WO2015028888A2 - Pharmaceutical compositions to treat viral infection - Google Patents

Abstract

The invention provides materials and methods for a pharmaceutical composition comprising a polyclonal antibody preparation or a monoclonal antibody preparation or an antibody fragment preparation with or without a drug to prevent the infection of, or treat an infection of a subject by a Human Enterovirus, including, a Human Enterovirus responsible for foot, hand and mouth disease. The pharmaceutical composition includes a polyclonal antibody preparation or a monoclonal antibody preparation or an antibody fragment preparation to protect against or treat an infection by a Human Enterovirus, including, a Human Enterovirus responsible for foot, hand and mouth disease. The invention further provides for a method to use the pharmaceutical composition to protect against or treat an infection by a Human Enterovirus, including, a Human Enterovirus responsible for foot, hand and mouth disease as well as kits to do the same.

Description

PHARMACEUTICAL COMPOSITIONS TO TREAT VIRAL INFECTION

[001] This U.S. Non-Provisional patent application claims the benefit of priority pursuant to 35 U.S.C. § 1 19(e) to U.S. Provisional Patent Application 61/ 869,744, filed on August 25, 2013, the content of which is hereby incorporated by reference in its entirety.

[002] Picornaviruses are a diverse family of viruses which cause a number of common illnesses. Of the Picornaviridae family, viruses of the genus Enterovirus, which are all very closely related, are significant for the number of diseases they cause.

[003] Viruses of the genus Enterovirus affect millions of people worldwide each year, and are often found in the respiratory secretions (e.g., saliva, sputum, or nasal mucus) and stool of an infected person. Enterovirus infects the gut, thus the derivation of their name from the root "enteric". There are 62 non- polio enteroviruses that can cause disease in humans: 23 Coxsackie A viruses, 6 Coxsackie B viruses, 28 echoviruses, and 5 other enteroviruses. Infection can result in a wide variety of symptoms ranging from mild respiratory illness (common cold), hand, foot and mouth disease, acute hemorrhagic conjunctivitis, aseptic meningitis, myocarditis, severe neonatal sepsis-like disease, and acute flaccid paralysis.

[004] Of the picornaviruses, Enterovirus represents a genus of a large and diverse group of small RNA viruses characterized by a single positive-strand genomic RNA. The enteroviruses are associated with several human and mammalian diseases. On the basis of their pathogenesis in humans and animals, enteroviruses were originally classified into four groups, polioviruses, Coxsackie A viruses (CA), Coxsackie B viruses (CB), and echoviruses, but it was quickly realized that there were significant overlaps in the biological properties of viruses in the different groups.

[005] The Enterovirus genus includes the following ten species: Bovine enterovirus, Human enterovirus A, Human enterovirus B, Human enterovirus C, Human enterovirus D, Human rhinovirus A, Human rhinovirus B, Human rhinovirus C, Porcine enterovirus B and Simian enterovirus A. Within these ten species are there are various serotypes.

[006] Diseases caused by enterovirus infection include poliomyelitis which is the most notable disease caused by an enterovirus infection. Nonspecific febrile illness is, however, the most common presentation of an enterovirus infection. Enteroviruses are the most common causes of aseptic meningitis in children. In the United States, enteroviruses are responsible for 30,000 to 50,000 cases of meningitis. Encephalitis is a rare manifestation of an enterovirus infection; when it occurs, the most frequent Enterovirus found to be causing the encephalitis is echovirus 9. Pleurodynia caused by enteroviruses is characterized by severe paroxysmal pain in the chest and abdomen, along with fever, and sometimes nausea, headache, and emesis. Pericarditis and/or myocarditis are typically caused by enteroviruses. Arrythmias, heart failure, and myocardial infarction have also been reported. Acute hemorrhagic conjunctivitis can be caused by enteroviruses. Hand, foot and mouth disease is a childhood illness most commonly caused by infection by Coxsackie A virus and/or HEV71 . A 2007 study suggested that acute respiratory or gastrointestinal infections associated with enteroviruses may be a factor in chronic fatigue syndrome.

[007] All picornaviruses share the same genomic structure, including 4 structural genes within the P1 gene: VP1 , VP2, VP3, and VP4, the VP4 and VP2 being expressed together as VPO, and viral proteases within the 3C and 3D genes. The viral protease will cleave the P1 gene, thereby allowing the virus to assemble into virus like particles (VLPs), virus capsomers, complexes and/or antigens of enteroviruses.

[008] All members of the genus Enterovirus, including HEV71 , polioviruses and Coxsackievirus A16 have a single stranded positive sense RNA genome which has a single open reading frame encoding a polyprotein, P1 , consisting of the capsid proteins VP4, VP2, VP3 and VP1 and several non-structural proteins including the viral proteases 3C and 3CD which are responsible for cleaving the polyprotein P1 into individual capsid proteins VP1 , VP3 and VPO, which VPO is eventually cleaved into VP2 and VP4. The capsid proteins may assemble into virus like particles (VLPs).

[009] Human enterovirus 71 (HEV71 ) and Coxsackievirus A16 are Enterovirus serotypes notable as the major causative agents for hand, foot and mouth disease (HFMD), and HEV71 is sometimes associated with severe central nervous system diseases. Hand Foot and Mouth Disease (HFMD) is a common, self-limiting illness of children caused by a group of species A enteroviruses (Picornaviridae family) such as human Coxsackievirus A16 (CVA16), Coxsackievirus A10 (CVA10) and Human enterovirus A 71 (HEV71 ).

[010] A solution for protection and/or treatment of a subject from a Human enterovirus includes the preparation of an effective preparation comprising a highly purified polyclonal antibody concentrate from individuals who have been exposed to or who have been immunized with a Human Enterovirus or a derivative thereof, including, without limitation, a virus like particle, a viral protein or a peptide of a viral protein, which provides protective immunity against a Human enterovirus infection, including, without limitation, without the use of antiviral compounds. The human enteroviruses for which protection and/or treatment with a highly purified immunoglobulin concentrate and/or antibody preparation include, for example, and without limitation, Human enterovirus A, including Coxsackievirus A16 and Human enterovirus 71 ; Human enterovirus B, including Coxsackievirus B serotypes, echoviruses and enterovirus serotypes; Human enterovirus C, including Human poliovirus 1 , Human poliovirus 2 and Human poliovirus 3; as well as Human enterovirus D, including EV 68.

[011] There is an increasing need to produce highly purified polyclonal antibody concentrates and/or antibodies for use to treat subjects. In this respect, highly purified polyclonal antibody concentrates, including, without limitation, those of polyclonal human IgGs contain immune antibodies directed against antigens that result from an immunization process using a Human Enterovirus, a Human Enterovirus virus like protein or antigens of a Human Enterovirus, including, without limitation, a causative agent of hand, foot and mouth disease. Such highly purified immunoglobulin concentrates and/or antibodies are useful for the treatment or prevention of a disease caused by a Human Enterovirus. Including, without limitation, hand, foot and mouth disease. Further, there is also a need to produce a monoclonal antibody or fragments of an antibody for use to treat subjects against a Human Enterovirus, including, without limitation, a causative agent of hand, foot and mouth disease. SUMMARY

[012] In an aspect, the present invention is a pharmaceutical composition comprising a polyclonal antibody preparation prepared from one or more subjects immunized with a vaccine for Human Enterovirus, including a causative agent responsible for foot, hand and mouth disease that is intended for the prevention and/or treatment of an infection by a Human Enterovirus, including a causative agent responsible for foot, hand and mouth disease. In an aspect, the present invention includes a polyclonal antibody preparation that is produced from plasma isolated from one or more subjects immunized against a Human Enterovirus, including a causative agent of hand, foot and mouth disease.

[013] In an aspect, the present invention is a pharmaceutical composition, wherein the immunization to produce the polyclonal antibody preparation against a Human Enterovirus, including a causative agent of hand, foot and mouth disease is with a virus like particle, a viral protein, a viral protein, virus capsomers, aggregates, complexes of antigens from viruses and/or a viral nucleic acid. In an aspect, the present invention is a causative agent of hand, foot and mouth disease is EV71 , CA5, CA6, CA10 and/or CA16.

[014] In an aspect, the present invention is a polyclonal antibody preparation that is a purified immunoglobulin preparation, further wherein, the purified immunoglobulin preparation is comprised primarily of IgG antibodies and even further wherein, the purified immunoglobulin preparation is comprised of one or more antibodies of the IgM, IgG and/or IgA classes. In an aspect, the present invention is IgG antibodies comprising a pooled polyspecific immunoglobulin G preparation. In an aspect, the present invention is a pharmaceutical composition that is in a liquid or a lyophilized form.

[015] In an aspect, the present invention is a pharmaceutical composition, wherein the pharmaceutical composition comprises a polyclonal antibody preparation that is co-administered with a drug. In an aspect, the present invention is a pharmaceutical composition is utilized as a prophylactic application. In an aspect, the present invention comprises a pharmaceutical composition wherein the polyclonal antibody preparation is in the form of a time-release, delayed release or sustained release delivery system. In an aspect, the present invention is a polyclonal antibody preparation that is administered one or more times prior to a subject suffering from an infection with a Human Enterovirus, including a causative agent responsible for hand, foot and mouth disease. In an aspect, the present invention is a polyclonal antibody preparation is administered prior to or during an infection with a Human Enterovirus, including a causative agent for hand, foot and mouth disease.

[016] In an aspect, the present invention is a kit comprising a pharmaceutical composition comprising a polyclonal antibody preparation for the prevention and/or treatment of an infection by a Human Enterovirus, including a causative agent for foot, hand and mouth disease. In an aspect, the present invention is a kit that comprises a plasma derived immunoglobulin, instructions for use to administer plasma-derived immunoglobulin in the treatment of a Human Enterovirus, including a causative agent of hand, foot and mouth disease in a subject. In an aspect, the present invention is a kit that comprises a plasma derived immunoglobulin, instructions for use to administer plasma-derived immunoglobulin in the treatment of a Human Enterovirus, including a causative agent of hand, foot and mouth disease in a subject and one or more drugs.

[017] In an aspect, the present invention is a method of use, wherein a subject is treated with a pharmaceutical composition comprising a polyclonal antibody preparation prepared from one or more subjects immunized with a vaccine for Human Enterovirus, including a causative agent for foot, hand and mouth disease for the prevention and/or treatment of disease.

[018] In an aspect, the present invention is a pharmaceutical composition comprising a monoclonal antibody preparation for the prevention and/or treatment of an infection by a Human Enterovirus, including a causative agent for foot, hand and mouth disease, wherein the enterovirus is EV71 , CA5, CA6, CA10 and/or CA16. In an aspect, the present invention a pharmaceutical composition is administered to a subject intravenously or subcutaneously. In an aspect, the present invention is a pharmaceutical composition comprises a monoclonal antibody preparation that is co-administered with a drug. In an aspect, the present invention is a time-release, delayed release or sustained release delivery system.

[019] In an aspect, the present invention is a kit comprising a pharmaceutical composition comprising a monocloani antibody preparation for the prevention and/or treatment of an infection by a Human Enterovirus, including a causative agent for foot, hand and mouth disease.

[020] In an aspect, the present invention is a method of use, wherein a subject is treated with a pharmaceutical composition comprising a monoclonal antibody.

[021] In an aspect, the present invention is a polyclonal antibody preparation that provides protection, treats and/or stops a neurological and/or cardiopulmonary condition resulting from a Human Enterovirus, including a causative agent of hand, foot and mouth disease.

[022] In an aspect, the present invention is a monoclonal antibody preparation that provides protection, treats and/or stops a neurological and/or cardiopulmonary condition resulting from a Human Enterovirus, including a causative agent of hand, foot and mouth disease.

BRIEF DESCRIPTION OF FIGURES

[023] Figure 1 . EV71 VLP expression cassette [P1 +IRES+3CD] and the pSN01 plasmid.

[024] Figure 2. EV71 VLP expression cassette [P1 +IRES+3C] and the pSN03 plasmid.

[025] Figure 3. EMCV IRES (SEQ ID NO:1 ) region of the EMCV genome. IRES region of the EMCV genome (SEQ ID NO:1 ). The IRES region represents the EMCV genomic sequence in GenBank accession number AF113968.2; nucleotidesl 666 to 2251 .

[026] Figure 4. Out framing of the EMCV start codon with 3CD protease coding sequence; native IRES sequence (SEQ ID NO:2) versus mutant IRES sequence (SEQ ID NO:3).

[027] Figure 5. Plasmid pSN01-M1 . [028] Figure 6. Plasmid pSN01 -M2.

[029] Figure 7. Plasmid pSN01 -M3.

[030] Figure 8. Plasmid pFastBac™ HT

[031] Figure 9. Prokaryotic expression construct for antigenic fusion proteins of Human enterovirus A and Human enterovirus C.

[032] Figure 10. EV71 VLP expression cassette with HEV71-IRES and HEV71 3CD protease (P1 +HEV71 IRES+3CD).

[033] Figure 1 1. EV71 VLP expression cassette with PV-IRES and HEV71 3CD protease (P1 +PV IRES+3CD).

DETAILED DESCRIPTION

[034] In an embodiment, the present specification discloses, in part, a pharmaceutical composition comprising a polyclonal antibody preparation that may include, without limitation, a highly purified antibody preparation capable of protecting and/or treating a subject suffering from a Human Enterovirus infection. In an embodiment, the Human Enterovirus is one that is capable of causing hand, foot and mouth disease in a subject. In a further embodiment, a polyclonal antibody preparation is one that is comprised primarily of IgG antibodies. In another embodiment, a polyclonal antibody preparation is one that is comprised of one or more antibodies of the IgM, IgG and/or IgA classes.

[035] In a further embodiment, the present specification discloses, in part, a pharmaceutical composition comprising a monoclonal antibody preparation and/or an antibody fragment capable of protecting and/or treating a subject suffering from a Human Enterovirus infection. In an embodiment, the Human Enterovirus is one that is capable of causing hand, foot and mouth disease in a subject.

[036] As used herein, the term Human Enterovirus, includes Human Enterovirus A and Human Enterovirus B, their subgroups, genotypes, mutants and variants.

[037] As used herein, the term "pharmaceutically acceptable" means any molecular entity or pharmaceutical composition that does not produce an adverse, allergic or other untoward or unwanted reaction when administered to a subject. As used herein, the term "pharmaceutically acceptable composition" is synonymous with "pharmaceutical composition" and means a therapeutically effective concentration of an active ingredient, such as, e.g., any of the therapeutic compounds disclosed herein. A pharmaceutical composition disclosed herein is useful for medical and/or veterinary applications. A pharmaceutical composition may be administered to a subject alone, or in combination with other supplementary active ingredients, agents, drugs or hormones.

[038] A pharmaceutical composition disclosed herein may optionally include a pharmaceutically- acceptable carrier that facilitates processing of an active ingredient into pharmaceutically-acceptable compositions. As used herein, the term "pharmacologically-acceptable carrier" is synonymous with "pharmacological carrier" and means any carrier that has substantially no long term or permanent detrimental effect when administered and encompasses terms such as "pharmacologically acceptable vehicle, stabilizer, diluent, additive, auxiliary or excipient." Such a carrier generally is mixed with an active compound or permitted to dilute or enclose the active compound and can be a solid, semi-solid, or liquid agent. It is understood that the active ingredients can be soluble or can be delivered as a suspension in the desired carrier or diluent. Any of a variety of pharmaceutically acceptable carriers can be used including, without limitation, aqueous media such as, e.g., water, saline, glycine, hyaluronic acid and the like; solid carriers such as, e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like; solvents; dispersion media; coatings; antibacterial and antifungal agents; isotonic and absorption delaying agents; or any other inactive ingredient. Selection of a pharmacologically acceptable carrier can depend on the mode of administration. Except insofar as any pharmacologically acceptable carrier is incompatible with the active ingredient, its use in pharmaceutically acceptable compositions is contemplated. Non-limiting examples of specific uses of such pharmaceutical carriers can be found in Pharmaceutical Dosage Forms and Drug Delivery Systems (Howard C. Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7th ed. 1999); REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (Alfonso R. Gennaro ed., Lippincott, Williams & Wilkins, 20th ed. 2000); Goodman & Gilman's The Pharmacological Basis of Therapeutics (Joel G. Hardman et al., eds., McGraw-Hill Professional, 10th ed. 2001 ); and Handbook of Pharmaceutical Excipients (Raymond C. Rowe et al., APhA Publications, 4th edition 2003). These protocols are routine procedures and any modifications are well within the scope of one skilled in the art and from the teaching herein.

[039] A pharmaceutical composition disclosed herein can optionally include, without limitation, other pharmaceutically acceptable components (or pharmaceutical components), including, without limitation, buffers, preservatives, tonicity adjusters, salts, antioxidants, osmolality adjusting agents, physiological substances, pharmacological substances, bulking agents, emulsifying agents, wetting agents, sweetening or flavoring agents, and the like. Various buffers and means for adjusting pH can be used to prepare a pharmaceutical composition disclosed herein, provided that the resulting preparation is pharmaceutically acceptable. Such buffers include, without limitation, acetate buffers, citrate buffers, phosphate buffers, neutral buffered saline, phosphate buffered saline and borate buffers. It is understood that acids or bases can be used to adjust the pH of a composition as needed. Pharmaceutically acceptable antioxidants include, without limitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene. Useful preservatives include, without limitation, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, a stabilized oxy chloro composition and chelants, such as, e.g., DTPA or DTPA-bisamide, calcium DTPA, and CaNaDTPA-bisamide. Tonicity adjusters useful in a pharmaceutical composition include, without limitation, salts such as, e.g., sodium chloride, potassium chloride, mannitol or glycerin and other pharmaceutically acceptable tonicity adjustor. The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. It is understood that these and other substances known in the art of pharmacology can be included in a pharmaceutical composition. [040] The term "antibody" or "immunoglobulin" as used interchangeably herein, is intended to refer to proteins comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, which has the ability to specifically bind antigen. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each variable region (VH or VL) contains 3 CDRs, designated CDR1 , CDR2 and CDR3. Each variable region also contains 4 framework sub-regions, designated FR1 , FR2, FR3 and FR4. It is intended that the term "antibody" encompass any Ig class or any Ig subclass (e.g. the lgG1 , lgG2, lgG3, and lgG4 subclasses of IgG) obtained from any source (e.g., in exemplary embodiments, humans and non-human primates, and in additional embodiments, mice, rodents, lagomorphs, caprines, bovines, equines, ovines, etc.).

[041] The term "Ig class" or "immunoglobulin class", as used herein, refers to the five classes of immunoglobulin that have been identified in humans and higher mammals, IgG, IgM, IgA, IgD, and IgE. The term "Ig subclass" refers to the two subclasses of IgM (H and L), three subclasses of IgA (lgA1 , lgA2, and secretory IgA), and four subclasses of IgG (lgG1 , lgG2, lgG3, and lgG4) that have been identified in humans and higher mammals.

[042] The term "IgG subclass" refers to the four subclasses of immunoglobulin class lgG-lgG1 , lgG2, lgG3, and lgG4 that have been identified in humans and higher mammals by the "(heavy chains of the immunoglobulins, "(1 -"(4, respectively)).

[043] As used herein, the terms "complementarity determining region" and "CDR" refer to the regions that are primarily responsible for antigen-binding. There are three CDRs in a light chain variable region (LCDR1 , LCDR2, and LCDR3), and three CDRs in a heavy chain variable region (HCDR1 , HCDR2, and HCDR3). The residues that make up these six CDRs have been characterized by Kabat and Chothia as follows: residues 24-34 (LCDR1 ), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and 31 -35 (HCDR1 ), 50-65 (HCDR2) and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., (1991 ) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., herein incorporated by reference; and residues 26-32 (LCDR1 ), 50-52 (LCDR2) and 9196(LCDR3) in the light chain variable region and 26-32 (HCDR1 ), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196: 901 917, herein incorporated by reference. Unless otherwise specified, the terms "complementarity determining region" and "CDR" as used herein, include the residues that encompass both the Kabat and Chothia definitions (i.e., residues 24-34 (LCDR1 ), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region; and 26-35 (HCDR1 ), 50-65 (HCDR2), and 95-102 (HCDR3)). Also, unless specified, as used herein, the numbering of CDR residues is according to Kabat.

[044] As used herein, the term "framework" refers to the residues of the variable region other than the CDR residues as defined herein. There are four separate framework sub-regions that make up the framework: FR1 , FR2, FR3, and FR4. In order to indicate if the framework sub-region is in the light or heavy chain variable region, an "L" or "H" may be added to the sub-region abbreviation (e.g., "FRL1 " indicates framework sub-region 1 of the light chain variable region). Unless specified, the numbering of framework residues is according to Kabat.

[045] The term "region" can also refer to a part or portion of an antibody chain or antibody chain domain (e.g., a part or portion of a heavy or light chain or a part or portion of a constant or variable domain, as defined herein), as well as more discrete parts or portions of said chains or domains. For example, light and heavy chains or light and heavy chain variable domains include "complementarity determining regions" or "CDRs" interspersed among "framework regions" or "FRs", as defined herein. Antibodies can exist in monomeric or polymeric form, for example, IgM antibodies which exist in pentameric form and/or IgA antibodies which exist in monomeric, dimeric or multimeric form.

[046] The term "Specific binding" of an antibody means that the antibody exhibits appreciable affinity for a particular antigen or epitope and, generally, does not exhibit significant crossreactivity. In exemplary embodiments, the antibody exhibits no crossreactivity. An antibody that "does not exhibit significant crossreactivity" is one that will not appreciably bind to an undesirable entity. Specific binding can be determined according to any art-recognized means for determining such binding.

[047] As used herein, the term "affinity" refers to the strength of the binding of a single antigen- combining site with an antigenic determinant. Affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, on the distribution of charged and hydrophobic groups, etc. Antibody affinity can be measured by equilibrium dialysis or by the kinetic BIACORE.TM. method. The BIACORE.TM. method relies on the phenomenon of surface plasmon resonance (SPR), which occurs when surface plasmon waves are excited at a metal/liquid interface. Light is directed at, and reflected from, the side of the surface not in contact with sample, and SPR causes a reduction in the reflected light intensity at a specific combination of angle and wavelength. Bimolecular binding events cause changes in the refractive index at the surface layer, which are detected as changes in the SPR signal.

[048] As used herein, the term "avidity" refers to the strength of the antigen-antibody bond after formation of reversible complexes.

[049] An "antigen" is an entity to which an immunoglobulin or antibody (or antigen binding fragment thereof) specifically binds.

[050] As used herein, the term "antigen binding site" refers to a site that specifically binds (immunoreacts with) an antigen (e.g., a cell surface or soluble antigen). Antibodies of the invention in an embodiment comprise at least two antigen binding sites. An antigen binding site commonly includes immunoglobulin heavy chain and light chain CDRs and the binding site formed by these CDRs determines the specificity of the antibody. An "antigen binding region" or "antigen binding domain" is a region or domain (e.g., an antibody region or domain that includes an antibody binding site as defined herein).

[051] As used herein, the term "immunotherapy" refers to a treatment, for example, a therapeutic or prophylactic treatment with, for example, and without limitation, a polyclonal antibody preparation, of a disease or disorder intended to and/or producing an immune response (e.g., an active or passive immune response).

[052] As used herein, the term "adjuvant" refers to any substance that can stimulate an immune response (e.g., a systemic immune response) and is included in the class of immunostimulatory drugs. Some adjuvants can cause activation of a cell of the immune system (e.g., an adjuvant can cause an immune cell to produce and secrete a cytokine). Examples of adjuvants that can cause activation of a cell of the immune system include, but are not limited to, saponins purified from the bark of the Q. saponaria tree, such as QS21 (a glycolipid that elutes in the 21 st peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.); poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl- muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.). Traditional adjuvants are well known in the art and include, for example, aluminum phosphate or hydroxide salts ("alum"). In some embodiments, compositions of the present invention are administered with one or more adjuvants (e.g., to skew the immune response towards a Th1 or Th2 type response).

[053] "Plasma-derived immunoglobulin", in accordance with the present invention, is intended to mean any polyclonal antibody fraction derived from mammalian, including, without limitation, human plasma. In this regard, the term "antibody" may be interchangeably used with the term "immunoglobulin". Donors of plasma should be healthy as defined in the art. In an embodiment, the plasma of several (more than 20, more than 100, more than 500, or more than 1000) donors is pooled and optionally further processed. In an embodiment, the immunoglobulin fraction is enriched from the pooled plasma. In an embodiment, donors are healthy donors. In a further embodiment, the immunoglobulin is purified from the pooled plasma, and then, in an embodiment, the immunoglobulin is purified and concentrated. In a further embodiment, purified and concentrated immunoglobulin G (IgG) is used.

[054] The term "antibody" comprises full length, unmodified antibodies or derivatives or fragments thereof which still retain the binding specificity. Such fragments comprise, inter alia, Fab fragments, F(ab')2 or Fv fragments. Also, the present invention contemplates the addition of non-plasma-derived antibodies to the plasma-derived immunoglobulin.

[055] The term " protective immune response" and/or "neutralizing immune response" as used herein is intended to mean that the vaccinated subject is capable of producing polyclonal antibodies against the antigens of the vaccine that allow an individual to resist or protect itself against an infection with the pathogenic agent against for which the vaccination was done.

[056] When describing the binding properties of an immunoglobulin or antibody chain, the chain can be described based on its ability to "direct antigen binding". A chain is said to "direct antigen binding" when it confers upon an intact immunoglobulin or antibody (or antigen binding fragment thereof) a specific binding property or binding affinity. A mutation (e.g., a backmutation) is said to substantially affect the ability of a heavy or light chain to direct antigen binding if it affects (e.g., decreases) the binding affinity of an intact immunoglobulin or antibody (or antigen binding fragment thereof) comprising said chain by at least an order of magnitude compared to that of the antibody (or antigen binding fragment thereof) comprising an equivalent chain lacking said mutation. A mutation "does not substantially affect (e.g., decrease) the ability of a chain to direct antigen binding" if it affects (e.g., decreases) the binding affinity of an intact immunoglobulin or antibody (or antigen binding fragment thereof) comprising said chain by only a factor of two, three, or four of that of the antibody (or antigen binding fragment thereof) comprising an equivalent chain lacking said mutation.

[057] Non-plasma derived antibodies, in an embodiment, which may be added to the plasma-derived immunoglobulin can be, for example, polyclonal or monoclonal. In a further embodiment, antibodies that can be employed here include those of Ig classes IgM, IgG and IgA. In an embodiment, an antibody is a bispecific antibody. In another embodiment, the term "antibody" also comprises derivatives or fragments thereof which still retain the binding specificity. Such fragments comprise, inter alia, Fab fragments, F(ab')2, Fv fragments or scFv derivatives. Techniques for the production of antibodies and fragments thereof are well known in the art and described, e.g. in Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 1998. The antibodies also include embodiments such as chimeric, humanized, carbohydrate-structure optimized and fully human antibodies. Various procedures are known in the art and may be used for the production of such antibodies and/or fragments. Further, techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies or fragments thereof etc. described above. Also, transgenic animals may be used to express humanized or even fully human antibodies or fragments thereof. In an embodiment, the added antibody is a monoclonal antibody. For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique originally described by Kohler and Milstein Nature 256 (1975), 495-497 and further developed by the art, the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). It is also envisaged in the context of this invention that the term "antibody" comprises antibody constructs that may be expressed in cells, e.g. antibody constructs which may be transfected and/or transduced via, amongst others, viruses or plasmid vectors. Once the antibody has been obtained, the antibody itself or the DNA encoding it can be sequenced providing for the information to produce the antibody by recombinant techniques in small or large scale. Methods of the production of a recombinant antibody are also known to the person skilled in the art. The recombinant antibody can also be further modified, e.g. by switching isotype, affinity maturation techniques, modifications to alter effector functions, modifications to alter glycosylation, etc. The skilled person will be well aware of these techniques.

[058] In an embodiment, the plasma-derived immunoglobulin is plasma-derived immunoglobulin G (IgG), for example, without limitation, human plasma-derived IgG, or further without limitation, intravenous immunoglobulin G (IVIG) or subcutaneous immunoglobulin G (SCIG).

[059] The term "intravenous immunoglobulin G", abbreviated "IVIG" denotes a therapeutic preparation of pooled polyspecific immunoglobulin G obtained from the plasma of a large number (typically at least 1000) of healthy individuals. It usually contains traces of immunoglobulins of different Ig classes such as IgA or IgM (typically less than 2% of IgM or IgA, and in an embodiment, less than 1 %). Typically the immunoglobulin will be >90% IgG, >95% IgG, even >98% IgG. The term "healthy individual" means an individual who is meeting current (at the time of donation) standard eligibility criteria for donating blood which the skilled person will be well aware of, bearing in mind that such eligibility criteria are subject to continuous improvement and change. IVIG denotes a product, as well as a route of administration, namely intravenously. On the other hand, IVIG may also be administered by other routes such as subcutaneously. In an embodiment, the IVIG is provided as a solution containing at least 0.01 % (w/v) immunoglobulin, 0.05% (w/v) immunoglobulin, 0.1 % (w/v) immunoglobulin, 0.5% (w/v) immunoglobulin, 1 % (w/v) immunoglobulin, 2% (w/v) immunoglobulin, 3% (w/v) immunoglobulin, 4% (w/v) immunoglobulin, 5% (w/v) immunoglobulin, 6% (w/v) immunoglobulin, 7% (w/v) immunoglobulin, 8% immunoglobulin, 9% (w/v) immunoglobulin, 10% (w/v) immunoglobulin, , 1 1 % (w/v) immunoglobulin, 12% (w/v) immunoglobulin, 13% (w/v) immunoglobulin, 14% (w/v) immunoglobulin, 15% (w/v) immunoglobulin, 16% (w/v) immunoglobulin, 17% (w/v) immunoglobulin, 18% (w/v) immunoglobulin, 19% (w/v) immunoglobulin, 20% (w/v) immunoglobulin, 21 % (w/v) immunoglobulin, 22% (w/v) immunoglobulin, 23% (w/v) immunoglobulin, 24% (w/v) immunoglobulin, 25% (w/v) immunoglobulin, 26% (w/v) immunoglobulin, 27% (w/v) immunoglobulin, 28% (w/v) immunoglobulin, 29% (w/v) immunoglobulin, 30% (w/v) immunoglobulin 35% (w/v) immunoglobulin, 40% (w/v) immunoglobulin, 45% (w/v) immunoglobulin, 50% (w/v) immunoglobulin, 55% (w/v) immunoglobulin, 60% (w/v) immu 65% (w/v) immunoglobulin, noglobulin, 70% (w/v) immunoglobulin, 75% (w/v) immunoglobulin or more. The solution may contain additional ingredients such as stabilizers, for example amino acids such as proline or glycine, or sucrose, maltose, sorbitol, albumin, nicotinamide, PEG or others.

[060] The term "subcutaneous immunoglobulin G", abbreviated SCIG, means in accordance with the present invention a therapeutic preparation of pooled immunoglobulin G, as IVIG, but formulated for subcutaneous administration. In another embodiment, the SCIG is provided as a solution containing at least 10% (w/v) immunoglobulin, at least 15% immunoglobulin, or at least 20% immunoglobulin. The solution may contain additional ingredients such as stabilizers, for example amino acids such as proline or glycine, or sucrose, maltose, sorbitol, albumin nicotinamide, PEG, polysorbate 80 or others.

[061] In an embodiment a polyclonal antibody preparation is comprised of principally IgG antibodies against a Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease. In a further embodiment a polyclonal antibody preparation is comprised principally of a purified IgG antibodies against a Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease.

[062] In an embodiment a polyclonal antibody preparation is comprised of principally IgM antibodies against a Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease. In a further embodiment a polyclonal antibody preparation is comprised principally of a purified IgM antibodies against a Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease.

[063] In an embodiment a polyclonal antibody preparation is comprised of principally IgA antibodies against a Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease. In a further embodiment a polyclonal antibody preparation is comprised principally of a purified IgA antibodies against a Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease.

[064] Practice of the present disclosure employs, unless otherwise indicated, standard methods and conventional techniques in the fields of cell biology, toxicology, molecular biology, biochemistry, cell culture, immunology, oncology, recombinant DNA and related fields as are within the skill of the art. Such techniques are described in the literature and thereby available to those of skill in the art. See, for example, Alberts, B. et al., "Molecular Biology of the Cell," 5^th edition, Garland Science, New York, N.Y., 2008; Voet, D. et al. "Fundamentals of Biochemistry: Life at the Molecular Level," 3 ^rd edition, John Wiley & Sons, Hoboken, N.J., 2008; Sambrook, J. et al., "Molecular Cloning: A Laboratory Manual," 3 ^rd edition, Cold Spring Harbor Laboratory Press, 2001 ; Ausubel, F. et al., "Current Protocols in Molecular Biology," John Wiley & Sons, New York, 1987 and periodic updates; Freshney, R.I., "Culture of Animal Cells: A Manual of Basic Technique," 4^th edition, John Wiley & Sons, Somerset, N.J., 2000; and the series "Methods in Enzymology," Academic Press, San Diego, Calif. See also, for example, "Current Protocols in Immunology," (R. Coico, series editor), Wiley, last updated August 2010.

[065] The present disclosure provides binding proteins, e.g., antibodies and antigen-binding fragments thereof, that bind to a Human enterovirus, including, without limitation, a causative agent of hand, foot and mouth disease. The binding proteins of the present disclosure generally comprise an immunoglobulin (Ig) heavy chain (or functional fragment thereof) and an Ig light chain (or functional fragment thereof). In some embodiments, the antigen-binding fragment may be selected from the group consisting of scFv, (scFv)2, Fab, Fab', and F(ab') 2, but is not limited thereto. [066] The term "antigen-binding fragment" used herein refers to fragments of an intact immunoglobulin, and any part of a polypeptide including antigen binding regions having the ability to specifically bind to the antigen. The antigen-binding fragment Fab, which includes the light-chain and heavy-chain variable regions, the light-chain constant region, and the heavy-chain constant region Cm, has one antigen- binding site. The antigen-binding fragment Fab' differs from Fab, in that Fab' includes a hinge region with at least one cysteine residue at a C-terminal of the heavy-chain constant domain Cm . The F(ab') 2 antibody is generated through disulfide bridging of the cysteine residue of the Fab' hinge region. Fv is a least antibody fragment with only variable regions of heavy chain and light chain. Recombination technologies of generating the Fv fragment are widely known in the art. Two-chain Fv includes heavy- chain and light-chain variable regions linked by non-covalent bonds. Single-chain Fv includes, in general, heavy-chain and light-chain variable regions linked by covalent bonding via a peptide linker, or may form a dimer structure, like the two-chain Fv, with heavy-chain and short-chain variable regions directly linked at C-terminals. These antigen-binding fragments may be attainable using protease (for example, the Fab fragment may be obtained by restriction-cleavage of a whole antibody with papain, and the F(ab') 2 fragment may be obtained by cleavage with pepsin), or may be constructed using gene recombination technologies.

[067] An "antibody fragment" comprises a portion of a full-length antibody, for example, the antigen binding or variable region of a full-length antibody. Such antibody fragments may also be referred to herein as "functional fragments: or "antigen-binding fragments". Examples of antibody fragments include Fab, Fab', F(ab') 2, and Fv fragments; diabodies; linear antibodies (Zapata et al. (1995) Protein Eng. 8(10):1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab') 2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

[068] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and - binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three complementarity-determining regions (CDRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or an isolated VH or VL region comprising only three of the six CDRs specific for an antigen) has the ability to recognize and bind antigen, although generally at a lower affinity than does the entire F_v fragment.

[069] The "Fab" fragment also contains, in addition to heavy and light chain variable regions, the constant domain of the light chain and the first constant domain (CHi) of the heavy chain. Fab fragments were originally observed following papain digestion of an antibody. Fab' fragments differ from Fab fragments in that F(ab') fragments contain several additional residues at the carboxy terminus of the heavy chain CHi domain, including one or more cysteines from the antibody hinge region. F(ab') 2 fragments contain two Fab fragments joined, near the hinge region, by disulfide bonds, and were originally observed following pepsin digestion of an antibody. Fab'-SH is the designation herein for Fab' fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Other chemical couplings of antibody fragments are also known.

[070] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to five major classes: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2.

[071] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. Diabodies are additionally described, for example, in EP 404,097; WO 93/11 161 and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[072] An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Components of its natural environment may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an isolated antibody is purified (1 ) to greater than 75%, 80%, 90%, 95% or 99% by weight of antibody as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, e.g., by use of a spinning cup sequenator, or (3) to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or nonreducing conditions, with detection by Coomassie blue or silver stain. The term "isolated antibody" includes an antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. In certain embodiments, isolated antibody is prepared by at least one purification step.

[073] In some embodiments, an antibody is a humanized antibody or a human antibody. Humanized antibodies include human immununoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. Thus, humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins which contain minimal sequence derived from non-human immunoglobulin. The non-human sequences are located primarily in the variable regions, particularly in the complementarity-determining regions (CDRs). In some embodiments, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. For the purposes of the present disclosure, humanized antibodies can also include immunoglobulin fragments, such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies.

[074] The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, for example, Jones et al. (1986) Nature 321 :522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

[075] Methods for humanizing non-human antibodies are known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" or "donor" residues, which are typically obtained from an "import" or "donor" variable domain. For example, humanization can be performed essentially according to the method of Winter and co-workers, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. See, for example, Jones et al., supra; Riechmann et al., supra and Verhoeyen et al. (1988) Science 239:1534-1536. Accordingly, such "humanized" antibodies include chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non- human species. In certain embodiments, humanized antibodies are human antibodies in which some CDR residues and optionally some framework region residues are substituted by residues from analogous sites in rodent antibodies (e.g., murine monoclonal antibodies).

[076] Human antibodies can also be produced, for example, by using phage display libraries. Hoogenboom et al. (1991 ) J. Mol. Biol, 227:381 ; Marks et al. (1991 ) J. Mol. Biol. 222:581 . Other methods for preparing human monoclonal antibodies are described by Cole et al. (1985) "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, p. 77 and Boerner et al. (1991 ) J. Immunol. 147:86-95.

[077] Human antibodies can be made by introducing human immunoglobulin loci into transgenic animals (e.g., mice) in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon immunological challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661 ,016, and in the following scientific publications: Marks et al. (1992) Bio/Technology 10:779-783 (1992); Lonberg et al. (1994) Nature 368: 856-859; Morrison (1994) Nature 368:812-813; Fishwald et al. (1996) Nature Biotechnology 14:845-851 ; Neuberger (1996) Nature Biotechnology 14:826; and Lonberg et al. (1995) Intern. Rev. Immunol. 13:65-93.

[078] Antibodies can be affinity matured using known selection and/or mutagenesis methods as described above. In some embodiments, affinity matured antibodies have an affinity which is five times or more, ten times or more, twenty times or more, or thirty times or more than that of the starting antibody (generally murine, rabbit, chicken, humanized or human) from which the matured antibody is prepared.

[079] An antibody can also be a bispecific antibody. Bispecific antibodies are monoclonal, and may be human or humanized antibodies that have binding specificities for at least two different antigens.

[080] As used herein, the term "polynucleotide" means a polymer of single-stranded or double-stranded deoxyribonucleic acid or ribonucleic acid. The polynucleotide includes RNA genome sequences, DNA (gDNA and cDNA), and RNA sequences transcribed therefrom, and additionally includes analogues of natural polynucleotides, unless specifically mentioned otherwise.

[081] An antibody as disclosed herein can also be an immunoconjugate. Such immunoconjugates comprise an antibody conjugated to a second molecule, such as a reporter. An immunoconjugate can also comprise an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[082] An antibody that "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope without substantially binding to any other polypeptide or polypeptide epitope.

[083] In some embodiments, an antibody of the present disclosure specifically binds to an antigen, including, without limitation, an antigen of a Human enterovirus, including, without limitation, a causative agent of hand, foot and mouth disease. In an embodiment, an antibody binds with a dissociation constant (Kd) equal to or lower than 100 nM, lower than 90 nM, lower than 80 nM, lower than 70 nM, lower than 60 nM, lower than 50 nM, lower than 40 nM, lower than 30 nM, lower than 20 nM, lower than 10 nM, lower than 5 nM, lower than 1 nM, lower than 0.5 nM, lower than 0.1 nM, lower than 0.01 nM, or lower than 0.005 nM. In a further embodiment, such an antibody is, without limitation, in the form of monoclonal antibody, scFv, Fab, or other form of antibody measured at a temperature of about 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 1 1 °C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42° C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, or more than about 50°C.

[084] In an embodiment, an antibody of the present disclosure binds to one or more antigens of a Human enterovirus, including, without limitation, a causative agent of hand, foot and mouth disease, thereby effectively blocking the ability of the one or more antigens, including, without limitation, an antigen of a Human enterovirus, further including, without limitation, a causative agent of hand, foot and mouth disease, to cause or continue the effects of a disease caused by a disease.

[085] In certain embodiments, an antibody according to the present disclosure binds to antigen, including, without limitation, an antigen of a Human enterovirus, including, without limitation, a causative agent of hand, foot and mouth disease, with an affinity at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 1 1 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 21 times, at least 22 times, at least 23 times, at least 24 times, at least 25 times, at least 26 times, at least 27 times, at least 28 times, at least 29 times, at least 30 times, at least 31 times, at least 32 times, at least 33 times, at least 34 times, at least 35 times, at least 36 times, at least 37 times, at least 38 times, at least 39 times, at least 40 times, at least 41 times, at least 42 times, at least 43 times, at least 44 times, at least 45 times, at least 46 times, at least 47 times, at least 48 times, at least 49 times, at least 50 times, at least 55 times, at least 60 times, at least 65 times, at least 70 times, at least 75 times, at least 80 times, at least 85 times, at least 90 times, at least 95 times, at least 100 times, at least 125 times, at least 150 times, at least 175 times, at least 200 times, at least 225 times, at least 250 times, at least 275 times, at least 300 times, at least 325 times, at least 350 times, at least 375 times, at least 400 times, at least 425 times, at least 450 times, at least 475 times, at least 500 times, or at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1000 times greater than its binding affinity for another antigen, including, without limitation, an antigen of a Human enterovirus, including, without limitation, a causative agent of hand, foot and mouth disease. Binding affinity can be measured by any method known in the art and can be expressed as, for example, on-rate, off-rate, dissociation constant (Kd), equilibrium constant (K_eq) or any term in the art.

[086] The polynucleotide also includes nucleotide sequences encoding the amino acid sequences of the heavy or light chain variable regions of the antibody specifically binding to c-Met protein and nucleotide sequences complementary thereto. The complementary sequences include completely complementary sequences and substantially complementary sequences.

[087] The term "vector" used herein refers to a polynucleotide for expressing a target gene in a host cell. For example, the vector may include a plasmid vector, a cosmid vector, and a virus vector, such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. Suitable recombinant vectors may be constructed by manipulating plasmids known in the art (for example, pSC101 , pGV1 106, pACYC177, ColE1 , pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, plJ61 , pLAFRI , pHV14, pGEX series, pET series, and pUC19), a phage (for example, .lamda.gt4.lamda.B, .lamda.-Charon, .lamda..DELTA.z1 , and M13), or a virus (for example, SV40).

[088] In the recombinant vector, the polynucleotides encoding the amino acid sequences of the heavy and light chain variable regions may be operatively linked to a promoter. The term "operatively linked" used herein refers to a functional linkage between a nucleotide expression regulating sequence (for example, a promoter sequence) and other nucleotide sequences. Thus, the nucleotide expression regulating sequence may regulate the transcription and/or translation of the other nucleotide sequences.

[089] The recombinant vector may be constructed for cloning or expression. The expression vector may be any vector known in the art for expressing an exogenous protein in plants, animals, or microorganisms. The recombinant vector may be constructed using various methods known in the art. [090] The recombinant vector may be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when a prokaryotic cell is used as a host cell, the expression vector used generally includes a strong promoter capable of initiating transcription (for example, .rho promoter, trp promoter, lac promoter, tac promoter, T7 promoter), a ribosome binding site for initiating translation, and a transcription/translation termination sequence. When a eukaryotic cell is used as a host cell, the vector used generally includes the origin of replication acting in the eukaryotic cell, for example, a l replication origin, a SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin, or a BBV replication origin, but is not limited thereto. A promoter in an expression vector for a eukaryotic host cell may be a promoter derived from the genomes of mammalian cells (for example, a metallothionein promoter) or a promoter derived from mammalian viruses (for example, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a cytomegalovirus promoter, and a tk promoter of HSV). A transcription termination sequence in an expression vector for a eukaryotic host cell may be, in general, a polyadenylation sequence.

[091] A vector system capable of expressing the heavy and light chain variable regions of the antibody may be a vector system in which the heavy and light chain variable regions are simultaneously expressed from a single vector, or a system in which the heavy and light chain variable regions are each independently expressed from separate vectors. In the latter case, the two vectors may be introduced into the host cell by co-transformation and targeted transformation.

[092] "Homology" or "identity" or "similarity" as used herein in the context of nucleic acids and polypeptides refers to the relationship between two polypeptides or two nucleic acid molecules based on an alignment of the amino acid sequences or nucleic acid sequences, respectively. Homology and identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. In comparing two sequences, the absence of residues (amino acids or nucleic acids) or presence of extra residues also decreases the identity and homology/similarity.

[093] As used herein, "identity" means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Sequences are generally aligned for maximum correspondence over a designated region, e.g., a region at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or more amino acids or nucleotides in length, and can be up to the full-length of the reference amino acid or nucleotide. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[094] Examples of algorithms that are suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Further exemplary algorithms include ClustalW (Higgins D., et al. (1994) Nucleic Acids Res 22: 4673-4680), available at www.ebi.ac.uk/Tools/clustalw/index.html.

[095] Residue positions which are not identical can differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.

[096] Sequence identity between two nucleic acids can also be described in terms of hybridization of two molecules to each other under stringent conditions. The hybridization conditions are selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). An example of stringent hybridization conditions is hybridization at 50° C. or higher and 0.1 XSSC (15 mM sodium chloride/1.5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42.degree. C. in a solution: 50% formamide, 5XSSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 XSSC at about 65° C. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, typically at least 90% as stringent as the above specific stringent conditions.

[097] Accordingly, the present disclosure provides, for example, antibodies or antigen binding fragments thereof, comprising a heavy chain variable region polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater amino acid sequence identity to an amino acid sequence of a heavy chain variable region and a variable light chain polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater amino acid sequence identity to an amino acid sequence of a light chain polypeptide. [098] Any host cell known in the art to enable stable and continuous cloning or expression of the recombinant vector may be used. Suitable prokaryotic host cells may include E. coli JM109, E. coli BL21 , E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus species strains such as Bacillus subtillis or Bacillus thuringiensis, intestinal bacteria and strains such as Salmonella typhymurum, Serratia marcescens, and various Pseudomonas species. Suitable eukaryotic host cells to be transformed may include yeasts, such as Saccharomyce cerevisiae, insect cells, plant cells, and animal cells, for example, Sp2/0, Chinese hamster ovary (CHO) K1 , CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines.

[099] The recombinant vector including the polynucleotide may be transferred into a host cell by using known transfer methods. Suitable transfer methods may be chosen according to the host cell. Suitable transfer methods for prokaryotic host cells may include a method using CaC and electroporation. Suitable transfer methods for eukaryotic host cells may include microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, and gene bombardment. However, any suitable transfer method may be used.

[0100] When a microorganism, such as E. coli, is used as the host cell, the production of antibodies is higher than that in an animal cell. However, a microorganism is not suitable for producing intact Ig-type antibodies due to lack of glycosylation of the antibodies produced, although a microorganism may be used for producing antigen-binding fragments of an antibody such as Fab and Fv.

[0101] Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common bacterial host is E. coli.

[0102] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including operably linked promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or other sequences as appropriate. Vectors can be plasmids, viral e.g. ^'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference in their entirety.

[0103] A transformed host cell may be selected using a phenotype expressed by a selected marker by any method known in the art. For example, if the selected marker is a gene that is resistant to a specific antibiotic, a transformant is cultured in a medium including the antibiotic, and thus the transformant may be easily selected.

[0104] According to an embodiment of the present disclosure, there is provided a pharmaceutical composition comprising a monoclonal, a polyclonal antibody and/or an antibody fragment, as described herein, and a pharmaceutically acceptable carrier, a diluent, or an excipient.

[0105] In a further embodiment, the plasma-derived immunoglobulin is administered prior to, concomitantly to, or after the onset of an infection caused by a Human Enterovirus.

[0106] The term "drug" as used herein includes chemically synthesized molecules as well as biologically produced molecules such as monoclonal or polyclonal antibodies or other therapeutically active proteins or nucleic acids. It may also include other therapeutic agents, such as cells, e.g. leukocytes, including stem cells, for example T cells including subpopulations thereof, such as regulatory T cells, T helper cells or cytotoxic T cells. Cells may be removed from a patient, modified, conditioned or enriched in vitro/ex vivo, and then reintroduced into the patient (e.g. autologous regulatory T cells). In an embodiment, the drug is an antiviral drug, including, without limitation an anti-retroviral drug, and further, without limitation, a combination of anti-retroviral drugs.

[0107] The term "specifically binds", interchangeably used with "specifically interacts with", in accordance with the present invention means that the antibody does not or essentially does not cross- react with an epitope of similar structure. Cross-reactivity of a panel of antibodies under investigation may be tested, for example, by assessing binding of said panel of antibodies under conventional conditions to the epitope of interest as well as to a number of more or less (structurally and/or functionally) closely related epitopes. Only those antibodies that bind to the epitope of interest in its relevant context (e.g. a specific motif in the structure of a protein) but do not or do not essentially bind to any of the other epitopes are considered specific for the epitope of interest and thus to be antibodies described in accordance with this invention. Corresponding methods are described e.g. in Harlow and Lane, 1988 and 1999, loc cit.

[0108] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest disease and its complications in a subject (e.g., preterm infant (e.g., low birth weight preterm infant)) already suffering from the disease. Amounts effective for this use depend upon the severity of the disease, the patient's general physiology, e.g., the patient's body mass, age, gender, the route of administration, and other factors well known to physicians and/or pharmacologists. Effective doses may be expressed, for example, as the total mass of antibody (e.g., in grams, milligrams or micrograms) or as a ratio of mass of antibody to body mass (e.g., as grams per kilogram (g/kg), milligrams per kilogram (mg/kg), or micrograms per kilogram (.mu.g/kg).

[0109] "Epitope" refers to the simplest form of an antigenic determinant, on a complex antigen molecule. This is the specific portion of an antigen that is recognized by an immunoglobulin or T-cell receptor. [0110] In an embodiment, a vaccine, including without limitation, a vaccine comprised of a viral nucleic acid, a virus like particle, a viral peptide, an attenuated or modified virus and/or a viral protein, can induce a protective immune response.

[0111] "B-cell" refers to a type of lymphocyte that produces immunoglobulins or antibodies that interact with antigens.

[0112] The language "consisting essentially of" means that in addition to those components which are mandatory, other components may also be present in compositions, provided that the essential, basic and/or novel characteristics of the compositions are not materially affected by their presence.

[0113] The language "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. Thus, for example, a promoter "operably linked" to a nucleic acid means that the promoter and the nucleic acids of a cistron, or more than one cistron, are combined in such a manner that a single cistronic, a single bicistronic, or a single multicistronic messenger RNA (mRNA) may be produced. Protein expression of the messenger RNA may be regulated according to transcriptional/translational elements of the nucleic acid sequence. An IRES sequence which is inserted into an expression cassette in an orientation which is upstream (5') to a cistron means that the IRES sequence and the nucleic acids of the cistron are ligated in such a manner that translation of the cistronic mRNA is regulated under the control of the IRES.

[0114] The terms "host" or "subject," as used herein, refer to an individual that is administered either prophylactic or therapeutic treatment with one or more compositions and/or methods of the invention. Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and including, without limitation, humans (e.g., preterm infant (e.g., low birth weight infant (e.g., very low birth weight infant))). In the context of the invention, the term "subject" generally refers to an individual who will be administered or who has been administered one or more compositions of the present invention.

[0115] As used herein, the terms "subject" and "patient" refer to any animal, such as a mammal like a dog, cat, bird, livestock, and a human.

[0116] A polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment may be in liquid or lyophilised form. In an embodiment, the polyclonal antibody preparation is in the presence of suitable stabilizers, and may be stored for later use. Said polyclonal antibody preparation, for therapeutic use, may in particular be injected via intravenous route as indicated previously. For this purpose, the polyclonal antibody preparation of the invention must be virally safe and may, in an embodiment, without limitation, use a conventional solvent-detergent treatment for example known in the prior art, e.g. using a mixture of Tween.RTM. 80/TnBP or Triton. RTM. X 100/TnBP, and/or filtering steps for optional removal of viruses and/or other macromolecules which may not have been removed by the solvent-detergent viricide treatment e.g. the prion-the agent responsible for transmissible spongiform encephalopathy.

[0117] The invention also relates to a kit of parts for the treatment of a Human Enterovirus, including, without limitation, an enterovirus capable of causing hand, foot and mouth disease in a subject, wherein, the kit comprises (a) plasma-derived immunoglobulin; (b) instructions for use to administer plasma- derived immunoglobulin in the treatment of an enterovirus capable of causing hand, foot and mouth disease in a subject and optionally (c) a therapeutically active compound/drug.

[0118] The components of the kit of parts may be contained in one or different containers such as one or more vials. The plasma-derived immunoglobulin may be in liquid or solid form (e.g. after freeze-drying through a process such as, without limitation, lyophilization) to enhance shelf-life. If in liquid form, the plasma-derived immunoglobulin may comprise additives such as stabilizers and/or preservatives such as proline, glycine or sucrose, also essentially in order to enhance shelf-life.

[0119] The kit may contain, without limitation, different compounds such as an active ingredient, such as, without limitation, a plasma-derived immunoglobulin (also interchangeably referred to as a polyclonal antibody preparation) along with any other drugs that are to be administered to a subject, at the same time or sequentially to, the plasma-derived immunoglobulin.

[0120] The kit may also contain, without limitation, different compounds such as an active ingredient, such as, without limitation, a monoclonal antibody or antibody fragment along with any other drugs that are to be administered to a subject, at the same time or sequentially to, the monoclonal antibody or antibody fragment.

[0121] A drug may be, without limitation, a vitamin, an antibiotic, an anti-viral agent, an immunostimulatory (including, without limitation, an adjuvant) and any other therapeutic compound.

[0122] A kit can also, without limitation, include, instructions for use, in any case, include directions to use the plasma-derived immunoglobulin in the treatment of a Human Enterovirus infection, including, without limitation, foot, hand and mouth disease. They may further contain information how to prepare (e.g. dilute or reconstitute, in the case of freeze-dried plasma-derived immunoglobulin) the plasma- derived immunoglobulin. They may further include guidance regarding to the dosage and frequency of administration.

[0123] The kit may, without limitation, be in the form of a pharmaceutical composition (with the mentioned instructions for use). In accordance with the present invention, the term "pharmaceutical composition" relates to a composition for administration to a subject, including, without limitation, a human subject to prevent or treat a Human Enterovirus, including, without limitation, the causative agent for hand, foot and mouth disease. In an embodiment, the pharmaceutical composition of the invention may comprise, without limitation, the compounds recited above, alone or in combination. In an embodiment, the composition may be in solid (again, to be reconstituted) or liquid form. The pharmaceutical composition of the present invention may, optionally and additionally, comprise a pharmaceutically acceptable carrier. The pharmaceutical composition can be administered systemically, such as intravenously or subcutaneously. The dosage regimen corresponding to a suitable dose for administration will be determined by the attending physician and clinical factors which may, inter alia, depend on the stage or severity of its condition.

[0124] In an embodiment of the kit, the plasma-derived immunoglobulin is plasma-derived IgG, for instance, without limitation, human plasma-derived IgG. In a further embodiment, the kit comprises, without limitation, IVIG or SCIG.

[0125] In an embodiment, the kit further provides a device for administering the pharmaceutical composition. The present invention is not limited by the type of device included in the kit.

[0126] In an embodiment, all kit components are present within a single container (e.g., vial or tube). In a further embodiment, each kit component is located in a single container (e.g., vial or tube). In an embodiment, one or more kit component are located in a single container (e.g., vial or tube) with other components of the same kit being located in a separate container (e.g., vial or tube). In a further embodiment, a kit comprises a buffer. In an embodiment, the kit further comprises written material comprising instructions for using the composition (e.g., providing instructions for dosing). In another embodiment, a kit contains labeling providing directions for use of the kit. In an embodiment, the term labeling refers to any written or recorded material that is attached to, or otherwise accompanies a kit at any time during its manufacture, transport, sale or use. For example, without limitation, the term labeling encompasses advertising leaflets and brochures, packaging materials, instructions, audio or videocassettes, computer discs, as well as writing imprinted directly on kits.

[0127] As used herein, the terms "co-administration" and "co-administering" refer to the administration of at least two agent(s) (e.g., a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment and one or more other drugs or therapies (e.g., a composition comprising one or more antivirals)) to a subject. In an embodiment, the co-administration of two or more drugs or therapies is concurrent. In another embodiment, a first drug/therapy is administered prior to a second drug/therapy. In an embodiment, co-administration can be via the same or different route of administration. Those of skill in the art understand that the formulations and/or routes of administration of the various drugs or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when drugs or therapies are co-administered, the respective drugs or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the drugs or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when coadministration of two or more drugs results in sensitization of a subject to beneficial effects of one of the drugs via co-administration of the other agent. [0128] The pharmaceutical compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, in an embodiment do not unduly interfere with the biological activities of the components of the pharmaceutical compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like) that do not deleteriously interact with a polyclonal antibody preparation. When used the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

[0129] Suitable buffering agents include, but are not limited to, acetic acid and a salt (1 -2% w/v); citric acid and a salt (1 -3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives may include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3- 0.9% w/v); parabens (0.01 -0.25% w/v) and thimerosal (0.004-0.02% w/v).

[0130] In an embodiment, a pharmaceutical composition comprises a polyconal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment that is co-administered with one or more antiviral drugs or antibiotics. For example, one or more antiviral drugs or antibiotics may be administered with, before and/or after administration of a polyclonal antibody preparation. The present invention is not limited by the type of antiviral drug or antibiotic co-administered.

[0131] In an embodiment, a variety of antibiotics may be co-administered including, but not limited to, .beta.-lactam antibiotics, penicillins (such as natural penicillins, aminopenicillins, penicillinase-resistant penicillins, carboxy penicillins, ureido penicillins), cephalosporins (first generation, second generation, and third generation cephalosporins), and other .beta.-lactams (such as imipenem, monobactams), .beta.-lactamase inhibitors, vancomycin, aminoglycosides and spectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin, metronidazole, polymyxins, doxycycline, quinolones (e.g., ciprofloxacin), sulfonamides, trimethoprim, and quinolines. In some embodiments, the antibiotic agent used is any antibiotic shown to have antimicrobial activity (e.g., anti- Staphylococcal activity). In some embodiments, the antibiotic is an anti-staphylococcal antibiotic (e.g., characterized as having anti-staphylococcal activity).

[0132] In another embodiment, a variety of antivirals may be co-administered including, but not limited to, amantadine, rimantadine, pleconaril, acyclovir, interferon, oseltamivir and/or zanamivir.

[0133] There are an enormous amount of antimicrobial drugs currently available for use in treating bacterial, fungal and viral infections. For a comprehensive treatise on the general classes of such drugs and their mechanisms of action, the skilled artisan is referred to Goodman & Gilman's "The Pharmacological Basis of Therapeutics" Eds. Hardman et al., 9th Edition, Pub. McGraw Hill, chapters 43 through 50, 1996, (herein incorporated by reference in its entirety). Generally, these drugs include agents that inhibit cell wall synthesis (e.g., penicillins, cephalosporins, cycloserine, vancomycin, bacitracin); and the imidazole antifungal agents (e.g., miconazole, ketoconazole and clotrimazole); agents that act directly to disrupt the cell membrane of the microorganism (e.g., detergents such as polmyxin and colistimethate and the antifungals nystatin and amphotericin B); drugs that affect the ribosomal subunits to inhibit protein synthesis (e.g., chloramphenicol, the tetracyclines, erthromycin and clindamycin); drugs that alter protein synthesis and lead to cell death (e.g., aminoglycosides); agents that affect nucleic acid metabolism (e.g., the rifamycins and the quinolones); the antimetabolites (e.g., trimethoprim and sulfonamides); and the nucleic acid analogues such as zidovudine, gangcyclovir, vidarabine, and acyclovir which act to inhibit viral enzymes essential for DNA synthesis. Various combinations of antimicrobials may be employed.

[0134] In some embodiments, co-administration of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment and one or more antibiotics is utilized as a prophylactic treatment (e.g., to prevent infection (e.g., caused by pathogenic bacteria)). In some embodiments, coadministration of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment and one or more antibiotics is utilized as a therapeutic treatment.

[0135] In some embodiments, co-administration of a polyclonal antibody preparation and one or more antiviral drugs is utilized as a prophylactic treatment (e.g., to prevent infection (e.g., caused by pathogenic bacteria)). In some embodiments, co-administration of a polyclonal antibody preparation and one or more antiviral drugs is utilized as a therapeutic treatment.

[0136] The present invention also includes methods involving co-administration of a composition comprising a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment with one or more drugs, including, without limitation, an immunostimulatory drug (e.g., an antibiotic, anti-oxidant, etc.). Indeed, in a further embodiment of this invention, methods are provided for enhancing prior art anti-microbial methods (e.g., antibacterial methods) and/or pharmaceutical compositions by co-administering a composition of the present invention. In co-administration procedures, the drugs may be administered concurrently or sequentially. In one embodiment, the compositions described herein are administered prior to the other active drug(s). The pharmaceutical formulations and modes of administration may be any of those described herein. In addition, the two or more co-administered drugs may each be administered using different modes (e.g., routes) or different formulations. The additional drugs to be co-administered (e.g., antibiotics, antivirals, adjuvants, etc.) can be any of the well-known agents in the art, including, but not limited to, those that are currently in clinical use. [0137] In some embodiments, a composition comprising a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment is administered to a subject via more than one route. For example, without limitation, a subject may benefit from receiving intravenous infusion and, additionally, receiving one or more other routes of administration (e.g., subcutaneous, parenteral, intrathecal, intraarterial, etc.).

[0138] Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compositions, increasing convenience to the subject and a physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhyd rides.

[0139] In one embodiment, a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein is capable of reducing the severity of a Human Enterovirus infection, including, without limitation, hand, foot and mouth disease by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a patient not receiving the same treatment. In other aspects of this embodiment, a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein is capable of reducing or stopping the severity or progression of a Human Enterovirus infection, including, without limitation, hand, foot and mouth disease by, e.g., about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% as compared to a patient not receiving the same treatment.

[0140] In a further embodiment, a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein that is capable of reducing the severity of a Human Enterovirus infection, including, without limitation, hand, foot and mouth disease has a half-life of 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, one month, two months, three months, four months or more.

[0141] In a further embodiment, a period of administration is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more. In a further embodiment, a period of during which administration is stopped is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, 12 months, or more.

[0142] In an embodiment, a polyclonal antibody preparation and/or a monoclonal antibody and/or an antibody fragment may be administered to a subject, without limitation, intravenously, orally, intravaginally, intraparentally, intra-anally, subcutaneously and/or intramuscularly.

[0143] In a further embodiment, a polyclonal antibody, a monoclonal antibody and/or a fragment of an antibody has specificity for one or more antigens of a Human Enterovirus 71 ("EV71"), a Coxsackie virus ("CA5"), a Coxsackie virus 6 ("CA6"), a Coxsackie virus ("CA10") and a Coxsackie virus 16 ("CA16"), including, without limitation all subgroups, genotypes and other forms or variants of each of EV71 , CA5 CA6, CAI O and CA16.

[0144] In an embodiment, a pharmaceutical composition comprises one or more drugs, with each drug present at a concentration of at least about 0.1 % (w/v), or alternatively at least about 0.01 %, 0.02%, 0.05%, 0.075%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 1.5%, 1 .75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, 6%,6.25%, 6.5%, 6.75%, 7%, 7.25%, 7.5%, 7.75%, 8%, 8.25%, 8.5%, 8.75%, 9%, 9.25%, 9.5%, 9.75%, 10%, 10.25%, 10.5%, 10.75%, 1 1 %, 1 1 .25%, 11 .5%, 11 .75%, 12%, 12.25%, 12.5%, 12.75%, 13%, 13.25%, 13.5%, 13.75%, 14%, 14.25%, 14.5%, 14.75%, 15%, 15.25%, 15.5%, 15.75%, 16%, 16.25%, 16.5%, 16.75%, 17%, 17.25%, 17.5%, 17.75%, 18%, 18.25%, 18.5%, 18.75%, 19%, 19.25%, 19.5%, 19.75%, 20%, 20.25%, 20.5%, 20.75%, 21 %, 21.25%, 21 .5%, 21 .75%, 22%, 22.25%, 22.5%,, 22.75%, 23%, 23.25%, 23.5%, 23.75%, 24%, 24.25%, 24.5%, 24.75%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 40%, or more (w/v) of the pharmaceutical composition.

[0145] A pharmaceutical composition includes both a sustained release delivery platform and an extended release delivery platform to deliver a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment with or without one or more other drugs. In some embodiments, co-administration of a polyclonal antibody preparation and one or more other drugs is utilized as a therapeutic treatment. In aspects of this embodiment, a sustained release pharmaceutical composition releases a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment and one or more other drugs disclosed herein with substantially zero order release kinetics over a period of, e.g., about 7 days after administration, about 15 days after administration, about 30 days after administration, about 45 days after administration, about 60 days after administration, about 75 days after administration, or about 90 days after administration. In other aspects of this embodiment, a sustained release pharmaceutical composition releases a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment and one or more other drugs disclosed herein with substantially zero order release kinetics over a period of, e.g., at least 7 days after administration, at least 15 days after administration, at least 30 days after administration, at least 45 days after administration, at least 60 days after administration, at least 75 days after administration, or at least 90 days after administration.

[0146] In aspects of this embodiment, a pharmaceutical composition releases a polyclonal antibody preparation and one or more other drugs disclosed herein with substantially zero order release kinetics over a period of, e.g., about 1 day after administration, about 2 days after administration, about 3 days after administration, about 4 days after administration, about 5 days after administration, or about 6 days after administration. In other aspects of this embodiment, a pharmaceutical composition releases a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment and one or more other drugs disclosed herein with substantially zero order release kinetics over a period of, e.g., at most 1 day after administration, at most 2 days after administration, at most 3 days after administration, at most 4 days after administration, at most 5 days after administration, or at most 6 days after administration.

[0147] In an embodiment, a pharmaceutical composition comprises a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment and one or more other drugs, with each ingredient present at a concentration of about 0.1 % (w/v) to about 40%, or alternatively at about 0.01 % to about 25%, 0.02% to about 25%, 0.05% to about 25%, 0.075% to about 25%, 0.2% to about 25%, 0.3% to about 25%, 0.4% to about 25%, 0.5% to about 25%, 0.6% to about 25%, 0.7% to about 25%, 0.8% to about 25%, 0.9% to about 25%, 1 % to about 25%, 1.5% to about 25%, 1.75% to about 25%, 2% to about 25%, 2.25% to about 25%,, 2.5% to about 25%,, 2.75% to about 25%,, 3% to about 25%, 3.25% to about 25%, 3.5% to about 25%, 3.75% to about 25%, 4% to about 25%, 4.25% to about 25%, 4.5% to about 25%, 4.75% to about 25%, 5% to about 25%, 5.25% to about 25%, 5.5% to about 25%, 5.75% to about 25%, 6% to about 25%, 6.25% to about 25%, 6.5% to about 25%, 6.75% to about 25%, 7% to about 25%, 7.25% to about 25%, 7.5% to about 25%, 7.75% to about 25%, 8% to about 25%, 8.25% to about 25%, 8.5% to about 25%, 8.75% to about 25%, 9% to about 25%, 9.25% to about 25%, 9.5% to about 25%, 9.75% to about 25%, 10% to about 25%, 10.25% to about 25%, 10.5% to about 25%, 10.75% to about 25%, 1 1 % to about 25%, 11 .25% to about 25%, 11 .5% to about 25%, 1 1 .75% to about 25%, 12% to about 25%, 12.25% to about 25%, 12.5% to about 25%, 12.75% to about 25%, 13% to about 25%, 13.25% to about 25%, 13.5% to about 25%, 13.75% to about 25%, 14% to about 25%, 14.25% to about 25%, 14.5% to about 25%, 14.75% to about 25%, 15% to about 25%, 15.25% to about 25%, 15.5% to about 25%, 15.75% to about 25%, 16% to about 25%, 16.25% to about 25%, 16.5% to about 25%, 16.75% to about 25%, 17% to about 25%, 17.25% to about 25%, 17.5% to about 25%, 17.75% to about 25%, 18% to about 25%, 18.25% to about 25%, 18.5% to about 25%, 18.75% to about 25%, 19% to about 25%, 19.25% to about 25%, 19.5% to about 25%, 19.75% to about 25%, 20% to about 25%, 20.25% to about 25%, 20.5% to about 25%, 20.75% to about 25%, 5% to about 20%, 6% to about 20%, 7% to about 20%, 8% to about 20%, 9% to about 20%, 10% to about 20%, 1 1 % to about 20%, 12% to about 20%, 13%, to about 20%, 14% to about 20%, 15% to about 20%, 5% to about 15%, 6% to about 15%, 7% to about 15%, 8% to about 15%, 9% to about 15%, 10% to about 15%, 1 1 % to about 15%, 12% to about 15%, 13%, to about 15%, 14% to about 15% (w/v).

[0148] In an embodiment, a formulation is considered stable when the pharmaceutical composition in the formulation (1 ) retains its physical stability, (2) retains its chemical stability and/or (3) retains it biological activity. In an embodiment, a pharmaceutical composition may be said to "retain its physical stability" in a formulation if, for example, without limitation, it shows no signs of aggregation, precipitation and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light scattering or by size exclusion chromatography (SEC) or electrophoresis, such as with reference to turbidity or aggregate formation.

[0149] In an embodiment, a pharmaceutical composition may be said to "retain its chemical stability" in a formulation, if, for example, without limitation, the chemical stability at a given time is such that there is no significant modification of the pharmaceutical composition by bond formation or cleavage resulting in a new chemical entity. In a further embodiment, chemical stability can be assessed by detecting and quantifying chemically altered forms of the therapeutic composition. Chemical alteration may involve, example, without limitation, size modification (e.g. clipping) which can be evaluated using size exclusion chromatography, SDS-PAGE and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS). Other types of chemical alteration include, for example, without limitation, charge alteration (e.g. occurring as a result of deamidation), which can be evaluated by ion-exchange chromatography, for example. Oxidation is another commonly seen chemical modification.

[0150] After contacting, the concentration of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein in the solution may be in any concentration desired. In an embodiment, the concentration of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein in the solution may be, e.g., at least 0.00001 mg/mL, at least 0.0001 mg/mL, at least 0.001 mg/mL, at least 0.01 mg/mL, at least 0.1 mg/mL, at least 1 mg/mL, at least 10 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least 100 mg/mL, at least 200 mg/mL, at least 500 mg/mL, at least 700 mg/mL, at least 1 ,000 mg/mL, or at least 1 ,200 mg/mL. In another aspect of this embodiment, the concentration of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein in the solution may be, e.g., at most 1 ,000 mg/mL, at most 1 , 100 mg/mL, at most 1 ,200 mg/mL, at most 1 ,300 mg/mL, at most 1 ,400 mg/mL, at most 1 ,500 mg/mL, at most 2,000 mg/mL, at most 2,000 mg/mL, or at most 3,000 mg/mL. In a further aspect of this embodiment, the concentration of a polyclonal antibody preparation disclosed herein in the solution may be in a range of, e.g., about 0.00001 mg/mL to about 3,000 mg/mL, about 0.0001 mg/mL to about 3,000 mg/mL, about 0.01 mg/mL to about 3,000 mg/mL, about 0.1 mg/mL to about 3,000 mg/mL, about 1 mg/mL to about 3,000 mg/mL, about 250 mg/mL to about 3,000 mg/mL, about 500 mg/mL to about 3,000 mg/mL, about 750 mg/mL to about 3,000 mg/mL, about 1 ,000 mg/mL to about 3,000 mg/mL, about 100 mg/mL to about 2,000 mg/mL, about 250 mg/mL to about 2,000 mg/mL, about 500 mg/mL to about 2,000 mg/mL, about 750 mg/mL to about 2,000 mg/mL, about 1 ,000 mg/mL to about 2,000 mg/mL, about 100 mg/mL to about 1 ,500 mg/mL, about 250 mg/mL to about 1 ,500 mg/mL, about 500 mg/mL to about 1 ,500 mg/mL, about 750 mg/mL to about 1 ,500 mg/mL, about 1 ,000 mg/mL to about 1 ,500 mg/mL, about 100 mg/mL to about 1 ,200 mg/mL, about 250 mg/mL to about 1 ,200 mg/mL, about 500 mg/mL to about 1 ,200 mg/mL, about 750 mg/mL to about 1 ,200 mg/mL, about 1 ,000 mg/mL to about 1 ,200 mg/mL, about 100 mg/mL to about 1 ,000 mg/mL, about 250 mg/mL to about 1 ,000 mg/mL, about 500 mg/mL to about 1 ,000 mg/mL, about 750 mg/mL to about 1 ,000 mg/mL, about 100 mg/mL to about 750 mg/mL, about 250 mg/mL to about 750 mg/mL, about 500 mg/mL to about 750 mg/mL, about 100 mg/mL to about 500 mg/mL, about 250 mg/mL to about 500 mg/mL, about 0.00001 mg/mL to about 0.0001 mg/mL, about 0.00001 mg/mL to about 0.001 mg/mL, about 0.00001 mg/mL to about 0.01 mg/mL, about 0.00001 mg/mL to about 0.1 mg/mL, about 0.00001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 0.01 mg/mL, about 0.001 mg/mL to about 0.1 mg/mL, about 0.001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 10 mg/mL, or about 0.001 mg/mL to about 100 mg/mL.

[0151] In a liquid or a lyophilized formulation, a concentration of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein typically may be between about 50 mg/mL to about 1 ,000 mg/mL. In aspects of this embodiment, a therapeutically effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein may be from, e.g., about 50 mg/mL to about 100 mg/mL, about 50 mg/mL to about 200 mg/mL, about 50 mg/mL to about 300 mg/mL, about 50 mg/mL to about 400 mg/mL, about 50 mg/mL to about 500 mg/mL, about 50 mg/mL to about 600 mg/mL, about 50 mg/mL to about 700 mg/mL, about 50 mg/mL to about 800 mg/mL, about 50 mg/mL to about 900 mg/mL, about 50 mg/mL to about 1 ,000 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about 300 mg/mL, about 100 mg/mL to about 400 mg/mL, about 100 mg/mL to about 500 mg/mL, about 100 mg/mL to about 600 mg/mL, about 100 mg/mL to about 700 mg/mL, about 100 mg/mL to about 800 mg/mL, about 100 mg/mL to about 900 mg/mL, about 100 mg/mL to about 1 ,000 mg/mL, about 200 mg/mL to about 300 mg/mL, about 200 mg/mL to about 400 mg/mL, about 200 mg/mL to about 500 mg/mL, about 200 mg/mL to about 600 mg/mL, about 200 mg/mL to about 700 mg/mL, about 200 mg/mL to about 800 mg/mL, about 200 mg/mL to about 900 mg/mL, about 200 mg/mL to about 1 ,000 mg/mL, about 300 mg/mL to about 400 mg/mL, about 300 mg/mL to about 500 mg/mL, about 300 mg/mL to about 600 mg/mL, about 300 mg/mL to about 700 mg/mL, about 300 mg/mL to about 800 mg/mL, about 300 mg/mL to about 900 mg/mL, about 300 mg/mL to about 1 ,000 mg/mL, about 400 mg/mL to about 500 mg/mL, about 400 mg/mL to about 600 mg/mL, about 400 mg/mL to about 700 mg/mL, about 400 mg/mL to about 800 mg/mL, about 400 mg/mL to about 900 mg/mL, about 400 mg/mL to about 1 ,000 mg/mL, about 500 mg/mL to about 600 mg/mL, about 500 mg/mL to about 700 mg/mL, about 500 mg/mL to about 800 mg/mL, about 500 mg/mL to about 900 mg/mL, about 500 mg/mL to about 1 ,000 mg/mL, about 600 mg/mL to about 700 mg/mL, about 600 mg/mL to about 800 mg/mL, about 600 mg/mL to about 900 mg/mL, or about 600 mg/mL to about 1 ,000 mg/mL.

[0152] In a liquid or a lyophilized formulation, an amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein typically may be between about 0. 01 % to about 45% by weight. In aspects of this embodiment, an amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein may be from, e.g., about 0.1 % to about 45% by weight, about 0.1 % to about 40% by weight, about 0.1 % to about 35% by weight, about 0.1 % to about 30% by weight, about 0.1 % to about 25% by weight, about 0.1 % to about 20% by weight, about 0.1 % to about 15% by weight, about 0.1 % to about 10% by weight, about 0.1 % to about 5% by weight, about 1 % to about 45% by weight, about 1 % to about 40% by weight, about 1 % to about 35% by weight, about 1 % to about 30% by weight, about 1 % to about 25% by weight, about 1 % to about 20% by weight, about 1 % to about 15% by weight, about 1 % to about 10% by weight, about 1 % to about 5% by weight, about 5% to about 45% by weight, about 5% to about 40% by weight, about 5% to about 35% by weight, about 5% to about 30% by weight, about 5% to about 25% by weight, about 5% to about 20% by weight, about 5% to about 15% by weight, about 5% to about 10% by weight, about 10% to about 45% by weight, about 10% to about 40% by weight, about 10% to about 35% by weight, about 10% to about 30% by weight, about 10% to about 25% by weight, about 10% to about 20% by weight, about 10% to about 15% by weight, about 15% to about 45% by weight, about 15% to about 40% by weight, about 15% to about 35% by weight, about 15% to about 30% by weight, about 15% to about 25% by weight, about 15% to about 20% by weight, about 20% to about 45% by weight, about 20% to about 40% by weight, about 20% to about 35% by weight, about 20% to about 30% by weight, about 20% to about 25% by weight, about 25% to about 45% by weight, about 25% to about 40% by weight, about 25% to about 35% by weight, or about 25% to about 30% by weight.

[0153] In another embodiment, a therapeutically effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein reduces the severity of illness caused by a Human Enterovirus, for example, without limitation, an enterovirus responsible for foot, hand and mouth disease in a subject by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%. In other aspects of this embodiment, a therapeutically effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein reduces the severity of illness caused by a Human Enterovirus for example, without limitation, an enterovirus responsible for foot, hand and mouth disease in a subject by, e.g., at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95% or at most 100%. In yet other aspects of this embodiment, a therapeutically effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein reduces the severity of illness caused by a Human Enterovirus for example, without limitation, an enterovirus responsible for foot, hand and mouth disease in a subject by, e.g., about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0154] In other aspects of this embodiment, a therapeutically effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein generally is in the range of about 0. 001 mg/kg/day to about 100 mg/kg/day. In aspects of this embodiment, an effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein may be, e.g., at least 0.001 mg/kg/day, at least 0.01 mg/kg/day, at least 0.1 mg/kg/day, at least 1 .0 mg/kg/day, at least 5.0 mg/kg/day, at least 10 mg/kg/day, at least 15 mg/kg/day, at least 20 mg/kg/day, at least 25 mg/kg/day, at least 30 mg/kg/day, at least 35 mg/kg/day, at least 40 mg/kg/day, at least 45 mg/kg/day, or at least 50 mg/kg/day. In other aspects of this embodiment, an effective amount of a polyclonal antibody preparation disclosed herein may be in the range of, e.g., about 0.001 mg/kg/day to about 10 mg/kg/day, about 0.001 mg/kg/day to about 15 mg/kg/day, about 0.001 mg/kg/day to about 20 mg/kg/day, about 0.001 mg/kg/day to about 25 mg/kg/day, about 0.001 mg/kg/day to about 30 mg/kg/day, about 0.001 mg/kg/day to about 35 mg/kg/day, about 0.001 mg/kg/day to about 40 mg/kg/day, about 0.001 mg/kg/day to about 45 mg/kg/day, about 0.001 mg/kg/day to about 50 mg/kg/day, about 0.001 mg/kg/day to about 75 mg/kg/day, or about 0.001 mg/kg/day to about 100 mg/kg/day. In yet other aspects of this embodiment, an effective amount of a polyclonal antibody preparation disclosed herein may be in the range of, e.g., about 0.01 mg/kg/day to about 10 mg/kg/day, about 0.01 mg/kg/day to about 15 mg/kg/day, about 0.01 mg/kg/day to about 20 mg/kg/day, about 0.01 mg/kg/day to about 25 mg/kg/day, about 0.01 mg/kg/day to about 30 mg/kg/day, about 0.01 mg/kg/day to about 35 mg/kg/day, about 0.01 mg/kg/day to about 40 mg/kg/day, about 0.01 mg/kg/day to about 45 mg/kg/day, about 0.01 mg/kg/day to about 50 mg/kg/day, about 0.01 mg/kg/day to about 75 mg/kg/day, or about 0.01 mg/kg/day to about 100 mg/kg/day. In still other aspects of this embodiment, an effective amount of a polyclonal antibody preparation disclosed herein may be in the range of, e.g., about 0.1 mg/kg/day to about 10 mg/kg/day, about 0.1 mg/kg/day to about 15 mg/kg/day, about 0.1 mg/kg/day to about 20 mg/kg/day, about 0.1 mg/kg/day to about 25 mg/kg/day, about 0.1 mg/kg/day to about 30 mg/kg/day, about 0.1 mg/kg/day to about 35 mg/kg/day, about 0.1 mg/kg/day to about 40 mg/kg/day, about 0.1 mg/kg/day to about 45 mg/kg/day, about 0.1 mg/kg/day to about 50 mg/kg/day, about 0.1 mg/kg/day to about 75 mg/kg/day, or about 0.1 mg/kg/day to about 100 mg/kg/day.

[0155] In other aspects of this embodiment, an effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein may be in the range of, e.g., about 1 mg/kg/day to about 10 mg/kg/day, about 1 mg/kg/day to about 15 mg/kg/day, about 1 mg/kg/day to about 20 mg/kg/day, about 1 mg/kg/day to about 25 mg/kg/day, about 1 mg/kg/day to about 30 mg/kg/day, about 1 mg/kg/day to about 35 mg/kg/day, about 1 mg/kg/day to about 40 mg/kg/day, about 1 mg/kg/day to about 45 mg/kg/day, about 1 mg/kg/day to about 50 mg/kg/day, about 1 mg/kg/day to about 75 mg/kg/day, or about 1 mg/kg/day to about 100 mg/kg/day. In yet other aspects of this embodiment, an effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein may be in the range of, e.g., about 5 mg/kg/day to about 10 mg/kg/day, about 5 mg/kg/day to about 15 mg/kg/day, about 5 mg/kg/day to about 20 mg/kg/day, about 5 mg/kg/day to about 25 mg/kg/day, about 5 mg/kg/day to about 30 mg/kg/day, about 5 mg/kg/day to about 35 mg/kg/day, about 5 mg/kg/day to about 40 mg/kg/day, about 5 mg/kg/day to about 45 mg/kg/day, about 5 mg/kg/day to about 50 mg/kg/day, about 5 mg/kg/day to about 75 mg/kg/day, or about 5 mg/kg/day to about 100 mg/kg/day.

[0156] In other aspects of this embodiment, a therapeutically effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein generally is in the range of about 1 mg/day to about 3,000 mg/day. In aspects of this embodiment, an effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein may be, e.g., at least 1 mg/day, at least 5 mg/day, at least 10 mg/day, at least 15 mg/day, at least 20 mg/day, at least 25 mg/day, at least 30 mg/day, at least 40 mg/day, at least 50 mg/day, at least 100 mg/day, at least 150 mg/day, at least 200 mg/day, at least 250 mg/day, at least 300 mg/day, at least 350 mg/day, at least 400 mg/day, at least 450 mg/day, at least 500 mg/day, at least 550 mg/day, at least 600 mg/day, at least 650 mg/day, at least 700 mg/day, at least 750 mg/day, at least 800 mg/day, at least 850 mg/day, at least 900 mg/day, at least 950 mg/day, at least 1 ,000 mg/day, at least 1 ,50 mg/day, at least 1 , 100 mg/day, at least 1 ,150 mg/day, at least 1 ,200 mg/day, at least 1 ,250 mg/day, at least 1 ,300 mg/day, at least 1 ,350 mg/day, at least 1 ,400 mg/day, at least 1 ,450 mg/day, at least 1 ,500 mg/day, at least 1 ,600 mg/day, at least 1 ,700 mg/day, at least 1 ,800 mg/day, at least 1 ,900 mg/day, at least 2,000 mg/day, at least 2,100 mg/day, at least 2,200 mg/day, at least 2,300 mg/day, at least 2,400 mg/day, at least 2,500 mg/day, at least 2,600 mg/day, at least 2,700 mg/day, at least 2,800 mg/day, at least 2,900 mg/day, or at least 3,000 mg/day. In yet other aspects of this embodiment, an effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment disclosed herein may be between, e.g., about 1 mg/day to about 1 ,000 mg/day, about 5 mg/day to about 1 ,000 mg/day, about 10 mg/day to about 1 ,000 mg/day, about 15 mg/day to about 1 ,000 mg/day, about 20 mg/day to about 1 ,000 mg/day, about 25 mg/day to about 1 ,000 mg/day, about 30 mg/day to about 1 ,000 mg/day, about 40 mg/day to about 1 ,000 mg/day, about 50 mg/day to about 1 ,000 mg/day, about 100 mg/day to about 1 ,000 mg/day, about 150 mg/day to about 1 ,000 mg/day, about 200 mg/day to about 1 ,000 mg/day, about 250 mg/day to about 1 ,000 mg/day, about 300 mg/day to about 1 ,000 mg/day, about 350 mg/day to about 1 ,000 mg/day, about 400 mg/day to about 1 ,000 mg/day, about 450 mg/day to about 1 ,000 mg/day, about 500 mg/day to about 1 ,000 mg/day, about 50 mg/day to about 1 ,500 mg/day, about 100 mg/day to about 1 ,500 mg/day, about 150 mg/day to about 1 ,500 mg/day, about 200 mg/day to about 1 ,500 mg/day, about 250 mg/day to about 1 ,500 mg/day, about 300 mg/day to about 1 ,500 mg/day, about 350 mg/day to about 1 ,500 mg/day, about 400 mg/day to about 1 ,500 mg/day, about 450 mg/day to about 1 ,500 mg/day, about 500 mg/day to about 1 ,500 mg/day, about 1 ,000 mg/day to about 3,000 mg/day, about 1 ,100 mg/day to about 3,000 mg/day, about 1 ,200 mg/day to about 3,000 mg/day, about 1 ,3000 mg/day to about 3,000 mg/day, about 1 ,400 mg/day to about 3,000 mg/day, about 1 ,500 mg/day to about 3,000 mg/day, about 1 ,600 mg/day to about 3,000 mg/day, about 1 ,700 mg/day to about 3,000 mg/day, about 1 ,800 mg/day to about 3,000 mg/day, about 1 ,900 mg/day to about 3,000 mg/day, or about 2,000 mg/day to about 3,000 mg/day.

[0157] In other aspects of this embodiment, a therapeutically effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment against the infectious agent responsible for foot, hand and mouth disease disclosed herein generally is in the range of about 0. 001 mg/kg/day to about 100 mg/kg/day. In aspects of this embodiment, an effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment against the infectious agent responsible for foot, hand and mouth disease disclosed herein may be, e.g., at least 0.001 mg/kg/day, at least 0.01 mg/kg/day, at least 0.1 mg/kg/day, at least 1 .0 mg/kg/day, at least 5.0 mg/kg/day, at least 10 mg/kg/day, at least 15 mg/kg/day, at least 20 mg/kg/day, at least 25 mg/kg/day, at least 30 mg/kg/day, at least 35 mg/kg/day, at least 40 mg/kg/day, at least 45 mg/kg/day, or at least 50 mg/kg/day. In other aspects of this embodiment, an effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment against the infectious agent responsible for foot, hand and mouth disease disclosed herein may be in the range of, e.g., about 0.001 mg/kg/day to about 10 mg/kg/day, about 0.001 mg/kg/day to about 15 mg/kg/day, about 0.001 mg/kg/day to about 20 mg/kg/day, about 0.001 mg/kg/day to about 25 mg/kg/day, about 0.001 mg/kg/day to about 30 mg/kg/day, about 0.001 mg/kg/day to about 35 mg/kg/day, about 0.001 mg/kg/day to about 40 mg/kg/day, about 0.001 mg/kg/day to about 45 mg/kg/day, about 0.001 mg/kg/day to about 50 mg/kg/day, about 0.001 mg/kg/day to about 75 mg/kg/day, or about 0.001 mg/kg/day to about 100 mg/kg/day.

[0158] In other aspects of this embodiment, an effective amount of a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment against the infectious agent responsible for foot, hand and mouth disease disclosed herein may be in the range of, e.g., about 1 mg/kg/day to about 10 mg/kg/day, about 1 mg/kg/day to about 15 mg/kg/day, about 1 mg/kg/day to about 20 mg/kg/day, about 1 mg/kg/day to about 25 mg/kg/day, about 1 mg/kg/day to about 30 mg/kg/day, about 1 mg/kg/day to about 35 mg/kg/day, about 1 mg/kg/day to about 40 mg/kg/day, about 1 mg/kg/day to about 45 mg/kg/day, about 1 mg/kg/day to about 50 mg/kg/day, about 1 mg/kg/day to about 75 mg/kg/day, or about 1 mg/kg/day to about 100 mg/kg/day.

[0159] In an embodiment, a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment preparation provides protection from or treatment of an infection with the causative agent of foot, hand and mouth disease.

[0160] In a further embodiment, a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment preparation provides protection against neurological conditions resulting from a Human Enterovirus infection, including Human Enterovirus A and/or Human Enterovirus B, and further including, without limitation the causative agent of hand, foot and mouth disease. In an embodiment, a neurological condition includes, without limitation, pulmonary adema, meningitis, meningoencephalomyelitis, poliomyelitis-like paralytic disease, Guillain-Barre syndrome, transverse myelitis, cerebellar ataxia, opsoclonus-myoclonus syndrome, benign intracranial hypertension, and brainstem encephalitis.

[0161] In a further embodiment, a polyclonal antibody preparation, a monoclonal antibody preparation and/or an antibody fragment preparation provides protection against cardiopulmonary conditions, including, without limitation, myocarditis, resulting from a Human Enterovirus infection, including Human Enterovirus A and/or Human Enterovirus B, and further including, without limitation the causative agent of hand, foot and mouth disease.

[0162] In another embodiment, a polyclonal antibody preparation is prepared through the immunization of a subject, including, without limitation, a human. The immunization may be done one or more times prior to the collection of a polyclonal antibody preparation. In an embodiment, a subject is immunized once, twice, three, four, five, six, seven, eight, nine or more times prior to the collection of the polyclonal antibody preparation. The immunization may be administered, without limitation, intravenously, orally, intravaginally, intraparentally, intra-anally, subcutaneously, intramuscularly and/or intravenously. The immunization may be done, without limitation, with a Human Enterovirus, including, without limitation, an enterovirus responsible for foot, hand and mouth disease.

[0163] In an embodiment, the immunization of a subject uses a formulation that comprises an antigen capable of stimulating an immune, including without limitation, a humoral polyclonal antibody response. In a further embodiment, a vaccine, including, without limitation, an antigen, is a virus like particle, a viral protein, a viral protein, virus capsomers, aggregates, complexes of antigens from viruses and/or a viral nucleic acid. In another embodiment, a vaccine, including, without limitation, an antigen is from a Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease, and further including, without limitation, Human Enterovirus 71 ("EV71 "), Coxsackie virus ("CA5"), Coxsackie virus 6 ("CA6"), Coxsackie virus ("CA10") and Coxsackie virus 16 ("CA16"), including, without limitation all subgroups, genotypes and other forms or variants of each of EV71 , CA5 CA6, CA10 and CA16. In an embodiment, a vaccine, including, without limitation, an antigen, is a virus like particle, a viral protein, an attenuated virus, a viral protein and/or a viral nucleic acid of or derived from EV71 or CA16.

[0164] In an embodiment, a vaccine is comprised of an inactivated Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease created by a method known to one of skill in the art. In an embodiment, a vaccine is comprised of an attenuated Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease created by a method known to one of skill in the art. In a further embodiment, an inactivated and/or attenuated virus is a complete virus and/or a partial virus. In another embodiment a partial virus is a virus that is missing one or more elements, including proteins or portions of a nucleic acid.

[0165] In an embodiment, a subject is administered at least 1 g, 10 g, 20 g, 30 g, 40 g, 50 g, 60 [ig, 70 [ig, 80 [ig, 90 [ig, 100 [ig, 125 [ig, 150 [ig, 175 [ig, 200 [ig, 225 [ig, 250 [ig, 275 [ig, 300 [ig, 325 [ig, 350 [ig, 375 [ig, 400 [ig, 425 [ig, 450 [ig, 475 [ig, 500 [ig, 525 [ig, 550 [ig, 575 [ig, 600 [ig, 625 [ig, 650 [ig, 675 [ig, 700 [ig, 725 [ig, 750 [ig, 775 [ig, 800 [ig, 825 [ig, 850 [ig, 875 [ig, 900 [ig, 925 [ig, 950 [ig, 975 [ig, 1000 [ig, 1 100 [ig, 1200 [ig, 1300 [ig, 1400 [ig, 1500 [ig, 1600 [ig, 1700 [ig, 1800 [ig, 1900 [ig, 2000 [ig, 2100 [ig, 2200 [ig, 2300 [ig, 2400 [ig, 2500 [ig, 2600 [ig, 2700 [ig, 2800 [ig, 2900 g, 3000 [ig, 3100 g, 3200 [ig, 3300 g, 3400 g, 3500 [ig, 3600 g, 3700 [ig, 3800 g, 3900 g, 4000 [ig, 4100 g, 4200 [ig, 4300 g, 4400 g, 4500 [ig, 4600 g, 4700 [ig, 4800 g, 4900 g, 5000 [ig, 5100 g, 5200 [ig, 5300 g, 5400 g, 5500 [ig, 5600 g, 5700 [ig, 5800 g, 5900 g, 6000 [ig, 6100 g, 6200 [ig, 6300 g, 6400 g, 6500 [ig, 6600 g, 6700 [ig, 6800 g, 6900 g, 7000 [ig, 7100 g, 7200 [ig, 7300 g, 7400 g, 7500 [ig, 7600 g, 7700 [ig, 7800 g, 7900 g, 8000 [ig, 8100 g, 8200 [ig, 8300 g, 8400 g, 8500 [ig, 8600 g, 8700 [ig, 88 g, 8900 g, 9000 [ig, 9100 g, 9200 [ig, 9300 g, 9400 g, 9500 [ig, 9600 g, 9700 [ig, 9800 g, 9900 g, 10000 [ig, or more. [0166] A subject administered a vaccine is administered the vaccine more than once. In an embodiment, a subject is administered a vaccine, two, three, four, five, six, seven, eight, nine, ten or more times. In a further embodiment, a follow on vaccination is administered to a subject, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more. Following immunization, plasma is collected from a subject and tested by standard methods to determine the subjects antibody titer against a Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease. From the results of the testing, in an embodiment, plasma containing antibodies against a Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease is pooled. In an embodiment, the pooled plasma is used to purify a polyclonal antibody preparation that includesa high titer of antibodies against a Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease.

[0167] In a further embodiment, pooled plasma from all subjects immunized one or more times with a vaccine against a Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease is collected and purified to form a polyclonal preparation that includes antibodies against a Human Enterovirus, including, without limitation, a causative agent of foot, hand and mouth disease.

[0168] In an embodiment, a subject is administered a polyclonal antibody preparation one or more times prior to infection or to treat an infection resulting from a Human Enterovirus. In another embodiment, a subject is administered a polyclonal antibody preparation one, two, three, four, five, six, seven, eight, nine, ten or more times prior to infection or to treat an infection resulting from a Human Enterovirus. In an embodiment, a subject is administered a polyclonal antibody preparation prior to, during or prior to and during an infection with a Human Enterovirus. In an embodiment, the Human Enterovirus that is administered to a subject is the causative agent for hand, foot and mouth disease.

[0169] Methods to collect and/or purify a polyclonal antibody preparation, including, without limitation from the plasma of one or more subjects, is known to one of skill in the art and such methods are hereby incorporated for the collection and/or purification of a polyclonal antibody preparation that provides protection from or treatment of an infection by a Human Enterovirus, including, without limitation, the causative agent of foot, hand and mouth disease.

[0170] The invention provides virus like particles (VLPs), virus capsomers, aggregates, and complexes of antigens from viruses of the Picornaviridae family as an immunogenic composition and/or vaccine for the protection against and/or treatment of a picornavirus infection. Representative examples may include an Enterovirus, a Coxsackie virus, and a poliovirus.

[0171] In an embodiment, an Enterovirus antigen may be a combination of Enterovirus coat/capsid proteins, or immunologically active fragments thereof. The virus coat/capsid proteins may be any combination of VP0, VP1 , VP2, VP3 and/or VP4 proteins, and may take the form of a virus-like particle (VLP), capsomer, complex and/or aggregate. The combination may be in the form of a fusion protein. [0172] The invention in an additional aspect includes a method for production of Picornaviridae virus like particles (VLPs), capsomers, complexes and/or aggregates which may include the steps of: (i) constructing an expression cassette operably linked to a promoter comprising one or more nucleic acids which each encode a picornavirus protein, for example, a P1 protein or a combination of picornavirus VPO protein, VP1 protein, VP2 protein, VP3 protein, and/or a VP4 protein, which is/are operably linked to an internal ribosome entry site (IRES), which IRES is operably linked to a 3C or 3CD protease; (ii) transfecting or transforming a suitable host cell with the expression cassette; (iii) culturing the host cells under conditions in which virus like particles (VLPs) and/or capsomers and/or antigens are produced by the cell after expression of the nucleic acids comprised in the cassette.

[0173] A nucleic acid or recombinant DNA molecule may be obtained whereby open reading frames which encode Coxsackievirus A16, HEV71 , Human enterovirus C (human polioviruses PV1 , PV2 and/or PV3), EV 68, or any other picornavirus proteins and proteases may be amplified by PCR amplification using suitably designed primers complementary to nucleic acid sequences of Coxsackievirus A16, HEV71 or Human enterovirus C or any other picornavirus. Suitable primers may be designed according to standard techniques from publicly available nucleic acid sequences of enteroviruses, including Coxsackievirus A16, HEV71 and Human enterovirus C or any other picornavirus. Complete genome sequences are available in GenBank and are accessible at the National Center for Biotechnology Information (NCBI).

[0174] In an embodiment, a picornavirus P1 protein, or any Enterovirus P1 protein, is expressed as a polypeptide which is subsequently cleaved by the 3C or 3CD protease into VPO, VP1 and VP3 virus protein, or immunologically or biologically active fragments thereof, which Enterovirus proteins elicit neutralizing antibodies directed against enteroviruses. The VPO protein may be further cleaved into VP2 and VP4 proteins, or immunologically or biologically active fragments thereof which elicit neutralizing antibodies directed against enteroviruses. The virus proteins may self-assemble into VLPs, capsomers and/or aggregates of enterovirus proteins. Further it will be appreciated that the protease genes may be included in the same DNA recombinant molecule of the VLP expression cassette or in different DNA recombinant molecules, and/or expressed from different promoters or translation elements.

[0175] Recombinant DNA molecules and nucleic acids of the VLP expression cassettes may be devised whereby open reading frames which encode picornavirus structural proteins or proteases may be obtained by PCR amplification using suitably designed primers complementary to nucleic acid sequences of human picornavi ruses.

[0176] In a further embodiment, the recombinant DNA molecule may encode a fusion protein having at least two enterovirus structural proteins, or portions thereof, which are expressed as a single polypeptide antigen.

[0177] The present invention encompasses a VLP expression cassette which harbors the gene sequences for Enterovirus structural proteins (P1 region) with a protease (3CD) which is necessary for the processing of P1 proteins into the proteins of the virus capsid, thus allowing the self-assembly of Enterovirus VLPs. The expression cassette is a bicistronic vector which uses a promoter upstream of the nucleic acid coding sequence for an Enterovirus P1 protein. Downstream from the cistron encoding the P1 protein is an internal ribosome entry site (IRES) sequence followed by the cistron containing a nucleotide sequence encoding the 3CD protease.

[0178] Expression of the P1 region and the 3CD protease proceeds from a single bicistronic message wherein the 3CD protease gene is translated in a cap-independent fashion under the control of the IRES. It is observed that expression of the protease 3CD may be moderately toxic leading to premature death of the host cells, thereby lowering the yield of the Enterovirus capsid proteins and VLPs. The activity of the protease may be reduced while maintaining the high level of P1 protein expression from the cassette. Different IRESs and IRES sequences comprising mutations were inserted into the expression cassettes to control expression/activity of the 3CD protease and to identify effective IRES to properly process the P1 without being toxic to the cell. For efficient production of VLPs, a number of recombinant baculoviruses which have the complete P1 coding sequence and the complete 3CD protease coding sequence whose expression is under the control of IRESs from different species or serotypes of viruses, were tested for efficient production of VLPs.

[0179] For example, the expression cassette of invention may comprise a promoter which is operably linked to a nucleic acid encoding a Human enterovirus A P1 polypeptide, an EMCV IRES, and a Human enterovirus A 3CD protease.

[0180] The expression cassette of invention may comprise a promoter which is operably linked to a nucleic acid encoding a Human enterovirus A P1 polypeptide, a Human enterovirus C IRES, and a Human enterovirus A 3CD protease.

[0181] The expression cassette of invention may comprise a promoter which is operably linked to a nucleic acid encoding a Human enterovirus C P1 polypeptide, an HEV71 IRES, and a Human enterovirus C 3CD protease.

[0182] Furthermore, the expression cassette of invention may comprise a promoter which is operably linked to a nucleic acid encoding a Human enterovirus C P1 polypeptide, an EMCV IRES, and a Human enterovirus C 3CD protease.

[0183] Moreover, making truncations and mutations of the 3CD protease in the expression cassette which comprises efficient IRES may achieve maximum yield of VLPs. For example, the Glycine of the HEV71 3C protease which is amino acid 1671 of GenBank accession number DQ341362.1 is changed to an Alanine using site directed mutagenesis for the expression of mutant HEV71 3C and subsequent processing of an HEV71 P1 polypeptide.

[0184] Counter to conventional wisdom in the art with respect to the goal of achieving high levels of expression and activity of a protein from an expression cassette, in an embodiment, the instant invention actually seeks to reduce the activity of a protein to achieve a maximum protein yield. Mutation of the IRES or 3C protease nucleic acid to reduce activity unexpectedly results in an increased yield of Enterovirus capsid proteins and VLPs.

[0185] The expression cassettes may be cloned into suitable vectors and transformed/transfected into appropriate host cells for expression and purification of antigens for vaccines and protection against infections from picornaviruses, including enteroviruses.

[0186] The expression cassettes encoding picornavirus antigens may be comprised in plasmids which may be transfected into eukaryotic host cells and expressed under the appropriate growth conditions. Suitable eukaryotic expression systems are known to those skilled in the art and include inducible expression systems and appropriate eukaryotic host cells.

[0187] Mammalian cell expression vectors comprising an expression cassette of the invention include those which may be transiently transfected into host cells and cell lines. Moreover, mammalian cell expression vectors may be vectors which are stably maintained within the host cell following transfection.

[0188] Furthermore, mammalian cell expression vectors may include vectors which are stably or transiently transfected into mammalian host cells or cell lines wherein expression of the protein of interest is induced by the addition of an inducing agent into the culture medium. Mammalian host cells and cell lines include, for example, CHO, HEK 293, COS-1 , HeLa, Vera and NIH3T3 cells. It will also be appreciated that other eukaryotic host cells may include yeast cells or other mammalian cell lines.

[0189] The expression cassette may be contained in recombinant viruses which may transfect the host cell. Suitable viruses that may be used for this purpose include baculovirus, vaccinia, sindbis virus, SV40, Sendai virus, retrovirus and adenovirus. Suitable host cells may include host cells that are compatible with the above viruses and these include insect cells such as Spodoptera frugiperda (e.g. Sf9 cells) Trichoplusia ni, CHO cells, chicken embryo fibroblasts, BHK cells, human SW13 cells, drosophila, mosquito cells derived from Aedes albopictus.

[0190] The expression cassette comprising Enterovirus nucleic acids may be introduced into an appropriate host cell by means known to those skilled in the art. The host cells are propagated and cultured under conditions which allow expression of Enterovirus genes and proteins.

[0191] A gene encoding an enterovirus VP2 protein, or immunologically or biologically active fragments thereof which elicit neutralizing antibodies against enteroviruses, may be inserted in a plasmid containing a suitable promoter and expressed in a host cell. The produced enterovirus VP2 protein will be isolated and used as the basis of an immunogenic composition for use as a vaccine or for diagnostic use.

[0192] A gene encoding an enterovirus VP4 protein, or immunologically or biologically active fragments thereof which elicit neutralizing antibodies against enteroviruses, may be inserted in a plasmid containing a suitable promoter and expressed in a host cell. The produced enterovirus VP4protein will be isolated and used as the basis of an immunogenic composition for use as a vaccine or for diagnostic use.

[0193] A gene encoding an enterovirus VPO protein, or immunologically or biologically active fragments thereof which elicit neutralizing antibodies against enteroviruses, may be inserted in a plasmid containing a suitable promoter and expressed in a host cell. The produced enterovirus VPO protein will be isolated and used as the basis of an immunogenic composition for use as a vaccine or for diagnostic use.

[0194] A gene encoding an enterovirus VPO protein may be operably linked to a suitable promoter and inserted into a plasmid, which plasmid exhibits an enterovirus protease linked to a suitable promoter to provide a doubly recombinant plasmid, which doubly recombinant plasmid may ultimately be expressed in a eukaryotic or prokaryotic cell expression system.

[0195] Suitable vectors for the cloning of genes and expression of enterovirus polypeptide antigens include cosmids or plasmids. Suitable expression systems include prokaryotic expression systems known to those skilled in the art and prokaryotic host cells, including E. coli, transformed with the cosmids or plasmids for expression of proteins in prokaryotic cells. Suitable expression systems include eukaryotic expression systems known to those skilled in the art and eukaryotic host cells transformed with plasmids for expression of proteins in various eukaryotic host cells and cell lines. Moreover, the Enterovirus polypeptide antigens may be obtained from host cells or culture supernatants by means known to those skilled in the art.

[0196] The Enterovirus VLPs, capsomers, antigens, immunologically active components thereof, and/or aggregates thereof, may be obtained from transfected and/or transformed host cells, or host cell culture medium, supernatants and lysates by any suitable means of purification known to those skilled in the art. Isolation of proteins released into the culture medium is a facile method of obtaining Enterovirus VLPs, capsomers, antigens and/or aggregates. The Enterovirus VLPs, capsomers, antigens, immunologically active components thereof, and/or aggregates thereof, may be further concentrated and purified by means known to those skilled in the art.

[0197] The invention in another aspect includes a vaccine containing picornavirus antigens, such as Enterovirus antigens, VLPs and/or capsomers in combination with a suitable adjuvant. The picornavirus antigens, immunologically active fragments thereof, VLPs and/or capsomers may be combined with any suitable adjuvant such as Modified Vaccinia Virus, ISCOMS, alum, aluminum hydroxide, aluminum phosphate, Freund's Incomplete or Complete Adjuvant, Quil A and other saponins or any other adjuvant as described for example in Vanselow (1987) S. Vet. Bull. 57 881 -896. The meaning of the terms "aluminum phosphate" and "aluminum hydroxide" as used herein includes all forms of aluminum hydroxide or aluminum phosphate which are suitable for adjuvanting vaccines.

[0198] A recombinant DNA molecule may be obtained whereby nucleic acids comprising open reading frames which encode Human enterovirus C structural proteins or proteases may be obtained by PCR amplification using suitably designed primers complementary to nucleic acid sequences of human Human enterovirus C. Suitable primers may be designed according to standard techniques from publicly available nucleic acid sequences of Human enterovirus C, such as those complete genome sequences available in GenBank and accessible at the National Center for Biotechnology Information (NCBI). Accession numbers for the complete genome of the Human enterovirus C poliovirus type I genome include V01149 and V01 150.

[0199] The expression cassettes of the invention may comprise nucleic acids which encode a Human enterovirus C P1 polypeptide. The P1 polypeptide is processed (cleaved) by the 3CD protease translated under the control of the IRES of the expression cassette to yield VP1 , VP3 and/or VPO polypeptides. Combinations of VPO, VP1 and VP3 may self-associate into virus-like particles.

[0200] Moreover, picornavirus antigens may be prepared by chemical synthesis of polypeptides based on the publicly available nucleic acid or protein sequences of human poliovirus or by chemical synthesis.

[0201] The Human enterovirus C polypeptide antigen may comprise a Human enterovirus C coat/capsid VP2 protein, a product of further processing of VPO, in combination with another poliovirus VPO, VP1 , VP3 and/or VP4 coat/capsid proteins. The combination of Human enterovirus C coat/capsid proteins may take the form of a virus-like particle (VLP), capsomer, complex and/or aggregate.

[0202] A gene encoding a Human enterovirus C coat/capsid VP2 protein may be inserted into a plasmid containing a suitable promoter and expressed in a host cell, the protein isolated and used as the basis of an immunogenic composition for use as a vaccine. Furthermore, the gene encoding human poliovirus VP2 protein may be inserted into a plasmid containing a suitable promoter and expressed in a host cell, the protein isolated, and used as the basis of an immunogenic composition for use as a vaccine.

[0203] A gene encoding a Human enterovirus C coat/capsid VP4 protein may be inserted into a plasmid containing a suitable promoter and expressed in a host cell, the protein isolated and used as the basis of an immunogenic composition for use as a vaccine. Furthermore, the gene encoding human poliovirus VP4 protein may be inserted into a plasmid containing a suitable promoter and expressed in a host cell, the protein isolated, and used as the basis of an immunogenic composition for use as a vaccine.

[0204] A gene encoding a Human enterovirus C coat/capsid VPO protein may be inserted into a plasmid containing a suitable promoter and expressed in a host cell, the protein isolated and used as the basis of an immunogenic composition for use as a vaccine. Furthermore, the gene encoding human poliovirus VPO protein may be inserted into a plasmid containing a suitable promoter and expressed in a host cell, the protein isolated, and used as the basis of an immunogenic composition for use as a vaccine.

[0205] The invention encompasses a vaccine comprising one or more immunologically active antigens comprising one or more Human enterovirus C VPO, VP1 , VP2, VP3, VP4 polypeptides, and immunologically active fragments thereof, which vaccine elicits a protective and/or neutralizing immune response directed against a human Enterovirus.

[0206] In an embodiment, the expression cassette consists essentially of a nucleic acid encoding a Human enterovirus C P1 polyprotein, an IRES and an enterovirus 3CD protease under the translational control of the IRES, which protease processes the Human enterovirus C P1 polyprotein into structural capsid proteins.

[0207] In another aspect, the invention provides a vaccine for administration to an individual for the production of an immune response. In a further aspect, the immune response is a humoral response. In an aspect the humoral response is an antibody response. In an aspect, the antibody response is a polyclonal antibody response. In another aspect, a monoclonal antibody is prepared from a vaccinated individual.

[0208] In a further aspect, comprising Human enterovirus A antigen(s), derived from a P1 polyprotein including, VP2, VP4 and/or VP0 proteins, and/or biologically or immunologically active fragments thereof. The Human enterovirus A antigens may be derived from HEV71 and/or Coxsackievirus A16. The HEV71 antigen may be a single human enterovirus virus coat/capsid protein. In particular, the HEV71 antigen may be an HEV71 P1 polyprotein, a VP4, VP2 or VP0 polypeptide, or a fragment thereof, which elicits an immune response upon administration to a human.

[0209] In an embodiment, the Human enterovirus A antigen may be a combination of Human enterovirus A coat/capsid proteins, or immunologically active fragments thereof. For example, the Human enterovirus A antigen may comprise a poliovirus VP2 protein, in combination with another poliovirus coat/capsid proteins selected from VP1 , VP3 and/or VP4 polypeptides. The combination of a VP2 polypeptide with other poliovirus coat/capsid proteins may take the form of a virus-like particle (VLP), capsomer, complex and/or aggregate. The combination may be in the form of a fusion protein.

[0210] More specifically the invention pertains to vaccines comprising a Human enterovirus A VP2 coat/capsid protein, or immunogenic fragment thereof, as antigen.

[0211] The invention in another aspect includes a vaccine comprising Human enterovirus A virus like particles (VLPs) and/or capsomers comprising VP1 , VP2, VP3 and/or VP4, or VP0 Human enterovirus A proteins.

[0212] A recombinant DNA molecule may be obtained whereby open reading frames which encode Human enterovirus A structural proteins and/or proteases may be amplified by PCR amplification using suitably designed primers complementary to nucleic acid sequences of Human enterovirus A. Suitably designed primers may be designed according to standard techniques from publicly available nucleic acid sequences of HEV71. Accession numbers for the complete genome of HEV71 include DQ341362, AB204852, AF302996 and AY465356. [0213] A recombinant DNA molecule may be obtained whereby open reading frames which encode Human enterovirus A P1 , VP1 , VP2, VP3 and/or VP4, or VPO proteins, and immunologically active fragments thereof, and proteases, may be obtained by PCR amplification using suitably designed primers complementary to nucleic acid sequences of Human enterovirus A.

[0214] In an aspect of the invention, a Human enterovirus A P1 protein is expressed to form a polyprotein or polypeptide which is subsequently cleaved by the 3C or 3CD protease into VPO, VP1 and VP3 proteins. VPO proteins may be further cleaved into VP2 and VP4 proteins. The enterovirus proteins may self- assemble into VLPs, capsomers, complexes and/or aggregates of enterovirus proteins. Further it will be appreciated that the non-structural genes and the protease genes may be included in the same DNA recombinant molecule or in different DNA recombinant molecules, and or expressed from different promoters or translation elements.

[0215] The expression cassettes of the invention may comprise nucleic acids which encode a Human enterovirus A P1 polypeptide. The P1 polypeptide is processed (cleaved) by the 3CD protease translated under the control of the IRES of the expression cassette to yield VP1 , VP3 and VPO polypeptides and immunologically active fragments thereof. Combinations of VPO, VP1 and VP3 polypeptides may self- associate into virus-like particles.

[0216] A gene encoding a Human enterovirus A VP2 protein, or immunologically active fragment thereof, may be inserted in a plasmid containing a suitable promoter and expressed in a host cell. The isolated Human enterovirus A antigen, for example, an HEV71 VP2 protein, may be isolated and used as the basis of an immunogenic composition for use as a vaccine or for diagnostic use.

[0217] A gene encoding a Human enterovirus A VP4 protein, or immunologically active fragment thereof, may be inserted in a plasmid containing a suitable promoter and expressed in a host cell. The isolated VP4 protein may be isolated and used as the basis of an immunogenic composition for use as a vaccine or for diagnostic use.

[0218] A gene encoding a Human enterovirus A VPO protein, or immunologically active fragment thereof, may be inserted in a plasmid containing a suitable promoter and expressed in a host cell. The isolated VPO protein may be isolated and used as the basis of an immunogenic composition for use as a vaccine or for diagnostic use.

[0219] A gene encoding a Human enterovirus A VPO protein, or immunologically active fragment thereof, may be operably linked to a suitable promoter and inserted into a plasmid, which plasmid exhibits a Human enterovirus A protease linked to a suitable promoter to provide a doubly recombinant plasmid, which doubly recombinant plasmid may ultimately be expressed in a eukaryotic or prokaryotic cell expression system.

[0220] The Human enterovirus A genes and nucleic acids comprised in the expression cassette may be introduced into an appropriate host cell by means known to those skilled in the art. The host cells are propagated and cultured under conditions which allow expression of Human enterovirus A genes and proteins.

[0221] The invention encompasses a vaccine comprising one or more immunologically active antigens comprising one or more Human enterovirus A VPO, VP1 , VP2, VP3, VP4 polypeptides, and immunologically active fragments thereof, which vaccine elicits a protective and/or neutralizing immune response directed against a Human Enterovirus that can be used to prepare polyclonal antibodies for use to prevent or treat an infection by a causative agent of foot, hand and mouth disease.

[0222] The in an embodiment, the expression cassette consists essentially of a nucleic acid encoding a Human enterovirus A P1 polyprotein, an IRES and an enterovirus 3CD protease under the translational control of the IRES, which protease processes the Enterovirus P1 polyprotein into enterovirus structural capsid proteins.

[0223] The expression of one or more of the Enterovirus proteins suggests that VP2 and/or VPO polypeptides contain epitopes recognized by neutralizing antisera. These functional antibodies surprisingly bind more strongly to Enterovirus VP2 and VPO polypeptides than to VP1 polypeptides, which VP1 polypeptide is understood in the art to be the major capsid protein required for the generation of neutralizing antibodies. It is unexpected that VP2 polypeptides are, in fact, important for generating neutralizing antibodies against HEV71 infection. The structural proteins may take the form of VLPs, capsomers, complexes and/or aggregates. Indeed, the expression of one or more of the Enterovirus proteins as described herein provides antigens which elicit antibodies, which antibodies are functional and able to neutralize enteroviruses selected from HEV71 , Coxsackievirus A16, Human enterovirus C or any other picornavirus to high titre.

[0224] Thus, it is surprising that an Enterovirus VP2 polypeptide is the dominant epitope, or antigenic determinant, of the capsid proteins for the generation of neutralizing antibodies against enterovirus infection. HEV71 VP2 polypeptides, either alone, or in combination with other HEV71 capsid proteins, for example VPO polypeptides, is the dominant antigen which elicits neutralizing antibodies directed against HEV71 .

[0225] In an aspect of the invention, prophylactic vaccinations for generation of a humoral response to raise a polyclonal antibody response are contemplated which vaccines incorporate VPO and/or VP2 structural proteins of human enteroviruses into an immunogenic composition. The immunogenic composition or vaccine may comprise VPO or VP2 structural proteins from Human enterovirus A, including HEV71 and Coxsackievirus A16, or a combination thereof. The immunogenic composition may be administered to a subject to elicit neutralizing antibodies directed against human enteroviruses. The immunogenic composition may be comprised in a vaccine which is administered to a subject for the prevention of hand, foot and mouth disease infection caused by Human enterovirus A, such as from viruses HEV71 and/or Coxsackievirus A16. [0226] In another aspect, the polyclonal antibodies are provided to prevent and/or relieve complications of HEV71 and/or Coxsackievirus A16 infection, for example, the neurologic and cardiovascular complications manifesting as syndromes such as meningitis, encephalitis, acute flaccid paralysis, pulmonary edema and cardiac failure.

[0227] In an aspect of the invention, polyclonal antibodies against HEV71 and Coxsackievirus A16 are provided for prevention of enterovirus infection wherein the vaccine used to immunize individuals from whom the polyclonal antibodies were collected incorporate VPO or VP2 structural proteins from Human enterovirus C, for example PV1 , PV2, PV3 structural proteins, or combinations thereof, or biologically or immunologically active fragments thereof. The immunogenic composition may be comprised in a vaccine which is administered to a subject for the prevention of polio caused by Human enterovirus C, including PV1 , PV2, and PV3.

[0228] Furthermore, the immunogenic composition or vaccine used to raise polyclonal antibodies against HEV71 and Coxsackievirus A16 may comprise a combination of antigens derived from both Human enterovirus C and Human enterovirus A.

[0229] It may be concluded that Enterovirus VP2 polypeptides are important to achieve neutralizing antibodies. VP2 polypeptides may be sufficient for formulating a vaccine against an infection with picornaviruses, such as Human enterovirus A, types HEV71 and Coxsackievirus A16; Human enterovirus C types 1 , 2 and 3: and Human enterovirus D type EV68.

[0230] In an aspect of the invention, vaccinations to raise polyclonal antibodies against HEV71 and Coxsackievirus A16 prevent picornavirus infection, which vaccinations incorporate at least VPO and/or VP2 and VP4 structural proteins of the virus into an immunogenic composition. The immunogenic composition may be administered to a subject to elicit neutralizing antibodies directed against a picornavirus for use to raise polyclonal antibodies against HEV71 and Coxsackievirus A16. The immunogenic composition may be comprised in a vaccine which is administered to a subject for the prevention of picornavirus infection.

[0231] In another aspect of the invention, polyclonal antibodies against HEV71 and Coxsackievirus A16 are provided to prevent and/or relieve complications of picornavirus infection, for example, the neurologic and cardiovascular complications manifesting as syndromes such as meningitis, encephalitis, acute flaccid paralysis, pulmonary edema and cardiac failure.

[0232] In a further aspect of the invention there is provided a polyclonal antibodies against HEV71 and Coxsackievirus A16 according to the invention for use in medicine.

[0233] In yet another aspect, the invention provides a bivalent, or multivalent vaccine comprising enterovirus VPO and/or VP2 and/or VP4 antigens for use to raise polyclonal antibodies against HEV71 and Coxsackievirus A16. For example, Human enterovirus A VPO and/or VP2 and/or VP4 antigens may be combined. Moreover, the aforementioned antigens from different serotypes of Human enterovirus A, such as antigens from Coxsackievirus A16 and HEV71 , may be combined in a vaccine to raise a polyclonal antibodies against HEV71 and Coxsackievirus A16, for example, directed against human foot- and-mouth disease.

[0234] The enterovirus antigens of bivalent or multivalent vaccines may be produced from the expression cassettes described herein. The enterovirus antigens may be in the form of virus-like particles, capsomers, complexes, and/or aggregates.

[0235] In yet another aspect, the invention provides a bivalent, or multivalent vaccine to raise polyclonal antibodies against HEV71 and Coxsackievirus A16 comprising enterovirus antigen(s), and an antigen providing immunity against one or more of the following pathogens: diphtheria (D); tetanus (T); pertussis (P); Haemophilus influenzae b (Hib); Hepatitis A (HA) Hepatitis B (HB), and Human Enterovirus 71 .

[0236] The amount of picornavirus antigen in each vaccine dose to raise polyclonal antibodies against HEV71 and Coxsackievirus A16 is selected as an amount which induces a humoral response without significant adverse side effects in typical vaccinees. Such amount will vary depending on which specific immunogens are employed. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titers and other responses in subjects. A primary vaccination course may include 2 or 3 doses of a vaccine, given at intervals optimal for providing an immunoprotective response.

[0237] The invention thus provides a method for preventing picornavirus infections in humans, which method comprises treating a human subject in need thereof with an effective dose of polyclonal antibodies against HEV71 and Coxsackievirus A16 according to any aspect of the invention as hereinabove described.

[0238] Reference may now be made to various embodiments of the invention as illustrated in the attached figures. In these embodiments it should be noted that the picornavirus VLPs, capsomers, antigens, and aggregates and specific constructs of DNA recombinant molecules are given by way of example.

[0239] Aspects of the present specification may also be described as follows:

1 . A pharmaceutical composition comprising a polyclonal antibody preparation prepared from one or more subjects immunized with a vaccine for a Human Enterovirus responsible for foot, hand and mouth disease for the prevention and/or treatment of an infection by a Human Enterovirus responsible for foot, hand and mouth disease.

2. The pharmaceutical composition according to embodiment 1 , wherein the polyclonal antibody preparation is produced from plasma isolated from one or more subjects immunized against a causative agent of hand, foot and mouth disease.

3. The pharmaceutical composition according to embodiment 2, wherein the immunization against a causative agent of hand, foot and mouth disease is with a virus like particle, a viral protein, virus capsomers, live attenuated virus, aggregates, complexes of antigens from viruses and/or a viral nucleic acid.

The pharmaceutical composition according to any one of embodiments 1 -3, wherein the causative agent is EV71 , CA5, CA6, CA10 and/or CA16.

The pharmaceutical composition according to any one of embodiments 1 -4, wherein the polyclonal antibody preparation is a purified immunoglobulin preparation.

The pharmaceutical composition according to embodiment 5, wherein the purified immunoglobulin preparation is comprised primarily of IgG antibodies.

The pharmaceutical composition according to embodiment 5, wherein the purified immunoglobulin preparation is comprised of one or more antibodies of the IgM, IgG and/or IgA classes.

The pharmaceutical composition of any one of embodiments 1-7, wherein the preparation comprises one or more pharmacologically acceptable carriers.

The pharmaceutical composition according to embodiment 8, wherein the one or more pharmaceutically acceptable carriers are selected from a vehicle, a stabilizer, a diluent, an additive, an auxiliary and/or an excipient.

The pharmaceutical composition according to embodiment 6, wherein the IgG antibodies comprise a pooled polyspecific immunoglobulin G preparation.

The pharmaceutical composition according to embodiment 10, wherein the pooled polyspecific immunoglobulin G preparation is an intravenous immunoglobulin G preparation.

The pharmaceutical composition according to embodiment 10, wherein the pooled polyspecific immunoglobulin G preparation is a subcutaneous immunoglobulin G preparation.

The pharmaceutical composition according to any one of embodiments 10-12, wherein the IgG comprises >90%, >95% or >98% of the pooled polyspecific immunoglobulin G preparation.

The pharmaceutical composition of any of the proceeding embodiments, wherein the pharmaceutical composition is administered to a subject intravenously or subcutaneously.

The pharmaceutical composition according to embodiment 12, wherein the subcutaneous immunoglobulin G preparation is at least 0.01 % (w/v) immunoglobulin, 0.05% (w/v) immunoglobulin, 0.1 % (w/v) immunoglobulin, 0.5% (w/v) immunoglobulin, 1 % (w/v) immunoglobulin, 2% (w/v) immunoglobulin, 3% (w/v) immunoglobulin, 4% (w/v) immunoglobulin, 5% (w/v) immunoglobulin, 6% (w/v) immunoglobulin, 7% (w/v) immunoglobulin, 8% immunoglobulin, 9% (w/v) immunoglobulin, 10% (w/v) immunoglobulin, , 1 1 % (w/v) immunoglobulin, 12% (w/v) immunoglobulin, 13% (w/v) immunoglobulin, 14% (w/v) immunoglobulin, 15% (w/v) immunoglobulin, 16% (w/v) immunoglobulin, 17% (w/v) immunoglobulin, 18% (w/v) immunoglobulin, 19% (w/v) immunoglobulin, 20% (w/v) immunoglobulin, 21 % (w/v) immunoglobulin, 22% (w/v) immunoglobulin, 23% (w/v) immunoglobulin, 24% (w/v) immunoglobulin, 25% (w/v) immunoglobulin, 26% (w/v) immunoglobulin, 27% (w/v) immunoglobulin, 28% (w/v) immunoglobulin, 29% (w/v) immunoglobulin, 30% (w/v) immunoglobulin 35% (w/v) immunoglobulin, 40% (w/v) immunoglobulin, 45% (w/v) immunoglobulin, 50% (w/v) immunoglobulin, 55% (w/v) immunoglobulin, 60% (w/v) immunoglobulin, 65% (w/v) immunoglobulin, 70% (w/v) immunoglobulin, 75% (w/v) immunoglobulin or more.

The pharmaceutical composition according to any one of embodiments 1 -15, wherein the composition is in a liquid or a lyophilized form.

The pharmaceutical composition according to any one of embodiments 1 -16, wherein the pharmaceutical composition comprises a polyclonal antibody preparation that is co-administered with a drug.

The pharmaceutical composition according to embodiment 17, wherein the drug is an antimicrobial, antibiotic, adjuvant, an immunostimulatory or antiviral drug.

The pharmaceutical composition according to embodiments 17 or 18, wherein the antibiotic is selected from penicillins, aminopenicillins, penicillinase-resistant penicillins, carboxy penicillins, ureido penicillins, cephalosporins, beta.-lactams (such as imipenem, monobactams), .beta. -lactamase inhibitors, vancomycin, aminoglycosides and spectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin, metronidazole, polymyxins, doxycycline, quinolones (e.g., ciprofloxacin), sulfonamides, trimethoprim, and quinolones.

The pharmaceutical composition according to embodiments 17 or 18, wherein the antiviral is selected from amantadine, rimantadine, pleconaril, acyclovir, interferon, oseltamivir and/or zanamivir.

The pharmaceutical composition according to any one of embodiments 1 -20, wherein the pharmaceutical composition is utilized as a prophylactic application.

The pharmaceutical composition according to any one of embodiments 1 -21 , wherein a delivery system for delivery of the pharmaceutical composition to a subject administered the pharmaceutical composition is a time-release, delayed release or sustained release delivery system.

The pharmaceutical composition according to any one of embodiments 1-22, wherein the polyclonal antibody preparation is capable of reducing the severity of a Human Enterovirus infection responsible for causing hand, foot and mouth disease by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a patient not receiving the same treatment.

The pharmaceutical composition according to any one of embodiments 1-23, wherein the polyclonal antibody preparation is capable of reducing or stopping the severity or progression of a Human Enterovirus infection responsible for causing hand, foot and mouth disease by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% as compared to a patient not receiving the same treatment.

The pharmaceutical composition according to any one of embodiments 1-24, wherein the polyclonal antibody preparation is capable of reducing the severity of a Human Enterovirus infection responsible for causing hand, foot and mouth disease has a half-life of 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, one month, two months, three months, four months or more.

The pharmaceutical composition according to any one of embodiments 1 -25, wherein the pharmaceutical composition is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

The pharmaceutical composition according to any one of embodiments 1 -26, wherein a pharmaceutical composition comprises a polyclonal antibody preparation, with the polyclonal antibody preparation present at a concentration of at least about 0.1 % (w/v), or alternatively at least about 0.01 %, 0.02%, 0.05%, 0.075%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, 6%,6.25%, 6.5%, 6.75%, 7%, 7.25%, 7.5%, 7.75%, 8%, 8.25%, 8.5%, 8.75%, 9%, 9.25%, 9.5%, 9.75%, 10%, 10.25%, 10.5%, 10.75%, 1 1 %, 11 .25%, 1 1 .5%, 1 1.75%, 12%, 12.25%, 12.5%, 12.75%, 13%, 13.25%, 13.5%, 13.75%, 14%, 14.25%, 14.5%, 14.75%, 15%, 15.25%, 15.5%, 15.75%, 16%, 16.25%, 16.5%, 16.75%, 17%, 17.25%, 17.5%, 17.75%, 18%, 18.25%, 18.5%, 18.75%, 19%, 19.25%, 19.5%, 19.75%, 20%, 20.25%, 20.5%, 20.75%, 21 %, 21.25%, 21 .5%, 21.75%, 22%, 22.25%, 22.5%,, 22.75%, 23%, 23.25%, 23.5%, 23.75%, 24%, 24.25%, 24.5%, 24.75%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 40%, or more (w/v) of the pharmaceutical composition.

The pharmaceutical composition according to any one of embodiments 1 -27, wherein a pharmaceutical composition comprises a polyclonal antibody preparation and one or more drugs, with the polyclonal antibody preparation and the one or more drugs present at a concentration of at least about 0.1 % (w/v), or alternatively at least about 0.01 %, 0.02%, 0.05%, 0.075%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 1 .5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, 6%,6.25%, 6.5%, 6.75%, 7%, 7.25%, 7.5%, 7.75%, 8%, 8.25%, 8.5%, 8.75%, 9%, 9.25%, 9.5%, 9.75%, 10%, 10.25%, 10.5%, 10.75%, 1 1 %, 1 1.25%, 1 1.5%, 1 1 .75%, 12%, 12.25%, 12.5%, 12.75%, 13%, 13.25%, 13.5%, 13.75%, 14%, 14.25%, 14.5%, 14.75%, 15%, 15.25%, 15.5%, 15.75%, 16%, 16.25%, 16.5%, 16.75%, 17%, 17.25%, 17.5%, 17.75%, 18%, 18.25%, 18.5%, 18.75%, 19%, 19.25%, 19.5%, 19.75%, 20%, 20.25%, 20.5%, 20.75%, 21 %, 21 .25%, 21.5%, 21.75%, 22%, 22.25%, 22.5%,, 22.75%, 23%, 23.25%, 23.5%, 23.75%, 24%, 24.25%, 24.5%, 24.75%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 40%, or more (w/v) of the pharmaceutical composition.

The pharmaceutical composition according to any one of embodiments 1 -27, wherein the pharmaceutical composition comprises a polyclonal antibody preparation at a concentration of about 0.1 % (w/v) to about 40%, or alternatively at about 0.01 % to about 25%, 0.02% to about 25%, 0.05% to about 25%, 0.075% to about 25%, 0.2% to about 25%, 0.3% to about 25%, 0.4% to about 25%, 0.5% to about 25%, 0.6% to about 25%, 0.7% to about 25%, 0.8% to about 25%, 0.9% to about 25%, 1 % to about 25%, 1 .5% to about 25%, 1 .75% to about 25%, 2% to about 25%, 2.25% to about 25%,, 2.5% to about 25%,, 2.75% to about 25%,, 3% to about 25%, 3.25% to about 25%, 3.5% to about 25%, 3.75% to about 25%, 4% to about 25%, 4.25% to about 25%, 4.5% to about 25%, 4.75% to about 25%, 5% to about 25%, 5.25% to about 25%, 5.5% to about 25%, 5.75% to about 25%, 6% to about 25%, 6.25% to about 25%, 6.5% to about 25%, 6.75% to about 25%, 7% to about 25%, 7.25% to about 25%, 7.5% to about 25%, 7.75% to about 25%, 8% to about 25%, 8.25% to about 25%, 8.5% to about 25%, 8.75% to about 25%, 9% to about 25%, 9.25% to about 25%, 9.5% to about 25%, 9.75% to about 25%, 10% to about 25%, 10.25% to about 25%, 10.5% to about 25%, 10.75% to about 25%, 1 1 % to about 25%, 11 .25% to about 25%, 11 .5% to about 25%, 1 1.75% to about 25%, 12% to about 25%, 12.25% to about 25%, 12.5% to about 25%, 12.75% to about 25%, 13% to about 25%, 13.25% to about 25%, 13.5% to about 25%, 13.75% to about 25%, 14% to about 25%, 14.25% to about 25%, 14.5% to about 25%, 14.75% to about 25%, 15% to about 25%, 15.25% to about 25%, 15.5% to about 25%, 15.75% to about 25%, 16% to about 25%, 16.25% to about 25%, 16.5% to about 25%, 16.75% to about 25%, 17% to about 25%, 17.25% to about 25%, 17.5% to about 25%, 17.75% to about 25%, 18% to about 25%, 18.25% to about 25%, 18.5% to about 25%, 18.75% to about 25%, 19% to about 25%, 19.25% to about 25%, 19.5% to about 25%, 19.75% to about 25%, 20% to about 25%, 20.25% to about 25%, 20.5% to about 25%, 20.75% to about 25%, 5% to about 20%, 6% to about 20%, 7% to about 20%, 8% to about 20%, 9% to about 20%, 10% to about 20%, 1 1 % to about 20%, 12% to about 20%, 13%, to about 20%, 14% to about 20%, 15% to about 20%, 5% to about 15%, 6% to about 15%, 7% to about 15%, 8% to about 15%, 9% to about 15%, 10% to about 15%, 1 1 % to about 15%, 12% to about 15%, 13%, to about 15%, 14% to about 15% (w/v). The pharmaceutical composition according to any one of embodiments 1 -27, wherein the pharmaceutical composition comprises a polyclonal antibody preparation and one or more other drugs, with each present at a concentration of about 0.1 % (w/v) to about 40%, or alternatively at about 0.01 % to about 25%, 0.02% to about 25%, 0.05% to about 25%, 0.075% to about 25%, 0.2% to about 25%, 0.3% to about 25%, 0.4% to about 25%, 0.5% to about 25%, 0.6% to about 25%, 0.7% to about 25%, 0.8% to about 25%, 0.9% to about 25%, 1 % to about 25%, 1 .5% to about 25%, 1.75% to about 25%, 2% to about 25%, 2.25% to about 25%,, 2.5% to about 25%,, 2.75% to about 25%,, 3% to about 25%, 3.25% to about 25%, 3.5% to about 25%, 3.75% to about 25%, 4% to about 25%, 4.25% to about 25%, 4.5% to about 25%, 4.75% to about 25%, 5% to about 25%, 5.25% to about 25%, 5.5% to about 25%, 5.75% to about 25%, 6% to about 25%, 6.25% to about 25%, 6.5% to about 25%, 6.75% to about 25%, 7% to about 25%, 7.25% to about 25%, 7.5% to about 25%, 7.75% to about 25%, 8% to about 25%, 8.25% to about 25%, 8.5% to about 25%, 8.75% to about 25%, 9% to about 25%, 9.25% to about 25%, 9.5% to about 25%, 9.75% to about 25%, 10% to about 25%, 10.25% to about 25%, 10.5% to about 25%, 10.75% to about 25%, 1 1 % to about 25%, 1 1 .25% to about 25%, 1 1.5% to about 25%, 1 1 .75% to about 25%, 12% to about 25%, 12.25% to about 25%, 12.5% to about 25%, 12.75% to about 25%, 13% to about 25%, 13.25% to about 25%, 13.5% to about 25%, 13.75% to about 25%, 14% to about 25%, 14.25% to about 25%, 14.5% to about 25%, 14.75% to about 25%, 15% to about 25%, 15.25% to about 25%, 15.5% to about 25%, 15.75% to about 25%, 16% to about 25%, 16.25% to about 25%, 16.5% to about 25%, 16.75% to about 25%, 17% to about 25%, 17.25% to about 25%, 17.5% to about 25%, 17.75% to about 25%, 18% to about 25%, 18.25% to about 25%, 18.5% to about 25%, 18.75% to about 25%, 19% to about 25%, 19.25% to about 25%, 19.5% to about 25%, 19.75% to about 25%, 20% to about 25%, 20.25% to about 25%, 20.5% to about 25%, 20.75% to about 25%, 5% to about 20%, 6% to about 20%, 7% to about 20%, 8% to about 20%, 9% to about 20%, 10% to about 20%, 1 1 % to about 20%, 12% to about 20%, 13%, to about 20%, 14% to about 20%, 15% to about 20%, 5% to about 15%, 6% to about 15%, 7% to about 15%, 8% to about 15%, 9% to about 15%, 10% to about 15%, 11 % to about 15%, 12% to about 15%, 13%, to about 15%, 14% to about 15% (w/v).

The pharmaceutical composition according to any one of embodiments 1 -26, wherein the concentration of a polyclonal antibody preparation in the solution is at least 0.00001 mg/mL, at least 0.0001 mg/mL, at least 0.001 mg/mL, at least 0.01 mg/mL, at least 0.1 mg/mL, at least 1 mg/mL, at least 10 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least 100 mg/mL, at least 200 mg/mL, at least 500 mg/mL, at least 700 mg/mL, at least 1 ,000 mg/mL, or at least 1 ,200 mg/mL.

The pharmaceutical composition of any of embodiments 1-26, wherein the concentration of a polyclonal antibody preparation in the solution is at most 1 ,000 mg/mL, at most 1 ,100 mg/mL, at most 1 ,200 mg/mL, at most 1 ,300 mg/mL, at most 1 ,400 mg/mL, at most 1 ,500 mg/mL, at most 2,000 mg/mL, at most 2,000 mg/mL, or at most 3,000 mg/mL.

The pharmaceutical composition according to any one of embodiments 1 -26, wherein the concentration of a polyclonal antibody preparation in the solution is in a range of about 0.00001 mg/mL to about 3,000 mg/mL, about 0.0001 mg/mL to about 3,000 mg/mL, about 0.01 mg/mL to about 3,000 mg/mL, about 0.1 mg/mL to about 3,000 mg/mL, about 1 mg/mL to about 3,000 mg/mL, about 250 mg/mL to about 3,000 mg/mL, about 500 mg/mL to about 3,000 mg/mL, about 750 mg/mL to about 3,000 mg/mL, about 1 ,000 mg/mL to about 3,000 mg/mL, about 100 mg/mL to about 2,000 mg/mL, about 250 mg/mL to about 2,000 mg/mL, about 500 mg/mL to about 2,000 mg/mL, about 750 mg/mL to about 2,000 mg/mL, about 1 ,000 mg/mL to about 2,000 mg/mL, about 100 mg/mL to about 1 ,500 mg/mL, about 250 mg/mL to about 1 ,500 mg/mL, about 500 mg/mL to about 1 ,500 mg/mL, about 750 mg/mL to about 1 ,500 mg/mL, about 1 ,000 mg/mL to about 1 ,500 mg/mL, about 100 mg/mL to about 1 ,200 mg/mL, about 250 mg/mL to about 1 ,200 mg/mL, about 500 mg/mL to about 1 ,200 mg/mL, about 750 mg/mL to about 1 ,200 mg/mL, about 1 ,000 mg/mL to about 1 ,200 mg/mL, about 100 mg/mL to about 1 ,000 mg/mL, about 250 mg/mL to about 1 ,000 mg/mL, about 500 mg/mL to about 1 ,000 mg/mL, about 750 mg/mL to about 1 ,000 mg/mL, about 100 mg/mL to about 750 mg/mL, about 250 mg/mL to about 750 mg/mL, about 500 mg/mL to about 750 mg/mL, about 100 mg/mL to about 500 mg/mL, about 250 mg/mL to about 500 mg/mL, about 0.00001 mg/mL to about 0.0001 mg/mL, about 0.00001 mg/mL to about 0.001 mg/mL, about 0.00001 mg/mL to about 0.01 mg/mL, about 0.00001 mg/mL to about 0.1 mg/mL, about 0.00001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 0.01 mg/mL, about 0.001 mg/mL to about 0.1 mg/mL, about 0.001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 10 mg/mL, or about 0.001 mg/mL to about 100 mg/mL. The pharmaceutical composition according to any one of embodiments 1 -26, wherein the concentration of a polyclonal antibody in a liquid or a lyophilized formulation is between about 50 mg/mL to about 1 ,000 mg/mL.

The pharmaceutical composition according to any one of embodiments 1 -26, wherein the therapeutically effective amount of a polyclonal antibody preparation is from about 50 mg/mL to about 100 mg/mL, about 50 mg/mL to about 200 mg/mL, about 50 mg/mL to about 300 mg/mL, about 50 mg/mL to about 400 mg/mL, about 50 mg/mL to about 500 mg/mL, about 50 mg/mL to about 600 mg/mL, about 50 mg/mL to about 700 mg/mL, about 50 mg/mL to about 800 mg/mL, about 50 mg/mL to about 900 mg/mL, about 50 mg/mL to about 1 ,000 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about 300 mg/mL, about 100 mg/mL to about 400 mg/mL, about 100 mg/mL to about 500 mg/mL, about 100 mg/mL to about 600 mg/mL, about 100 mg/mL to about 700 mg/mL, about 100 mg/mL to about 800 mg/mL, about 100 mg/mL to about 900 mg/mL, about 100 mg/mL to about 1 ,000 mg/mL, about 200 mg/mL to about 300 mg/mL, about 200 mg/mL to about 400 mg/mL, about 200 mg/mL to about 500 mg/mL, about 200 mg/mL to about 600 mg/mL, about 200 mg/mL to about 700 mg/mL, about 200 mg/mL to about 800 mg/mL, about 200 mg/mL to about 900 mg/mL, about 200 mg/mL to about 1 ,000 mg/mL, about 300 mg/mL to about 400 mg/mL, about 300 mg/mL to about 500 mg/mL, about 300 mg/mL to about 600 mg/mL, about 300 mg/mL to about 700 mg/mL, about 300 mg/mL to about 800 mg/mL, about 300 mg/mL to about 900 mg/mL, about 300 mg/mL to about 1 ,000 mg/mL, about 400 mg/mL to about 500 mg/mL, about 400 mg/mL to about 600 mg/mL, about 400 mg/mL to about 700 mg/mL, about 400 mg/mL to about 800 mg/mL, about 400 mg/mL to about 900 mg/mL, about 400 mg/mL to about 1 ,000 mg/mL, about 500 mg/mL to about 600 mg/mL, about 500 mg/mL to about 700 mg/mL, about 500 mg/mL to about 800 mg/mL, about 500 mg/mL to about 900 mg/mL, about 500 mg/mL to about 1 ,000 mg/mL, about 600 mg/mL to about 700 mg/mL, about 600 mg/mL to about 800 mg/mL, about 600 mg/mL to about 900 mg/mL, or about 600 mg/mL to about 1 ,000 mg/mL.

The pharmaceutical composition according to any one of embodiments 1 -35, wherein immunologically active antigens used to immunize a subject from whom a polyclonal antibody preparation is collected comprise one or more Human Enterovirus polypeptides selected from VP0, VP1 , VP2, VP3, VP4, and immunologically active fragments thereof.

The pharmaceutical composition according to any one of embodiments 1-26, wherein a liquid or a lyophilized formulation comprises a polyclonal antibody preparation of between about 0.01 % to about 45% by weight of the pharmaceutical composition.

The pharmaceutical composition according to any one of embodiments 1-26, wherein a liquid or a lyophilized formulation comprises a polyclonal antibody preparation of between about 0.1 % to about 45% by weight, about 0.1 % to about 40% by weight, about 0.1 % to about 35% by weight, about 0.1 % to about 30% by weight, about 0.1 % to about 25% by weight, about 0.1 % to about 20% by weight, about 0.1 % to about 15% by weight, about 0.1 % to about 10% by weight, about 0.1 % to about 5% by weight, about 1 % to about 45% by weight, about 1 % to about 40% by weight, about 1 % to about 35% by weight, about 1 % to about 30% by weight, about 1 % to about 25% by weight, about 1 % to about 20% by weight, about 1 % to about 15% by weight, about 1 % to about 10% by weight, about 1 % to about 5% by weight, about 5% to about 45% by weight, about 5% to about 40% by weight, about 5% to about 35% by weight, about 5% to about 30% by weight, about 5% to about 25% by weight, about 5% to about 20% by weight, about 5% to about 15% by weight, about 5% to about 10% by weight, about 10% to about 45% by weight, about 10% to about 40% by weight, about 10% to about 35% by weight, about 10% to about 30% by weight, about 10% to about 25% by weight, about 10% to about 20% by weight, about 10% to about 15% by weight, about 15% to about 45% by weight, about 15% to about 40% by weight, about 15% to about 35% by weight, about 15% to about 30% by weight, about 15% to about 25% by weight, about 15% to about 20% by weight, about 20% to about 45% by weight, about 20% to about 40% by weight, about 20% to about 35% by weight, about 20% to about 30% by weight, about 20% to about 25% by weight, about 25% to about 45% by weight, about 25% to about 40% by weight, about 25% to about 35% by weight, or about 25% to about 30% by weight of the pharmaceutical composition.

The pharmaceutical composition according to any one of embodiments 1 -39, wherein a therapeutically effective amount of the polyclonal antibody preparation reduces the severity of illness caused by a Human Enterovirus responsible for foot, hand and mouth disease in a subject by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%.

The pharmaceutical composition according to any one of embodiments 1 -39, wherein a therapeutically effective amount of a polyclonal antibody preparation reduces the severity of illness caused by a Human Enterovirus responsible for foot, hand and mouth disease in a subject by at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95% or at most 100%.

The pharmaceutical composition according to any one of embodiments 1 -39, wherein a therapeutically effective amount of a polyclonal antibody preparation reduces or stops the severity or progression of illness caused by a Human Enterovirus responsible for foot, hand and mouth disease in a subject by about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

The pharmaceutical composition according to any one of embodiments 1 -41 , wherein a therapeutically effective amount of a polyclonal antibody preparation is in the range of about 0. 001 mg/kg/day to about 100 mg/kg/day.

The pharmaceutical composition according to any one of embodiments 1-41 , wherein an effective amount of a polyclonal antibody preparation is at least 0.001 mg/kg/day, at least 0.01 mg/kg/day, at least 0.1 mg/kg/day, at least 1.0 mg/kg/day, at least 5.0 mg/kg/day, at least 10 mg/kg/day, at least 15 mg/kg/day, at least 20 mg/kg/day, at least 25 mg/kg/day, at least 30 mg/kg/day, at least 35 mg/kg/day, at least 40 mg/kg/day, at least 45 mg/kg/day, or at least 50 mg/kg/day.

The pharmaceutical composition according to any one of embodiments 1-41 , wherein an effective amount of a polyclonal antibody preparation is in the range of about 0.001 mg/kg/day to about 10 mg/kg/day, about 0.001 mg/kg/day to about 15 mg/kg/day, about 0.001 mg/kg/day to about 20 mg/kg/day, about 0.001 mg/kg/day to about 25 mg/kg/day, about 0.001 mg/kg/day to about 30 mg/kg/day, about 0.001 mg/kg/day to about 35 mg/kg/day, about 0.001 mg/kg/day to about 40 mg/kg/day, about 0.001 mg/kg/day to about 45 mg/kg/day, about 0.001 mg/kg/day to about 50 mg/kg/day, about 0.001 mg/kg/day to about 75 mg/kg/day, or about 0.001 mg/kg/day to about 100 mg/kg/day.

The pharmaceutical composition according to any one of embodiments 1-41 , wherein an effective amount of a polyclonal antibody preparation is in the range of about 0.01 mg/kg/day to about 10 mg/kg/day, about 0.01 mg/kg/day to about 15 mg/kg/day, about 0.01 mg/kg/day to about 20 mg/kg/day, about 0.01 mg/kg/day to about 25 mg/kg/day, about 0.01 mg/kg/day to about 30 mg/kg/day, about 0.01 mg/kg/day to about 35 mg/kg/day, about 0.01 mg/kg/day to about 40 mg/kg/day, about 0.01 mg/kg/day to about 45 mg/kg/day, about 0.01 mg/kg/day to about 50 mg/kg/day, about 0.01 mg/kg/day to about 75 mg/kg/day, or about 0.01 mg/kg/day to about 100 mg/kg/day.

The pharmaceutical composition according to any one of embodiments 1-41 , wherein an effective amount of a polyclonal antibody preparation is in the range of about 0.1 mg/kg/day to about 10 mg/kg/day, about 0.1 mg/kg/day to about 15 mg/kg/day, about 0.1 mg/kg/day to about 20 mg/kg/day, about 0.1 mg/kg/day to about 25 mg/kg/day, about 0.1 mg/kg/day to about 30 mg/kg/day, about 0.1 mg/kg/day to about 35 mg/kg/day, about 0.1 mg/kg/day to about 40 mg/kg/day, about 0.1 mg/kg/day to about 45 mg/kg/day, about 0.1 mg/kg/day to about 50 mg/kg/day, about 0.1 mg/kg/day to about 75 mg/kg/day, or about 0.1 mg/kg/day to about 100 mg/kg/day.

The pharmaceutical composition according to any one of embodiments 1 -46, wherein a subject immunized to provide a polyclonal antibody preparation is immunized once, twice, three, four, five, six, seven, eight, nine or more times prior to the collection of the polyclonal antibody preparation from the subject.

The pharmaceutical composition according to any one of embodiments 1 -46, wherein a subject is administered a polyclonal antibody preparation one or more times prior to infection with a Human Enterovirus responsible for hand, foot and mouth disease.

The pharmaceutical composition according to any one of embodiments 1 -46, wherein a subject is administered a polyclonal antibody preparation one, two, three, four, five, six, seven, eight, nine, ten or more times prior to infection with a Human Enterovirus responsible for hand, foot and mouth disease.

The pharmaceutical composition according to any one of embodiments 1 -46, wherein a subject is administered a polyclonal antibody preparation prior to or during an infection with a Human Enterovirus responsible for hand, foot and mouth disease.

A kit comprising a pharmaceutical composition comprising a polyclonal antibody preparation for the prevention and/or treatment of an infection by a Human Enterovirus responsible for foot, hand and mouth disease.

The kit according to embodiment 51 , wherein the kit comprises a plasma derived immunoglobulin, instructions for use to administer plasma-derived immunoglobulin in the treatment of an a Human Enterovirus capable of causing hand, foot and mouth disease in a subject.

The kit of any according to embodiments 51 or 52, wherein the components of the kit may be contained in one or different containers.

The kit according to embodiment 53, wherein the one or different containers are one or more vials. The kit according to any one of embodiments 51 -54, wherein the plasma derived immunoglobulin is in a liquid or a solid form.

The kit according to any one of embodiments 51 -55, wherein the kit comprises a comprises a plasma derived immunoglobulin, instructions for use to administer plasma-derived immunoglobulin in the treatment of a Human Enterovirus capable of causing hand, foot and mouth disease in a subject and one or more drugs.

The kit according to any one of embodiments 51-56, wherein the one or more drugs are an antimicrobial, antibacterial, immunostimulatory and/or antiviral.

The kit according to any one of embodiments 51 -57, wherein the kit includes instructions for the use of the components of the kits.

The kit according to any one of embodiments 56-58, wherein the instructions contain information how to prepare the plasma-derived immunoglobulin.

The kit according to any one of embodiments 56-59, wherein the instructions contain information on how to dilute or reconstitute the lyophilized plasma-derived immunoglobulin.

The kit according to any one of embodiments 56-60, wherein the instructions provide guidance regarding the dosage and frequency of administration or the pharmaceutical composition.

The kit according to any one of embodiments 51 -61 , wherein the kit comprises an IVIG and/or SCIG. A method of use, wherein a subject is treated with the pharmaceutical composition as defined in any one of embodiments 1 -50.

A method of use according to embodiment 63, wherein the polyclonal antibody preparation is produced from plasma isolated from one or more subjects immunized against a causative agent of hand, foot and mouth disease.

A method of use according to embodiment 64, wherein the immunization against a causative agent of hand, foot and mouth disease is with a virus like particle, a viral protein, a viral protein, virus capsomers, aggregates, complexes of antigens from viruses and/or a viral nucleic acid.

A method of use according to any one of embodiments 63-65, wherein the causative agent is EV71 and/or CA16.

A method of use according to any one of embodiments 63-66, wherein the polyclonal antibody preparation is a purified immunoglobulin preparation.

A method of use according to embodiment 67, wherein the purified immunoglobulin preparation is comprised primarily of IgG antibodies.

A method of use according to embodiment 67, wherein the purified immunoglobulin preparation is comprised of one or more antibodies of the IgM, IgG and/or IgA classes.

A method of use according to any one of embodiments 63-69, wherein the preparation comprises one or more pharmacologically acceptable carriers.

A method of use according to embodiment 70, wherein the one or more pharmaceutically acceptable carriers are selected from a vehicle, a stabilizer, a diluent, an additive, an auxiliary and/or an excipient.

A method of use according to embodiment 68, wherein the IgG antibodies comprise a pooled polyspecific immunoglobulin G preparation.

A method of use according to embodiment 72, wherein the pooled polyspecific immunoglobulin G preparation is an intravenous immunoglobulin G preparation.

A method of use according to embodiment 72, wherein the pooled polyspecific immunoglobulin G preparation is a subcutaneous immunoglobulin G preparation.

A method of use according to embodiment 72-74, wherein the IgG comprises >90%, >95% or >98% of the pooled polyspecific immunoglobulin G preparation.

A method of use according to any one of embodiments 63-75, wherein the pharmaceutical composition is administered to a subject intravenously or subcutaneously.

A method of use according to embodiment 76, wherein the subcutaneous immunoglobulin G preparation is at least 0.01 % (w/v) immunoglobulin, 0.05% (w/v) immunoglobulin, 0.1 % (w/v) immunoglobulin, 0.5% (w/v) immunoglobulin, 1 % (w/v) immunoglobulin, 2% (w/v) immunoglobulin, 3% (w/v) immunoglobulin, 4% (w/v) immunoglobulin, 5% (w/v) immunoglobulin, 6% (w/v) immunoglobulin, 7% (w/v) immunoglobulin, 8% immunoglobulin, 9% (w/v) immunoglobulin, 10% (w/v) immunoglobulin, , 1 1 % (w/v) immunoglobulin, 12% (w/v) immunoglobulin, 13% (w/v) immunoglobulin, 14% (w/v) immunoglobulin, 15% (w/v) immunoglobulin, 16% (w/v) immunoglobulin, 17% (w/v) immunoglobulin, 18% (w/v) immunoglobulin, 19% (w/v) immunoglobulin, 20% (w/v) immunoglobulin, 21 % (w/v) immunoglobulin, 22% (w/v) immunoglobulin, 23% (w/v) immunoglobulin, 24% (w/v) immunoglobulin, 25% (w/v) immunoglobulin, 26% (w/v) immunoglobulin, 27% (w/v) immunoglobulin, 28% (w/v) immunoglobulin, 29% (w/v) immunoglobulin, 30% (w/v) immunoglobulin 35% (w/v) immunoglobulin, 40% (w/v) immunoglobulin, 45% (w/v) immunoglobulin, 50% (w/v) immunoglobulin, 55% (w/v) immunoglobulin, 60% (w/v) immu 65% (w/v) immunoglobulin, noglobulin, 70% (w/v) immunoglobulin, 75% (w/v) immunoglobulin or more.

A method of use according to any one of embodiments 63-77, wherein the composition is in a liquid or a lyophilized form.

A method of use according to any one of embodiments 63-78, wherein the pharmaceutical composition comprises a polyclonal antibody preparation that is co-administered with a drug.

A method of use according to embodiment 79, wherein the drug is an antimicrobial, antibiotic, adjuvant, an immuostimulatory or antiviral drug.

A method of use according to embodiments 79 or 80, wherein the antibiotic is selected from penicillins, aminopenicillins, penicillinase-resistant penicillins, carboxy penicillins, ureido penicillins, cephalosporins, beta.-lactams (such as imipenem, monobactams), .beta.-lactamase inhibitors, vancomycin, aminoglycosides and spectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin, metronidazole, polymyxins, doxycycline, quinolones (e.g., ciprofloxacin), sulfonamides, trimethoprim, and quinolones.

A method of use according to any one of embodiments 63-80, wherein the antiviral is selected from amantadine, rimantadine, pleconaril, acyclovir, interferon, oseltamivir and/or zanamivir.

A method of use according to any one of embodiments 63-82, wherein the pharmaceutical composition is utilized as a prophylactic treatment.

A method of use according to any one of embodiments 63-83, wherein a delivery system for delivery of the pharmaceutical composition to a subject administered the pharmaceutical composition is a time-release, delayed release or sustained release delivery system.

A pharmaceutical composition comprising a monoclonal antibody preparation for the prevention and/or treatment of an infection by a Human Enterovirus responsible for foot, hand and mouth disease.

The pharmaceutical composition according to embodiment 85, wherein the Human Enterovirus is EV71 , CA5, CA6, CA10 and/or CA16. The pharmaceutical composition according to embodiments 85 or 86, wherein the preparation comprises one or more pharmacologically acceptable carriers.

The pharmaceutical composition according to embodiment 87, wherein the one or more pharmaceutically acceptable carriers are selected from a vehicle, a stabilizer, a diluent, an additive, an auxiliary and/or an excipient.

The pharmaceutical composition according to any one of embodiments 85-88, wherein the pharmaceutical composition is administered to a subject intravenously or subcutaneously.

The pharmaceutical composition of any one of embodiments 85-89, wherein the pharmaceutical composition comprises a monoclonal antibody preparation that is co-administered with a drug.

The pharmaceutical composition according to embodiment 90, wherein the drug is an antimicrobial, antibiotic, adjuvant an immunostimulatory or antiviral drug.

The pharmaceutical composition according to embodiments 90 or 91 , wherein the antibiotic is selected from penicillins, aminopenicillins, penicillinase-resistant penicillins, carboxy penicillins, ureido penicillins, cephalosporins, beta.-lactams (such as imipenem, monobactams), .beta. -lactamase inhibitors, vancomycin, aminoglycosides and spectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin, metronidazole, polymyxins, doxycycline, quinolones (e.g., ciprofloxacin), sulfonamides, trimethoprim, and quinolones.

The pharmaceutical composition according to embodiments 90 or 91 , wherein the antiviral is selected from amantadine, rimantadine, pleconaril, acyclovir, interferon, oseltamivir and/or zanamivir.

The pharmaceutical composition according to any one of embodiments 85-93, wherein the pharmaceutical composition is utilized as a prophylactic application.

The pharmaceutical composition according to any one of embodiments 85-94, wherein a delivery system for delivery of the pharmaceutical composition to a subject administered the pharmaceutical composition is a time-release, delayed release or sustained release delivery system.

The pharmaceutical composition according to any one of embodiments 85-95, wherein the monoclonal antibody preparation is capable of reducing the severity of a Human Enterovirus infection responsible for causing hand, foot and mouth disease by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a patient not receiving the same treatment.

The pharmaceutical composition according to any one of embodiments 85-96, wherein the monoclonal antibody preparation is capable of reducing or stopping the severity or progression of a Human Enterovirus infection responsible for causing hand, foot and mouth disease by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% as compared to a patient not receiving the same treatment. The pharmaceutical composition according to any one of embodiments 85-97, wherein the monoclonal antibody preparation is capable of reducing the severity of a Human Enterovirus infection responsible for causing hand, foot and mouth disease has a half-life of 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, one month, two months, three months, four months or more.

The pharmaceutical composition according to any one of embodiments 85-98, wherein the pharmaceutical composition is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

. The pharmaceutical composition according to any one of embodiments 85-99, wherein a pharmaceutical composition comprises a monoclonal antibody preparation, with the monoclonal antibody preparation present at a concentration of at least about 0.1 % (w/v), or alternatively at least about 0.01 %, 0.02%, 0.05%, 0.075%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, 6%,6.25%, 6.5%, 6.75%, 7%, 7.25%, 7.5%, 7.75%, 8%, 8.25%, 8.5%, 8.75%, 9%, 9.25%, 9.5%, 9.75%, 10%, 10.25%, 10.5%, 10.75%, 1 1 %, 1 1 .25%, 1 1 .5%, 11 .75%, 12%, 12.25%, 12.5%, 12.75%, 13%, 13.25%, 13.5%, 13.75%, 14%, 14.25%, 14.5%, 14.75%, 15%, 15.25%, 15.5%, 15.75%, 16%, 16.25%, 16.5%, 16.75%, 17%, 17.25%, 17.5%, 17.75%, 18%, 18.25%, 18.5%, 18.75%, 19%, 19.25%, 19.5%, 19.75%, 20%, 20.25%, 20.5%, 20.75%, 21 %, 21.25%, 21.5%, 21 .75%, 22%, 22.25%, 22.5%,, 22.75%, 23%, 23.25%, 23.5%, 23.75%, 24%, 24.25%, 24.5%, 24.75%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 40%, or more (w/v) of the pharmaceutical composition.

. The pharmaceutical composition according to any one of embodiments 85-99, wherein a pharmaceutical composition comprises a monoclonal antibody preparation and one or more drugs, with the monoclonal antibody preparation and the one or more drugs present at a concentration of at least about 0.1 % (w/v), or alternatively at least about 0.01 %, 0.02%, 0.05%, 0.075%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 1 .5%, 1 .75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, 6%,6.25%, 6.5%, 6.75%, 7%, 7.25%, 7.5%, 7.75%, 8%, 8.25%, 8.5%, 8.75%, 9%, 9.25%, 9.5%, 9.75%, 10%, 10.25%, 10.5%, 10.75%, 1 1 %, 1 1.25%, 11 .5%, 1 1.75%, 12%, 12.25%, 12.5%, 12.75%, 13%, 13.25%, 13.5%, 13.75%, 14%, 14.25%, 14.5%, 14.75%, 15%, 15.25%, 15.5%, 15.75%, 16%, 16.25%, 16.5%, 16.75%, 17%, 17.25%, 17.5%, 17.75%, 18%, 18.25%, 18.5%, 18.75%, 19%, 19.25%, 19.5%, 19.75%, 20%, 20.25%, 20.5%, 20.75%, 21 %, 21 .25%, 21.5%, 21 .75%, 22%, 22.25%, 22.5%,, 22.75%, 23%, 23.25%, 23.5%, 23.75%, 24%, 24.25%, 24.5%, 24.75%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 40%, or more (w/v) of the pharmaceutical composition.

. The pharmaceutical composition according to any one of embodiments 85-99, wherein the pharmaceutical composition comprises a monoclonal antibody preparation at a concentration of about 0.1 % (w/v) to about 40%, or alternatively at about 0.01 % to about 25%, 0.02% to about 25%, 0.05% to about 25%, 0.075% to about 25%, 0.2% to about 25%, 0.3% to about 25%, 0.4% to about 25%, 0.5% to about 25%, 0.6% to about 25%, 0.7% to about 25%, 0.8% to about 25%, 0.9% to about 25%, 1 % to about 25%, 1 .5% to about 25%, 1 .75% to about 25%, 2% to about 25%, 2.25% to about 25%,, 2.5% to about 25%,, 2.75% to about 25%,, 3% to about 25%, 3.25% to about 25%, 3.5% to about 25%, 3.75% to about 25%, 4% to about 25%, 4.25% to about 25%, 4.5% to about 25%, 4.75% to about 25%, 5% to about 25%, 5.25% to about 25%, 5.5% to about 25%, 5.75% to about 25%, 6% to about 25%, 6.25% to about 25%, 6.5% to about 25%, 6.75% to about 25%, 7% to about 25%, 7.25% to about 25%, 7.5% to about 25%, 7.75% to about 25%, 8% to about 25%, 8.25% to about 25%, 8.5% to about 25%, 8.75% to about 25%, 9% to about 25%, 9.25% to about 25%, 9.5% to about 25%, 9.75% to about 25%, 10% to about 25%, 10.25% to about 25%, 10.5% to about 25%, 10.75% to about 25%, 1 1 % to about 25%, 1 1 .25% to about 25%, 1 1 .5% to about 25%, 1 1 .75% to about 25%, 12% to about 25%, 12.25% to about 25%, 12.5% to about 25%, 12.75% to about 25%, 13% to about 25%, 13.25% to about 25%, 13.5% to about 25%, 13.75% to about 25%, 14% to about 25%, 14.25% to about 25%, 14.5% to about 25%, 14.75% to about 25%, 15% to about 25%, 15.25% to about 25%, 15.5% to about 25%, 15.75% to about 25%, 16% to about 25%, 16.25% to about 25%, 16.5% to about 25%, 16.75% to about 25%, 17% to about 25%, 17.25% to about 25%, 17.5% to about 25%, 17.75% to about 25%, 18% to about 25%, 18.25% to about 25%, 18.5% to about 25%, 18.75% to about 25%, 19% to about 25%, 19.25% to about 25%, 19.5% to about 25%, 19.75% to about 25%, 20% to about 25%, 20.25% to about 25%, 20.5% to about 25%, 20.75% to about 25%, 5% to about 20%, 6% to about 20%, 7% to about 20%, 8% to about 20%, 9% to about 20%, 10% to about 20%, 1 1 % to about 20%, 12% to about 20%, 13%, to about 20%, 14% to about 20%, 15% to about 20%, 5% to about 15%, 6% to about 15%, 7% to about 15%, 8% to about 15%, 9% to about 15%, 10% to about 15%, 1 1 % to about 15%, 12% to about 15%, 13%, to about 15%, 14% to about 15% (w/v).

The pharmaceutical composition according to any one of embodiments 85-99, wherein the pharmaceutical composition comprises a monoclonal antibody preparation and one or more other drugs, with each present at a concentration of about 0.1 % (w/v) to about 40%, or alternatively at about 0.01 % to about 25%, 0.02% to about 25%, 0.05% to about 25%, 0.075% to about 25%, 0.2% to about 25%, 0.3% to about 25%, 0.4% to about 25%, 0.5% to about 25%, 0.6% to about 25%, 0.7% to about 25%, 0.8% to about 25%, 0.9% to about 25%, 1 % to about 25%, 1 .5% to about 25%,

I .75% to about 25%, 2% to about 25%, 2.25% to about 25%,, 2.5% to about 25%,, 2.75% to about 25%,, 3% to about 25%, 3.25% to about 25%, 3.5% to about 25%, 3.75% to about 25%, 4% to about 25%, 4.25% to about 25%, 4.5% to about 25%, 4.75% to about 25%, 5% to about 25%, 5.25% to about 25%, 5.5% to about 25%, 5.75% to about 25%, 6% to about 25%, 6.25% to about 25%, 6.5% to about 25%, 6.75% to about 25%, 7% to about 25%, 7.25% to about 25%, 7.5% to about 25%, 7.75% to about 25%, 8% to about 25%, 8.25% to about 25%, 8.5% to about 25%, 8.75% to about 25%, 9% to about 25%, 9.25% to about 25%, 9.5% to about 25%, 9.75% to about 25%, 10% to about 25%, 10.25% to about 25%, 10.5% to about 25%, 10.75% to about 25%, 1 1 % to about 25%,

I I .25% to about 25%, 1 1 .5% to about 25%, 1 1 .75% to about 25%, 12% to about 25%, 12.25% to about 25%, 12.5% to about 25%, 12.75% to about 25%, 13% to about 25%, 13.25% to about 25%, 13.5% to about 25%, 13.75% to about 25%, 14% to about 25%, 14.25% to about 25%, 14.5% to about 25%, 14.75% to about 25%, 15% to about 25%, 15.25% to about 25%, 15.5% to about 25%, 15.75% to about 25%, 16% to about 25%, 16.25% to about 25%, 16.5% to about 25%, 16.75% to about 25%, 17% to about 25%, 17.25% to about 25%, 17.5% to about 25%, 17.75% to about 25%, 18% to about 25%, 18.25% to about 25%, 18.5% to about 25%, 18.75% to about 25%, 19% to about 25%, 19.25% to about 25%, 19.5% to about 25%, 19.75% to about 25%, 20% to about 25%, 20.25% to about 25%, 20.5% to about 25%, 20.75% to about 25%, 5% to about 20%, 6% to about 20%, 7% to about 20%, 8% to about 20%, 9% to about 20%, 10% to about 20%, 1 1 % to about 20%, 12% to about 20%, 13%, to about 20%, 14% to about 20%, 15% to about 20%, 5% to about 15%, 6% to about 15%, 7% to about 15%, 8% to about 15%, 9% to about 15%, 10% to about 15%, 1 1 % to about 15%, 12% to about 15%, 13%, to about 15%, 14% to about 15% (w/v).

The pharmaceutical composition according to any one of embodiments 85-99, wherein the concentration of a monoclonal antibody preparation in the solution is at least 0.00001 mg/mL, at least 0.0001 mg/mL, at least 0.001 mg/mL, at least 0.01 mg/mL, at least 0.1 mg/mL, at least 1 mg/mL, at least 10 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least 100 mg/mL, at least 200 mg/mL, at least 500 mg/mL, at least 700 mg/mL, at least 1 ,000 mg/mL, or at least 1 ,200 mg/mL. The pharmaceutical composition according to any one of embodiments 85-99, wherein the concentration of a monoclonal antibody preparation in the solution is at most 1 ,000 mg/mL, at most 1 ,100 mg/mL, at most 1 ,200 mg/mL, at most 1 ,300 mg/mL, at most 1 ,400 mg/mL, at most 1 ,500 mg/mL, at most 2,000 mg/mL, at most 2,000 mg/mL, or at most 3,000 mg/mL.

The pharmaceutical composition according to any one of embodiments 85-99, wherein the concentration of a monoclonal antibody preparation in the solution is in a range of about 0.00001 mg/mL to about 3,000 mg/mL, about 0.0001 mg/mL to about 3,000 mg/mL, about 0.01 mg/mL to about 3,000 mg/mL, about 0.1 mg/mL to about 3,000 mg/mL, about 1 mg/mL to about 3,000 mg/mL, about 250 mg/mL to about 3,000 mg/mL, about 500 mg/mL to about 3,000 mg/mL, about 750 mg/mL to about 3,000 mg/mL, about 1 ,000 mg/mL to about 3,000 mg/mL, about 100 mg/mL to about 2,000 mg/mL, about 250 mg/mL to about 2,000 mg/mL, about 500 mg/mL to about 2,000 mg/mL, about 750 mg/mL to about 2,000 mg/mL, about 1 ,000 mg/mL to about 2,000 mg/mL, about 100 mg/mL to about 1 ,500 mg/mL, about 250 mg/mL to about 1 ,500 mg/mL, about 500 mg/mL to about 1 ,500 mg/mL, about 750 mg/mL to about 1 ,500 mg/mL, about 1 ,000 mg/mL to about 1 ,500 mg/mL, about 100 mg/mL to about 1 ,200 mg/mL, about 250 mg/mL to about 1 ,200 mg/mL, about 500 mg/mL to about 1 ,200 mg/mL, about 750 mg/mL to about 1 ,200 mg/mL, about 1 ,000 mg/mL to about 1 ,200 mg/mL, about 100 mg/mL to about 1 ,000 mg/mL, about 250 mg/mL to about 1 ,000 mg/mL, about 500 mg/mL to about 1 ,000 mg/mL, about 750 mg/mL to about 1 ,000 mg/mL, about 100 mg/mL to about 750 mg/mL, about 250 mg/mL to about 750 mg/mL, about 500 mg/mL to about 750 mg/mL, about 100 mg/mL to about 500 mg/mL, about 250 mg/mL to about 500 mg/mL, about 0.00001 mg/mL to about 0.0001 mg/mL, about 0.00001 mg/mL to about 0.001 mg/mL, about 0.00001 mg/mL to about 0.01 mg/mL, about 0.00001 mg/mL to about 0.1 mg/mL, about 0.00001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 0.01 mg/mL, about 0.001 mg/mL to about 0.1 mg/mL, about 0.001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 10 mg/mL, or about 0.001 mg/mL to about 100 mg/mL.

The pharmaceutical composition according to any one of embodiments 85-99, wherein the concentration of a monoclonal antibody in a liquid or a lyophilized formulationis between about 50 mg/mL to about 1 ,000 mg/mL.

The pharmaceutical composition according to any one of embodiments 85-99, wherein the therapeutically effective amount of a monoclonal antibody preparation is from about 50 mg/mL to about 100 mg/mL, about 50 mg/mL to about 200 mg/mL, about 50 mg/mL to about 300 mg/mL, about 50 mg/mL to about 400 mg/mL, about 50 mg/mL to about 500 mg/mL, about 50 mg/mL to about 600 mg/mL, about 50 mg/mL to about 700 mg/mL, about 50 mg/mL to about 800 mg/mL, about 50 mg/mL to about 900 mg/mL, about 50 mg/mL to about 1 ,000 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about 300 mg/mL, about 100 mg/mL to about 400 mg/mL, about 100 mg/mL to about 500 mg/mL, about 100 mg/mL to about 600 mg/mL, about 100 mg/mL to about 700 mg/mL, about 100 mg/mL to about 800 mg/mL, about 100 mg/mL to about 900 mg/mL, about 100 mg/mL to about 1 ,000 mg/mL, about 200 mg/mL to about 300 mg/mL, about 200 mg/mL to about 400 mg/mL, about 200 mg/mL to about 500 mg/mL, about 200 mg/mL to about 600 mg/mL, about 200 mg/mL to about 700 mg/mL, about 200 mg/mL to about 800 mg/mL, about 200 mg/mL to about 900 mg/mL, about 200 mg/mL to about 1 ,000 mg/mL, about 300 mg/mL to about 400 mg/mL, about 300 mg/mL to about 500 mg/mL, about 300 mg/mL to about 600 mg/mL, about 300 mg/mL to about 700 mg/mL, about 300 mg/mL to about 800 mg/mL, about 300 mg/mL to about 900 mg/mL, about 300 mg/mL to about 1 ,000 mg/mL, about 400 mg/mL to about 500 mg/mL, about 400 mg/mL to about 600 mg/mL, about 400 mg/mL to about 700 mg/mL, about 400 mg/mL to about 800 mg/mL, about 400 mg/mL to about 900 mg/mL, about 400 mg/mL to about 1 ,000 mg/mL, about 500 mg/mL to about 600 mg/mL, about 500 mg/mL to about 700 mg/mL, about 500 mg/mL to about 800 mg/mL, about 500 mg/mL to about 900 mg/mL, about 500 mg/mL to about 1 ,000 mg/mL, about 600 mg/mL to about 700 mg/mL, about 600 mg/mL to about 800 mg/mL, about 600 mg/mL to about 900 mg/mL, or about 600 mg/mL to about 1 ,000 mg/mL.

A kit comprising a pharmaceutical composition comprising a monoclonal antibody preparation for the prevention and/or treatment of an infection by a Human Enterovirus responsible for foot, hand and mouth disease.

The kit according to embodiment 109, wherein the kit comprises a monoclonal antibody, instructions for use to administer monoclonal antibody in the treatment of a Human Enterovirus capable of causing hand, foot and mouth disease in a subject.

The kit according to any of embodiments 109 or 1 10, wherein the components of the kit may be contained in one or different containers.

The kit according to embodiment 11 1 , wherein the one or different containers are one or more vials. The kit according to any one of embodiments 109-1 12, wherein the monoclonal antibody is in a liquid or a solid form.

The kit according to any one of embodiments 109-1 13, wherein the kit comprises a comprises a monoclonal antibody, instructions for use to administer the monoclonal antibody in the treatment of an enterovirus capable of causing hand, foot and mouth disease in a subject and one or more drugs. The kit according to embodiment 1 14, wherein the one or more drugs are an antimicrobial, antibacterial, immunostimulatory and/or antiviral.

The kit according to any one of embodiments 109-1 15, wherein the kit includes instructions for the use of the components of the kits.

1 17. The kit according to embodiment 1 16, wherein the instructions contain information how to prepare the monoclonal antibody.

1 18. The kit according to embodiments 116 or 117, wherein the instructions contain information on how to dilute or reconstitute the lyophilized monoclonal antibody.

1 19. The kit according to any one of embodiments 109-1 18, wherein the instructions provide guidance regarding the dosage and frequency of administration or the pharmaceutical composition.

120. A method of use, wherein a subject is treated with the pharmaceutical composition of embodiment 85.

121 . The pharmaceutical composition according to any one of embodiments 1 -50, wherein the polyclonal antibody preparation provides protection, treats and/or stops a neurological and/or cardiopulmonary condition resulting from hand, foot and mouth disease.

122. The pharmaceutical composition according to embodiment 121 wherein the neurological condition is pulmonary adema, meningitis, meningoencephalomyelitis, poliomyelitis-like paralytic disease, Guillain-Barre syndrome, transverse myelitis, cerebellar ataxia, opsoclonus-myoclonus syndrome, benign intracranial hypertension, and/or brainstem encephalitis.

123. The pharmaceutical composition of embodiment 121 , wherein the cardiopulmonary condition is myocarditis.

124. The pharmaceutical composition according to any one of embodiments 85-108, wherein the monoclonal antibody preparation provides protection, treats and/or stops a neurological and/or cardiopulmonary condition resulting from hand, foot and mouth disease.

125. The pharmaceutical composition according to embodiment 124 wherein the neurological condition is pulmonary adema, meningitis, meningoencephalomyelitis, poliomyelitis-like paralytic disease, Guillain-Barre syndrome, transverse myelitis, cerebellar ataxia, opsoclonus-myoclonus syndrome, benign intracranial hypertension, and/or brainstem encephalitis.

126. The pharmaceutical composition according to embodiment 124, wherein the cardiopulmonary condition is myocarditis.

127. The pharmaceutical composition according to any one of embodiments 1-10, 15, 18-20, 51 -61 or 63- 72, wherein the pharmaceutical composition is administered to a subject orally, intravaginally, intraparentally, intra-anally, into the spinal fluid, into the cranial fluid, subcutaneously, intramuscularly and/or intravenously.

128. The pharmaceutical composition according to any one of embodiments 85-88 or 109-108, wherein the pharmaceutical composition is administered to a subject orally, intravaginally, intraparentally, intra-anally, into the spinal fluid, into the cranial fluid, subcutaneously, intramuscularly and/or intravenously.

EXAMPLE 1. Description of HEV71 VLP Expression Cassettes and Vectors. [00187] Expression cassettes may be constructed through means understood in the art

1 .1 HEV71 VLP expression cassette ΓΡ1 +IRES+3CD1

Features: Cassette size: 5172 bp

prPs: Pox virus strong early/late synthetic promoter, 43 bp

P1 : P1 protein coding sequence from EV71-SB12736-SAR-03 (GenBank Accession: DQ341362) with the addition of a stop codon, 2588 bp

IRES: Internal Ribosome Binding site, 585 bp

3CD: C and D protein coding sequence of P3 from EV71-SB12736-SAR- 03 (GenBank Accession:

DQ341362) with the addition of a ATG start codon and stop codon, 1940 bp

Pac I: Rare cutters, enables cassette to be cloned into pSNXOI (MVA del 3 integration vector)

The cassette was cloned into pDONR221 Gateway entry vector (Invitrogen) to produce pSN01 .

See Figure 1 for a diagram of the expression cassette and the pSN01 plasmid.

1 .2 HEV71 VLP expression cassette fP1 +IRES+3C1

Features:

Cassette size: 3773 bp

prPS: Pox virus strong early/late synthetic promoter, 43 bp

P1 : P1 protein coding sequence from EV71-SB12736-SAR-03 (GenBank Accession: DQ341362) with the addition of a stop codon, 2588 bp

IRES: Internal Ribosome Binding site, 585 bp

3CD: C protein coding sequence of P3 from EV71-SB12736-SAR- 03 (GenBank Accession: DQ341362) with the addition of a ATG a start codon and stop codon, 551 bp

Pac I: Rare cutters, enables cassette to be cloned into pSNXOI (MVA del 3 integration vector)

The cassette was cloned into pDONR221 Gateway entry vector (Invitrogen) to produce pSN03.

See Figure 2 for a diagram of the expression cassette and the pSN03 plasmid.

1 .1 Methods for obtaining Recombinant Baculoviruses containing the insert HEV71 [P1 +IRES+3CD1 or [P1 +IRES+3C1

The source material for HEV71 P1 plus 3CD was pSN01 and for P1 plus 3C was pSN03. The aim was to introduce the HEV71-VLP cassette from pSN01 and pSN03 (Entry vectors) into the baculovirus expression plasmid pDEST8 (Destination vector) by attL/aaR in vitro recombination using LR CLONASE®, following the instructions in the Invitrogen BAC-TO-BAC® manual (2009).

Two recombinase reactions were set up:

1 . pSN01 (EV71 -P1 +3CD) x pDEST8 to produce pSN07;

2. pSN03 (EV71 -P1 +3C) x pDEST8 to produce pSN08

pSN07 and pSN08 were used to produce recombinant bacmids bacSN07 and bacSN08 by transforming DHIObac as described in the Invitrogen BAC-TO-BAC® manual. The recombinant bacmids were transfected into Sf9 cells to rescue recombinant baculoviruses SN07 and SN08.

The recombinant baculoviruses SN07 and SN08 were used to further infect Sf9 cells in 6 well plates to evaluate for expression of processed capsid proteins. A polyclonal rabbit antiserum specific for VP1 was used to identify VP1 protein in Western blots of lysates and supernatants from recombinant baculovirus infected Sf9 cells.

1 .3 Supernatants of infected Sf9 cells were harvested daily from day 3 to day 7 post infection and the proteins were resolved on a 12% SDS-PAGE and transferred to nitrocellulose membranes which were then probed with polyclonal rabbit anti-VP1 antisera (1 :4000 dilution) overnight followed by anti-rabbit conjugated with HRP (1 :1000 dilution) for 1 hour at room temperature. The Western blots were subsequently developed using TMB. The results are shown in Figure 3. It was observed that the HEV71 VP1 was processed and that expression of the protein was observed in the supernatant at day 3 and day 4 post infection and that the amount of VP1 expression diminished thereafter. Recombinant baculovirus SN07 on infection of Sf9 cells generates antigens which are found in the supernatant.

EXAMPLE 2. Processed VP1 in both the supernatants and the lysates

[0240] Sf9 cells were infected at a Multiplicity of Infection (MOI) of 10 with different recombinant baculovirus isolates, including SN07, SN08, a control baculovirus bacGUS and mock infected. Supernatants and lysates were harvested on days 3 and 4 post infection and expression of the proteins evaluated by Western blots using rabbit anti-VP1 antisera (1 :4000 dilution) to compare yields of proteins produced by SN07 and SN08. As shown in Figure 4, expression construct SN07 produced more cleaved VP1 than expression construct SN08 both in the supernatant and in the lysate on both days 3 and 4 post infection.

EXAMPLE 3. VP1 and VP0 is in the retentate after ultrafiltration over a 100kD molecular weight cut off (MWCO) membrane

[0241] Supernatants from SN07 infected Sf9 cells at day 3 post-infection were clarified and were passed through AMICON® filters (Millipore Corp.) with a 100 kDa MWCO. The retentate was tested for the presence of processed VP1 and VP0. Since the molecular weight of VP1 is approximately 33 kDa, and the molecular weight of VP0 is 36 KDa, these proteins would not be expected to remain in the retentate unless they were in an oligomeric form. As shown in Figure 5, these antigens remain in the retentate on passing the supernatants through a 100kDa MWCO ultrafilter. This suggests that the antigens are associated in an oligomeric form. Thus, it may be concluded that VP1 and VP0 are processed and are in an oligomeric association with other capsid proteins.

EXAMPLE 4. Production of VLPs of HEV71, as well as Human enterovirus C, by means of reducing the protease 3CD mediated killing of baculovirus infected cells

[0242] This experiment describes the construction of recombinant baculovirus vector for the expression of the P1 region and the protease 3CD from a single bicistronic message. The protease gene 3CD is translated in a cap-independent fashion under control of the EMCV IRES, shown in Figure 6. This system provides the leverage to regulate the expression of protease 3CD, i.e., evaluate the mutant IRES sequences to find weakest IRES so that a lesser amount of the protease is produced compared to the P1 proteins. A bicistronic vector was constructed in which a plasmid contains a polyhedrin promoter upstream of the coding sequence for the P1 . Downstream from the cistrons encoding P1 is an Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence followed by the cistrons containing nucleotide sequence encoding the protease 3CD, see Figure 1 . The IRES used in Example 1 contains native EMCV IRES sequence as there are altered forms. The native EMCV IRES sequence shows the A6 bifurcation loop in the JK segment. Adding one nucleotide, for example an adenine (A7), reduces the expression. Also, in the construct used in Example 1 , the 3CD protease is fused with Encephalomyocarditis virus IRES at the amino-terminus. Importantly out framing the EMCV start codon with 3CD protease coding sequence should considerably reduce the expression of downstream genes. Both modifications are incorporated into the EMCV IRES sequence of pSNOIand named pSN01 -M1 , as shown in Figure 8, and synthesized by DNA2.0.

[0243] Experiments were designed to characterize the VLPs expressed from the mutant EMCV IRES of pSN01 -M1 . Recombinant baculoviruses were generated using the BAC-TO-BAC® system according to the manufacturer's instructions (Invitrogen). Briefly, LR CLONASE® was used to introduce the HEV71 VLP cassette into the baculovirus expression plasmid pDEST8 (Destination using vector) by attL/aaR in vitro recombination. The LR CLONASE® reaction was carried out at 25°C for 1 hr followed by incubation with proteinase K. The LR CLONASE® reaction mix was transformed into Library Efficiency DH5a competent cells to obtain expression clones. DNA was isolated from the resultant colonies and confirmed for the presence of the HEV71/poliovirus cassette by restriction enzyme analysis. Recombinant bacmids are constructed by introducing the expression cassette of pSN07-M1 , into the baculovirus genome harbored in DHI Obac cells by T7 transposition recombinase to give bacSN07- M1. The recombinant bacmids are verified by their white phenotype on LB agar plates supplemented with 50 pg/ml kanamycin, 7 pg/ml gentamicin, 10 pg/ml tetracycline, 100 pg/ml X-gal, and 40 pg/ml IPTG. The PureLink HiPure Plasmid DNA Miniprep Kit (Invitrogen) was used to purify high quality bacmid DNA from DHIOBac E. coli. M13 forward, M13 reverse and internal primers from the insert were used to confirm the existence of the HEV71/poliovirus cassette. EFFECTENE® transfection reagent (Qiagen) was used to rescue recombinant baculoviruses by transfecting the DNAs into Sf9 insect cells. Briefly, Sf9 cells were seeded at 2 million per T25 flask and incubated to adhere for 6hr at 28°C. One microgram of recombinant bacmid DNA was resuspended in 150 μΙ of DNA condensation buffer and 8μΙ of enhancer solution is mixed and incubated at room temperature for 5 minutes. Then 25μΙ of EFFECTENE® reagent was added into DNA mix and incubated for 10 minutes at room temperature. One ml culture medium was added into the tubes containing the transfection complexes and transferred into cell culture flasks and uniformly distributed. At day 3 the supernatant was harvested by centrifugation at 500g for 5 minutes. Following transfection, a high titer viral stock is prepared. Once a high viral stock is obtained, it is employed to determine the optimal times for target protein expression. For the analysis of the protein of interest, Sf9 cells grown in 10% Grace's insect cell medium (Invitrogen) were resuspended in 1 % FBS Sf900-ll SFM medium (Invitrogen) to get single cells and were seeded at a million per ml density in the flasks and incubated for 4 hr at 28°C. Viral stocks were added into the PBS washed cells at an MOI of 10 and rocked gently for 1 hr. The infected cells were washed three times with PBS and the cells were grown in Sf900ll-SFM for different time points. Cells were lysed with hypotonic douncing buffer /1 %TRITON® X-100 (TX-100) (1.5mM MgCl2, 50mM KCI, 20mM HEPES, 1 % TX-100) by rocking the flask for 30 minutes at room temperature and cell lysates were prepared by collecting the lysed cells from the flask and centrifuging at 4°C for 30 minutes at 7000 rpm. The components of the cell lysates and supernatants were analyzed by immunoblotting and ELISA using specific antibodies. EXAMPLE 5. Efficient production of VLPs of HEV71, as well as Human enterovirus C (poliovirus) by means of reducing the protease 3CD mediated killing of baculovirus infected cells

[0244] The construction of a recombinant baculovirus vector for expression of the P1 region and the protease 3CD from a single bicistronic message is shown in Figure 1. The 3CD protease gene is translated in a cap-independent fashion under control of the EMCV IRES. This system provides the leverage to regulate the expression of 3CD protease, i.e., evaluate the mutant IRES sequences to find the weakest IRES so that a lesser amount of protease is produced compared to the P1 proteins.

[0245] A bicistronic vector was constructed and the plasmid contains a polyhedrin promoter upstream of the coding sequence for the P1 . Downstream from the cistrons encoding P1 is an Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence followed by the cistrons containing a nucleotide sequence encoding the 3CD protease, see Figure 1 . The IRES used in Example 1 contains native EMCV IRES sequence as there are altered forms. The native EMCV IRES sequence has the A6 bifurcation loop in the JK segment, indeed by adding one nucleotide (A7) known to reduce the expression. The A6 bifurcation loop was modified into A7 in the EMCV IRES sequence of pSN01 and named pSN01 - M2, see Figure 6, which is synthesized by DNA2.0.

[0246] VLPs expressed by the mutant EMCV IRES of pSN01 -M2 were characterized. Recombinant baculoviruses were generated using the BAC-TO-BAC® system according to the manufacturer's instructions (Invitrogen). Briefly, LR CLONASE® was used to introduce the HEV71 VLP cassette into the baculovirus expression plasmid pDEST8 (Destination using vector) by attL/aaR in vitro recombination. The LR CLONASE® reaction was carried out at 25°C for 1 hr followed by incubation with proteinase K. The LR CLONASE® reaction mix was transformed into Library Efficiency DH5a competent cells to obtain expression clones. DNA was isolated from the resultant colonies and confirmed for the presence of the HEV71 /poliovirus cassette by restriction enzyme analysis. Recombinant bacmids were constructed by introducing the expression cassette of pSN07-M2, into the baculovirus genome harbored in DHI Obac cells by T7 transposition recombinase to give bacSN07- M2. The recombinant bacmids were verified by their white phenotype on LB agar plates supplemented with 50 g/ml kanamycin, 7 g/ml gentamicin, 10 g/ml tetracycline, 100 g/ml X-gal, and 40 g/ml IPTG. The PureLink HiPure Plasmid DNA Miniprep Kit (Invitrogen) was used to purify high quality bacmid DNA from DHI OBac E. coli. M13 forward, M13 reverse and internal primers from the insert were used to confirm the existence of the HEV71 /poliovirus cassette. EFFECTENE® transfection reagent (Qiagen) was used to rescue recombinant baculoviruses by transfecting the DNAs into Sf9 insect cells. Briefly, Sf9 cells were seeded at 2 million per T25 flask and incubated to adhere for 6hr at 28°C.

[0247] One microgram of recombinant bacmid DNA was resuspended in 150 μΙ of DNA condensation buffer and 8μΙ of enhancer solution is mixed and incubated at room temperature for 5 minutes. Then 25μΙ of EFFECTENE® reagent was added into the DNA mix and incubated for 10 minutes at room temperature. One ml culture medium was added into the tubes containing the transfection complexes and transferred into cell culture flasks and uniformly distributed. At day 3, the supernatant was harvested by centrifugation at 500g for 5 minutes. Following transfection, a high titer viral stock was prepared. Once a high viral stock was obtained, it was employed to determine the optimal times for target protein expression. For the analysis of the protein of interest, Sf9 cells grown in 10% Grace's insect cell medium (Invitrogen) was resuspended in 1 % FBS Sf900-ll SFM medium (Invitrogen) to get single cells and were seeded at a million per ml density in the flasks and incubated for 4 hr at 28°C. Viral stocks were added into the PBS washed cells at an MOI of 10 and rocked gently for 1 hr. The infected cells were washed three times with PBS and cells were grown in Sf900ll-SFM for different time points. Cells were lysed with hypotonic douncing buffer /1 %TX-100 (1.5mM MgCl2, 50mM KCI, 20mM HEPES, 1 % TX-100) by rocking the flask for 30 minutes at room temperature and the cell lysates were prepared by collecting the lysed cells from the flask and centrifuging at 4°C for 30 minutes at 7000rpm. The components of the cell lysates and supernatants were analyzed by immunoblotting and ELISA using specific antibodies.

EXAMPLE 6. Efficient production of VLPs of HEV71 as well as Human enterovirus C (poliovirus) by means of reducing the protease 3CD mediated killing of baculovirus infected cells

[0248] The construction of a recombinant baculovirus vector for expression of the P1 region and the protease 3CD from a single bicistronic message is shown in Figure 1. The 3CD protease gene is translated in a cap-independent fashion under control of the EMCV IRES, shown in Figure 6. This system provides the leverage to regulate the expression of protease 3CD, i.e., evaluate the mutant IRES sequences to find the weakest IRES so that a lesser amount of protease is produced compared to the P1 proteins.

[0249] A bicistronic vector was constructed in which the plasmid contains a polyhedrin promoter upstream of the coding sequence for the P1 . Downstream from the cistrons encoding P1 is an Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence followed by the cistrons containing a nucleotide sequence encoding the protease 3CD (Figure 1 ). The IRES used in Example 1 contains native EMCV IRES sequence as there are altered forms. In the pSN01 construct the 3CD protease is fused with Encephalomyocarditis virus polyprotein at the amino-terminus. Out framing the EMCV start codon with 3CD protease coding sequence should considerably reduce the expression of downstream genes, see Figure 7. This modification was incorporated into EMCV IRES sequence of pSN01 and named pSN01- M3, and is synthesized by DNA2.0.

[0250] The VLPs expressed by the mutant EMCV IRES of pSN01 -M3 were analyzed. Recombinant baculoviruses were generated using the BAC-TO-BAC® system according to the manufacturer's instructions (Invitrogen). Briefly, LR CLONASE® was used to introduce the HEV71 VLP cassette into the baculovirus expression plasmid pDEST8 (Destination using vector), by attL/aaR in vitro recombination. The LR CLONASE® reaction was carried out at 25°C for 1 hr followed by incubation with proteinase K. The LR CLONASE® reaction mix was transformed into Library Efficiency DH5a competent cells to obtain expression clones. DNA was isolated from the resultant colonies and confirmed for the presence of HEV71/poliovirus cassette by restriction enzyme analysis. Recombinant bacmids were constructed by introducing the expression cassette of pSN07-M3, into the baculovirus genome harbored in DHI Obac cells by T7 transposition recombinase to give bacSN07-M3. The recombinant bacmids were verified by their white phenotype on LB agar plates supplemented with 50 pg/ml kanamycin, 7 pg/ml gentamicin, 10 pg/ml tetracycline, 100 g/ml X-gal, and 40 g/ml IPTG. The PureLink HiPure Plasmid DNA Miniprep Kit (Invitrogen) was used to purify high quality bacmid DNA from DHIOBac E. coli. M13 forward, M13 reverse and internal primers from the insert were used to confirm the existence of the HEV71/poliovirus cassette. EFFECTENE® transfection reagent (Qiagen) was used to rescue recombinant baculoviruses by transfecting the DNAs into Sf9 insect cells. Briefly, Sf9 cells were seeded at 2 million per T25 flask and incubated to adhere for 6hr at 28°C. One microgram of recombinant bacmid DNA was resuspended in 150 μΙ of DNA condensation buffer and 8μΙ of enhancer solution is mixed and incubated at room temperature for 5 minutes. Then 25μΙ of EFFECTENE® reagent was added into the DNA mix and incubated for 10 minutes at room temperature. One ml culture medium was added into the tubes containing the transfection complexes and transferred into cell culture flasks and uniformly distributed. After day 3, supernatant was harvested by centrifugation at 500g for 5 minutes. Following transfection, a high titer viral stock is prepared. Once a high viral stock is obtained, it is employed to determine the optimal times for target protein expression.

[0251] For the analysis of the protein of interest, Sf9 cells grown in 10% Grace's insect cell medium (Invitrogen) were resuspended in 1 % FBS Sf900-ll SFM medium (Invitrogen) to get single cells and were seeded at a million per ml density in the flasks and incubated for 4 hr at 28°C. Viral stocks were added into the PBS washed cells at a MOI of 10 and rocked gently for 1 hr. The infected cells were washed three times with PBS and cells were grown in Sf900ll-SFM for different time points. Cells were lysed with hypotonic douncing buffer /1 %TX-100 (1 .5mM MgCl2, 50mM KCI, 20mM HEPES, 1 %TX-100) by rocking the flask for 30 minutes at room temperature and cell lysates were prepared by collecting the lysed cells from the flask and centrifuging at 4°C for 30 minutes at 7000 rpm. The components of the cell lysates and supernatants were analyzed by immunoblotting and ELISA using specific antibodies.

EXAMPLE 7. Mutant IRES construct M2 expresses higher levels of VP1 in the supernatant

[0252] Recombinant baculoviruses expressing HEV71 capsid proteins under the control of the wild type or mutant EMCV IRES's were evaluated with respect to the level of expression of the HEV71 capsid proteins from the ECMV IRES's. Baculovirus produced VLPs which are expressed under the control of the wild type EMCV IRES from SN07 of Example 1 , and the 3 mutant IRES's, M1 , M2 and M3 from Examples 10, 11 , and 12, respectively, were analyzed with respect to the level of baculovirus expression of the HEV71 capsid proteins. A recombinant baculovirus expressing P1 and 3CD under different promoters (F) and a control recombinant baculovirus expressing bacGUS (G) were also included in the study. Sf9 cells were infected at an MOI of 5 and both lysates and supernatants were harvested on day 3 as described in Examples 10-12 above. The lysates and supernatants were probed with an anti-VP1 antibody to detect the expression of HEV71 capsid proteins.

[0253] Immunoblots show that when lysates were probed with antibodies to VP1 , the mutants M1 and M2 express higher levels of VP1 than the mutant M3 or the construct driven by 2 promoters. However, the mutant M2 produced more VP1 in the supernatant. These data are shown in the top panel.

[0254] The blots which were probed with a control anti-gp64 antibody which is directed to the coat protein of baculovirus. The immunoblot shows that equivalent amounts of baculovirus were produced in each sample.

EXAMPLE 8. Cloning, expression and purification of subunit vaccines using a baculovirus expression system

[0255] The present invention is intended for the generation and use of recombinant HEV71 and poliovirus structural proteins which are fused as single immunogens to elicit a protective immune response in vaccinated individuals. The present invention relates generally to preparing recombinant HEV71 and/or poliovirus fusion protein vaccine compositions comprising HEV71 and/or poliovirus subunit protein, or an immunogenic fragment thereof, and an adjuvant in combination with the recombinant HEV71 and/or poliovirus subunit fusion protein. HEV71 and poliovirus subunit fusion proteins may comprise capsid proteins selected from VP1 , VP2, VP3, and VP4, combinations thereof, and combinations of immunogenic fragments thereof. In one aspect of this embodiment, the recombinant HEV71 and/or poliovirus fusion protein comprises HEV71 or poliovirus subunit protein and a fusion partner protein in genetic association with the HEV71 or poliovirus subunit protein. The present invention contemplates methods to generate the constructs to express the following subunit vaccines in E.coli as well as baculovirus: VPO, VP4-VP2-VP3 fusion, VP2-VP3-VP1 fusion.

[0256] To generate cDNAs from HEV71 and/or poliovirus encoding VPO, VP4- VP2-VP3 fusion, or VP2- VP3-VP1 , reverse transcription/polymerase chain reaction (The High Pure Nucleic Acid Kit; Roche) was carried out using purified genomic viral RNA. A forward and a reverse primer was made from the 5'and 3' end of the genes and which primers incorporate a start and stop codon. The amplified PCR products were digested with EcoRI and Notl restriction enzymes and cloned into the pFastBac HT vector (Invitrogen). The BAC-TO-BAC® expression system from Invitrogen is commercially available and methods were used according to the manufacturer's instructions. The fusion genes are cloned into pFastBac HT donor plasmid and the production of recombinant proteins was based upon the BAC-TO- BAC® to baculovirus expression system (Invitrogen). The pFastBac HT donor plasmid carrying the fusion genes was transferred into a baculovirus shuttle vector (bacmid) by site-specific recombination by T7 transposition recombinase. This was accomplished in E. coli strain DHI OBac. The DHIOBac cells contain the bacmid, which conferred kanamycin resistance and a helper plasmid, which encoded the transposase and conferred resistance to tetracycline. The recombinant pFastBac HT plasmids with the gene of interest were transformed into DHI OBac cells for the transposition to generate recombinant bacmids. The transformed cells were serially diluted and each dilution was plated on LB agar plates supplemented with 50pg/ml kanamycin, 7 g /ml gentamicin, 10pg/ml tetracycline, 100 pg/ml X-gal, and 40 pg/ml IPTG and incubated for at least 48 hours at 37°C. The white colonies were picked and re-streaked to confirm a white phenotype. Recombinant bacmids were isolated by the PureLink HiPure Plasmid DNA Miniprep Kit (Invitrogen) and the DNA samples were dissolved in 40 lof TE (10 mM Tris-HCI pH 8, 1 mM EDTA) and used for transfections.

[0257] The isolated bacmid DNA was screened for the inserted gene of interest by PCR. EFFECTENE® transfection reagent (Qiagen) was used to rescue recombinant baculoviruses by transfecting the DNAs into Sf9 insect cells. Briefly, Sf9 cells were seeded at 2 million per T25 flask and incubated to adhere for 6hr at 28°C. One microgram of recombinant bacmid DNA was resuspended in 150 μΙ of DNA condensation buffer and 8μΙ of enhancer solution is mixed and incubated at room temperature for 5 minutes. Then 25μΙ of EFFECTENE® reagent was added into DNA mix and incubated for 10 minutes at room temperature. One ml culture medium was added into the tubes containing the transfection complexes and transferred into cell culture flasks and uniformly distributed. At day 3, the supernatant was harvested by centrifugation at 500g for 5 minutes. Following transfection, a high titer viral stock is prepared. Once a high viral stock is obtained, it is employed to determine the optimal times for target protein expression. For the analysis of the protein of interest, Sf9 cells grown in 10% Grace's insect cell medium (Invitrogen) was resuspended in 1 % FBS Sf900-ll SFM medium (Invitrogen) to get single cells and are seeded at a million per ml density in the flasks and incubated for 4 hr at 28°C. Viral stocks were added into PBS washed cells at a MOI of 10 and rocked gently for 1 hr. The infected cells were washed three times with PBS and cells are grown in Sf900ll-SFM for different time points. Cells were lysed with hypotonic douncing buffer /1 %TX- 100 (1 .5mM MgCl2, 50mM KCI, 20mM HEPES, 1 %TX-100) by rocking the flask for 30 minutes at room temperature and the cell lysates were prepared by collecting the lysed cells from the flask and centrifuging at 4°C for 30 minutes at 7000rpm. The expression of the heterologous protein in the cells was verified by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots using the His Probe-HRP antibody (Thermo Scientific) as the probe. Once production of baculovirus and the expression of protein were confirmed, the virus stock was amplified to produce a concentrated stock of the baculovirus that carry the gene of interest. The most appropriate concentration of the virus to infect insect cells and the optimum time point for the production of the desired protein was also established. For purification under denaturing conditions, the cells were lysed in a lysis buffer containing 6 M guanidinium-HCI in 100 mM NaH2P04, 10 mM Tris, 300mM NaCI, 10 mM imidazole, pH 8.0 (lysis buffer). The suspension was sonicated on ice with 5 pulses of 1 minute per pulse at a power setting of 60 watts, and was mixed at room temperature for 1 hour. The lysate was centrifuged at 27K g for 30 min to eliminate cell debris. The supernatant was loaded on to a HisTrap (GE healthcare life sciences) column pre-equilibrated with lysis buffer. Following loading, the column was washed with 20 column volumes of 6 M guanidinium-HCI in 100 mM NaH 2PO4, 10 mM Tris, 300 mM NaCI, 40 mM Imidazole, pH 8.0 (wash buffer 1 ), followed by washes with 20 column volumes of 8 M urea in 100 mM NaH2PO4,10 mM Tris, 300 mM NaCI, 40 mM imidazole, pH 8.0 (wash buffer 2). The bound protein was eluted with a buffer containing 8 M urea, 100 mM NaH2P04, 10 mM Tris, 300 mM NaCI, 250 mM imidazole, pH 8 (Elution Buffer). The fractions containing the protein were pooled and dialyzed against PBS, overnight at 4° C. TEV protease was used for removal of the histidine tag following protein purification according to manufacturer's instructions.

EXAMPLE 9. Expression and purification of Human enterovirus A and Human enterovirus C (poliovirus) subunit vaccines in E.coli

[0258] The Champion™ pET SUMO Expression System (Invitrogen) produces the highest levels of soluble protein in E. coli. It utilizes a small ubiquitin-related modifier (SUMO) fusion to enhance the solubility of expressed fusion proteins. After expression, the 11 kD SUMO moiety can be cleaved by the highly specific and active SUMO (ULP-1 ) protease at the carboxyl terminal, producing a native protein. Also it contains N-terminal 6xHis tag for protein detection and purification.

[0259] The construction of pET SUMO-VP0, pET SUMO- VP4-VP2-VP3 and pET SUMO-VP2-VP3-VP1 expression vector for antigenic fusion proteins of HEV71 and poliovirus, , is as follows. The fragments of VPO, VP4-VP2-VP3 fusion and VP2-VP3-VP1 fusion were used as the antigens for HEV71 and poliovirus subunit vaccines. A SUMO motif and the 6XHistag were conjugated to the N-terminus of fusions to aid in solubilization of the protein and purification of the protein, respectively. The antigenic fusion proteins were created by a gene cloning technology comprising cloning cDNA sequences encoding respective proteins into an expression vector to form expression vectors of pET SUMO- VPO, pET SUMO- VPO, pET SUMO- VP4-VP2-VP3 and pET SUM 0-VP2-VP3-VP1 . The DNA fragments encoding fusion partners were PCR amplified using specific primers which consist of a start codon and a stop codon in the forward and reverse primers, respectively. Ligation of the PCR product was carried out as follows: fresh PCR product, 10X ligation buffer, pET SUMO vector (25 ng/μΙ) 2 μΙ, sterile water added to a total volume of 9 μΙ , and 1 μΙ T4 DNA ligase (4.0 Weiss units) was added and the ligation reaction incubated at 15°C for overnight then proceeded to transforming One Shot® Mach1™-T1 R (Invitrogen) competent cells. Ten (10) colonies were selected and plasmid DNA isolated from them using the PureLink™ HQ Mini Plasmid Purification Kit (Invitrogen). The plasmids were analyzed by restriction analysis to confirm the presence and the correct orientation of the insert. From the recombinants, plasmid DNA was isolated as earlier and the plasmids were transformed into BL21 (DE3) One Shot® cells (Invitrogen). The transformants were grown and induction of expression with IPTG at several time points was carried out to determine the optimal time of expression. For each time point, 500 μΙ was removed from the induced and uninduced cultures and each cell pellet was resuspended in 80 μΙ of SDS-PAGE sample buffer. After centrifuging the boiled samples, 10 μΙ of each sample was loaded onto an SDS-PAGE gel and electrophoresed.

[0260] To scale-up the purification of recombinant fusion protein using a HisTrap nickel column (GE Healthcare Life Sciences), the following procedure was adapted. An overnight culture (5%) was inoculated into 100-300 ml LB plus 50μg/ml kanamycin and induced after 2hrs with 1 mM IPTG. After 2hrs the cells were harvested by centrifuging at 3000g for 10 minutes. The pellet was resuspended in 10% (total volume of the culture) the binding buffer (20mM sodium phosphate, 0.5M NaCI and 20mM imidazole at pH7.4). The cells were sonicated with Misonic UltraSonicate Liquid processor for five times for a minute with a minute gap in an ice bucket. The sonicated samples were separated into soluble and insoluble form by centrifuging at 4000 rpm for 1 hr at 4°C. The insoluble fraction was resuspended with binding buffer containing 6M urea. Both the soluble and insoluble fractions were centrifuged at 4000 rpm for 1 hr at 4°C then filtered through 0.22μηι filter unit. A HisTrap column was equilibrated with the binding buffer and filtered samples were loaded onto the column. Next, the column was washed with binding buffer with 40mM imidazole and the recombinant protein was eluted with binding buffer containing 0.5M imidazole (6M urea for insoluble fraction). All the collected samples were tested using Coomassie blue staining protocol for proteins. Pure recombinant protein containing eluted fraction were dialysed using Merck tubing in Tris-HCI buffer. After the dialysis protein concentrations were estimated using a Bradford reagent. The native protein was generated by using SUMO protease to cleave the N-terminal peptide containing the 6xHis tag and SUMO according to manufacturer's instructions.

EXAMPLE 10. Construction of HEV71 VLP Expression Cassettes with HEV71 IRES (P1+HEV71 IRES+3CD)

[0261] The schematic structure of a HEV71 VLP cassette with HEV71 -IRES is shown in Figure 11 . The expression cassette is similar to the construct shown in Example 1 (pSN01 ) except that the expression of the 3CD protease is driven by the HEV71 IRES rather than the EMCV IRES. The HEV71 IRES sequence is found in GenBank, Accession Number DQ341362.1 ; nucleotides 1 to 747. An HEV71 expression cassette containing vector is introduced into the baculovirus expression plasmid pDEST8 (Destination vector) by attL/aaR in vitro recombination using LR CLONASE®, following the instructions in the Invitrogen BAC-TO-BAC® manual (2009). Recombination between the entry vector and pDEST8 produces an expression clone. Expression clones give rise to recombinant bacmid by transforming DHIObac as described in the Invitrogen BAC-TO-BAC® manual. Transfection of the recombinant bacmid into Sf9 cells rescues the recombinant baculovirus carrying expression cassette which harbors P1 , HEV71 IRES and 3CD.

[0262] Further infection of Sf9 cells can be used to evaluate expression of processed capsid proteins with rescued recombinant baculoviruses in 6 well plates. A polyclonal rabbit antiserum specific for VP1 , VPO and VP3 will identify the assembled VLPs by Western blotting of lysates and supernatants from recombinant baculovirus infected Sf9 cells.

EXAMPLE 11. Construction of HEV71 VLP Expression Cassettes with PV-IRES (P1 +PV-IRES+3CD).

[0263] The expression cassette is similar to the expression cassette in Example 24 except that the expression of the 3CD protease is driven by a poliovirus IRES (PV- IRES) rather than an HEV71 -IRES. The poliovirus IRES sequence is found in GenBank, Accession Number V01 150.12; nucleotides 1 to 628.

[0264] An HEV71 expression cassette containing vector is introduced into the baculovirus expression plasmid pDEST8 (Destination vector) by attL/aaR in vitro recombination using LR CLONASE®, following the instructions in the Invitrogen BAC- TO-BAC® manual (2009). Recombination reaction between entry vector and pDEST8 is set up to produce an expression clone. An expression clone give rises to recombinant bacmid by transforming DHI Obac as described in the Invitrogen BAC-TO- BAC® manual. Transfection of the recombinant bacmid into Sf9 cells rescues the recombinant baculovirus carrying expression cassette which harbors P1 , PV IRES and 3CD.

[0265] Further infection of Sf9 cells can be used to evaluate for expression of processed capsid proteins with rescued recombinant baculoviruses in 6 well plates. A polyclonal rabbit antiserum specific for VP1 , VPO and VP3 will identify the assembled VLPs by Western blotting of lysates and supernatants from recombinant baculovirus infected Sf9 cells.

[0266] One of skill will understand that the material produced as set forth in EXAMPLES 1 -10 can be used to immunize a subject to produce a polyclonal antibody preparation for use as to prevent or treat a causative agent of foot, hand and mouth disease, including, without limitation, EV71 and CA16.

EXAMPLE 12 Therapeutic use of polyclonal antibodies

[0267] A six year old male child suffering from foot, hand and mouth disease is administered a polyclonal antibody preparation that was purified from immunized humans who were administered a vaccine comprising virus like particles of EV71 and CA16. Following administration of the polyclonal antibody preparation, the child began to recover from the disease.

EXAMPLE 13 Therapeutic use of polyclonal antibodies

[0268] An eight year old female child in a town where an outbreak of foot, hand and mouth disease is occurring is administered a polyclonal antibody preparation that was purified from immunized humans who were administered a vaccine comprising virus like particles of EV71 and CA16 and an antiviral drug. Three weeks following administration, the child does not show symptoms of an infection with hand, foot and mouth disease.

EXAMPLE 14 Therapeutic use of a monoclonal antibody

[0269] A seven year old boy infected with a causative agent of hand, foot and mouth disease is administered a monoclonal antibody to treat the disease. The boy is administered multiple therapeutically effective dose of the monoclonal antibody over several weeks after which, the severity of the disease was reduced.

[0270] In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular methodology, protocol, and/or reagent, etc., described herein. As such, various modifications or changes to or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of the present specification. Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not limited to that precisely as shown and described.

[0271] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the present invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0272] Groupings of alternative embodiments, elements, or steps of the present invention are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0273] Unless otherwise indicated, all numbers expressing a characteristic, item, quantity, parameter, property, term, and so forth used in the present specification and claims are to be understood as being modified in all instances by the term "about." As used herein, the term "about" means that the characteristic, item, quantity, parameter, property, or term so qualified encompasses a range of plus or minus ten percent above and below the value of the stated characteristic, item, quantity, parameter, property, or term. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of numerical ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the present specification as if it were individually recited herein.

[0274] The terms "a," "an," "the" and similar referents used in the context of describing the present invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the present invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the present specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0275] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term "consisting of" excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the present invention so claimed are inherently or expressly described and enabled herein.

[0276] All patents, patent publications, and other publications referenced and identified in the present specification are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Claims

1 . A pharmaceutical composition comprising an antibody preparation for the prevention and/or treatment of an infection by a Human Enterovirus responsible for foot, hand and mouth disease.

2. The pharmaceutical composition according to Claim 1 , wherein the antibody preparation is a monoclonal antibody preparation or a polyclonal antibody preparation.

3. The pharmaceutical composition according to Claim 2, wherein the polyclonal antibody preparation is prepared from one or more subjects immunized with a vaccine against a Human Enterovirus responsible for foot, hand and mouth disease.

4. The pharmaceutical composition according to any one of Claims 1-3, wherein the Human Enterovirus is EV71 , CA5, CA6, CA10 and/or CA16.

5. The pharmaceutical composition according to any one of Claims 1 -4, wherein the antibody preparation comprises one or more immunologically active antigens comprising one or more Human Enterovirus VPO polypeptide, VP1 polypeptide, VP2 polypeptide, VP3 polypeptide, VP4 polypeptide, an immunologically active fragment thereof, or any combination thereof.

6. The pharmaceutical composition according to any one of Claims 1 -5, wherein the antibody preparation comprises at least 0.01 %, at least 0.02%, at least 0.05%, at least 0.075%, at least 0.1 %, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1 %, at least 1 .5%, at least 1.75%, at least 2%, at least 2.25%, at least 2.5%, at least 2.75%, at least 3%, at least 3.25%, at least 3.5%, at least 3.75%, at least 4%, at least 4.25%, at least 4.5%, at least 4.75%, at least 5%, at least 5.25%, at least 5.5%, at least 5.75%, at least 6%,6.25%, at least 6.5%, at least 6.75%, at least 7%, at least 7.25%, at least 7.5%, at least 7.75%, at least 8%, at least 8.25%, at least 8.5%, at least 8.75%, at least 9%, at least 9.25%, at least 9.5%, at least 9.75%, at least 10%, at least 10.25%, at least 10.5%, at least 10.75%, at least 1 1 %, at least 1 1.25%, at least 1 1 .5%, at least 1 1.75%, at least 12%, at least 12.25%, at least 12.5%, at least 12.75%, at least 13%, at least 13.25%, at least 13.5%, at least 13.75%, at least 14%, at least 14.25%, at least 14.5%, at least 14.75%, at least 15%, at least 15.25%, at least 15.5%, at least 15.75%, at least 16%, at least 16.25%, at least 16.5%, at least 16.75%, at least 17%, at least 17.25%, at least 17.5%, at least 17.75%, at least 18%, at least 18.25%, at least 18.5%, at least 18.75%, at least 19%, at least 19.25%, at least 19.5%, at least 19.75%, at least 20%, at least 20.25%, at least 20.5%, at least 20.75%, at least 21 %, at least 21 .25%, at least 21 .5%, at least 21.75%, at least 22%, at least 22.25%, at least 22.5%,, at least 22.75%, at least 23%, at least 23.25%, at least 23.5%, at least 23.75%, at least 24%, at least 24.25%, at least 24.5%, at least 24.75%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 40%, or more (w/v) of the pharmaceutical composition.

7. The pharmaceutical composition according to any one of Claims 1 -6, wherein the concentration of the antibody preparation is at least 0.00001 mg/mL, at least 0.0001 mg/mL, at least 0.001 mg/mL, at least 0.01 mg/mL, at least 0.1 mg/mL, at least 1 mg/mL, at least 10 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least 100 mg/mL, at least 200 mg/mL, at least 500 mg/mL, at least 700 mg/mL, at least 1 ,000 mg/mL, or at least 1 ,200 mg/mL.

8. The pharmaceutical composition of any of Claims 1-7, wherein the concentration of the antibody preparation is at most 1 ,000 mg/mL, at most 1 ,100 mg/mL, at most 1 ,200 mg/mL, at most 1 ,300 mg/mL, at most 1 ,400 mg/mL, at most 1 ,500 mg/mL, at most 2,000 mg/mL, at most 2,000 mg/mL, or at most 3,000 mg/mL.

The pharmaceutical composition according to any one of Claims 1 -8, wherein the concentration of the antibody preparation is in a range of about 0.00001 mg/mL to about 3,000 mg/mL, about 0.0001 mg/mL to about 3,000 mg/mL, about 0.01 mg/mL to about 3,000 mg/mL, about 0.1 mg/mL to about 3,000 mg/mL, about 1 mg/mL to about 3,000 mg/mL, about 250 mg/mL to about 3,000 mg/mL, about 500 mg/mL to about 3,000 mg/mL, about 750 mg/mL to about 3,000 mg/mL, about 1 ,000 mg/mL to about 3,000 mg/mL, about 100 mg/mL to about 2,000 mg/mL, about 250 mg/mL to about 2,000 mg/mL, about 500 mg/mL to about 2,000 mg/mL, about 750 mg/mL to about 2,000 mg/mL, about 1 ,000 mg/mL to about 2,000 mg/mL, about 100 mg/mL to about 1 ,500 mg/mL, about 250 mg/mL to about 1 ,500 mg/mL, about 500 mg/mL to about 1 ,500 mg/mL, about 750 mg/mL to about 1 ,500 mg/mL, about 1 ,000 mg/mL to about 1 ,500 mg/mL, about 100 mg/mL to about 1 ,200 mg/mL, about 250 mg/mL to about 1 ,200 mg/mL, about 500 mg/mL to about 1 ,200 mg/mL, about 750 mg/mL to about 1 ,200 mg/mL, about 1 ,000 mg/mL to about 1 ,200 mg/mL, about 100 mg/mL to about 1 ,000 mg/mL, about 250 mg/mL to about 1 ,000 mg/mL, about 500 mg/mL to about 1 ,000 mg/mL, about 750 mg/mL to about 1 ,000 mg/mL, about 100 mg/mL to about 750 mg/mL, about 250 mg/mL to about 750 mg/mL, about 500 mg/mL to about 750 mg/mL, about 100 mg/mL to about 500 mg/mL, about 250 mg/mL to about 500 mg/mL, about 0.00001 mg/mL to about 0.0001 mg/mL, about 0.00001 mg/mL to about 0.001 mg/mL, about 0.00001 mg/mL to about 0.01 mg/mL, about 0.00001 mg/mL to about 0.1 mg/mL, about 0.00001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 0.01 mg/mL, about 0.001 mg/mL to about 0.1 mg/mL, about 0.001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 10 mg/mL, or about 0.001 mg/mL to about 100 mg/mL.

0. The pharmaceutical composition according to any one of Claims 1 -10, wherein the concentration of the antibody preparation is from about 50 mg/mL to about 100 mg/mL, about 50 mg/mL to about 200 mg/mL, about 50 mg/mL to about 300 mg/mL, about 50 mg/mL to about 400 mg/mL, about 50 mg/mL to about 500 mg/mL, about 50 mg/mL to about 600 mg/mL, about 50 mg/mL to about 700 mg/mL, about 50 mg/mL to about 800 mg/mL, about 50 mg/mL to about 900 mg/mL, about 50 mg/mL to about 1 ,000 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about 300 mg/mL, about 100 mg/mL to about 400 mg/mL, about 100 mg/mL to about 500 mg/mL, about 100 mg/mL to about 600 mg/mL, about 100 mg/mL to about 700 mg/mL, about 100 mg/mL to about 800 mg/mL, about 100 mg/mL to about 900 mg/mL, about 100 mg/mL to about 1 ,000 mg/mL, about 200 mg/mL to about 300 mg/mL, about 200 mg/mL to about 400 mg/mL, about 200 mg/mL to about 500 mg/mL, about 200 mg/mL to about 600 mg/mL, about 200 mg/mL to about 700 mg/mL, about 200 mg/mL to about 800 mg/mL, about 200 mg/mL to about 900 mg/mL, about 200 mg/mL to about 1 ,000 mg/mL, about 300 mg/mL to about 400 mg/mL, about 300 mg/mL to about 500 mg/mL, about 300 mg/mL to about 600 mg/mL, about 300 mg/mL to about 700 mg/mL, about 300 mg/mL to about 800 mg/mL, about 300 mg/mL to about 900 mg/mL, about 300 mg/mL to about 1 ,000 mg/mL, about 400 mg/mL to about 500 mg/mL, about 400 mg/mL to about 600 mg/mL, about 400 mg/mL to about 700 mg/mL, about 400 mg/mL to about 800 mg/mL, about 400 mg/mL to about 900 mg/mL, about 400 mg/mL to about 1 ,000 mg/mL, about 500 mg/mL to about 600 mg/mL, about 500 mg/mL to about 700 mg/mL, about 500 mg/mL to about 800 mg/mL, about 500 mg/mL to about 900 mg/mL, about 500 mg/mL to about 1 ,000 mg/mL, about 600 mg/mL to about 700 mg/mL, about 600 mg/mL to about 800 mg/mL, about 600 mg/mL to about 900 mg/mL, or about 600 mg/mL to about 1 ,000 mg/mL.

1 1 . The pharmaceutical composition according to any one of Claims 1-10, wherein the pharmaceutical composition further comprises one or more pharmacologically acceptable carriers.

12. The pharmaceutical composition according to any one of embodiments 1 -1 1 , wherein the pharmaceutical composition is in a liquid, a lyophilized form, a time-release delivery system, delayed release delivery system, or sustained release delivery system.

13. The pharmaceutical composition according to any one of Claims 1-12, wherein the pharmaceutical composition further comprises one or more additional drugs.

14. The pharmaceutical composition according to Claim 13, wherein the one or more additional drugs is an antimicrobial, antibiotic, adjuvant, an immunostimulatory, antiviral drug, or any combination thereof.

15. The pharmaceutical composition according to Claim 13 or 14, wherein the one or more additional drugs comprises at least 0.01 %, at least 0.02%, at least 0.05%, at least 0.075%, at least 0.1 %, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1 %, at least 1.5%, at least 1 .75%, at least 2%, at least 2.25%, at least 2.5%, at least 2.75%, at least 3%, at least 3.25%, at least 3.5%, at least 3.75%, at least 4%, at least 4.25%, at least 4.5%, at least 4.75%, at least 5%, at least 5.25%, at least 5.5%, at least 5.75%, at least 6%,6.25%, at least 6.5%, at least 6.75%, at least 7%, at least 7.25%, at least 7.5%, at least 7.75%, at least 8%, at least 8.25%, at least 8.5%, at least 8.75%, at least 9%, at least 9.25%, at least 9.5%, at least 9.75%, at least 10%, at least 10.25%, at least 10.5%, at least 10.75%, at least 1 1 %, at least 1 1.25%, at least 1 1 .5%, at least 1 1 .75%, at least 12%, at least 12.25%, at least 12.5%, at least 12.75%, at least 13%, at least 13.25%, at least 13.5%, at least 13.75%, at least 14%, at least 14.25%, at least 14.5%, at least 14.75%, at least 15%, at least 15.25%, at least 15.5%, at least 15.75%, at least 16%, at least 16.25%, at least 16.5%, at least 16.75%, at least 17%, at least 17.25%, at least 17.5%, at least 17.75%, at least 18%, at least 18.25%, at least 18.5%, at least 18.75%, at least 19%, at least 19.25%, at least 19.5%, at least 19.75%, at least 20%, at least 20.25%, at least 20.5%, at least 20.75%, at least 21 %, at least 21 .25%, at least 21 .5%, at least 21.75%, at least 22%, at least 22.25%, at least 22.5%,, at least 22.75%, at least 23%, at least 23.25%, at least 23.5%, at least 23.75%, at least 24%, at least 24.25%, at least 24.5%, at least 24.75%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 40%, or more (w/v) of the pharmaceutical composition.

16. The pharmaceutical composition according to any one of Claims 13-15, wherein the concentration of the one or more additional drugs is at least 0.00001 mg/mL, at least 0.0001 mg/mL, at least 0.001 mg/mL, at least 0.01 mg/mL, at least 0.1 mg/mL, at least 1 mg/mL, at least 10 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least 100 mg/mL, at least 200 mg/mL, at least 500 mg/mL, at least 700 mg/mL, at least 1 ,000 mg/mL, or at least 1 ,200 mg/mL.

17. The pharmaceutical composition of any of Claims 13-16, wherein the concentration of the one or more additional drugs is at most 1 ,000 mg/mL, at most 1 ,100 mg/mL, at most 1 ,200 mg/mL, at most 1 ,300 mg/mL, at most 1 ,400 mg/mL, at most 1 ,500 mg/mL, at most 2,000 mg/mL, at most 2,000 mg/mL, or at most 3,000 mg/mL.

18. The pharmaceutical composition according to any one of Claims 13-17, wherein the concentration of the one or more additional drugs is in a range of about 0.00001 mg/mL to about 3,000 mg/mL, about 0.0001 mg/mL to about 3,000 mg/mL, about 0.01 mg/mL to about 3,000 mg/mL, about 0.1 mg/mL to about 3,000 mg/mL, about 1 mg/mL to about 3,000 mg/mL, about 250 mg/mL to about 3,000 mg/mL, about 500 mg/mL to about 3,000 mg/mL, about 750 mg/mL to about 3,000 mg/mL, about 1 ,000 mg/mL to about 3,000 mg/mL, about 100 mg/mL to about 2,000 mg/mL, about 250 mg/mL to about 2,000 mg/mL, about 500 mg/mL to about 2,000 mg/mL, about 750 mg/mL to about 2,000 mg/mL, about 1 ,000 mg/mL to about 2,000 mg/mL, about 100 mg/mL to about 1 ,500 mg/mL, about 250 mg/mL to about 1 ,500 mg/mL, about 500 mg/mL to about 1 ,500 mg/mL, about 750 mg/mL to about 1 ,500 mg/mL, about 1 ,000 mg/mL to about 1 ,500 mg/mL, about 100 mg/mL to about 1 ,200 mg/mL, about 250 mg/mL to about 1 ,200 mg/mL, about 500 mg/mL to about 1 ,200 mg/mL, about 750 mg/mL to about 1 ,200 mg/mL, about 1 ,000 mg/mL to about 1 ,200 mg/mL, about 100 mg/mL to about 1 ,000 mg/mL, about 250 mg/mL to about 1 ,000 mg/mL, about 500 mg/mL to about 1 ,000 mg/mL, about 750 mg/mL to about 1 ,000 mg/mL, about 100 mg/mL to about 750 mg/mL, about 250 mg/mL to about 750 mg/mL, about 500 mg/mL to about 750 mg/mL, about 100 mg/mL to about 500 mg/mL, about 250 mg/mL to about 500 mg/mL, about 0.00001 mg/mL to about 0.0001 mg/mL, about 0.00001 mg/mL to about 0.001 mg/mL, about 0.00001 mg/mL to about 0.01 mg/mL, about 0.00001 mg/mL to about 0.1 mg/mL, about 0.00001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 0.01 mg/mL, about 0.001 mg/mL to about 0.1 mg/mL, about 0.001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 10 mg/mL, or about 0.001 mg/mL to about 100 mg/mL.

19. The pharmaceutical composition according to any one of Claims 13-18, wherein the concentration of the one or more additional drugs is in a range of about 50 mg/mL to about 100 mg/mL, about 50 mg/mL to about 200 mg/mL, about 50 mg/mL to about 300 mg/mL, about 50 mg/mL to about 400 mg/mL, about 50 mg/mL to about 500 mg/mL, about 50 mg/mL to about 600 mg/mL, about 50 mg/mL to about 700 mg/mL, about 50 mg/mL to about 800 mg/mL, about 50 mg/mL to about 900 mg/mL, about 50 mg/mL to about 1 ,000 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about 300 mg/mL, about 100 mg/mL to about 400 mg/mL, about 100 mg/mL to about 500 mg/mL, about 100 mg/mL to about 600 mg/mL, about 100 mg/mL to about 700 mg/mL, about 100 mg/mL to about 800 mg/mL, about 100 mg/mL to about 900 mg/mL, about 100 mg/mL to about 1 ,000 mg/mL, about 200 mg/mL to about 300 mg/mL, about 200 mg/mL to about 400 mg/mL, about 200 mg/mL to about 500 mg/mL, about 200 mg/mL to about 600 mg/mL, about 200 mg/mL to about 700 mg/mL, about 200 mg/mL to about 800 mg/mL, about 200 mg/mL to about 900 mg/mL, about 200 mg/mL to about 1 ,000 mg/mL, about 300 mg/mL to about 400 mg/mL, about 300 mg/mL to about 500 mg/mL, about 300 mg/mL to about 600 mg/mL, about 300 mg/mL to about 700 mg/mL, about 300 mg/mL to about 800 mg/mL, about 300 mg/mL to about 900 mg/mL, about 300 mg/mL to about 1 ,000 mg/mL, about 400 mg/mL to about 500 mg/mL, about 400 mg/mL to about 600 mg/mL, about 400 mg/mL to about 700 mg/mL, about 400 mg/mL to about 800 mg/mL, about 400 mg/mL to about 900 mg/mL, about 400 mg/mL to about 1 ,000 mg/mL, about 500 mg/mL to about 600 mg/mL, about 500 mg/mL to about 700 mg/mL, about 500 mg/mL to about 800 mg/mL, about 500 mg/mL to about 900 mg/mL, about 500 mg/mL to about 1 ,000 mg/mL, about 600 mg/mL to about 700 mg/mL, about 600 mg/mL to about 800 mg/mL, about 600 mg/mL to about 900 mg/mL, or about 600 mg/mL to about 1 ,000 mg/mL.

20. A kit comprising a pharmaceutical composition as defined in any one of Claims 1 -19.

21 . The kit according to Claim 21 , further comprising one or more additional components.

22. The kit according to Claim 20 or 21 , further comprising instructions for use to administer the pharmaceutical composition in the treatment of a Human Enterovirus capable of causing hand, foot and mouth disease in a subject.

23. The kit according to any one of Claims 20-22, further comprising one or more additional drugs.

24. The kit according to Claim 23, wherein the one or more additional drugs is an antimicrobial, antibacterial, immunostimulatory and/or antiviral.

25. Use of a pharmaceutical composition as defined in any one of Claims 1-19 in the manufacture of a medicament for the treatment of hand, foot and mouth disease.

26. The use according to Claim 25, wherein the medicament is a liquid, a lyophilized form, a time-release delivery system, delayed release delivery system, or sustained release delivery system.

27. Use of a pharmaceutical composition as defined in any one of Claims 1 -19 of a kit as defined in Claims 20-24 to reduce a symptom associated with hand, foot and mouth disease.

28. A method of treating hand, foot and mouth disease in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a pharmaceutical composition as defined in any one of Claims 1 -19, wherein administration of the pharmaceutical composition reduces a symptom associated with hand, foot and mouth disease, thereby treating the subject.

29. The use according to Claim 27 or the method according to Claim 28, wherein the pharmaceutical composition is utilized as a prophylactic application.

30. The use according to Claim 27 or 29 or the method according to Claim 28 or 29, wherein the pharmaceutical composition is co-administered with one or more additional drugs.

31 . The use or method according to Claim 30, wherein the drug is an antimicrobial, antibiotic, adjuvant, an immunostimulatory, antiviral drug, or any combination thereof.

32. The use according to any one of Claims 27, 29 or 30 or the method according to any one of Claims 28-30, wherein the pharmaceutical composition is administered to the subject orally, intravaginally, intraparentally, intra-anally, into the spinal fluid, into the cranial fluid, subcutaneously, intramuscularly and/or intravenously.

33. The use according to any one of Claims 27 or 29-31 or the method according to any one of Claims 28-31 , wherein the symptom reduced is a severity of a Human Enterovirus infection causing the hand, foot and mouth disease or a progression of a Human Enterovirus infection causing the hand, foot and mouth disease.

34. The use or method according to Claim 32, wherein the severity and/or the progression of a Human Enterovirus infection is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a subject not receiving the same treatment.

35. The use or method according to Claim 32, wherein the severity and/or the progression of a Human Enterovirus infection is reduced by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% as compared to a subject not receiving the same treatment.

36. The use according to any one of Claims 33-35 or the method according to any one of Claims 33-35, wherein the severity and/or the progression of a Human Enterovirus infection is reduced by a half-life of at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 1 1 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least one month, at least two months, at least three months, at least four months or more.

37. The use according to any one of Claims 27 or 29-36 or the method according to any one of Claims 28-36, wherein the symptom reduced is a neurological condition and/or cardiopulmonary condition resulting from hand, foot and mouth disease.

38. The use or method according to Claim 37, wherein the neurological condition is pulmonary adema, meningitis, meningoencephalomyelitis, poliomyelitis-like paralytic disease, Guillain-Barre syndrome, transverse myelitis, cerebellar ataxia, opsoclonus-myoclonus syndrome, benign intracranial hypertension, and/or brainstem encephalitis.

39. The use or method according to Claim 37, wherein the cardiopulmonary condition is myocarditis.

40. The use according to any one of Claims 27 or 29-39 or the method according to any one of Claims 28-39, wherein a therapeutically effective amount of the antibody preparation is at least 0.001 mg/kg/day, at least 0.01 mg/kg/day, at least 0.1 mg/kg/day, at least 1 .0 mg/kg/day, at least 5.0 mg/kg/day, at least 10 mg/kg/day, at least 15 mg/kg/day, at least 20 mg/kg/day, at least 25 mg/kg/day, at least 30 mg/kg/day, at least 35 mg/kg/day, at least 40 mg/kg/day, at least 45 mg/kg/day, or at least 50 mg/kg/day.

41. The use according to any one of Claims 27 or 29-39 or the method according to any one of Claims 28-39, wherein a therapeutically effective amount of the antibody preparation is in the range of about 0.001 mg/kg/day to about 10 mg/kg/day, about 0.001 mg/kg/day to about 15 mg/kg/day, about 0.001 mg/kg/day to about 20 mg/kg/day, about 0.001 mg/kg/day to about 25 mg/kg/day, about 0.001 mg/kg/day to about 30 mg/kg/day, about 0.001 mg/kg/day to about 35 mg/kg/day, about 0.001 mg/kg/day to about 40 mg/kg/day, about 0.001 mg/kg/day to about 45 mg/kg/day, about 0.001 mg/kg/day to about 50 mg/kg/day, about 0.001 mg/kg/day to about 75 mg/kg/day, or about 0.001 mg/kg/day to about 100 mg/kg/day.

42. The use according to any one of Claims 27 or 29-39 or the method according to any one of Claims 28-39, wherein a therapeutically effective amount of the antibody preparation is in the range of about 0.01 mg/kg/day to about 10 mg/kg/day, about 0.01 mg/kg/day to about 15 mg/kg/day, about 0.01 mg/kg/day to about 20 mg/kg/day, about 0.01 mg/kg/day to about 25 mg/kg/day, about 0.01 mg/kg/day to about 30 mg/kg/day, about 0.01 mg/kg/day to about 35 mg/kg/day, about 0.01 mg/kg/day to about 40 mg/kg/day, about 0.01 mg/kg/day to about 45 mg/kg/day, about 0.01 mg/kg/day to about 50 mg/kg/day, about 0.01 mg/kg/day to about 75 mg/kg/day, or about 0.01 mg/kg/day to about 100 mg/kg/day.

43. The use according to any one of Claims 27 or 29-39 or the method according to any one of Claims 28-39, wherein a therapeutically effective amount of the antibody preparation is in the range of about 0.1 mg/kg/day to about 10 mg/kg/day, about 0.1 mg/kg/day to about 15 mg/kg/day, about 0.1 mg/kg/day to about 20 mg/kg/day, about 0.1 mg/kg/day to about 25 mg/kg/day, about 0.1 mg/kg/day to about 30 mg/kg/day, about 0.1 mg/kg/day to about 35 mg/kg/day, about 0.1 mg/kg/day to about 40 mg/kg/day, about 0.1 mg/kg/day to about 45 mg/kg/day, about 0.1 mg/kg/day to about 50 mg/kg/day, about 0.1 mg/kg/day to about 75 mg/kg/day, or about 0.1 mg/kg/day to about 100 mg/kg/day.

44. A method of making a pharmaceutical composition comprising a polyclonal antibody preparation or the prevention and/or treatment of an infection by a Human Enterovirus responsible for foot, hand and mouth disease, the method comprising the step of collecting a polyclonal antibody preparation from one or more subjects immunized with a vaccine for a Human Enterovirus responsible for foot, hand and mouth disease.

45. The method of making according to Claim 45, wherein the polyclonal antibody preparation is prepared from plasma isolated from one or more subjects immunized against a causative agent of hand, foot and mouth disease.

46. The method of making according to Claim 44 or 45, wherein the one or more subjects is immunized once, twice, three, four, five, six, seven, eight, nine or more times prior to the collection of the polyclonal antibody preparation from the subject.

47. The method of making according to any one of Claims 44-46, wherein the one or more subjects is administered a polyclonal antibody preparation one, two, three, four, five, six, seven, eight, nine, ten or more times prior to infection with a Human Enterovirus responsible for hand, foot and mouth disease.

48. The method of making according to any one of Claims 44-47, wherein the Human Enterovirus is EV71 , CA5, CA6, CA10 and/or CA16.

49. The pharmaceutical composition according to any one of Claims 44-48, wherein the antibody preparation comprises one or more immunologically active antigens comprising one or more of a virus like particle, a viral protein, a viral protein, virus capsomers, aggregates, complexes of antigens from viruses and/or a viral nucleic acid from a Human Enterovirus.

50. The pharmaceutical composition according to any one of Claims 44-49, wherein the antibody preparation comprises one or more immunologically active antigens comprising one or more Human Enterovirus VP0 polypeptide, VP1 polypeptide, VP2 polypeptide, VP3 polypeptide, VP4 polypeptide, an immunologically active fragment thereof, or any combination thereof.