then incubated with enterovirus D68-VP1 and VP2 specific primary antibodies (GeneTex) for 90 minutes. Soon after, the blot was washed four times with phosphate buffered saline containing Tween 20 (PBST) and then incubated with secondary antibodies conjugated with horseradish peroxidase (HRP). The expression signal was detected using the chromogenic substrate-metal enhanced DAB (Thermo Scientific).

[0109] Example 6: Preparation and formulation of vaccines for immunization

[0110] The propagated and harvested EVD68 virus encoding the cDNA in SEQ ID No.1 was sterile filtered; host nucleic acids were removed using nuclease treatment and concentrated by tangential flow filtration using a 100 kDa filter. Subsequently, the concentrated virus was inactivated using 1 / 2000-1 / 4000 formalin at temperatures varying from 25-37°C for up to 3 weeks, or using BPL at 4-25°C for up to 120 hours. The formalin was neutralized by using thiosulfate. Alternatively, the virus was inactivated using 0.005% to 3% hydrogen peroxide at 25±5°C for up to 6 hours. The reaction was stopped by adding 10U / ml of catalase to hydrolyze the residual hydrogen peroxide. At the end of the inactivation experiment, the inactivation was confirmed by serial passage of the inactivated virus in Vero cells for up to three times and without cytopathic effect. The inactivated virus antigen was tested and confirmed for in vitro antigenicity using enterovirus 68 antigen-specific VP1 antibodies. Subsequently, Sepharose CL-4B gel filtration is used and then DEAE resin is used or anion exchange chromatography is carried out by Capto Core700 resin (GE Healthcare Life Sciences) to purify the inactivated virus. Purification is also carried out by double step / two rounds of size exclusion chromatography. Cellulose sulfate can also be used and then anion exchange chromatography is carried out as required to purify the inactivated virus. Purification is also carried out by mixed mode resin-CHT II type (Biorad) alone or in combination with the aforementioned resin.

[0111] Alternatively, inactivation as described above is performed after virus purification. The buffer for the inactivated vaccine is 10-30 mM phosphate buffer. Alternatively, Tris buffer, histidine buffer, sucrose phosphate glutamate or any pharmaceutically acceptable buffer is used for formulation.

[0112] Suitable excipients for inactivated vaccines include, but are not limited to, stabilizers selected from sugar alcohols such as glycerol, sorbitol, mannitol (up to 20%). The formulation also contains stabilizers such as sucrose and / or trehalose and / or polysorbate 40 or polysorbate 80. The vaccine formulation contains an amino acid selected from glycine, glutamic acid, arginine or arginine hydrochloride (up to 2%). The vaccine formulation also contains human or animal serum albumin.

[0113] The vaccine formulation comprises a preservative selected from thimerosal and 2-phenoxyethanol. For mercury-free vaccine formulations, 2-phenoxyethanol (2.5-20 mg / ml) will be used. In another embodiment, the formulation does not contain any preservative.

[0114] The inactivated antigen is formulated with or without an aluminum adjuvant (in a concentration range of 100 μg to 1 mg); more precisely with 200-500 μg of aluminum adjuvant for immunization. The inactivated enterovirus 68 vaccine can be formulated with other adjuvants, such as emulsions such as oil-in-water adjuvants or water-in-oil adjuvants, Toll-like receptor (TLR) ligands such as monophosphoryl lipid A (MPL), intact or truncated flagellin, adjuvants including bacterial cell components, squalene-based adjuvants such as MF59 or AddaVax, montanide, etc., Ribi adjuvants, CpG and non-CpG containing oligonucleotides, saponins such as QS-21, immunostimulating complexes (ISCOM), ISCOMATRIX, etc., vitamins, immunomodulators including cytokines.

[0115] Example 7: Combination vaccine

[0116] EVD68, EV71, and polioviruses cause neurological symptoms and are often associated with acute flaccid myelitis—weakness associated with the spinal cord that causes weakness of muscles and body reflexes. Therefore, a combination vaccine against EVD68, EV71, and polioviruses would reduce neurological complications associated with these viruses.

[0117] In one embodiment, formalin-inactivated EVD68 antigen (up to 5 micrograms) and formalin-inactivated EV71 inactivated antigen (up to 5 micrograms) are formulated in phosphate buffer (10-30mM) (antigen ratio of 1:1). Other components of the formulation include adjuvants (including but not limited to aluminum adjuvants or MPLA up to 0.9mg / ml or combined aluminum adjuvants and MPLA), sugars and / or sugar alcohols. The formulation may include amino acids such as glycine and / or glutamate and / or arginine (up to 2%). The formulation may or may not contain a preservative, such as thimerosal or 2-phenoxyethanol.

[0118] In another embodiment, formalin-inactivated EVD68 antigen (up to 5 micrograms of antigen) is formulated with formalin-inactivated sabin type 1 polio (up to 40D antigen units) and sabin type 3 polio vaccine (up to 32D antigen units). The combination vaccine may or may not include formalin-inactivated EV71 antigen (up to 5 micrograms of antigen).

[0119] EV71 virus inactivation and poliovirus type 1 and 3 inactivation are performed using formalin at 36°C ± 1°C for up to 4 days. In another embodiment, inactivated EVD68, EV71 and poliovirus type 1 and 3 are adsorbed in aluminum adjuvant at room temperature for 2-3 hours or up to 6 hours or adsorbed for 16 hours at 4°C under stirring conditions. Subsequently, the adsorbed antigens are mixed together under stirring conditions.

[0120] Alternatively, EVD68 antigen or EV71 or polio antigen is first adsorbed in aluminum adjuvant, followed by other antigens.

[0121] Formulations were also prepared without adjuvant by mixing antigens.

[0122] The table provided below is for illustrative purposes only. The range of possible formulations of monovalent inactivated EVD68 vaccine and combination vaccine with inactivated EVD68, inactivated EV71, inactivated sabin type 1 polio vaccine and inactivated sabin type 3 polio vaccine is not limited to the formulations mentioned in the table. The pH value of the vaccine formulation is maintained in the range of 6.5 to 7.5.

[0123]

[0124] Table 2: Formulation of EVD68 monovalent vaccine

[0125]

[0126]

[0127] Table 3: Combination vaccine formulations

[0128] Example 8: Vaccine stability

[0129] The stability of the EVD68 antigen was tested by exposing the inactivated EVD68 antigen to 2-8°C for up to 6 months. Alternatively, an accelerated stability study was performed at 37°C for up to 1 month. The inactivated antigen was found to maintain >75% stability at 37°C for up to 1 month in the absence of any stabilizer. In the presence of 10% sucrose or a combination of 10% sucrose and 10% sorbitol, the stability was ≥90% for up to 1 month at 37°C. The stability was greater than 85% when stored at 2-8°C for 6 months in the absence of stabilizers and greater than 95% ( Figure 6 ).

[0130]

[0131] Table 4: Stability study of inactivated enterovirus D68 antigen

[0132] Example 9: Immunogenicity of vaccine antigens

[0133] The vaccine containing inactivated antigen was used to immunize mice aged 6-8 weeks three times at three-week intervals. Serum was collected before and after each immunization. ELISA was performed as follows: After coating with enterovirus D68-specific antigen, it was then blocked in 1% BSA and the ELISA plate was subsequently treated with immune mouse serum and anti-mouse secondary antibody.

[0134] It was found that formalin-inactivated antigens induced very high enterovirus D68-specific antibody titers (up to 200,000) and Figure 7 BPL-inactivated antigens induced relatively small but significant amounts of enterovirus D68-specific antibody titers (up to 20,000), as shown in Figure 8 shown.

[0135] In addition, the enterovirus D68-specific IgG1 / IgG2a ratio was determined from the mouse sera collected two weeks after the third immunization to detect whether the immune response pattern was Th1 or Th2 by ELISA ( Fig. 9 ). If IgG1 is higher than IgG2a, the immune response in Balb / C mice is considered to be mainly associated with Th2 lymphocytes, while when IgG2a is higher than IgG1, it is mainly associated with Th1 lymphocytes. When IgG1 / IgG2a is close to 1, it is generally considered to be a balanced Th1 and Th2 immune response. It has been found that the EVD68 antigen inactivated by β-propiolactone (BPL) without adjuvant elicited high IgG1-type virus-specific antibodies (IgG1 / IgG2a=3) compared to IgG2a, indicating a Th2-type immune response. The EVD68 antigen inactivated by BPL with aluminum adjuvant elicited even higher IgG1-type virus-specific antibodies (IgG1 / IgG2a=9.8) compared to IgG2a, indicating a further polarization toward a Th2-type immune response. In contrast, it was found that unadjuvanted formalin-inactivated EVD68 antigen elicited balanced IgG1 and IgG2a antibody responses (IgG2a / IgG1=1.22), indicating a more balanced Th1 and Th2 type immune response, which was slightly more Th2 type in the presence of aluminum adjuvant as adjuvant (IgG1 / IgG2a=1.67).

[0136] For neutralization titer detection, sera from mice immunized with EVD68 inactivated antigen vaccine (prepared with or without adjuvant) were serially diluted and incubated with enterovirus D68 virus at a ratio of 1:1 for 2 hours. The serum-virus mixture was added to Vero cells and incubated for 2 hours. Subsequently, the cells were covered with 1.2% Avicel in DMEM medium. Four days after infection, the cells were fixed in 10% formalin and stained with 0.8% crystal violet solution to observe plaques. PRNT50 was calculated as the highest serum dilution that showed a 50% or more reduction in the number of plaques.

[0137] Alternatively, a microneutralization assay was performed in a 96-well format. In the microneutralization method, 2.5 million cells were inoculated per 96-well plate. Serum from mice immunized with an inactivated EVD68 antigen vaccine (prepared with or without an adjuvant) was serially diluted and incubated with enterovirus D68 virus at a ratio of 1:1 for 2 hours. Subsequently, the serum-virus mixture was added to the Vero cells and waited for up to 4 days to observe the inhibition of the cytopathic effect of Vero cells in the wells with the serum-virus mixture. The neutralization titer was considered to be the last dilution of serum that inhibited 50% of the cytopathic effect compared to the wells with the control virus or the negative control serum-virus mixture.

[0138] Both formalin- and BPL-inactivated enterovirus D68 antigen induced significant neutralizing antibody titers in Balb / c mice.

[0139] Antigen preparation Neutralization titer Formalin-inactivated EVD68 antigen 64 Formalin-inactivated EVD68 antigen + aluminum adjuvant 64 BPL-inactivated EVD68 antigen 16 BPL inactivated EVD68 antigen + aluminum adjuvant 16

[0140] Table 5: Neutralization titers induced by formalin- and BPL-inactivated Vero cell-adapted enterovirus D68 antigen. Sequence Listing <110> Bharat Biotech International Limited <120> Adaptation of enterovirus to VERO cells and its vaccine preparation <130> P-18-753-IN <140> 201841049814 <141> 2018-12-29 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 7348 <212> DNA <213> Complementary DNA sequence of the whole genome of the Vero-adapted enterovirus 68 / enterovirus D68 strain US / KY-18953 (referred to as US / KY / 14-18953-Vero) <400> 1 ataaaacagc cttggggttg ttcccacccc aagggcccac gtggcggcta gtactctggt 60 atttcggtac ctttgtacgc ctgttttatc tccctcccca atgtaactta gaagctttca 120 aatcaaagct caataggtgg agcgcaagcc agcgcttccg tgagcaagca ctcctgtttc 180 cccggtgcgg ttgtatagac tgttcccacg gttgaaaaca acctatccgt tatccgctat 240 agtacttcga gaaacctagt atcacctttg gattgttgat gcgttgcgct cagcacacta 300 acccgtgtgt agcttgggtc gatgagtctg gacatacccc actggtgaca gtggtccagg 360 ctgcgttggc ggcctactca tggtgaaaac catgagacgc taaacatgaa caaggtgtga 420 agagtctatt gagctactat agagtcctcc ggcccctgaa tgcggctaat cttaaccatg 480 gagcaagtgc tcacaaacca gtgagttgct tgtcgtaatg cgcaagtccg tggcggaacc 540 gactactttg ggtgtccgtg tttcactttt tacttttatg actgcttatg gtgacaattt 600 aatattgtta ccatttagct agtcaaatta atcatataaa actttaaatc ctatctaaca 660 attcacattt ggatagcttc attttgagga ctttatacct acttacaaag atgggggctc 720 aagttactag acagcaaacc ggaacccatg agaacgccaa cattgccaca aatggatccc 780 atatcacata caaccagata atttttaca agatagtta tgcggcttca gctagtaaac 840 aagatttttc tcaggaccca tcaaattta ctgaaccagt ggtagaaggc ttaaagcag 900 gggctccagt tttgaaatcc cctagtgctg aggcatgtgg ctacagtgat agagtattac 960 aactcaaatt aggtattcg gctattgtca ctcaggaagc agcgaactat tgttgcgctt 1020 atggtgaatg gcccaattat ttaccagatc acgaggcagt agctattgac aaacccacac 1080 aaccagaaac tgctacggac agatctata ccttgaatc ggtcaatgg gaactgaaa 1140 gtacaggatg gtggtggaaa ttacctgatg cactaacaa cataggtag tttggacaa 1200 atgtacagca tcactactta tataggtctg gtttcttaat tcatgtgcag tgtaacgcca 1260 caaaattcca tcaggtgcc ctattagtgg tagcaatccc agacaccaa aggggggcgc 1320 acacaccac tacttaccca ggttttgatg atatcatgaa gggtgaagaa ggagggacat 1380 ttaccaccc atatgtcctt gatgatggga cgtcactagc ctgcgcaaca atatttccac 1440 accagtggat aaatttgaga accaatatt cagcacaat agttctccc tggatgatg 1500 ccgctccaat ggacttccca ctcaggcaca atcagtggac actagcaata attccagtag 1560 taccactagg tacgcgcaca gtgtcaagta tggttccaat aacagtttct attgctccaa 1620 tgtgttgtga gttaatgga ctcagacacg ccattactca aggtgtccca acataccttc 1680 taccaggctc aggacaattt ctaactactg atgatcacag ctctgcgccg cttcttccat 1740 gcttcaaccc aactccagaa atgcacattc cagggcaagt ccgcaacatg ctagaagtga 1800 ttcaggtgga atcaatgatg gagattaata acacagaaaa tgcagttggc atgcagcgtc 1860 tcaaagttga tatatcagta ctaacagacg ttgatcaatt gttattcaac atcccactag 1920 acatacagtt agatgggcca cttagaaaca ctctagtagg aaacatatct agatattata 1980 ctcactggtc tggatctcta gaaatgacat tttgttttg tgggagcttc atggcaacag 2040 gaaaattaat tctgtgttac actcctccag gcgggtcatg cccaacaacc agagaaactg 2100 ctatgttagg tacacatatt gtttgggatt ttggattaca atccagtgtc actctggtaa 2160 taccttggat cagtggatcc cactacagga tgttcaacaa tgatgctaag tcaaccaatg 2220 ccaatgttgg ctatgttacc tgttttatgc aaaccaattt aatagttccc agtgagtctt 2280 ctaacacatg ttccttaata gggttcgtag cagcaaaaga tgacttttcc ctcaggctaa 2340 tgagagatag ccctgacatt ggacaactag aacatttaca tgaggcagag gcagcctacc 2400 agattgagag catcatcaag acagcaactg acaccgtgaa aagtgagatt aacgctgaac 2460 ttggcgtggt ccctagtttg aatgcagttg aaacaggtgc aagttctaac actgaaccag 2520 aggaagccat acaaactcgc acggtgataa atcagcatgg tgtatctgaa acattagtgg 2580 agaattttct tagcagggca gccttagtat caaagagaag tttcgaatac aaaaatcaca 2640 cctcatctgg agcacgaaca gacaagaact tttataaatg gacaattaac accaagtcct 2700 ttgtccagtt gaggagaaag ctggaactgt tcacgtacct tagatttgat gctgaaatta 2760 ccatacttac aactgtggca gtaggtagca acaacagcac atacaagggc cttcctgact 2820 tgacacttca agcaatgttt gtacctactg gtgcccttac tccaaaaaaa caggattcat 2880 tccattggca gtccggtagt aatgctagtg tattcttcaa agtttctgac cccccagcca 2940 gaatgactat accttttatg tgtatcaact cagcatactc agttttctat gatggttttg 3000 ctggatttga aaagaatggt ctttatggga taaatccagc tgataccatc ggcaacttat 3060 gtgtcagaat agtgaatgag caccagccaa ttggtttcac agtgaccgtc agggtttaca 3120 tgaagcctaa acacataaaa gcgtgggcac ccagaccacc acgaaccctg ccatatatga 3180 gcattgcaaa tgcaaactat aagggtaaag aaagggcacc aaatgcgctc aatgccataa 3240 ttggcaatag agagagtgtc aaaaccatgc ctcatgatat ccggttagtg aatactggtc 3300 caggcttcgg aggggtcttt gtaggatctt ttaaaataat taactaccac ttagccacta 3360 tagaggaaag acagtcagct atctatgtag actggcaatc agatgttctg gttactccca 3420 ttgctgccca tggaagacac caaatagcaa gatgtaagtg caatacaggg gtttattatt 3480 gcagacacag agataagagc tacccagttt gttttgaagg cccagggatt caatggattg 3540 aacaaaatga atattaccca gcaaggtacc agactaatgt gcttctagca gctggtcctg 3600 cagaagcagg agattgtggt ggtttactgg tctgtccaca tggggtaatt ggtcttctca 3660 cagcaggggg gggtggaatt gtagctttca ctgatatcag gatttacta tggttagata 3720 ctgatgctat ggacaaggc attactgatt acattcaaa tctcggtaat gccttttggag 3780 cggggtttac agaaacaatc tctagtaaag ctaggagt gcaagacatg tgattggag 3840 agagttcact attagagaaa ttgttaaaag ctctaatcaa attackctca gcattggtga 3900 ttgtaattag aaattcagaa gatttaatta cagtcacagc cacactagca ttgctaggtt 3960 gccatgattc accatggagc tattgaaac ataaagtgtg ctcatattta ggtattcctt 4020 atgtacctag acagagtgaa tcatggctca agaagttcac agaagcatgc aatgctctca 4080 ggggtttaga ttggttatca cagagatag aataattcat siactggctt aaaaaaaaaa 4140 tattaccaga ggctagggag aaatatgaat ttgtacaag actcaacag ttaccagtca 4200 tagagaacca agttagcact attgaacata gctgcccac aagaaacaacaggcct 4260 tgtttaacaa tgttcaat tactcacact attgtagaaa gtatgcacca ctttacgcag 4320 tggaggcaa gagagtgaca gctcttgaaa agaaaataa taactacac cagttcaagt 4380 ccaaatctcg cattgaaccg gtttgttaa tatacatgg ttccccaggg actggcaaat 4440 cagtagcttc aaatctgatt gccagggcta tcacagaaaa gttaggaggg gatgtttatt 4500 ctctaccccc agacccaaaa tattttgatg ggtataaaca gcaaacagta gtccttatgg 4560 atgatttaat gcaaaatcca gatggaaacg acatatctat gttttgccaa atggtttcca 4620 ccgtggactt tatacctcca atggccagtt tagaagaaaa gggaactcta tacaccagtc 4680 cattttgat agccactact aatgctggct caatacatgc accaaccgtc tcagactcca 4740 aggctttgtc acgcagattt aaatttgatg tgaacattga ggtcacagat tcatacaaag 4800 actcaaataa attggacatg tcaagagcag tggagatgtg taaaccagat gactgtgctc 4860 ccattaatta taaaagatgc tgcccattga tttgtggaaa ggccattcaa tttagagatc 4920 gtagatctaa tgcaagatcc actatagata tgttagtaac tgacattatt aaggagtata 4980 gaatcagaaa cagtacacaa gataaactgg aggctctgtt ccagggacct ccacagttta 5040 aagagatcaa aatttcagtt actccagaca caccagctcc tgatgctata aacgatcttc 5100 tcaggtcagt ggattctcaa gaagttaggg actattgcca aaagaaaggg tggattgtaa 5160 tacacccatc aaatgaatta ttagtggaaa aacacattag cagggctttt atcactctac 5220 aggccgttgc tacctttgta tcaatagcag gtgtagttta tgttatatat aaactttttg 5280 ctggtatcca aggcccttac acaggaattc ccaatcctaa acctaaggta ccctctctta 5340 gaacagctaa agtgcagggg ccagggtttg atttgcaca agctataatg aagaaaaaca 5400 ccgtcattgc caggactgaa aagggtgagt tcaccatgtt gggtatacat gatagagtgg 5460 cagtatccc cacacatgca tctgttggag aaaccattta catcaacgat gtagagacta 5520 aagttttaga tgcatgtgct ctcagagact tgactgacac aaatttagag attaccatag 5580 tcaaactgga tcgtaaccaa aagtttagag atatcagaca ttttctgccc agatatgagg 5640 atgattataa tgatgctgtg cttagcgtgc atacatcaaa atttccaaat atgtatcc 5700 cagtcgggca agtcaccaat tatggtttcc taaacctagg tggcacacca actcaccgca 5760 ttttgatgta taactttcca acaagagctg gtcagtgtgg tggtgtggtg acaaccacag 5820 gtaaagtgat agggatacat gtaggtggaa atggagctca agggtttgca gcaatgctac 5880 tccattctta cttattgat acacaaggtg aaatagttag caatgagaaa agtggggtgt 5940 gtattaacgc accggcaaag accaaacttc aacccagtgt cttccatcaa gtttttgaag 6000 gttcaaagga accagcagtt ctcaattcaa aagatcctag gcttaagaca gattttgagg 6060 aagctatctt ttcaaaatat acaggtaata aaattatgtt aatggatgaa tacatggaag 6120 aagcagtgga tcattatgta ggatgtttag aaccattaga tattagtata gatcctatac 6180 cccttgaaag tgccatgtat gggatggatg gtctcgaggc attagactta accaccagtg 6240 cagggttccc ctacttgctg caagggaaga agaaaaggga tatattcaac agacaaacca 6300 gggacaccac tgaaatgaca aggatgttag aaaaatatgg agttgatcta ccttttgtaa 6360 cttttgtaaa agatgaactt agatcaagag aaaaagttga aaaaggaaag tcacgcttaa 6420 ttgaagccag ttccttgaat gattcagttg ctatgagggt tgcttttgga aatctttacg 6480 ctacattcca taacaatcca ggtacagcaa ctggaagtgc agttggttgt gatccagata 6540 tattctggtc aaaaatccct attttgctag atggagaaat ctttgctttt gattacactg 6600 gttatgatgc cagcttatca ccagtatggt ttgcctgttt aaagaaagtt ttaattaaat 6660 taggttacac ccatcagaca tctttcatag attatctgtg tcactcggta catttgtaca 6720 aggacagaaa atatatagtc aatggtggaa tgccctctgg ttcttcaggc accagcatat 6780 tcaatactat gatcaataat ataatcatga ggactttgtt gattagggtt tataaaggca 6840 tagacttaga ccaattcaag atgatagcat atggagatga tgttattgct agttatccac 6900 acaagattga tccaggttta ctagcagaag caggcaaaca ttatggatta ctaatgacac 6960 cagcagacaa aggaaccagc tttgttgata ctaattggga aaatgtaact ttcctgaaaa 7020 gatactttag agcagatcag caatatccct ttcttataca tccagtaatg ccaatgaagg 7080 agatacatga gtccattaga tggactaaag accccagaaa tacacaggat catgttagat 7140 cttgtgtta tctcgcatgg cataacgggg aggaggctta caatgaattt tgtagaaaaa 7200 ttagaagtgt gcctgtggga agggcattga cactacctgc atactctagt cttagacgaa 7260 aatggttaga ttcgttctag ataactctaa ttgaaaccca agttgtagtt actttcattt 7320 agaggtaaat tttggtcact tgggggcc 7348 <210> 2 <211> 6570 <212> DNA <213> Nucleotide sequence encoding the polyprotein of US / KY / 14-18953-Vero <400> 2 atgggggctc aagttactag acagcaaacc ggaacccatg agaacgccaa cattgccaca 60 aatggatccc atatcacata caaccagata aatttttaca aagatagtta tgcggcttca 120 gctagtaaac aagatttttc tcaggaccca tcaaaattta ctgaaccagt ggtagaaggc 180 ttaaaagcag gggctccagt tttgaaatcc cctagtgctg aggcatgtgg ctacagtgat 240 agagtattac aactcaaatt aggtaattcg gctattgtca ctcaggaagc agcgaactat 300 tgttgcgctt atggtgaatg gcccaattat ttaccagatc acgaggcagt agctattgac 360 aaacccacac aaccagaaac tgctacggac agattctata ccttgaaatc ggtcaaatgg 420 gaaactgaaa gtacaggatg gtggtggaaa ttacctgatg cactaaacaa cataggtatg 480 tttggacaaa atgtacagca tcactactta tataggtctg gtttcttaat tcatgtgcag 540 tgtaacgcca caaattcca tcaggtgcc ctattagtgg tagcaatccc agaacaccaa 600 aggggggcgc acacac tactaccca ggttttgatg atatcatgaa gggtgaagaa 660 ggagggacat ttaccaccc atatgtcctt gatgatggga cgtcactagc ctgcgcaaca 720 atatttccac accagtggat aaatttgaga accaatatt cagcaacaat agttcttccc 780 tggatgaatg ccgctccaat ggactccca ctcaggcaca atcagtggac actagcaata 840 attccagtag taccactagg tacgcgcaca gtgtcaagta tggttccaat aacagttctct 900 attgctccaa tgtgttgtga gtttaatgga ctcagacacg ccattactca aggtgtcca 960 acataccttc taccaggctc aggacaattt actaactactg atgatcacag ctctgcgccg 1020 cttcttccat gcttcaaccc aactccagaa atgcacattc caggcaagt ccgcaacatg 1080 ctagaagtga ttcaggtgga atcaatgatg gagattaata acacagaaaa tgcagttggc 1140 atgcagcgtc tcaagttga tatatcagta ccacagacg ttgatcatt gttattcaac 1200 atcccactag acatacagtt agatgggcca cttagaaca cttagtagg aaacatatct 1260 agatattata ctcactggtc tggatctcta gaaatgacat tttgttttg tgggagcttc 1320 atggcaacag gaaaattaat tctgtgttac actcctccag gcgggtcatg cccaacaacc 1380 agagaaactg ctatgttagg tacacatatt gtttgggatt ttggattaca atccagtgtc 1440 actctggtaa taccttggat cagtggatcc cactacagga tgttcaacaa tgatgctaag 1500 tcaaccaatg ccaatgttgg ctatgttacc tgttttatgc aaaccaattt aatagttccc 1560 agtgagtctt ctaacacatg ttccttaata gggttcgtag cagcaaaaga tgactttttcc 1620 ctcaggctaa tgagagatag ccctgacatt ggacaactag aacatttaca tgaggcagag 1680 gcagcctacc agattgagag catcatcaag acagcaactg acaccgtgaa aagtgagatt 1740 aacgctgaac ttggcgtggt ccctagttg aatgcagttg aaacaggtgc aagttctaac 1800 actgaaccag aggaagccat acaaactcgc acggtgataa atcagcatgg tgtatctgaa 1860 acattagtgg agaatttct tagcagggca gccttagtat caaagagaag tttcgaatac 1920 aaaaatcaca cctcatctgg agcacgaaca gacaagaact tttataaatg gacaattaac 1980 accaagtcct ttgtccagtt gaggagaaag ctggaactgt tcacgtacct tagatttgat 2040 gctgaaatta ccatacttac aactgtggca gtaggtagca acaacagcac atacaagggc 2100 cttcctgact tgacacttca agcaatgttt gtacctactg gtgcccttac tccaaaaaaa 2160 caggattcat tccattggca gtccggtagt aatgctagtg tattcttcaa agtttctgac 2220 cccccagcca gaatgactat acctttatg tgtatcaact cagcatactc agttttctat 2280 gatggttttg ctggatttga aaagaatggt ctttatggga taaatccagc tgataccatc 2340 ggcaacttat gtgtcagaat agtgaatgag caccagccaa ttggtttcac agtgaccgtc 2400 agggtttaca tgaagcctaa acacataaaa gcgtgggcac ccagaccacc acgaaccctg 2460 ccatatatga gcattgcaaa tgcaaactat aagggtaaag aaagggcacc aaatgcgctc 2520 aatgccataa ttggcaatag agagagtgtc aaaaccatgc ctcatgatat ccggttagtg 2580 aatactggtc caggcttcgg aggggtcttt gtaggatctt ttaaataat taactaccac 2640 ttagccacta tagaggaaag acagtcagct atctatgtag actggcaatc agatgttctg 2700 gttactccca ttgctgccca tggaagacac caaatagcaa gatgtaagtg caatacaggg 2760 gtttattatt gcagacacag agataagagc tacccagttt gttttgaagg cccagggatt 2820 caatggattg aacaaaatga atattaccca gcaaggtacc agactaatgt gcttctagca 2880 gctggtcctg cagaagcagg agattgtggt ggtttactgg tctgtccaca tggggtaatt 2940 ggtcttctca cagcaggggg gggtggaatt gtagctttca ctgatatcag gaatttacta 3000 tggttagata ctgatgctat ggaacaaggc attactgatt acattcaaaa tctcggtaat 3060 gcctttggag cggggtttac agaaacaatc tctagtaaag ctaaggaagt gcaagacatg 3120 ttgattggag agagttcact attagagaaa ttgttaaaaag ctctaatcaa aattatctca 3180 gcattggtga ttgtaattag aaattcagaa gatttaatta cagtcacagc cacactagca 3240 ttgctaggtt gccatgattc accatggagc tatttgaaac ataaagtgtg ctcatattta 3300 ggtattcctt atgtacctag acagagtgaa tcatggctca agaagttcac agaagcatgc 3360 aatgctctca ggggtttaga ttggttatca cagaagatag ataaattcat caactggctt 3420 aaaaaaaaaa tattaccaga gggctagggag aaatatgaat ttgtacaag actcaacag 3480 ttaccagtca tagagaacca agttaagcact attgaacata gctgcccaac aacagaaca 3540 caacaggcct tgtttaacaa tgttcaat tactcacact attgtagaaa gtatgcacca 3600 ctttacgcag tggaggcaa gagagtgaca gctcttgaaa agaaaataa taactacatc 3660 cagttcaagt ccaaatctcg cattgaaccg gtttgtta taatacatgg ttccccaggg 3720 actggcaaat cagtagcttc aaatctgatt gccagggcta tcacagaaaa gttaggaggg 3780 gatgtttatt cttacccccc agacccaaaa tattttg ggtataaaca gcaacagta 3840 gtccttatgg atgatttaat gcaaatcca gatggaaacg acatactct gttttgccaa 3900 atggtttcca ccgtggactt tatacctcca atggccagtt tagagaaaa gggaactcta 3960 tacaccagtc cattttgat agccactact aatgctggct caatacatgc accaccgtc 4020 tcagactcca aggctttgtc acgcagattt aaatttgatg tgaacattga ggtcacagat 4080 tcatacaaag actcaaataa attggacatg tcacagcag tggagatgtg tcaaccagat 4140 gactgtgctc ccattaatta taaaagatgc tgcccattga tttgtggaaa ggccattcaa 4200 tttagagatc gtagatctaa tgcaagatcc actatagata tgttagtaac tgacattatt 4260 aaggagtata gaatcagaaa cagtacacaa gataaactgg aggctctgtt ccagggacct 4320 ccacagttta aagagatcaa aatttcagtt actccagaca caccagctcc tgatgctata 4380 aacgatcttc tcaggtcagt ggattctcaa gaagttaggg actattgcca aaagaaaggg 4440 tggattgtaa tacacccatc aaatgaatta ttagtggaaa aacacattag cagggctttt 4500 atcactctac aggccgttgc tacctttgta tcaatagcag gtgtagttta tgttatatat 4560 aaacttttg ctggtatcca aggcccttac acaggaattc ccaatcctaa acctaaggta 4620 ccctcttta gaacagctaa agtgcagggg ccagggtttg attttgcaca agctataatg 4680 aagaaaaaca ccgtcattgc caggactgaa aagggtgagt tcaccatgtt gggtatacat 4740 gatagagtgg cagttatccc cacacatgca tctgttggag aaaccattta catcaacgat 4800 gtagagacta aagttttaga tgcatgtgct ctcagagact tgactgacac aaatttagag 4860 attaccatag tcaaactgga tcgtaaccaa aagtttagag atatcagaca ttttctgccc 4920 agatatgagg atgattataa tgatgctgtg cttagcgtgc atacatcaaa atttccaaat 4980 atgtatatcc cagtcgggca agtcaccaat tatggttttcc taaacctagg tggcacacca 5040 actcaccgca ttttgatgta taactttcca acaagagctg gtcagtgtgg tggtgtggtg 5100 acaaccacag gtaaagtgat agggatacat gtaggtggaa atggagctca agggtttgca 5160 gcaatgctac tccattctta cttattgat acacaaggtg aaatagttag caatgagaaa 5220 agtggggtgt gtattaacgc accggcaaag accaaacttc aacccagtgt cttccatcaa 5280 gttttgaag gttcaaagga accagcagtt ctcaattcaa aagatcctag gcttaagaca 5340 gattttgagg aagctatctt ttcaaaatat acaggtaata aaattatgtt aatggatgaa 5400 tacatggaag aagcagtgga tcattatgta ggatgtttag aaccattaga tattagtata 5460 gatcctatac cccttgaaag tgccatgtat gggatggatg gtctcgaggc attagactta 5520 accaccagtg cagggttccc ctacttgctg caagggaaga agaaaaggga tatattcaac 5580 agacaaacca gggacaccac tgaaatgaca aggatgttag aaaaatatgg agttgatcta 5640 ccttttgtaa cttttgtaaa agatgaactt agatcaagg aaaagttga aaaaggaag 5700 tcacgcttaa tgagccag ttccttgaat gattcagttg ctatgaggt tgcttttgga 5760 aatctttacg ctacattcca taacaatcca ggtacagcaa ctggaagtgc agttggttgt 5820 gatccagata tattctggtc aaaaatccct atttgctag atggagaat ctttgctttt 5880 gattacactg gttatgatgc cagcttatca ccagtatggt ttgctgttt aaagaaagtt 5940 ttaattaat taggttacac ccatcagaca tcttcatag attatctgtg tcactcggta 6000 cattgtaca aggacagaaa atatatagtc aatggtggaa tgccctgg ttctcaggc 6060 accagcatat tcaatactat gatcataat ataatcatga ggactttgtt gattaggtt 6120 tataaaggca tagacttaga ccaattcaag atgatagcat atggagatga tgttattgct 6180 agttatccac acagattga tccaggttta ctagcagaag caggcaaca tttaggatta 6240 ctaatgacac cagcagacaa aggaaccagc ttgttgata ctaatttggga aaatgtaact 6300 ttcctgaaaa gatactttag agcagatcag caatatccct ttcttataca tccagtaatg 6360 ccaatgaagg agatacatga gtccattaga tggactaaag accccagaaa tacacaggat 6420 catgttagat ctttgtgtta tctcgcatgg cataacgggg aggaggctta caatgaattt 6480 tgtagaaaaa ttagaagtgt gcctgtggga agggcattga cactacctgc atactctagt 6540 cttagacgaa aatggttaga ttcgttctag 6570 <210> 3 <211> 2189 <212> PRT <213> Amino acid sequence of the polyprotein of US / KY / 14-18953-Vero <400> 3 Met Gly Ala Gln Val Thr Arg Gln Gln Thr Gly Thr His Glu Asn Ala 1 5 10 15 Asn Ile Ala Thr Asn Gly Ser His Ile Thr Tyr Asn Gln Ile Asn Phe 20 25 30 Tyr Lys Asp Ser Tyr Ala Ala Ser Ala Ser Lys Gln Asp Phe Ser Gln 35 40 45 Asp Pro Ser Lys Phe Thr Glu Pro Val Val Glu Gly Leu Lys Ala Gly 50 55 60 Ala Pro Val Leu Lys Ser Pro Ser Ala Glu Ala Cys Gly Tyr Ser Asp 65 70 75 80 Arg Val Leu Gln Leu Lys Leu Gly Asn Ser Ala Ile Val Thr Gln Glu 85 90 95 Ala Ala Asn Tyr Cys Cys Ala Tyr Gly Glu Trp Pro Asn Tyr Leu Pro 100 105 110 Asp His Glu Ala Val Ala Ile Asp Lys Pro Thr Gln Pro Glu Thr Ala 115 120 125 Thr Asp Arg Phe Tyr Thr Leu Lys Ser Val Lys Trp Glu Thr Glu Ser 130 135 140 Thr Gly Trp Trp Trp Lys Leu Pro Asp Ala Leu Asn Asn Ile Gly Met 145 150 155 160 Phe Gly Gln Asn Val Gln His His Tyr Leu Tyr Arg Ser Gly Phe Leu 165 170 175 Ile His Val Gln Cys Asn Ala Thr Lys Phe His Gln Gly Ala Leu Leu 180 185 190 Val Val Ala Ile Pro Glu His Gln Arg Gly Ala His Asn Thr Thr Thr 195 200 205 Ser Pro Gly Phe Asp Asp Ile Met Lys Gly Glu Glu Gly Gly Thr Phe 210 215 220 Asn His Pro Tyr Val Leu Asp Asp Gly Thr Ser Leu Ala Cys Ala Thr 225 230 235 240 Ile Phe Pro His Gln Trp Ile Asn Leu Arg Thr Asn Asn Ser Ala Thr 245 250 255 Ile Val Leu Pro Trp Met Asn Ala Ala Pro Met Asp Phe Pro Leu Arg 260 265 270 His Asn Gln Trp Thr Leu Ala Ile Ile Pro Val Val Pro Leu Gly Thr 275 280 285 Arg Thr Val Ser Ser Met Val Pro Ile Thr Val Ser Ile Ala Pro Met 290 295 300 Cys Cys Glu Phe Asn Gly Leu Arg His Ala Ile Thr Gln Gly Val Pro 305 310 315 320 Thr Tyr Leu Leu Pro Gly Ser Gly Gln Phe Leu Thr Thr Asp Asp His 325 330 335 Ser Ser Ala Pro Leu Leu Pro Cys Phe Asn Pro Thr Pro Glu Met His 340 345 350 Ile Pro Gly Gln Val Arg Asn Met Leu Glu Val Ile Gln Val Glu Ser 355 360 365 Met Met Glu Ile Asn Asn Thr Glu Asn Ala Val Gly Met Gln Arg Leu 370 375 380 Lys Val Asp Ile Ser Val Leu Thr Asp Val Asp Gln Leu Leu Phe Asn 385 390 395 400 Ile Pro Leu Asp Ile Gln Leu Asp Gly Pro Leu Arg Asn Thr Leu Val 405 410 415 Gly Asn Ile Ser Arg Tyr Tyr Thr His Trp Ser Gly Ser Leu Glu Met 420 425 430 Thr Phe Met Phe Cys Gly Ser Phe Met Ala Thr Gly Lys Leu Ile Leu 435 440 445 Cys Tyr Thr Pro Pro Gly Gly Ser Cys Pro Thr Thr Arg Glu Thr Ala 450 455 460 Met Leu Gly Thr His Ile Val Trp Asp Phe Gly Leu Gln Ser Ser Val 465 470 475 480 Thr Leu Val Ile Pro Trp Ile Ser Gly Ser His Tyr Arg Met Phe Asn 485 490 495 Asn Asp Ala Lys Ser Thr Asn Ala Asn Val Gly Tyr Val Thr Cys Phe 500 505 510 Met Gln Thr Asn Leu Ile Val Pro Ser Glu Ser Ser Asn Thr Cys Ser 515 520 525 Leu Ile Gly Phe Val Ala Ala Lys Asp Asp Phe Ser Leu Arg Leu Met 530 535 540 Arg Asp Ser Pro Asp Ile Gly Gln Leu Glu His Leu His Glu Ala Glu 545 550 555 560 Ala Ala Tyr Gln Ile Glu Ser Ile Ile Lys Thr Ala Thr Asp Thr Val 565 570 575 Lys Ser Glu Ile Asn Ala Glu Leu Gly Val Val Pro Ser Leu Asn Ala 580 585 590 Val Glu Thr Gly Ala Ser Ser Asn Thr Glu Pro Glu Glu Ala Ile Gln 595 600 605 Thr Arg Thr Val Ile Asn Gln His Gly Val Ser Glu Thr Leu Val Glu 610 615 620 Asn Phe Leu Ser Arg Ala Ala Leu Val Ser Lys Arg Ser Phe Glu Tyr 625 630 635 640 Lys Asn His Thr Ser Ser Gly Ala Arg Thr Asp Lys Asn Phe Tyr Lys 645 650 655 Trp Thr Ile Asn Thr Lys Ser Phe Val Gln Leu Arg Arg Lys Leu Glu 660 665 670 Leu Phe Thr Tyr Leu Arg Phe Asp Ala Glu Ile Thr Ile Leu Thr Thr 675 680 685 Val Ala Val Gly Ser Asn Asn Ser Thr Tyr Lys Gly Leu Pro Asp Leu 690 695 700 Thr Leu Gln Ala Met Phe Val Pro Thr Gly Ala Leu Thr Pro Lys Lys 705 710 715 720 Gln Asp Ser Phe His Trp Gln Ser Gly Ser Asn Ala Ser Val Phe Phe 725 730 735 Lys Val Ser Asp Pro Pro Ala Arg Met Thr Ile Pro Phe Met Cys Ile 740 745 750 Asn Ser Ala Tyr Ser Val Phe Tyr Asp Gly Phe Ala Gly Phe Glu Lys 755 760 765 Asn Gly Leu Tyr Gly Ile Asn Pro Ala Asp Thr Ile Gly Asn Leu Cys 770 775 780 Val Arg Ile Val Asn Glu His Gln Pro Ile Gly Phe Thr Val Thr Val 785 790 795 800 Arg Val Tyr Met Lys Pro Lys His Ile Lys Ala Trp Ala Pro Arg Pro 805 810 815 Pro Arg Thr Leu Pro Tyr Met Ser Ile Ala Asn Ala Asn Tyr Lys Gly 820 825 830 Lys Glu Arg Ala Pro Asn Ala Leu Asn Ala Ile Ile Gly Asn Arg Glu 835 840 845 Ser Val Lys Thr Met Pro His Asp Ile Arg Leu Val Asn Thr Gly Pro 850 855 860 Gly Phe Gly Gly Val Phe Val Gly Ser Phe Lys Ile Ile Asn Tyr His 865 870 875 880 Leu Ala Thr Ile Glu Glu Arg Gln Ser Ala Ile Tyr Val Asp Trp Gln 885 890 895 Ser Asp Val Leu Val Thr Pro Ile Ala Ala His Gly Arg His Gln Ile 900 905 910 Ala Arg Cys Lys Cys Asn Thr Gly Val Tyr Tyr Cys Arg His Arg Asp 915 920 925 Lys Ser Tyr Pro Val Cys Phe Glu Gly Pro Gly Ile Gln Trp Ile Glu 930 935 940 Gln Asn Glu Tyr Tyr Pro Ala Arg Tyr Gln Thr Asn Val Leu Leu Ala 945 950 955 960 Ala Gly Pro Ala Glu Ala Gly Asp Cys Gly Gly Leu Leu Val Cys Pro 965 970 975 His Gly Val Ile Gly Leu Leu Thr Ala Gly Gly Gly Gly Ile Val Ala 980 985 990 Phe Thr Asp Ile Arg Asn Leu Leu Trp Leu Asp Thr Asp Ala Met Glu 995 1000 1005 Gln Gly Ile Thr Asp Tyr Ile Gln Asn Leu Gly Asn Ala Phe Gly 1010 1015 1020 Ala Gly Phe Thr Glu Thr Ile Ser Ser Lys Ala Lys Glu Val Gln 1025 1030 1035 Asp Met Leu Ile Gly Glu Ser Ser Leu Leu Glu Lys Leu Leu Lys 1040 1045 1050 Ala Leu Ile Lys Ile Ile Ser Ala Leu Val Ile Val Ile Arg Asn 1055 1060 1065 Ser Glu Asp Leu Ile Thr Val Thr Ala Thr Leu Ala Leu Leu Gly 1070 1075 1080 Cys His Asp Ser Pro Trp Ser Tyr Leu Lys His Lys Val Cys Ser 1085 1090 1095 Tyr Leu Gly Ile Pro Tyr Val Pro Arg Gln Ser Glu Ser Trp Leu 1100 1105 1110 Lys Lys Phe Thr Glu Ala Cys Asn Ala Leu Arg Gly Leu Asp Trp 1115 1120 1125 Leu Ser Gln Lys Ile Asp Lys Phe Ile Asn Trp Leu Lys Asn Lys 1130 1135 1140 Ile Leu Pro Glu Ala Arg Glu Lys Tyr Glu Phe Val Gln Arg Leu 1145 1150 1155 Lys Gln Leu Pro Val Ile Glu Asn Gln Val Ser Thr Ile Glu His 1160 1165 1170 Ser Cys Pro Thr Thr Glu Gln Gln Gln Ala Leu Phe Asn Asn Val 1175 1180 1185 Gln Tyr Tyr Ser His Tyr Cys Arg Lys Tyr Ala Pro Leu Tyr Ala 1190 1195 1200 Val Glu Ala Lys Arg Val Thr Ala Leu Glu Lys Lys Ile Asn Asn 1205 1210 1215 Tyr Ile Gln Phe Lys Ser Lys Ser Arg Ile Glu Pro Val Cys Leu 1220 1225 1230 Ile Ile His Gly Ser Pro Gly Thr Gly Lys Ser Val Ala Ser Asn 1235 1240 1245 Leu Ile Ala Arg Ala Ile Thr Glu Lys Leu Gly Gly Asp Val Tyr 1250 1255 1260 Ser Leu Pro Pro Asp Pro Lys Tyr Phe Asp Gly Tyr Lys Gln Gln 1265 1270 1275 Thr Val Val Leu Met Asp Asp Leu Met Gln Asn Pro Asp Gly Asn 1280 1285 1290 Asp Ile Ser Met Phe Cys Gln Met Val Ser Thr Val Asp Phe Ile 1295 1300 1305 Pro Pro Met Ala Ser Leu Glu Glu Lys Gly Thr Leu Tyr Thr Ser 1310 1315 1320 Pro Phe Leu Ile Ala Thr Thr Asn Ala Gly Ser Ile His Ala Pro 1325 1330 1335 Thr Val Ser Asp Ser Lys Ala Leu Ser Arg Arg Phe Lys Phe Asp 1340 1345 1350 Val Asn Ile Glu Val Thr Asp Ser Tyr Lys Asp Ser Asn Lys Leu 1355 1360 1365 Asp Met Ser Arg Ala Val Glu Met Cys Lys Pro Asp Asp Cys Ala 1370 1375 1380 Pro Ile Asn Tyr Lys Arg Cys Cys Pro Leu Ile Cys Gly Lys Ala 1385 1390 1395 Ile Gln Phe Arg Asp Arg Arg Ser Asn Ala Arg Ser Thr Ile Asp 1400 1405 1410 Met Leu Val Thr Asp Ile Ile Lys Glu Tyr Arg Ile Arg Asn Ser 1415 1420 1425 Thr Gln Asp Lys Leu Glu Ala Leu Phe Gln Gly Pro Pro Gln Phe 1430 1435 1440 Lys Glu Ile Lys Ile Ser Val Thr Pro Asp Thr Pro Ala Pro Asp 1445 1450 1455 Ala Ile Asn Asp Leu Leu Arg Ser Val Asp Ser Gln Glu Val Arg 1460 1465 1470 Asp Tyr Cys Gln Lys Lys Gly Trp Ile Val Ile His Pro Ser Asn 1475 1480 1485 Glu Leu Leu Val Glu Lys His Ile Ser Arg Ala Phe Ile Thr Leu 1490 1495 1500 Gln Ala Val Ala Thr Phe Val Ser Ile Ala Gly Val Val Tyr Val 1505 1510 1515 Ile Tyr Lys Leu Phe Ala Gly Ile Gln Gly Pro Tyr Thr Gly Ile 1520 1525 1530 Pro Asn Pro Lys Pro Lys Val Pro Ser Leu Arg Thr Ala Lys Val 1535 1540 1545 Gln Gly Pro Gly Phe Asp Phe Ala Gln Ala Ile Met Lys Lys Asn 1550 1555 1560 Thr Val Ile Ala Arg Thr Glu Lys Gly Glu Phe Thr Met Leu Gly 1565 1570 1575 Ile His Asp Arg Val Ala Val Ile Pro Thr His Ala Ser Val Gly 1580 1585 1590 Glu Thr Ile Tyr Ile Asn Asp Val Glu Thr Lys Val Leu Asp Ala 1595 1600 1605 Cys Ala Leu Arg Asp Leu Thr Asp Thr Asn Leu Glu Ile Thr Ile 1610 1615 1620 Val Lys Leu Asp Arg Asn Gln Lys Phe Arg Asp Ile Arg His Phe 1625 1630 1635 Leu Pro Arg Tyr Glu Asp Asp Tyr Asn Asp Ala Val Leu Ser Val 1640 1645 1650 His Thr Ser Lys Phe Pro Asn Met Tyr Ile Pro Val Gly Gln Val 1655 1660 1665 Thr Asn Tyr Gly Phe Leu Asn Leu Gly Gly Thr Pro Thr His Arg 1670 1675 1680 Ile Leu Met Tyr Asn Phe Pro Thr Arg Ala Gly Gln Cys Gly Gly 1685 1690 1695 Val Val Thr Thr Thr Gly Lys Val Ile Gly Ile His Val Gly Gly 1700 1705 1710 Asn Gly Ala Gln Gly Phe Ala Ala Met Leu Leu His Ser Tyr Phe 1715 1720 1725 Ile Asp Thr Gln Gly Glu Ile Val Ser Asn Glu Lys Ser Gly Val 1730 1735 1740 Cys Ile Asn Ala Pro Ala Lys Thr Lys Leu Gln Pro Ser Val Phe 1745 1750 1755 His Gln Val Phe Glu Gly Ser Lys Glu Pro Ala Val Leu Asn Ser 1760 1765 1770 Lys Asp Pro Arg Leu Lys Thr Asp Phe Glu Glu Ala Ile Phe Ser 1775 1780 1785 Lys Tyr Thr Gly Asn Lys Ile Met Leu Met Asp Glu Tyr Met Glu 1790 1795 1800 Glu Ala Val Asp His Tyr Val Gly Cys Leu Glu Pro Leu Asp Ile 1805 1810 1815 Ser Ile Asp Pro Ile Pro Leu Glu Ser Ala Met Tyr Gly Met Asp 1820 1825 1830 Gly Leu Glu Ala Leu Asp Leu Thr Thr Ser Ala Gly Phe Pro Tyr 1835 1840 1845 Leu Leu Gln Gly Lys Lys Lys Arg Asp Ile Phe Asn Arg Gln Thr 1850 1855 1860 Arg Asp Thr Thr Glu Met Thr Arg Met Leu Glu Lys Tyr Gly Val 1865 1870 1875 Asp Leu Pro Phe Val Thr Phe Val Lys Asp Glu Leu Arg Ser Arg 1880 1885 1890 Glu Lys Val Glu Lys Gly Lys Ser Arg Leu Ile Glu Ala Ser Ser 1895 1900 1905 Leu Asn Asp Ser Val Ala Met Arg Val Ala Phe Gly Asn Leu Tyr 1910 1915 1920 Ala Thr Phe His Asn Asn Pro Gly Thr Ala Thr Gly Ser Ala Val 1925 1930 1935 Gly Cys Asp Pro Asp Ile Phe Trp Ser Lys Ile Pro Ile Leu Leu 1940 1945 1950 Asp Gly Glu Ile Phe Ala Phe Asp Tyr Thr Gly Tyr Asp Ala Ser 1955 1960 1965 Leu Ser Pro Val Trp Phe Ala Cys Leu Lys Lys Val Leu Ile Lys 1970 1975 1980 Leu Gly Tyr Thr His Gln Thr Ser Phe Ile Asp Tyr Leu Cys His 1985 1990 1995 Ser Val His Leu Tyr Lys Asp Arg Lys Tyr Ile Val Asn Gly Gly 2000 2005 2010 Met Pro Ser Gly Ser Ser Gly Thr Ser Ile Phe Asn Thr Met Ile 2015 2020 2025 Asn Asn Ile Ile Met Arg Thr Leu Leu Ile Arg Val Tyr Lys Gly 2030 2035 2040 Ile Asp Leu Asp Gln Phe Lys Met Ile Ala Tyr Gly Asp Asp Val 2045 2050 2055 Ile Ala Ser Tyr Pro His Lys Ile Asp Pro Gly Leu Leu Ala Glu 2060 2065 2070 Ala Gly Lys His Tyr Gly Leu Leu Met Thr Pro Ala Asp Lys Gly 2075 2080 2085 Thr Ser Phe Val Asp Thr Asn Trp Glu Asn Val Thr Phe Leu Lys 2090 2095 2100 Arg Tyr Phe Arg Ala Asp Gln Gln Tyr Pro Phe Leu Ile His Pro 2105 2110 2115 Val Met Pro Met Lys Glu Ile His Glu Ser Ile Arg Trp Thr Lys 2120 2125 2130 Asp Pro Arg Asn Thr Gln Asp His Val Arg Ser Leu Cys Tyr Leu 2135 2140 2145 Ala Trp His Asn Gly Glu Glu Ala Tyr Asn Glu Phe Cys Arg Lys 2150 2155 2160 Ile Arg Ser Val Pro Val Gly Arg Ala Leu Thr Leu Pro Ala Tyr 2165 2170 2175 Ser Ser Leu Arg Arg Lys Trp Leu Asp Ser Phe 2180 2185 <210> 4 <211> 207 <212> DNA <213> Complementary DNA sequence of VP4 protein of US / KY / 14-18953-Vero <400> 4 atgggggctc aagttactag acagcaaacc ggaacccatg agaacgccaa cattgccaca 60 aatggatccc atatcacata caaccagata aatttttaca aagatagtta tgcggcttca 120 gctagtaaac aagatttttc tcaggaccca tcaaaattta ctgaaccagt ggtagaaggc 180 ttaaaagcag gggctccagttttgaaa 207 <210> 5 <211> 69 <212> PRT <213> Amino acid sequence of US / KY / 14-18953-Vero VP4 protein <400> 5 Met Gly Ala Gln Val Thr Arg Gln Gln Thr Gly Thr His Glu Asn Ala 1 5 10 15 Asn Ile Ala Thr Asn Gly Ser His Ile Thr Tyr Asn Gln Ile Asn Phe 20 25 30 Tyr Lys Asp Ser Tyr Ala Ala Ser Ala Ser Lys Gln Asp Phe Ser Gln 35 40 45 Asp Pro Ser Lys Phe Thr Glu Pro Val Val Glu Gly Leu Lys Ala Gly 50 55 60 Ala Pro Val Leu Lys 65 <210> 6 <211> 744 <212> DNA <213> US / KY / 14-18953-Complementary DNA sequence of Vero VP2 protein <400> 6 tccctagtg ctgaggcatg tgctacagt gatagagtat tacactca attagtaat 60 tcggctattg tcactcagga agcagcgaac tattgttgcg cttatggtga atggcccaat 120 tattaccag atcacgaggc agtagctatt vakaaaccca cacaaccaga aactgctacg 180 gabagattct ataccttgaa atcggtcaa tgggaactg aaagtacagg atggtggtgg 240 aaattacctg atgcactaaa cacataggt atgtttggac aaatgtaca gcatcactac 300 ttatataggt ctggttctt aattcatgtg cagtgtaacg ccacaaattt ccatcaggt 360 gccctattag tggtagcaat cccacacac caagggggg cgcacacac cactactagc 420 ccaggttttg atgatatcat gaagggtgaa gaaggaggga catttaacca cccatatgtc 480 cttgatgatg ggacgtcact agcctgcgca acaatatttc cacaccagtg gataaatttg 540 agaaccaata attcagcac atagttctt ccctggatga atgccgctcc atggacttc 600 ccactcaggc acatcagtg vakactagca atattccag tagtaccact aggtacgcgc 660 acagtgtcaa gtatggttcc ataacagtt tctattgctc caatgtgttg tgagtttaat 720 ggactcagac acgccattac tcaa 744 <210> 7 <211> 248 <212> PRT <213> Amino acid sequence of VP2 protein of US / KY / 14-18953-Vero <400> 7 Ser Pro Ser Ala Glu Ala Cys Gly Tyr Ser Asp Arg Val Leu Gln Leu 1 5 10 15 Lys Leu Gly Asn Ser Ala Ile Val Thr Gln Glu Ala Ala Asn Tyr Cys 20 25 30 Cys Ala Tyr Gly Glu Trp Pro Asn Tyr Leu Pro Asp His Glu Ala Val 35 40 45 Ala Ile Asp Lys Pro Thr Gln Pro Glu Thr Ala Thr Asp Arg Phe Tyr 50 55 60 Thr Leu Lys Ser Val Lys Trp Glu Thr Glu Ser Thr Gly Trp Trp Trp 65 70 75 80 Lys Leu Pro Asp Ala Leu Asn Asn Ile Gly Met Phe Gly Gln Asn Val 85 90 95 Gln His His Tyr Leu Tyr Arg Ser Gly Phe Leu Ile His Val Gln Cys 100 105 110 Asn Ala Thr Lys Phe His Gln Gly Ala Leu Leu Val Val Ala Ile Pro 115 120 125 Glu His Gln Arg Gly Ala His Asn Thr Thr Thr Ser Pro Gly Phe Asp 130 135 140 Asp Ile Met Lys Gly Glu Glu Gly Gly Thr Phe Asn His Pro Tyr Val 145 150 155 160 Leu Asp Asp Gly Thr Ser Leu Ala Cys Ala Thr Ile Phe Pro His Gln 165 170 175 Trp Ile Asn Leu Arg Thr Asn Asn Ser Ala Thr Ile Val Leu Pro Trp 180 185 190 Met Asn Ala Ala Pro Met Asp Phe Pro Leu Arg His Asn Gln Trp Thr 195 200 205 Leu Ala Ile Ile Pro Val Val Pro Leu Gly Thr Arg Thr Val Ser Ser 210 215 220 Met Val Pro Ile Thr Val Ser Ile Ala Pro Met Cys Cys Glu Phe Asn 225 230 235 240 Gly Leu Arg His Ala Ile Thr Gln 245 <210> 8 <211> 705 <212> DNA <213> Complementary DNA sequence of VP3 protein of US / KY / 14-18953-Vero <400> 8 ggtgtcccaa cataccttct accaggctca ggacaatttc taactactga tgatcacagc 60 tctgcgccgc ttcttccatg cttcaaccca actccagaaa tgcacattc agggcaagtc 120 cgcaacatgc tagaagtgat tcaggtggaa tcaatgatgg agattaataa cacagaaaat 180 gcagttggca tgcagcgtct caaagttgat atatcagtac taacagacgt tgatcaattg 240 ttattcaaca tcccactaga catacagtta gatgggccac ttagaaacac tctagtagga 300 aacatatcta gatattatac tcactggtct ggatctctag aaatgacatt tatgttttgt 360 gggagcttca tggcaacagg aaaattaatt ctgtgttaca ctcctccagg cgggtcatgc 420 ccaacaacca gagaaactgc tatgttaggt acacatattg tttgggattt tggattacaa 480 tccagtgtca ctctggtaat accttggatc agtggatccc actacaggat gttcaacaat 540 gatgctaagt caaccaatgc caatgttggc tatgttacct gttttatgca aaccaattta 600 atagttccca gtgagtcttc taacacatgt tccttaatag ggttcgtagc agcaaaagat 660 gactttccc tcaggctaat gagagatagc cctgacattg gacaa 705 <210> 9 <211> 235 <212> PRT <213> US / KY / 14-18953-Vero VP3 Registered <400> 9 Gly Val Pro Thr Tyr Leu Leu Pro Gly Ser Gly Gln Phe Leu Thr Thr 1 5 10 15 Asp Asp His Ser Ser Ala Pro Leu Leu Pro Cys Phe Asn Pro Thr Pro 20 25 30 Glu Met His Ile Pro Gly Gln Val Arg Asn Met Leu Glu Val Ile Gln 35 40 45 Val Glu Ser Met Met Glu Ile Asn Asn Thr Glu Asn Ala Val Gly Met 50 55 60 Gln Arg Leu Lys Val Asp Ile Ser Val Leu Thr Asp Val Asp Gln Leu 65 70 75 80 Leu Phe Asn Ile Pro Leu Asp Ile Gln Leu Asp Gly Pro Leu Arg Asn 85 90 95 Thr Leu Val Gly Asn Ile Ser Arg Tyr Tyr Thr His Trp Ser Gly Ser 100 105 110 Leu Glu Met Thr Phe Met Phe Cys Gly Ser Phe Met Ala Thr Gly Lys 115 120 125 Leu Ile Leu Cys Tyr Thr Pro Pro Gly Gly Ser Cys Pro Thr Thr Arg 130 135 140 Glu Thr Ala Met Leu Gly Thr His Ile Val Trp Asp Phe Gly Leu Gln 145 150 155 160 Ser Ser Val Thr Leu Val Ile Pro Trp Ile Ser Gly Ser His Tyr Arg 165 170 175 Met Phe Asn Asn Asp Ala Lys Ser Thr Asn Ala Asn Val Gly Tyr Val 180 185 190 Thr Cys Phe Met Gln Thr Asn Leu Ile Val Pro Ser Glu Ser Ser Asn 195 200 205 Thr Cys Ser Leu Ile Gly Phe Val Ala Ala Lys Asp Asp Phe Ser Leu 210 215 220 Arg Leu Met Arg Asp Ser Pro Asp Ile Gly Gln 225 230 235 <210> 10 <211> 930 <212> DNA <213> Complementary DNA sequence of VP1 protein of US / KY / 14-18953-Vero <400> 10 ctagaacatt tacatgaggc agaggcagcc taccagattg agagcatcat caagacagca 60 actgacaccg tgaaaagtga gattaacgct gaacttggcg tggtccctag tttgaatgca 120 gttgaaacag gtgcaagttc taacactgaa ccagaggaag ccatacaaac tcgcacggtg 180 ataaatcagc atggtgtatc tgaaacatta gtggagaatt ttcttagcag ggcagcctta 240 gtatcaaaga gaagtttcga atacaaaaat cacacctcat ctggagcacg aacagacaag 300 aacttttata aatggacaat taacaccaag tcctttgtcc agttgaggag aaagctggaa 360 ctgttcacgt accttagatt tgatgctgaa attaccatac ttacaactgt ggcagtaggt 420 agcaacaaca gcacatacaa gggccttcct gacttgacac ttcaagcaat gtttgtacct 480 actggtgcccc ttactccaaa aaaacaggat tcattccatt ggcagtccgg tagtaatgct 540 agtgtattct tcaaagtttc tgacccccca gccagaatga ctataccttt tatgtgtatc 600 aactcagcat actcagtttt ctatgatggt tttgctggat ttgaaaagaa tggtctttat 660 gggataaatc cagctgatac catcggcaac ttatgtgtca gaatagtgaa tgagcaccag 720 ccaattggtt tcacagtgac cgtcagggtt tacatgaagc ctaaacacat aaaagcgtgg 780 gcacccagac caccacgaac cctgccatat atgagcattg caaatgcaaa ctataagggt 840 aaagaaaggg caccaaatgc gctcaatgcc ataattggca atagagagag tgtcaaaacc 900 atgcctcatg ataccggtt agtgaatact 930 <210> 11 <211> 310 <212> PRT <213> Amino acid sequence of VP1 protein of US / KY / 14-18953-Vero <400> 11 Leu Glu His Leu His Glu Ala Glu Ala Ala Tyr Gln Ile Glu Ser Ile 1 5 10 15 Ile Lys Thr Ala Thr Asp Thr Val Lys Ser Glu Ile Asn Ala Glu Leu 20 25 30 Gly Val Val Pro Ser Leu Asn Ala Val Glu Thr Gly Ala Ser Ser Asn 35 40 45 Thr Glu Pro Glu Glu Ala Ile Gln Thr Arg Thr Val Ile Asn Gln His 50 55 60 Gly Val Ser Glu Thr Leu Val Glu Asn Phe Leu Ser Arg Ala Ala Leu 65 70 75 80 Val Ser Lys Arg Ser Phe Glu Tyr Lys Asn His Thr Ser Ser Gly Ala 85 90 95 Arg Thr Asp Lys Asn Phe Tyr Lys Trp Thr Ile Asn Thr Lys Ser Phe 100 105 110 Val Gln Leu Arg Arg Lys Leu Glu Leu Phe Thr Tyr Leu Arg Phe Asp 115 120 125 Ala Glu Ile Thr Ile Leu Thr Thr Val Ala Val Gly Ser Asn Asn Ser 130 135 140 Thr Tyr Lys Gly Leu Pro Asp Leu Thr Leu Gln Ala Met Phe Val Pro 145 150 155 160 Thr Gly Ala Leu Thr Pro Lys Lys Gln Asp Ser Phe His Trp Gln Ser 165 170 175 Gly Ser Asn Ala Ser Val Phe Phe Lys Val Ser Asp Pro Pro Ala Arg 180 185 190 Met Thr Ile Pro Phe Met Cys Ile Asn Ser Ala Tyr Ser Val Phe Tyr 195 200 205 Asp Gly Phe Ala Gly Phe Glu Lys Asn Gly Leu Tyr Gly Ile Asn Pro 210 215 220 Ala Asp Thr Ile Gly Asn Leu Cys Val Arg Ile Val Asn Glu His Gln 225 230 235 240 Pro Ile Gly Phe Thr Val Thr Val Arg Val Tyr Met Lys Pro Lys His 245 250 255 Ile Lys Ala Trp Ala Pro Arg Pro Pro Arg Thr Leu Pro Tyr Met Ser 260 265 270 Ile Ala Asn Ala Asn Tyr Lys Gly Lys Glu Arg Ala Pro Asn Ala Leu 275 280 285 Asn Ala Ile Ile Gly Asn Arg Glu Ser Val Lys Thr Met Pro His Asp 290 295 300 Ile Arg Leu Val Asn Thr 305 310

Claims

1. An enterovirus D68 adapted to be propagated to high titers in Vero cells, wherein the enterovirus D68 is encoded by a cDNA molecule, the nucleotide sequence of which is SEQ ID No.

1.

2. A nucleic acid molecule encoding an enterovirus D68 polyprotein adapted to Vero cells, wherein the nucleotide sequence of the nucleic acid molecule is SEQ ID No.

2.

3. An enterovirus D68 polyprotein adapted to Vero cells, wherein the amino acid sequence of the polyprotein is SEQ ID No.

3.

4. A method for inactivating enterovirus D68 containing cDNA encoded by SEQ ID No. 1, comprising the following steps: (a) sterile filtering the harvested enterovirus D68 virus; (b) removal of host nucleic acids using nuclease treatment and concentration by tangential flow filtration using a 100 kDa filter; (c) inactivation using 1 / 2000-1 / 4000 formalin at temperatures varying from 25-37°C for up to 3 weeks or BPL at 4-25°C for up to 120 hours or 0.005% to 3% hydrogen peroxide at 25 ± 5°C for up to 6 hours; and (d) Purifying the inactivated antigen or purifying the inactivated viral antigen prior to inactivating the viral antigen by using chromatography, resins or a combination thereof.

5. The method of claim 4, wherein the purification is performed by gel filtration using Sepharose CL-4B followed by anion exchange chromatography using DEAE resin, or by two steps / two rounds of size exclusion chromatography, or by cellulose sulfate followed by anion exchange chromatography, or by mixed mode resins alone or in combination with other resins.

6. An immunogenic composition comprising the inactivated enterovirus D68 viral antigen obtained as claimed in claim 4 in a physiologically acceptable vehicle, and optionally one or more pharmaceutically acceptable excipients selected from adjuvants, stabilizers or preservatives.

7. The immunogenic composition of claim 6, wherein the composition is stable at 2-8°C for at least 6 months and at 37°C for at least 1 month.

8. The immunogenic composition of claim 6, wherein the composition further comprises inactivated other enteroviruses, the other enteroviruses comprising EV71, poliovirus, or a combination thereof.

9. An immunogenic composition comprising an inactivated enterovirus D68 viral antigen in a physiologically acceptable vehicle, wherein the enterovirus D68 is encoded by a cDNA molecule whose nucleotide sequence is SEQ ID No. 1.

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