WO2015179979A1 - Viral particles as immunogens against enterovirus infection and production thereof - Google Patents

Abstract

The present invention relates to viral particles as immunogens against enterovirus infection and a method of producing the same. Specifically, the present invention features that human embryo kidney 293 (HEK 293) cells are used to produce viral particles of Enterovirus A, particularly Coxsackievirus A6 (CVA6) particles or Coxsackievirus A10 (CVA10) particles or both and optionally additional viral particles of other Enterovirus A e.g. Coxsackievirus A16 (CVA16) and/or Enterovirus A71 (EV71). The yield of the viral particles in HEK 293 cells are unexpectedly high and effective to induce an immune response against enterovirus infection, especially CVA6 and CVA10. The present invention also relates to an immunogenic composition against enterovirus infection for human use comprising the viral particles as described herein and a method of preventing enterovirus infection or a disease as caused, particularly Hand-Foot-Mouth diseases (HFMD), by administering the immunogenic composition to a subject in need thereof.

Description

VIRAL PARTICLES AS IMMUNOGENS AGAINST ENTEROVIRUS INFECTION AND

PRODUCTION THEREOF

FIELD OF THE INVENTION

[0001] The present invention relates to viral particles as immunogens against enterovirus infection and a method of producing the same by using human embryo kidney 293 (HEK 293) cells. The present invention also relates to an immunogenic composition against enterovirus infection for human use, and a method of inducing an immune response against enterovirus infection or a disease as caused, particularly Hand-Foot-Mouth diseases (HFMD).

BACKGROUND OF THE INVENTION

[0002] Enterovirus, within the Picornaviridae family, is a genus of small, non-enveloped viruses containing positive-strand RNAs. The Enterovirus genus now comprises 12 species: Enterovirus A, Enterovirus B, Enterovirus C, Enterovirus D, Enterovirus E, Enterovirus F, Enterovirus Q Enterovirus H, Enterovirus J Rhinovirus A, Rhinovirus B and Rhinovirus C. These viruses infect the intestinal tract but can cause various types of diseases. Typical enterovirus diseases are meningitis, paralysis, myocarditis, hand, foot and mouth-disease (HFMD), herpangina, pleurodynia, hepatitis, rash and respiratory diseases including pneumonia. The only enterovirus vaccine for use in human beings is vaccine of poliovirus which belongs to Enterovirus C. Currently, vaccines against non-polio enteroviruses are not available for human use.

[0003] The Enteroviruses have a RNA genome including the 5' untranslated region (UTR), the protein coding regions, the 3 ' UTR and a variable length poly-A tract is located at the terminus of the 3 ' end. The RNA genome size is 7.4 Kbp and the single open reading frame (ORF) encodes a polyprotein. The polyprotein is subdivided into three regions, PI, P2 and P3. PI encodes four viral structural proteins VP4, VP2, VP3 and VP1, while P2 and P3 encode seven non- structural proteins 2A to 2C and 3A to 3D. Coxsackieviruses are divided into 23 serotypes (1-22, 24) of group A and six serotypes (1-6) of group B (Knipe and Howley, 2001). Recently, human scavenger receptor class B, member 2 (SCARB2) was identified to be important receptor for EV71 and CVA16 infection (Yamayoshi et al. , 2009).

[0004] Based on the Taiwan Sentinel Physician Surveillance, the epidemiology of major Enterovirus serotypes was systematically examined and monitored Enterovirus infections in Taiwan (Tseng et al , 2007). The information shows that there are different prevalent

Enterovirus serotypes every year, especially CVA16 and EV71 which primarily manifest as hand-foot-and-mouth disease (HFMD). In contrast to CVA16 and EV71, other common circulating serotypes, including E30, E6, El l, CB3, CB4, CB5, CVA4, CVA6 and CVA10, had been identified to cause HFMD outbreaks during 2000-2005. This study also demonstrates the repeated cyclic epidemic pattern of these serotypes that have important public health

implications. It is unclear what levels of cross-protection existing among these different geno- and/or serotype subgroups (Tseng et al, 2007). According to the data from Center for Diseases Control of Taiwan, besides CVA16 and EV71, CVA6 is usually among the top five most common enterovirus serotypes in Taiwan from 2001 to 2008 (Lo et al., 2011).

[0005] In Singapore, some high peaks of non-EV71 HFMD activity were found to be caused by CVA6, and/or CVA10 or CVA16 during 2001-2007 (Ang et al, 2009). The predominant serotypes were CVA10 (39.9%) and CVA6 (28%), followed by CVA16 (17.5%) and EV71 (6.3%)). In Spain, several outbreaks and sporadic cases of HFMD were occurred between 2010 and 2012. Enteroviruses were detected in 53 of the patients (66%>). CVA6 was the most frequent genotype, followed by CVA16 and EV71, but other minority types were also identified.

Interestingly, during 2010, CVA16 was the only causative agent of HFMD at the beginning, but by the end of the year, CVA6 became the predominant and CVA16 was not detected during 2011. In 2012, however, both CVA6 and CVA16 co-circulated. EV-71 was associated with HFMD symptoms only in three cases during 2012 (Cabrerizo et al., 2013). A recent outbreak of CVA6 HFMD occurred in Taiwan and some patients were found to have onychomadesis and

desquamation following HFMD (Wei et al., 2011). Taking the current epidemiological results, the most common pathogens of HFMD are CVA16, EV71, CVA6 and CVA10 (Kaminska et al., 2013).

[0006] Although CVA6 and CVAIO have poor tendency to cause neurological disease during infection, these viruses could induce erythematous, enanthem of the oral cavity and

onychomadesis. The prior art has developed formalin-inactivated EV71 vaccines (Chou et al., 2013 and Zhu et al., 2013) that are found to be non-protective against CVA16 infections (Chong et al., 2012). For CVA6 and CVAIO, no prior art references report a suitable cell line for propagating viral particles for human vaccine production. [0007] WO 99/53034 provides modified viral genomes for use as vaccines or vectors, which are improved in their ability to retain attenuating mutations. WO2010139193 Al discloses a hand- foot-mouth disease vaccine obtained by inactivating purified EV71 virus type B and type C, and CoxA16 virus. US20120045468 Al provides immunogenic compositions (e.g., vaccines) against EV71 infection and related methods.

[0008] There is still a need to develop vaccine candidates against Enterovirus A, especially CVA6 or CVAIO or both.

BRIEF SUMMARY OF THE INVENTION

[0009] The present invention develops a technology for producing viral particles of Enterovirus A, especially CVA6 or CVAIO, by culturing the enterovirus in human embryo kidney 293 (HEK 293) cells. The yield of the viral particles in HEK 293 cells are unexpectedly high, particularly in comparison to prior art using Vero cells and effective to induce an immune response against enterovirus infection. The viral particles thus produced can be used as effective immunogens and are useful for preparing an immunogenic composition against enterovirus infection, especially for human use.

[0010] In one aspect, the present invention provides a method of producing an immunogen against enterovirus infection, comprising

(a) producing viral particles of Coxsackievirus A6 (CVA6) in first cultures of human embryo kidney 293 (HEK 293) cells and collecting the CVA6 viral particles from the first cultures; or

(b) producing viral particles of Coxsackievirus A10 (CVAIO) in second cultures of human embryo kidney 293 (HEK 293) cells and collecting the CVAIO viral particles from the second cultures; or

(c) conducting steps (a) and (b).

[0011] In another aspect, the present invention provides an immunogenic composition against enterovirus infection, comprising CVA6 viral particles or CVAIO viral particles or both.

[0012] Also provided is use of an immunogenic composition as described herein for manufacturing a human vaccine against enterovirus infection or a disease as caused.

[0013] In a further aspect, the present invention provides a method of inducing an immune response to enterovirus infection, comprising administering to a subject in need thereof an effective amount of an immunogenic composition comprising CVA6 viral particles or CVA10 viral particles or both as described herein.

[0014] In some embodiments, the CVA6 viral particles and the CVA10 viral particles, produced and collected from cultures of HEK 293 cells, are combined together to form a multivalent immunogenic composition.

[0015] In some embodiments, the CVA6 viral particles and the CVA10 viral particles as described herein are further combined with additional viral particles of Enterovirus A other than CVA6 and CVA10 to form a multivalent immunogenic composition.

[0016] In some embodiments, the Enterovirus A other than CVA6 and CVA10 is selected from the group consisting of

Coxsackievirus A2 (CVA2), Coxsackievirus A3 (CVA3), Coxsackievirus A4 (CVA4),

Coxsackievirus A5 (CVA5), Coxsackievirus A7 (CVA7), Coxsackievirus A8 (CVA8),

Coxsackievirus A12 (CVA12), Coxsackievirus A14 (CVA14), Coxsackievirus A16 (CVA16), Enterovirus A71 (EVA71), Enterovirus A76 (EVA76), Enterovirus A89 (EVA89), Enterovirus A90 (EVA90), Enterovirus A91 (EVA91), Enterovirus A92 (EVA92), Enterovirus A114

(EVA114) and Enterovirus Al 19 (EVA119).

[0017] In certain examples, the Enterovirus A other than CVA6 and CVA10 is selected from the group consisting of CVA16 and EV71 and a combination thereof.

[0018] In certain examples, the viral particles according to the present invention are produced and collected from cultures of HEK 293 cells.

[0019] In some embodiments, the viral particles according to the present invention after collected from cell cultures are subjected to purification and/or inactivation.

[0020] In some embodiments, the purification is conducted by a sucrose gradient zonal ultracentrifugati on .

[0021] In some embodiments, the inactivation is conducted by a formalin treatment.

[0022] Also provided is an antibody directed to viral particles of Enterovirus A produced from cultures of HEK293 cells according to the present invention.

[0023] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims. BRIEF DESCRIPTION OF THE DRAWING

[0024] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

[0025] In the drawings:

[0026] Fig. 1 shows that RD and Vero cell cultures are infected by CVA6, CVAIO, CVA16 and EV71 (5 days post-infection).

[0027] Fig. 2 shows detection of viral proteins expressed in the Vero cell cultures infected either with CVA6, or CVAIO, or CVA16 or EV71 with monoclonal antibody (Nl and MAB979) by dot-blot assay.

[0028] Fig. 3 shows that both CVA6 and CVAIO only infected HEK293 cells (6 days postinfection).

[0029] Fig. 4 shows (A) CVA6 virus purification by sucrose gradient zonal ultracentrifugation; (B) Silver-stain of each fractions analyzed by SDS-PAGE.

[0030] Fig. 5 shows (A) CVAIO virus purification by sucrose gradient zonal ultracentrifugation; (B) Silver-stain of each fractions analyzed by SDS-PAGE.

[0031] Fig. 6 shows that some irregular icosahedral particle structures in the Empty -particles of CVA6 and CVAIO (6A and 6B) and Formalin-inactivated Full-particles (6C and 6D) were similar.

[0032] Fig. 7 shows protein bands of CVA6, CVAIO and EVA71 viral particles.

[0033] Fig. 8 shows the recognition between virus and mice anti-sera by dot blotting assay. Four formalin-inactivated viral particles (CVA6, CVAIO, CVA16 and EV71) were directly dropped onto the nitrocellulose membrane for dot blotting.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as is commonly understood by one of skill in the art to which this invention belongs.

[0035] The present invention is at least in part based on the unexpected findings that

coxsackievirus strains CVA6 and CVAIO unlike EVA71 and CVA16 would not infect cell lines used in human vaccine production such as Vero cells, MRC-5 cells and MDCK cells; and in contrast, these viruses (CVA6 and CVAIO) replicated well in HEK293 cells. The virus title can reach 10⁶ to 10⁸ TCID50/ml. Thus, we firstly in the world describe the production, purification and characterization of different strains of CV propagated in HEK293 cells and thus provides a technology for producing viral particles of Enterovirus A, especially CVA6 and CVAIO, in HEK293 cells. The viral particles of the invention can be used as effective immunogens and are useful for preparing an immunogenic composition, especially for human use, against enterovirus infections, including CVA6 or CVAIO or both or further Enterovirus A other than CVA6 or CVAIO, e.g. CVA16 or EVA71.

[0036] Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers or steps.

[0037] As used herein, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise.

[0038] As used herein, the species Enterovirus A includes the serotypes as follows:

Coxsackievirus A2 (CVA2), Coxsackievirus A3 (CVA3), Coxsackievirus A4 (CVA4),

Coxsackievirus A5 (CVA5), Coxsackievirus A6 (CVA6), Coxsackievirus A7 (CVA7),

Coxsackievirus A8 (CVA8), Coxsackievirus A10 (CVAIO), Coxsackievirus A12 (CVA12), Coxsackievirus A14 (CVA14), Coxsackievirus A16 (CVA16), Enterovirus A71 (EVA71), Enterovirus A76 (EVA76), Enterovirus A89 (EVA89), Enterovirus A90 (EVA90), Enterovirus A91 (EVA91), Enterovirus A92 (EVA92), Enterovirus A114 (EVA 114) and Enterovirus A119 (EVA119).

[0039] As used herein, the term "viral particle" can mean the fully or partially assembled capsid of a virus that may include an empty particle, a full particle or a sub-particle.

[0040] As used herein, the term "empty particle" refers to a viral particle that does not contain a nucleic acid, vector or plasmid, and is therefore not infectious.

[0041] As used herein, the term "full particle" refers to a virus particle containing genetic materials and four capsid proteins (i.e., VP1, VP2, VP3 and VP4). In general, VP1 has a molecular weight of 32-35 kDa (SEQ ID NO: 4, 5 or 6), VP2 has a molecular weight of 24-28 kDa (SEQ ID NO: 7, 8 or 9), VP3 has a molecular weight of 24-28 kDa (SEQ ID NO: 10, 11 or 12), VP4 has a molecular weight of 6-8 kDa (SEQ ID NO: 13, 14 or 15).

[0042] As used herein, the term "sub-particle" refers to noninfectious subparticles of a virus empty particle. Particularly, the sub-particle refers to a virus particle having a different capsid protein composition as compared to that of a full particle. For example, a sub-particle can (1) contain less capsid protein than VPl, VP2, VP3 and VP4, (2) contain more capsid proteins than VPl, VP2, VP3 and VP4, and/or (3) contain one or more incompletely processed capsid proteins.

[0043] As used herein, the "antigen" refers to a particle or a molecule containing one or more epitopes that will stimulate a host's immune system to make a humoral and/or cellular antigen- specific response. The term "antigen" is used interchangeably with "immunogen. " As a result of coming in contact with appropriate cells, an antigen induces a state of sensitivity or immune responsiveness and reacts in a demonstrable way with antibodies or immune cells of the sensitized subject in vivo or in vitro. An antigen can be specifically recognized and bound by antibodies in an organism. An antigen in association with a major histocompatibility complex (MHC) can also be recognized and bound by receptors on the surface of T lymphocytes (T-cells), leading to the activation of the T-cells. The term "epitope" as used herein refers to the site on an antigen to which a specific antibody molecule or a T-cell receptor binds. The term is used herein interchangeably with "antigenic determinant" or "antigenic determinant site."

[0044] As used herein, the term "immune response" or "immunogenic response" refers to any reaction of the immune system in response to an antigen in a subject. Examples of an immune response in a vertebrate include, but are not limited to, antibody production, induction of cell- mediated immunity, and complement activation. The immune response to a subsequent stimulus by the same antigen, also named the secondary immune response, is more rapid than in the case of the primary immune response. The term "immunogenic" refers to a capability of producing an immune response in a host animal against an antigen or antigens. This immune response forms the basis of the protective immunity elicited by a vaccine against a specific infectious organism.

[0045] As used herein, the term "antibody" refers to an immunoglobulin molecule or at least one immunologically active portion of an immunoglobulin molecule that has a specific amino acid sequence and binds only to an antigen or a group of antigens that are closely related.

Examples of antibodies include IgQ IgM, IgA, IgD and IgE. Examples of immunologically active portions of immunoglobulin molecules include Fab and F(ab)'2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. An antibody can be a monoclonal antibody or a polyclonal antibody. A "monoclonal antibody" refers to a population of antibody molecules that contains only one species of an antigen binding site and that is capable of immunoreacting with a particular epitope. A "polyclonal antibody" refers to a population of antibody molecules that contains more than one species of antigen binding sites and that is capable of immunoreacting with more than one epitope on the polypeptide.

[0046] As used herein, the term "adjuvant" refers to a substance added to an immunogenic composition, such as a vaccine, that while not having any specific antigenic effect in itself, can stimulate the immune system and increase the immune response to the immunogenic composition. Examples of adjuvants include, but are not limited to, alum-precipitate, Freund's complete adjuvant, Freund's incomplete adjuvant, monophosphoryl-lipid A/trehalose dicorynomycolate adjuvant, water in oil emulsion containing Cory neb acterium parvum and tRNA, and other substances that accomplish the task of increasing immune response by mimicking specific sets of evolutionarily conserved molecules including liposomes, lipopolysaccharide (LPS), molecular cages for antigen, components of bacterial cell walls, and endocytosed nucleic acids such as double-stranded RNA, single-stranded DNA, and

unmethylated CpG dinucleotide-containing DNA. Other examples include cholera toxin, E. coli heat-labile enterotoxin, liposome, immune-stimulating complex (ISCOM), immunostimulatory sequences oligodeoxynucleotide, and aluminum hydroxide. The composition can also include a polymer that facilitates in vivo delivery. See Audran et al. Vaccine 21 : 1250-5, 2003; and Denis- Mize et al. Cell Immunol., 225 : 12-20, 2003. Alternatively, the antigen described herein can be used in a vaccine without any adjuvant.

[0047] As used herein, the term "effective amount" refers to an amount that imparts a desired effect, which is optionally a therapeutic or prophylactic effect. For example, the effective amount is an amount of an active agent sufficient to generate or induce an immune response against a pathogen (e.g. enterovirus) in the recipient thereof. The therapeutically effective amount may change depending on various reasons, such as administration route and frequency, body weight and species of the individual receiving said pharmaceutical, and purpose of administration. Persons skilled in the art may determine the dosage in each case based on the disclosure herein, established methods, and their own experience.

[0048] In one aspect, the present invention provides a method of producing an immunogen against enterovirus infection, comprising:

(a) producing viral particles of Coxsackievirus A6 (CVA6) in first cultures of human embryo kidney 293 (HEK 293) cells and collecting CVA6 viral particles from the first cultures; or

(b) producing viral particles of Coxsackievirus A10 (CVA10) in second cultures of human embryo kidney 293 (HEK 293) cells and collecting CVA10 viral particles from the second cultures; or

(c) conducting steps (a) and (b).

[0049] Particularly, to produce viral particles in the HEK 293 cells, the cells are contacted with the desired enteroviruses which result in viral infection in the HEK 293 cells; the infected cells are cultured for a period of time sufficient to allow for production of the viral particles; and then the viral particles thus produced are collected, which can act as an immunogen against enteroviral infection. More particularly, before infection, cells are cultured to reach a density of about 10⁵ to 10⁶ cells per mL after about 3 to 7 days of cultivation and then the cells are infected with the virus at a MOI (multiplicity of infection) of about 10^"2 to 10^"5 and cultivated for about 3 to 7 days. The virus particles are then harvested and collected from the culture supernatants, which are subjected to a subsequent procedure, such as concentration, purification and/or inactivation.

[0050] In some embodiments, the cell culture are conducted in a serum-free medium.

[0051] In some embodiments, the cell culture is conducted in a roller bottle, cell-factory and/or a bioreactor.

[0052] In particular, the method of the present invention further comprises combining the CVA6 viral particles and the CVA10 viral particles to form a multivalent immunogenic composition.

[0053] In some embodiments, the method of the present invention comprises combining the CVA6 viral particles or the CVA10 viral particles or both with additional viral particles of Enterovirus A other than CVA6 and CVA10 to form a multivalent immunogenic composition.

[0054] In certain embodiments, the Enterovirus A other than CVA6 and CVA10 is selected from the group consisting of:

Coxsackievirus A2 (CVA2), Coxsackievirus A3 (CVA3), Coxsackievirus A4 (CVA4),

Coxsackievirus A5 (CVA5), Coxsackievirus A7 (CVA7), Coxsackievirus A8 (CVA8),

Coxsackievirus A12 (CVA12), Coxsackievirus A14 (CVA14), Coxsackievirus A16 (CVA16), Enterovirus A71 (EVA71), Enterovirus A76 (EVA76), Enterovirus A89 (EVA89), Enterovirus A90 (EVA90), Enterovirus A91 (EVA91), Enterovirus A92 (EVA92), Enterovirus A114 (EVA114) and Enterovirus Al 19 (EVA119).

[0055] In certain embodiments, the Enterovirus A of the additional viral particles other than CVA6 and CVAIO is selected from the group consisting of CVA16 and EV71 and a combination thereof.

[0056] In particular embodiments, the viral particles of CVA6 or CVAIO or other Enterovirus A are produced from cultures of HEK293 cells. Specifically, these viral particles of different enterovirus are separately produced and collected from individual cultures of HEK293 cells and then the collected viral particles are combined together to form a multivalent immunogenic composition.

[0057] In one certain embodiment, the present invention provides a method for preparing a multivalent immunogenic composition against enterovirus infection, comprising the steps as follows:

(a) producing viral particles of CVA6 in first cultures of HEK 293 cells and collecting CVA6 viral particles from the first cultures;

(b) producing viral particles of CVAIO in second cultures of HEK 293 cells and collecting CVAIO viral particles from the second cultures;

(c) producing viral particles of CVA16 in third cultures of HEK 293 cells and collecting CVA16 viral particles from the third cultures;

(d) producing viral particles of EVA71 in fourth cultures of HEK 293 cells and collecting EVA71 viral particles from the fourth cultures; and

(e) combining the CVA6 viral particles, the CVAIO viral particles, the CVA16 viral particles, and the EVA71 viral particles to form the multivalent immunogenic composition.

[0058] In some examples, the viral particles of each type of enterovirus are combined in substantially equal proportion of weight. For example, a multivalent immunogenic composition of the present invention can comprise viral particles of four types of enterovirus, CVA6 viral particles, CVAIO viral particles, CVA16 viral particles, and EVA71 viral particles, at a weight ratio of about 1 : 1 : 1 : 1. The ratio can be adjusted as needed.

[0059] The term a "multivalent immunogenic composition" mean that it can stimulate a host's immune response to produce specific immune responses against two or more virus strains or serotypes.

[0060] In some embodiments, the CVA6 or CVAIO viral particles or other viral particles (e.g. CVA16 or EVA71 viral particles) are further subjected to purification or inactivation or both.

[0061] In some embodiments, the purification is conducted by liquid chromatography purification, sucrose gradient ultracentrifuge purification, or a combination thereof. Preferably, the purification is conducted by sucrose gradient ultracentrifuge purification. More preferably, 10 to 60% sucrose density gradient is used in the sucrose gradient ultracentrifuge purification.

[0062] In particular, the purification is conducted to obtain a fraction of full particles, a fraction of empty particles, and/or a fraction of sub-particle of the viruses.

[0063] In some embodiments, a fraction of full particles can be identified at 35-45% sucrose gradient.

[0064] In some embodiments, a fraction of empty particles can be identified at 25-35%) sucrose gradient.

[0065] In some embodiments, a fraction of sub-particles can be identified at less than 25% sucrose gradient.

[0066] In some embodiments, the immunogenic composition of the invention can comprise (i) a fraction of full particles, a fraction of empty particles, a fraction of sub-particles of CVA6 or any combination thereof; (ii) a fraction of full particles, a fraction of empty particles, a fraction of sub-particles of CVAIO or any combination thereof; (iii) a fraction of full particles, a fraction of empty particles, a fraction of sub-particles of Enterovirus A other than CVA6 and CVAIO (e.g. CVA16 or EVA71) or any combination thereof; or (iv) any combination of (i), (ii) and (iii).

[0067] In particular embodiments, a fraction of sub-particle of the viruses can be normally removed and a fraction of full particles and a fraction of empty particles are collected.

[0068] In some embodiments, the empty particles of the enterovirus are detected to have PI polypeptide that is incompletely processed during viral assembly and packaging, having a molecular weight of 65-95 kDa. In some embodiments, the full particles of the enterovirus are detected to have VP1 (32-35 kDa), VP2 (24-28 kDa), VP3 (24-28 kDa) and VP4 (6-8 kDa).

[0069] In particular, the collected viral particles are inactivated, for example, by formalin treatment. In certain examples, the treatment with formaldehyde is at about 20-45°C for 2 to 20 days.

[0070] In a further embodiment, the method of the invention further comprises a step of determining the amount of the purified enterovirus particles.

[0071] An effective amount of the immunogen or composition described above may be administered parenterally, e.g., subcutaneous injection or intramuscular injection. Alternatively, other modes of administration including suppositories and oral formulations may be desirable. For suppositories, binders and carriers may include, for example, polyalkalene glycols or triglycerides. Oral immunogens or compositions may include normally employed excipients such as pharmaceutical grades of saccharine, cellulose, magnesium carbonate and the like. These immunogens or compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0072] A vaccine prepared from the immunogen or immunogenic composition of the invention can be administered in a manner compatible with the dosage formulation, and in an amount that is therapeutically effective, protective and immunogenic. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies, and if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of micrograms of the polypeptide of this invention. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage of the vaccine may also depend on the route of administration and varies according to the size of the host.

[0073] A subject susceptible to virus infection (especially young children) can be identified by methods known in the art and administered a composition of the invention. The dose of the composition depends, for example, on the particular antigen, whether an adjuvant is coadministered, and the type of adjuvant co-administered, the mode and frequency of

administration, as can be determined by one skilled in the art. Administration is repeated as necessary, as can be determined by one skilled in the art. For example, a priming dose can be followed by three booster doses at weekly intervals. A booster shot can be given at 4 to 8 weeks after the first immunization, and a second booster can be given at 8 to 12 weeks, using the same formulation. Sera or T-cells can be taken from the subject for testing the immune response elicited by the composition against the virus. Methods of assaying antibodies or cytotoxic T cells against a protein or infection are well known in the art. Additional boosters can be given as needed. By varying the amount of polypeptide/protein, the dose of the composition, and frequency of administration, the immunization protocol can be optimized for eliciting a maximal immune response. Before a large scale administering, efficacy testing is desirable. In an efficacy testing, a non-human subject (e.g., mouse, rat, rabbit, house, pig, cow, or monkey) can be administered via an oral or parenteral route with a composition of the invention. After the initial administration or after optional booster administration, both the test subject and the control subject (receiving mock administration) can be challenged with virus to test the efficacy of the composition.

[0074] In a further embodiment, the immunogenic composition of the invention further comprises a pharmaceutically acceptable adjuvant. Preferably, the adjuvant contains aluminum phosphate.

[0075] In another aspect, the invention provides a method of inducing an immune response to enterovirus infection, comprising administering to a subject in need thereof an effective amount of the immunogen or immunogenic composition of the invention.

[0076] In certain embodiments, the enterovirus infection is caused by Enterovirus A, selected from the group consisting of: Coxsackievirus A2 (CVA2), Coxsackievirus A3 (CVA3),

Coxsackievirus A4 (CVA4), Coxsackievirus A5 (CVA5), Coxsackievirus A6 (CVA6),

Coxsackievirus A7 (CVA7), Coxsackievirus A8 (CVA8), Coxsackievirus A10 (CVA10),

Coxsackievirus A12 (CVA12), Coxsackievirus A14 (CVA14), Coxsackievirus A16 (CVA16), Enterovirus A71 (EVA71), Enterovirus A76 (EVA76), Enterovirus A89 (EVA89), Enterovirus A90 (EVA90), Enterovirus A91 (EVA91), Enterovirus A92 (EVA92), Enterovirus A114

(EVA114) and Enterovirus Al 19 (EVA119).

[0077] A "subject" as used herein is a human or non-human mammal. Non-human mammals include, but are not limited to, primates, ungulates, canines and felines.

[0078] In particular, the method of the present invention is effective in providing protective effects against enterovirus infection and thus prevent or treat a disease caused by the enterovirus infection, especially Hand-Foot-Mouth diseases (HFMD).

[0079] Also provided is an isolated antibody that selectively binds to a peptide having one of the sequences mentioned above or viral particles as described herein. Further provided is a method of producing the antibody by immunizing an animal with the above-described

immunogen or immunogenic composition, which elicits an immune response in the animal to produce the antibody; and isolating the antibody or a cell producing the antibody from the animal.

[0080] Examples [0081] In this study, it is found that Coxsackievirus strains CVA6 and CVA10 unlike EV71 and CVA16 would not infect cell lines used in human vaccine production such as Vero cells, MRC-5 cells and MDCK cells. In contrast, these viruses (CVA6 and CVA10) replicated well in HEK293 cells. Thus, we firstly in the world describe the production, purification and characterization of different strains of CV propagated in HEK293 cells. The CV viral titer was found to be >10⁶ the tissue culture's infectious dose (TCID₅₀) per mL within 6 days post-infection when a multiplicity

2 5

of infection (MOI) of 10^"" to 10^"" was used for infection. Two CV viral fractions were separated and detected when the harvested viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 25-35% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35-45% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin- inactivated and the infectious particle fraction was found to be capable of inducing CV strain- specific neutralizing antibody responses in mouse immunogenicity studies. But these antisera failed to neutralize EV71 and CVA16. On the other hand, rabbits vaccinated with either infectious particles or non-infectious particles of both CVA6 and CVAIO could generate neutralizing antibody responses, but these antibodies still failed to neutralize EV71 and CVA16 infections. These results suggest that crossreaction among differ serotypes of enteroviruses are week and components from different viruses are needed to develop and mix as a useful and potent multivalent enterovirus vaccine.

[0082] Materials and Methods

[0083] Ethics Statement

[0084] All experiments were conducted in accordance with the guidelines of the Laboratory Animal Center of NHRI. Animal use protocols have been reviewed and approved by the NHRI Institutional Animal Care and Use Committee (Approved protocol No. NHRI-IACUC-098033-A, NHRI-IACUC- 101042- A and NHRI-IACUC- 101050- AC).

[0085] Cells, media and viruses

[0086] The human embryonic kidney 293 (HEK293) cells are obtained by Life Technologies™. African green monkey kidney (Vero), MDCK and rhabdomyosarcoma (RD) cells are kindly provided by either the Taiwan Centers of Disease Control (Taiwan CDC) or NHRI Vaccine Center. The original cell lines are obtained from the American Type Culture Collection (ATCC). We use VP-SFM, Dulbecco's modified Eagle's medium + 10%FBS and other appropriate serum- free medium to culture these cell lines. The E59 strain (genotype B4), the clinical isolate of the EV71 virus, was obtained from the Taiwan CDC. The CVA6, CVA10 and CVA16 isolates were either obtained from the Taiwan CDC or Professor Jen-Ren Wang (NCKU). CVA6, CVA10 and CVA16 virus stocks are collected from the supernatants of infected RD cells three days post infection (DPI). The titers of virus stocks are determined by TCID₅₀ assay, and these stocks are stored at -80 °C. Since RD cells are not for human vaccine production, so a GMP -grade certified HEK293 cell-line was used to propagate CVA6 and CVA10 that could not infect Vero and MDCK cell lines. The extracted viral RNA was amplified using one-step RT-PCR (Promega, Madison, WI USA). Oligonucleotide primer sequences used in this invention are rationally designed and available in the text. The amplified DNA was sequenced using an ABI 3730 XL DNA Analyzer (Applied Biosystem Inc., Foster City, CA USA). Nucleotide sequences of VP1 and amino acid sequences of all four structural viral proteins reported here are reported in the text.

[0087] Virus cultivation.

[0088] Enterovirus (EV71, CVA16, CVA6 and CVA10) was cultivated in either Vero cell or HEK293 cell using serum-free VP-SFM medium, Dulbecco's modified Eagle's medium + 10%FBS and other appropriate serum-free medium in T-flak. Cell density reached 1 to 2.5 ^χ 10⁶ cells per mL after six days of cultivation. The cells were infected with virus at a MOI of 10^"2 to 10^"5. Virus was harvested and collected from the culture supernatants at 6 days post-infection (DPI).

[0089] Purification of virus particles using sucrose gradient ultracentrifugation

[0090] The virus culture supernatant was harvested from the T-flask culture. The cell debris was removed by passage through a 0.65 μπι filter (Sartorius, Germany), and the supernatant was concentrated 20-fold with a 100K TFF capsule (Pall). The crude virus concentrate (-50 mL) was loaded onto a 10-60% continuous sucrose gradient and centrifuged at 32,000 rpm for three hours using a zonal rotor in a Hitachi CP80 ultracentrifuge. The fractions (50 mL per fraction) at 10 to 60%) sucrose were collected and individually dialyzed against three exchanges of 1 L PBS at pH 7.4 (Gibco/Life Technologies, Taipei, Taiwan), then stored at 4°C. The infectivity of the purified virus fraction was assessed by a tissue culture's infectious dose (TCID₅₀) assay. The fractions were also subjected to SDS-PAGE and Western blot analyses. The fractions identified to contain virus were pooled and concentrated by diafiltration using an Amicon 100K tube (Millipore, Belerica, MA USA) and centrifuged at 3,000 x g, then stored at 4°C. The total protein

concentration of the purified virus fractions was determined by a BCA protein assay. Half of the purified virus fractions (15 mL) was stored at -80°C in 0.5 mL aliquots; the other half was inactivated by 1/4000 (v/v) formalin at 37°C for 3 days and stored at 4°C.

[0091] The inactivated EV-71 particles in Tables 4 and 5 below were prepared as described in Liu et al., PLos One. 6(5): e20005 and the inactivated CVA 16 particles in Tables 4 and 5 below were prepared as described in Chong et al., PLoS One, 2012. 7(l l):e49973.

[0092] Determination of viral titer

[0093] Viral titers were determined using the TCID₅₀ median endpoint. Serially-diluted virus samples (from 10^"1 to 10^"8) were added to RD cells grown in 96-well plates, and 6 replicate samples were used for each dilution. The 96-well plates were incubated for six days at 37 C, and TCID₅₀ values were measured by counting cytopathic effects (CPE) on infected RD cells. The TCID₅₀ values were calculated using the Reed-Muench method.

[0094] SDS-PAGE analysis and Dot blotting

[0095] SDS-PAGE analysis of purified virus fractions was performed in a NuPAGE 4-12% Bis- Tris Gel (Invitrogen, CA USA) according to the protocol suggested by the manufacturer. For dot blotting, four formalin-inactivated viral particles (CVA6, CVAIO, CVA16 and EV71) were directly dropped onto the BA85 nitrocellulose membrane (Whatman). The membrane was subsequently soaked overnight at 4°C in 5% skim milk in PBS. MAB979 (Millipore, USA) and mouse anti-virus sera were bound for 2 hours at room temperature. The membranes were then washed five times with 15 mL assay buffer. Binding of the respective antibodies to the viral particles was detected by adding 1 mL PBS buffer containing a horseradish peroxidase (HRP)- conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch) at a dilution of 1 : 5,000. Afterl-hour incubation at room temperature, the membrane was washed 6 times with assay buffer and blotted dry. The dots were revealed by adding TMB substrate solution (KPL).

[0096] Animal immunogenicity studies

[0097] Different amounts of inactivated viral particles were adsorbed with aluminum phosphate at room temperature for 3 hours before immunization. A group of 6 female BALB/c mice (6-8 weeks old) were immunized intramuscularly (i.m.) with either 0.2 mL (0^g + 60μg Alum). Rabbits were immunized intramuscularly (i.m.) with 0.5 mL {2.5 ig + 300μg Alum). All animals were boosted twice with the same dose at two-week intervals after priming. The immunized mice and rabbits were bled one week after the final boost, and the serum was collected and used to analyze virus neutralization.

[0098] Virus neutralizing assay

[0099] Serum samples were collected from immunized mice and inactivated at 56 C for 30 minutes. Each serum sample was added to a microtube and serially diluted two-fold with fresh cell culture medium; 400 μΐ. of a 200-TCID₅₀ virus suspension was then added to each tube containing 400 μΐ. of serially diluted serum. After incubation at 4 C for 18-24 hours, 100 μΐ. of serially diluted samples were added to the 96-well plates containing RD cells. The cultures in the 96-well plates were incubated for 7 days at 37 C, and TCID₅₀ values were measured by counting CPE in infected cells. The 50% neutralization inhibition dose (ID₅₀) was calculated as the reciprocal of the serum dilution that yielded a 50% reduction in the viral titer using the Reed- Muench method.

[00100] Characterization of viral particles by transmission electron microscopy (TEM)

[00101] Inactivated viral particles were deposited on a Formvar-coated and carbon-vaporized 200-mesh copper grid. The sample was kept on the copper grid for 15 minutes at room temperature, and excess sample was then removed using filter paper. After washing twice with ddH₂0, the copper grid was stained with 2% phosphotungstic acid solution for 2 minutes, which was then removed using filter paper. The stained grid was dried for 3-7 days and observed under a JEM-2100F transmission electron microscope.

[00102] Example 1: Rationale of multi-valent immunogenic components for HFMD vaccine development

[00103] The serum-free, Vero cell-based formalin-inactivated whole-virus EV71 vaccines have been developed, produced and tested in human clinical trial (Wu et al., 2004; Liu et al., 2007; Liu et al., 2011; Chang et al., 2012; Chou et al., 2012; Li et al., 2012; Cheng et al., 2013; Zhu et al., 2013). To our surprise, unexpected results have shown that EV71 vaccine could not prevent CVA16 infections in the cell culture assay (Chou et al., 2013). Our recent studies of C VA16 vaccine candidate also showed that both mouse and rabbit anti-CVA16 antisera failed to neutralize EV71 (Chong et al., 2012). When the protein sequence of these virus strains (EV71, CVA6, CVAIO and C VA16) were aligned and analyzed; the similarity rate base on PI peptides are found to be 65-80% similarity rate on PI sequences (Table 1). In order to overcome HFMD caused by other most common Enterovirus strains such as CVA6 and CVAIO, A multivalent immunogenic components of HFMD vaccine containing different Enterovirus strains is urgently needed.

[00104] Table 1. The similarity rate (%) of Enterovirus' PI peptides alignment

CVA6 CVAIO CVA16 EV71

(M0746) (M2014) (N5079) (E59)

CVA6 (M0746) 100 73.67 67.40 65.89

CVAIO (M2014) 73.67 100 68.91 66.24

CVA16 (N5079) 67.40 68.91 100 79.93

EV71 (E59) 65.89 66.24 79.93 100

[00105] Example 2: Establishment of a novel bioprocess for Coxsackievirus group A strains

[00106] A manufacturing bioprocess for human multivalent HFMD vaccine development was investigated. Based on the current bioprocess for EV71 vaccine development published in literature (Liu et al., 2007; Chou et al., 2012), to our big surprise CVA6 and CVAIO would infect and replicate in RD cells, but not in Vero cell grown in the serum-free culture (Fig. 1). However, RD cells are not as good as HEK 293 cell for human vaccine production. See also Table 2 and Table 3.

[00107] Table 2: Virus titers of enterovirus produced in different cells.

Virus titer i l^'CID5^{( )}/mI . ι

Control CVA6 CVAIO CVA16 EV71

RD cell No titer 1.6 x 10⁵ 2.0 x 10⁷ 2.8 x XO³ 8.9 x 1Ό⁷

No CPE CPE CPE CPE CPE

Vero cell No titer No titer No titer 8.9 x 1 · ι · 1.5 x 10⁷

No CPE No CPE No CPE CPE CPE

CPE: cytopathic effect

[00108] To confirm no viral proteins had been expressed and produced in the Vero cell culture, two monoclonal antibodies (Nl and MAB979 specific against VP1 and VP2 of EV71 and CVA16, respectively) were used to detect viral components in the Vero cell cultures infected either by CVA6 or CVAIO. The results showed that no viral protein of CVA6 and CVAIO was detected in the dot-blot analysis (Fig. 2). Thus, the serum-free Vero cell-based vaccine concept may not be applied to manufacture multivalent HFMD vaccines.

[00109] Example 3: irus cultivation using HEK293 cell culture [00110] Since CVA6 and CVAIO that could not infect Vero cells and RD cells are not for human vaccine production, so other potential GMP-grade certified cell-lines such as MDCK, MRC-5, CHO and HEK293 were tested and used to propagate CVA6 and/or CVAIO. To our big surprise, both CVA6 and CVAIO only infected HEK293 cells (Fig. 3).

5 [00111] To obtain sufficient CVA6 and CVAl 0 for biochemical and immunological

characterizations, these viruses were cultivated in HEK293 cells. Cell density reached 1 to 2.5 ^χ 10^ϋ cells per mL after six days of cultivation. The cells were infected with virus at a MOI of 10^" to 10^"5. Virus was harvested and collected from the cell culture supernatants at 6 days postinfection (DPI). See Table 3.

l o [00112] Table 3 : Virus titers of enterovirus produced in HEK293 cells.

Virus titer

CPE: cytopathic effect

[00113] When EV71 infected HEK293 cells, the virus titer reached 0.5-1.6xl0⁸ TCID50/mL.

[00114] Based on the results, the manufacture bioprocess could use several bioreactor systems that include suspension bioreactor, microcarrier bioreactor and wave bioreactor.

[00115] Example 4: CV viral particles purification by sucrose gradient zonal

ultracentrifugation

[00116] Virus was harvested and collected from the culture supernatants at either 7 or 8 DPI. Cell debris were removed by micro-filtration through 0.65 μπι and 0.22 μπι membrane, and the virus bulk was 20-fold concentrated using a 100 kDa cut-off diafiltration membrane in a tangential flow filter (TFF) cassette. Then the concentrated virus bulk was loaded onto a 10- 60% continuous sucrose gradient using a zonal rotor and centrifuged at 32,000 rpm for three hours in a Hitachi CP80 ultracentrifuge. The sucrose-gradient fractions were collected and analyzed using infectivity RD cell (virus TCID₅₀) assay and SDS-PAGE as shown in Fig. 4 and 5. The first region containing viral antigens was in fractions 10 to 16, which contained 25-35% sucrose and had either low or no infectivity as determined by TCID₅₀ assay as shown in Fig. 4A and 5A for CVA6 and CVAIO, respectively. Based on biochemical, viral and immunological properties, these virus particles were defined as pseudo/defective viral particles or empty- particles. The second region containing viral proteins was found to co-locate within infectious viral fractions 17 to 22. Based on biochemical, viral and immunological properties, these infectious virus particles were defined as real viral particles or Full-particles. The empty viral particle fractions at 25-35% sucrose gradient and full particle fractions at 35-45% sucrose gradient were identified. Infectivity of the purified virus fraction was assessed again by the virus TCID₅₀ assay, SDS-PAGE and western blot analyses. The zonal centrifugation-purified CV viral particles were pooled and concentrated by diafiltration using an Amicon 100K tube and centrifuged at 3000xg. Each pooled viral particle fractions were individually dialyzed with PBS. The total protein concentration of the purified virus bulk is determined by the BCA protein assay. The purified virus is stored at 4°C in 0.5mL aliquots.

[00117] Example 5: Biophysical characterization of CV viral particles by transmission electron microscopy (TEM)

[00118] The physical structures of CV Empty- and Full-particles were revealed by TEM analysis. For biosafety reasons, purified virus solutions were individually inactivated by formalin solution (v/v 1 :4000 dilution) at 37°C for 3 days. After preparation as described in Materials and Methods, both samples were analyzed by TEM and found to have some irregular icosahedral particle structures in the Empty -particles of CVA6 and CVAIO (Fig. 6A and 6B). Formalin-inactivated Full-particles (Fig. 6C and 6D) were similar; the icosahedral structure of both viral Full-particles might be destroyed by formalin-inactivation. Both CV viral particles were found to have diameters of approximately 30-35 nm, which are very similar to EV71 and CVA16 of the

Piconaviridae family [30-35].

[00119] Example 6: The viral protein compositions of CVA16 viral particles

[00120] Both the sucrose gradient zonal ultracentrifugation and TEM biophysical analysis had demonstrated that there were two types of CV viral particles. Empty-particles were shown to contain many protein bands with different molecular weights (MWs) (Fig. 7, lanes 1 and 3).

Some high molecular weight proteins indicate that the PI polypeptide was most likely

incompletely processed during viral assembly and packaging. Interestingly, many protein bands with low MWs <17 kDa were observed that were not seen in EV71 and CVA16 Empty -particles. The Full-particles viral proteins were separated and analyzed by SDS-PAGE, and found to contain four major protein bands with MWs similar to those found in EV71 infectious particles (Fig 7, lanes 2 & 4). These four major protein bands correspond with human enterovirus capsid proteins VPl (32-35 kDa), VP2 (24-28 kDa), VP3 (24-28 kDa) and VP4 (6-8 kDa) based on their predicted protein sequences (Fig. 7, lanes 2 and 4). Taken together, these results indicate that the two viral particles have different protein compositions. Furthermore, the immature capsid constructed by incompletely-processed viral proteins can still form the particle structure (see below).

[00121] Example 7: Immunogenicity studies of CV viral particles.

[00122] It is of interest to investigate whether these two types of formalin-inactivated C V viral particles could generate strong and efficacious immune responses. Different amounts of inactivated CV particles were adsorbed with aluminum phosphate at room temperature for 3 hours before immunization. A group of 6 female BALB/c mice (6-8 weeks old) were immunized intramuscularly (i.m.) with either 0.2 mL (0^g + 6C^g Alum). Four rabbits were immunized intramuscularly (i.m.) with 0.5 mL (2.5μ§ + 300μg Alum). All animals were boosted twice with the same dose at two-week intervals after priming. The immunized mice and rabbits were bled one week after the final boost, and the serum was collected and used to analyze virus

neutralization.

[00123] Mouse anti-sera from the groups of mice immunized with either formalin-inactivated Full-particles or empty-particles of CVA6 generated CVA6 viral-specific neutralizing antibody responses (Table 4). This means CVA6-specific antisera only neutralize CVA6 infections and not against EV71, or CVAIO or CVA16 infections in the RD TCID₅₀ assay. As expected, the average virus neutralization titer of mouse antisera elicited from Empty-particles (1/32) was 10-fold lower than those obtained from Full-particles antisera (1/427). Unlike CVA6, mice immunized either with formalin-inactivated Empty-particles or Full-particles of CVAIO induced no (<l/8) or very low (1/11) CVAlO-specific virus neutralization, respectively (Table 4). This was a surprise since formalin-inactivated CVA16 Full-particles were found to be capable of eliciting strong CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. In the previous EV71 study, the EV71 -specific neutralization titer of mouse anti-sera generated from formalin-inactivated EV71 Full-particles was also found to be around 1/2000. Based on the current results, formalin inactivation may destroy some neutralization epitopes of CVAIO that could cause poor virus neutralizing antibody responses. This is supported by our previous study that the virus neutralization epitopes of some EV71 virus strain were very sensitive to chemical inactivation such as formalin, UV and heat-treatment. Based on the current results, the dosage of CVAIO should be increased to enhance the neutralizing antibody responses.

[00124] To investigate whether formalin-inactivated CVAIO is truly a poor immunogen or if the poor antibody responses are due to mouse immunogenicity problems, two groups of rabbits (3 rabbits per group) were immunized i.m. 3 times every 2 weeks with 2.5 μg of formalin- inactivated CVA16 particles formulated with alum. Anti-sera obtained from two rabbits immunized with formalin-inactivated Full-particles were found to have 1/32 and 1/16

neutralization titers (the average titer 1/26 as shown in Table 5), and anti-sera from rabbits immunized with the same amount of formalin-inactivated Empty-particles formulated with alum were found to have similar neutralization titers (1/16 and 1/32) against CVAIO virus (Table 5). These neutralization titers were far too low compared to those titers obtained from EV71 Full- particles in previous studies. These anti-EV71 titers were found to be approximately 1/2000 and 1/32,000 from mouse and rabbit immunogenicity studies, respectively.

[00125] To investigate what factors caused these poor neutralizing antibody responses, we can rule out vaccine precipitation since no obvious viral particle aggregates were observed in the solution containing formalin-treated CVAIO virion. It seems most likely that the formalin inactivation process destroys some critical virus neutralization epitopes.

[00126] It is of interest to determine whether rabbit neutralizing antibodies generated against CVA6 virus can cross-neutralize EV71. Like mouse anti-CVA6 antibodies, rabbit antisera were tested in the neutralization assay and found to be inactive against EV71 at 1/8 dilution (Tables 5 and 6). The current results indicate that neutralizing antibody responses to formalin-inactivated CVA6 vaccine candidates are virus-specific. Our previous results also demonstrated that formalin-inactivated EV71 vaccine candidates induced EV71 -specific neutralizing antibody responses that had no or poor cross-neutralization activity against CVA16. Taking these results together, a multivalent EV71/CVA6/CVA10/CVA16 vaccine should be developed and tested against human enteroviruses causing HFMD.

[00127] Example 8: Immunogenicity studies of multivalent HFMD vaccine candidate containing viral particles from EV71, CVA6, CVAIO and CVA16

[00128] It is of interest to investigate whether formalin-inactivated CVA6 and CVAIO Full- particles formulated with EV71 and CVA16 viral particles could generate strong and efficacious immune responses against all four viruses. Different amounts of inactivated-particles were adsorbed with aluminum phosphate at room temperature for 3 hours before immunization. A group of 6 female BALB/c mice (6-8 weeks old) were immunized intramuscularly (i.m.) with either 0.2 mL (O^g of each viral particles + 60μg Alum). Four rabbits were immunized intramuscularly (i.m.) with 0.5 mL (2.5μ§ of each viral particles + 300μg Alum). All animals were boosted twice with the same dose at two-week intervals after priming. The immunized mice and rabbits were bled one week after the final boost, and the serum was collected and used to analyze virus neutralization.

[00129] Mouse anti-sera from the groups of mice immunized with multivalent formalin- inactivated Full-particles generated virus neutralizing antibody responses against all four viruses (Table 4). This means antisera not only neutralize CVAIO infections, but also against EV71, CVA6 and CVA16 infections in the RD TCrD₅₀ assay. This result also indicates that multivalent formalin-inactivated Full-particles could enhance anti-CVAlO virus neutralizing antibody responses (Table 4).

[00130] Table 4. Enterovirus neutralization titers of a pool of 6 mouse anti-sera generated against either CVA6, CVAO, CVA16 and EV71 formalin inactivated viral particles as measured b TCID50 neutralization assa .

[00131] Table 5. Enterovirus neutralization titers of rabbit anti-sera generated against either CVA6 or CVAIO formalin-inactivated particles as measured by TCID50 neutralization assay.

[00132] Example 9: The recognition of mice anti-sera that immunized with viral particles

[00133] We used the dot-blot analysis to confirm the recognition between virus and anti-sera. Five groups of mice anti-sera were used to detect CVA6, CVAIO, CVA16 and EV71 viral particles. Two monoclonal antibodies (Nl and MAB979 specific against VP1 and VP2 of EV71 and CVA16, respectively) were used to detect viral components for the control group. The results showed at Fig. 8. The mice anti-sera that immunized with CVA6 E-particle were recognized CVA6 and CVAIO viral particles. However, the mice anti-sera that immunized with CVA6 F- particle were only recognized CVA6 viral particles. The similar results were also observed in CVAIO. The mice anti-sera that immunized with CVAIO E-particle were recognized CVA6 and CVAIO viral particles, and the mice anti-sera that immunized with CVAIO F-particle were only recognized CVAIO viral particles. The mice anti-sera that immunized with multivalent F-particle were recognized CVA6, CVAIO, CVA16 and EV71 viral particles. However, this mice anti-sera was weakness to recognize CVA16 viral particle. Nl monoclonal antibody highly recognized EV71 viral particles without other strains. MAB979 monoclonal antibody highly recognized EV71 and CVA16 viral particles. These results showed the recognition of mice anti-sera that immunized with different viral particles presented very different characterization.

[00134] The inventions provide important information for cell-based Enterovirus vaccine (preferably HFMD vaccine) development. Particularly, to eliminate HFMD, a multivalent EV71/CVA6/CVA10/CVA16 vaccine formulation is necessary.

[00135] In summary, the invention is based, at least in part, on that mouse antibodies elicited against EV71 and CVA16 could not neutralize CVA6 and CVAIO infections in cell culture assay. To develop CVA6 and/or CVAIO vaccine candidates, the invention found that the manufacturing bioprocess used in EV71 vaccine development to be not useful since both CVA6 and CVAIO would not replicate in the Vero cell culture with and without serum. Different cell substrates such as MDCK, MRC-5 and CHO cell-line used for human vaccine manufacturing have been tested and found to be very poor for CVA6 and CVAIO replications. In this invention, HEK293 cells which has been used for recombinant adenovirus vaccine production is tested and found to be the excellent cell substrate for both CVA6 and CVAIO replications. The present invention therefore develops a technology for producing viral particles of CVA6 or CVAIO or other enterovirus A from HEK293 cells and provides an immunogenic composition against enterovirus infection for human use comprising viral particles of CVA6 or CVAIO or both, optionally additional viral particles of Enterovirus A other than CVA6 and CVAIO, particularly CVA16 or EVA71 or both.

[00136] In particular, it is found that infectious virus particles purified from either CVA6 or CVAIO could elicit strong neutralizing antibody response against homologous virus, but fail to neutralize other viruses CVA16 and/or EV71. We therefore particularly develop a multivalent vaccine comprising EV71, CVA6, CVAIO and CVA16 viral particles, which is useful to induce protective immune responses against EV71, CVA6, CVAIO and CVA16 infections and prevent a disease as caused, particularly HFMD.

Sequence Information

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Claims

What is claimed is: 1. A method of producing an immunogen against enterovirus infection, comprising

(a) producing viral particles of Coxsackievirus A6 (CVA6) in first cultures of human embryo kidney 293 (HEK 293) cells and collecting the CVA6 viral particles from the first cultures; or

(b) producing viral particles of Coxsackievirus A10 (CVAIO) in second cultures of human embryo kidney 293 (HEK 293) cells and collecting the CVAIO viral particles from the second cultures; or

(c) conducting steps (a) and (b).

2. The method of claim 1, comprising combining the CVA6 viral particles and the CVAIO viral particles to form a multivalent immunogenic composition.

3. The method of claim 1, comprising combining the CVA6 viral particles or the CVAIO viral particles or both with additional viral particles of Enterovirus A other than CVA6 and CVAIO to form a multivalent immunogenic composition.

4. The method of claim 3, wherein the Enterovirus A other than CVA6 and CVAIO is selected from the group consisting of

Coxsackievirus A2 (CVA2), Coxsackievirus A3 (CVA3), Coxsackievirus A4 (CVA4), Coxsackievirus A5 (CVA5), Coxsackievirus A7 (CVA7), Coxsackievirus A8 (CVA8), Coxsackievirus A12 (CVA12), Coxsackievirus A14 (CVA14), Coxsackievirus A16 (CVA16),

Enterovirus A71 (EVA71), Enterovirus A76 (EVA76), Enterovirus A89 (EVA89), Enterovirus A90 (EVA90), Enterovirus A91 (EVA91), Enterovirus A92 (EVA92), Enterovirus Al 14 (EVA114) and Enterovirus Al 19 (EVA119). 5. The method of claim 3, wherein the Enterovirus A other than CVA6 and CVAIO is selected from the group consisting of CVA16 and EV71 and a combination thereof.

6. The method of claim 3, wherein the additional viral particles are produced and collected from third cultures of HEK293 cells.

7. The method of claim 1, wherein the CVA6 or CVAIO viral particles are further subjected to purification or inactivation or both.

8. The method of claim 7, wherein the purification is conducted by a sucrose gradient zonal ultracentrifugati on .

9. The method of claim 7, wherein the inactivation is conducted by a formalin treatment.

10. The method of claim 6, wherein the additional viral particles are purified and/or inactivated.

11. A method for preparing a multivalent immunogenic composition against enterovirus infection, comprising

(a) producing viral particles of CVA6 in first cultures of HEK 293 cells and collecting the CVA6 viral particles from the first cultures;

(b) producing viral particles of CVAIO in second cultures of HEK 293 cells and collecting the CVAIO viral particles from the second cultures;

(c) producing viral particles of CVA16 in third cultures of HEK 293 cells and collecting the CVA16 viral particles from the third cultures;

(d) producing viral particles of EVA71 in fourth cultures of HEK 293 cells and collecting the EVA71 viral particles from the fourth cultures; and

(e) combining the CVA6 viral particles, the CVAIO viral particles, the CVA16 viral particles, and the EVA71 viral particles to form the multivalent immunogenic composition.

12. The method of claim 11, wherein the CVA6 viral particles, the CVAIO viral particles, the CVA16 viral particles, and the EVA71 viral particles are purified and inactivated.

13. An immunogenic composition against enterovirus infection, comprising CVA6 viral particles or CVAIO viral particles or both.

14. The immunogenic composition of claim 13, which is for human use.

15. The immunogenic composition of claim 13, wherein the enterovirus infection is caused by CVA6 or CVAIO or both.

16. The immunogenic composition of claim 13, wherein the viral particles are produced and collected from cultures of HEK293 cells.

18. The immunogenic composition of claim 13, further comprising additional viral particles of Enterovirus A other than CVA6 and CVAIO.

19. The immunogenic composition of claim 18, wherein the Enterovirus A other than CVA6 and CVAIO is selected from the group consisting of

Coxsackievirus A2 (CVA2), Coxsackievirus A3 (CVA3), Coxsackievirus A4 (CVA4), Coxsackievirus A5 (CVA5), Coxsackievirus A7 (CVA7), Coxsackievirus A8 (CVA8), , Coxsackievirus A12 (CVA12), Coxsackievirus A14 (CVA14), Coxsackievirus A16 (CVA16), Enterovirus A71 (EVA71), Enterovirus A76 (EVA76), Enterovirus A89 (EVA89), Enterovirus A90 (EVA90), Enterovirus A91 (EVA91), Enterovirus A92 (EVA92), Enterovirus Al 14 (EVA114) and Enterovirus Al 19 (EVA119).

20. The immunogenic composition of claim 18, wherein the Enterovirus A is selected from the group consisting of CVA16 and EV71 and a combination thereof.

21. The immunogenic composition of claim 18, wherein the additional viral particles are produced and collected from cultures of HEK293 cells.

22. A multivalent immunogenic composition against enterovirus infection comprising CVA6 viral particles, CVAIO viral particles, CVA16 viral particles, and EVA71 viral particles.

23. The multivalent immunogenic composition of claim 22, wherein the viral particles are produced and collected from cultures of HEK293 cells.

24. Use of a multivalent immunogenic composition of claim 20 for manufacturing a human vaccine against enterovirus infection or a disease as caused.

25. Use of claim 24, wherein the enterovirus infection is caused by CVA6, CVAIO, CVA16 or EVA71 or any combination thereof.

26. The use of claim 24, wherein the disease as caused is hand, foot and mouth disease (HFMD).

27. A method of inducing an immune response to enterovirus infection, comprising administering to a subject in need thereof an effective amount of an immunogenic composition comprising CVA6 viral particles or CVAIO viral particles or both.

28. The method of claim 27, wherein the immunogenic composition further comprises additional viral particles of Enterovirus A other than CVA6 and CVAIO.

29. The method of claim 27, wherein the viral particles are produced and collected from cultures of HEK293 cells.

30. The method of claim 28, wherein the viral particles are produced and collected from cultures of HEK293 cells.

31. The method of claim 27, wherein the subject is a human.

32. An antibody directed to viral particles of Enterovirus A produced from cultures of HEK293 cells.