WO2016145087A1

WO2016145087A1 - High-growth enterovirus 71 strains and vaccines

Info

Publication number: WO2016145087A1
Application number: PCT/US2016/021572
Authority: WO
Inventors: Min-Shi Lee
Original assignee: National Health Research Institutes
Priority date: 2015-03-09
Filing date: 2016-03-09
Publication date: 2016-09-15
Also published as: CN108055827B; TW201636424A; CN108055827A; MY188494A; TWI638048B

Abstract

The present invention relates to adapted Enterovirus 71 (EV71) vaccine strains suitable for increased vaccine production for mammals. The EV71 vaccine strains contain at least one modified enterovirus protein that results in increased production of the EV71 virus from a mammalian host cell, such as a Vero cell. The present invention also relates to the vaccines produced from the adapted EV71 virus vaccine strains, and the producing method of the vaccines.

Description

HIGH-GROWTH ENTEROVIRUS 71 STRAINS AND VACCINES

BACKGROUND OF THE INVENTION

Technical Field of the Invention

[0001 ] The present invention relates to an isolated Enterovirus 71 (EV71) vaccine strain. In particular, the present invention relates to an Enterovirus 71 (EV71) genotype B5 vaccine virus strain suitable for high growth in a mammalian host cell, such as Vero cell, and induction of cross-reactive neutralizing antibody titers against other EV71 genotypes.

Background

[0002] Enterovirus 71 (EV71) is a positive-sense RNA virus that belongs to the enterovirus genus in the family Picornaviridae along with other important human pathogens, such as polioviruses and rhinoviruses. EV71 was first isolated in 1969 in USA from a child with neurological disease, but a recent retrospective study suggested that EV71 was circulating in the Netherland as early as 1963 (Van Der Saden et al. 2009). The EV-71 genome encodes a long polyprotein with a single open reading frame followed by a poly A tract. The single polyprotein is flanked by untranslated regions at both the 5'and 3' ends and can be divided into three different genomic regions (PI, P2 and P3).

[0003 ] Based on phylogenetic analysis of the most variable VPl gene, EV71 could be classified into 3 major genogroups (A, B and C) including 11 genotypes (A, B1-B5, and C1-C5) by analyzing the most variable capsid protein sequences (VPl) (Solomon et al. 2010). Since 1997, different EV71 genotypes have caused life-threatening epidemics with severe neurologic complications in Asian countries, including Malaysia, Taiwan, Singapore, Brunei, Vietnam, Cambodia and China. Therefore, development of EV71 vaccines is a national priority in these countries.

[0004] Based on historical experiences with inactivated poliovirus vaccines (IPV), inactivated EV71 whole virus vaccines have been perceived as feasible by following the regulatory pathway of IPV. After the Bulgaria epidemic in 1975, an inactivated EV71 whole virus vaccine candidate was produced in Moscow using the similar manufacturing process of IPV and was evaluated in Bulgaria in 1976. This EV71 vaccine candidate was well tolerated and immunogenic in children 1-4 years of age. For practical reason of having no further outbreaks of EV71, the Bulgaria vaccine candidate was not further evaluated for its clinical efficacy, and no potency assay to quantify vaccine antigens had been developed (Huang et al. 2011).

[0005 ] With this background, the inactivated EV71 vaccine is the most likely candidate to be launched and indeed five vaccine candidates are evaluated in clinical trials. Currently, five vaccine candidates have been evaluated in clinical trials in China (3 candidates in phase three), Singapore (1 candidate in phase one), Taiwan (1 candidate in phase one), which are listed in Table 1.

Table 1. EV71 vaccine candidates in clinical trials

KMB-17 cell Inactivated virus

PUMC (China) Liang Z, et al. 2012

(C4) (viral protein)

National Health

Vero cell Inactivated virus

Research institutes Chou AH, et al. 2012

(B4) (total protein)

(Taiwan)

Inviragen/Takeda Vero cell Inactivated virus Hwa SH, et al. 2013

(Singapore) (B2) (total protein)

CNBG: China National Biotech Group; PUMC: Peking Union Medical College

[0006] Four of these five vaccine candidates were produced using Vero cells (a popular cell line for human vaccine production) but their peak virus titers could only reach above 10⁷ TCID50/ml (Chou et al. 2012; Liang et al. 2012). Overall, these 5 vaccine candidates could not grow very well (~10⁷ PFU/ml) in cells qualified for vaccine production. In addition, genotypes of these 5 candidates include B2 (Singapore), B4 (Taiwan), and three C4 (China) strains, which are different from the current predominant genotype B5 in Taiwan and South-Eastern Asia.

SUMMARY OF INVENTION

[0007] In this invention, an EV71 genotype B5 vaccine virus is isolated to be adapted to grow efficiently in Vero cells using serum-free medium.

[0008] Accordingly, in one aspect, the present invention relates to an isolated EV71 genotype B5 vaccine virus that is able to grow efficiently in a mammalian host cell and is suitable for increasing production of EV71 vaccine from the mammalian host cell.

[0009] In some embodiments, the isolated EV71 genotype B5 vaccine virus comprises at least one EV71 gene that encodes at least one modified enterovirus protein results in an increased production of the EV71 virus from a mammalian host cell. Preferably, the EV71 gene is originally isolated and/or identified from an EV71 genotype B5 virus, such as EV71 B5-141.

[0010] In certain embodiments, the isolated EV71 genotype B5 vaccine virus comprises at least one variation in a coding sequence of EV71 protein selected from P1-VP4, P1-VP1, P3-3A and P3-3D proteins.

[0011 In another aspect, the present invention relates to an EV71 vaccine. The vaccine comprises the isolated EV71 genotype B5 vaccine virus strain according to the invention and a pharmaceutically acceptable carrier.

[0012] In a further aspect, the present invention relates to a method of preparing the EV71 vaccine. Isolated nucleic acid molecules that encode a modified EV71 B5 protein and the isolated modified EV71 B5 proteins are also provided. Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and the appended claims.

BREIF DESCRIPTION OF THE DRAWINGS

[0013 ] Figure 1 shows a flowchart of adapting high-growth enterovirus 71 virus in Vero cells using serum-free medium (VP-SFM).

[0014] Figure 2 shows the plaque morphology of EV71 B5-141 and two Vero cell-adapted viruses. Figure 2A shows the results of plaque assay in 6-well plate of viruses EV71 B5-141, 4-2 and 6-5 on Vero cells in VP-SFM. Figure 2B shows the results of immune-plaque assay in 12-well plate of viruses EV71 B5-141, 4-2 and 6-5 on Vero cells in VP-SFM. DETAILED DESCRIPTION OF THE INVENTION

[0015 ] The other characteristics and advantages of the present invention will be further illustrated and described in the following examples. The examples described herein are using for illustrations, not for limitations of the invention.

[0016] As used herein, the term "modified strain" means a strain of a virus strain obtained by serial passages and/ or selection of the parental virus and its progenies in a mammalian host cell. In one embodiment of the present invention, the modified strain has increased virus production from the mammalian host cell compared to that of the parental virus strain.

[0017] As used herein, the term "enterovirus protein" refers to any polypeptide or protein encoded by an EV71 virus gene.

[0018] As used herein, an "EV71 virus gene" can be any gene originally isolated and/or identified from an EV71 virus. The EV71 virus gene can be a gene originally isolated and/or identified from any EV71 genotype B5 virus. The full length nucleotide sequence of the genome of EV71 B5-141 virus is listed in SEQ ID NO. 2.

[0019] As used herein, the term "modified enterovirus protein" refers to a protein that includes one or more modifications to the amino acid sequence of an unmodified or reference enterovirus protein. The modification includes, for example, an insertion, substitution, or deletion of one or more amino acid residues of an unmodified or reference enterovirus protein. Therefore, the modified enterovirus protein can have at least one modified amino acid residue and/ or codon occupying an identified location of an unmodified or reference protein. [ 0020 ] EXPERIMENTAL EXAMPLES

[ 0021 ] Adaptation of high-growth vaccine virus in Vero cells

[0022] Methods

[0023 ] Cells, culture medium and virus: Human rhabdomyosarcoma (RD) cell and African green monkey kidney (Vero) cells were used to grow enterovirus 71 following the standard procedures (Wu et al. 2001; Huang et al. 2010).

[0024] Virological assays: Virus infectious titers were measured using the 50% tissue culture infectious doses (TCID50) assay based on cytopathic effect (CPE) and the plaque assay based on plaque forming unit (PFU) in Vero and RD cells (Huang et al. 2010). A positive control with pre-specified acceptable range was included for conducting TCID50, and plaque assays.

[0025 ] Determination of virus growth curves in cell cultures: EV71 viruses were grown in 6-well plates and supernatant was harvested at day 1-6 post infection for measuring infectious virus titers. Potential of using the Veo cell-adapted EV71 virus strain as a vaccine seed virus was evaluated in a microcarrier-based cell culture system using serum-containing medium. Cytodex 1 microcarriers (GE Healthcare, USA) were hydrated, autoclaved, and preconditioned according to manufacturer's instruction. Growth curves of Vero cells with different culture conditions were tested in 100-ml spinner flasks (50 ml working volume) and microcarriers were sampled to count cell density every day. When the cells grew confluent on microcarriers, the EV71 viruses were added to infect cells.

[0026] Purification of virus particles: Ultracentrifugation was used to purify vaccine antigens. Purity of the vaccine antigens was evaluated using SDS-PAGE, Western Blot, and electron microscopy (Chia et al. 2014). [0027] Immunogenecity study in rabbits: Recently, we have developed a rabbit model which could induce cross-reactive neutralizing antibody profiles similar to those observed in EV71-infected young children (Chia M, et al 2014). The rabbit model was used to evaluate immunogenicity of Vero cell-adapted vaccine viruses.

[0028] Serologic assays: Laboratory methods for measuring EV71 serum neutralizing antibody titers followed standard protocols (Huang et al. 2010). Twofold serially diluted sera (1 :8 - 1 :512) and a virus working solution containing 100 TCID₅₀ of EV71 strain were mixed on 96-well microplates and incubated with rhabdomyosarcoma cells. A cytopathic effect was observed in a monitor linked with an inverted microscope after an incubation period of 4 to 5 days. The neutralization titers were read as the highest dilution that could result in a 50% reduction in the cytopathic effect. Each test sample was run simultaneously with cell control, serum control, and virus back titration. The starting dilution was 1 :8; the cutoff level of seropositivity was set at 8.

[0029] Results

[0030] As shown in Figure 1, the B5-141 virus was isolated from throat swab of a 20-month-old herpangina patient in 2008. The parental B5-141 virus could not cause cytopathic effect (CPE) and form a clear plaque in Vero cells. After 23 passages in Vero cells, the adapted B5-141 could form clear plaque and 2 plaques (B5-141-4, and B5-141-6) were selected in the 24^th (B5-141-4) and 25^th passage (B5-141-6) in Vero cells.

[0031 ] After another plaque selection and three passages, two virus strains (B5-141-4-2 and B5-141-6-5) were further selected to generate virus stocks (Figure 1). Comparing with the parental B5-141 virus, the B5-141-4-2 and B5-141-6-5 viruses could form very clear plaques and reach >10⁸ PFU/ml (Figure 2) (Table 2). Table 2. Virus titers of the parental and Vero cell-adapted B5-141 virus stocks amplified and measured in Vero cells

[0032] Growth efficiency of these two virus stocks were evaluated in Vero cells in 24-well plates using different multiplicities of infection (MOI). As shown in Table 3, peak virus titers of the B5-141-6-5 virus could reach over 10⁸ TCID50/ml under the MOI 0.001 and 0.0001.

Table 3. Daily virus titers of the Vero cell-adapted B5-141 viruses amplified in Vero cells using 24-well plates under different multiplicity of infection (MOI)

[0033 ] Therefore, growth efficiency of the B5-141-6-5 virus was further evaluated using microcarrier-based serum-free Vero cell culture systems in spinner flasks. As shown in Table 4, virus titers could reach about 10⁸ TCID50/ml in three runs, which confirm the commercial potential of the B5-141-6-5 virus.

Table 4. Production of the Vero cell-adapted B5-141-6-5 viruses using

microcarrier-based serum-free Vero cell culture systems in spinner flasks

[ 0034 ] Immunogenicity of EV71 B5-141-6-5 vaccine antigens in rabbits

[0035 ] EV71 B5-141-6-5 virus particles were purified using ultracentrifugation. Two types of virus particles, including complete and empty particles, were separated after ultracentrifugation and the complete virus particles were used to evaluate immunogenicity of the B5-141-6-5 vaccine antigens in rabbits. Two groups of rabbits (two rabbits for each group) were intramuscularly immunized with two doses of vaccines at day 0 and 14 using two dosages (0.05 and 0.25 g of total protein) adjuvanted with alum hydroxide. Sera were collected for measuring neutralizing antibody titers before the first vaccination (Day 0) and 14 days after the first (Day 14) and second vaccinations (Day 28).

[0036] As shown in Table 5, neutralizing antibody titers against the vaccine virus could be detected at 14 days after the second vaccination (Day 28) in both treatment groups, which indicates that the vaccine antigen is immunogenic.

Table 5. Neutralizing antibody titers in rabbits immunized with the EV71 B5-141-6-5 vaccine antigens adjuvanted with alum hydroxide

[0037] Sequence variations of the Vero cell-adapted viruses

[0038] To identify genetic mutations occurring during Vero cell adaptation, complete genomes of the B5-141, B5-141-4-2, and B5-141-6-5 were sequenced and analyzed. Virus RNA was extracted by a commercial kit (Geneaid, Taoyuan, Taiwan). The extracted virus RNA was amplified using RT-PCR (Promega, Madison,WI). Sequences of the oligonucleotide primers used in this study are available upon request. The amplified DNA was sequenced using the ABI 3730 XL DNA Analyzer (Applied Biosystem Inc., Foster City, CA). [0039] Comparing with the parental B5-141 virus (the full length of amino acid and the coding NT sequence of EV71 B5-141 are listed in SEQ ID Nos. 1 and 2, respectively), 5 and 4 nucleotide changes were detected in the B5-141-4-2 and B5-141-6-5 viruses (Table 6). Therefore, the isolated EV71 genotype B5 vaccine viruses of present invention comprise at least one variation in an EV71 gene selected from P1-VP4 (SEQ ID No. 4), P1-VP1 (SEQ ID No. 6), P3-3A (SEQ ID No. 8) and P3-3D (SEQ ID No. 10) genes.

Table 6. Genetic variations among the parental B5-141 and Vero cell-adapted viruses

* Based on numbering of 5511-SIN-00 (accession no. DQ341364).

Table 7. Summary of the sequences in Sequence Listing

P1-VP4 gene segment 748..954 of EV71 B5-141 genome

P1-VP1 protein 566..862 of CDS

P1-VP1 gene 2443..3333 of EV71 B5-141 genome

P3 -3 A protein 1441..1526 of CDS

P3 -3 A gene 5068..5325 of EV71 B5-141 genome

P3-3D protein 1732..2193 of CDS

P3-3D gene 5941..7326 of EV71 B5-141 genome

Claims

C LA IM S

1. An isolated EV71 genotype B5 vaccine virus, comprising at least one EV71 gene that encodes at least one modified enterovirus protein, wherein the isolated EV71 genotype B5 vaccine virus is able to grow efficiently in a mammalian host cell and is suitable for increased production of EV71 vaccine from the mammalian host cell.

2. The isolated EV71 genotype B5 vaccine virus of claim 1, wherein the isolated EV71 genotype B5 vaccine virus comprises at least one amino substitution at an amino acid residue corresponding to the amino acid residue of an EV71 B5 protein selected from P1-VP4 (SEQ ID No. 3), Pl-VPl (SEQ ID No. 5), P3-3A (SEQ ID No. 7) and P3-3D (SEQ ID No. 9) proteins.

3. The isolated EV71 genotype B5 vaccine virus of claim 2, wherein the isolated EV71 genotype B5 vaccine virus comprises :

(a) a modified enterovirus P1-VP4 protein having a substitution at an amino acid residue corresponding to amino acid residue Thr7 of EV71 B5 protein (SEQ ID No. 1), wherein the substitution in the modified P1-VP4 protein is from Thr (T) to Ala (A);

(b) a modified enterovirus Pl-VPl protein having a substitution at an amino acid residue corresponding to amino acid residue Thr802 of EV71 B5 protein (SEQ ID No. 1), wherein the substitution in the modified Pl-VPl protein is from Thr (T) to Asn (N); and

(c) a modified enterovirus Pl-VPl protein having a substitution at an amino acid residue corresponding to amino acid residue Pro811 of EV71 B5 protein (SEQ ID No. 1), wherein the substitution in the modified Pl-VPl protein is from Pro (P) to Ala (A);. The isolated EV71 genotype B5 vaccine virus of claim 2, wherein the isolated EV71 genotype B5 vaccine virus comprises :

(a) a modified enterovirus P1-VP4 genome having a substitution at nucleotide 766 of EV71 B5 genome (SEQ ID No. 2), wherein the substitution in the modified P1-VP4 genome is from A to G;

(b) a modified enterovirus Pl-VPl genome having a substitution at nucleotide 3152 of EV71 B5 genome (SEQ ID No. 2), wherein the substitution in the modified Pl-VPl genome is from C to A;

(c) a modified enterovirus Pl-VPl genome having a substitution at nucleotide 3178 of EV71 B5 genome (SEQ ID No. 2), wherein the substitution in the modified Pl-VPl genome is from C to G;

(d) a modified enterovirus P3-3A genome having a substitution at nucleotide 5097 of EV71 B5 genome (SEQ ID No. 2), wherein the substitution in the modified P3-3A genome is from C to T; and

(e) optionally a modified enterovirus P3-3D genome having a substitution at nucleotide 5097 of EV71 B5 genome (SEQ ID No. 2), wherein the substitution in the modified P3-3D genome is from A to G.

The isolated EV71 genotype B5 vaccine virus of claim 1, which is adapted for high-growth in Vero cells using serum-free medium.

The isolated EV71 genotype B5 vaccine virus of claim 2, which is adapted for high-growth in Vero cells using serum-free medium. The isolated EV71 genotype B5 vaccine vims of claim 5, wherein the EV71 genotype B5 vaccine vims is derived from EV71 genotype B5-141 strain.

An EV71 vaccine, comprising the isolated EV71 genotype B5 vaccine vims strain of claim 1, and a pharmaceutically acceptable carrier.

An EV71 vaccine, comprising the isolated EV71 genotype B5 vaccine vims strain of claim 2, and a pharmaceutically acceptable carrier.