WO2015037992A1

WO2015037992A1 - Parechovirus specific antibodies

Info

Publication number: WO2015037992A1
Application number: PCT/NL2014/050624
Authority: WO
Inventors: Adrianus Quirinus Bakker; Tim Beaumont; Brenda Mirande WESTERHUIS; Klazine Christine WOLTHERS
Original assignee: Aimm Therapeutics B.V.
Priority date: 2013-09-11
Filing date: 2014-09-11
Publication date: 2015-03-19

Abstract

The invention relates to isolated, synthetic or recombinant antibodies and functional parts thereof specific for Parechovirus, and to nucleic acid sequences encoding such antibodies. The invention further relates to the use of such antibodies for diagnosis of a Parechovirus infection and as a medicament and/or prophylactic agent for at least in part treating or alleviating symptoms of a Parechovirus infection.

Description

Title: Parechovirus specific antibodies

The invention relates to the fields of biology, immunology and medicine. In particular, the invention relates to parechovirus specific antibodies.

The genus Parechovirus, belonging to the family of Picornaviridae, currently includes 16 human (sero)types, human parechovirus (HPeV) 1- 16 and the rodent Ljungan virus (LV). HPeVl and HPeV2 (previously known as echovirus 22 and 23 respectively) have been designated to the Parechovirus genus in view of their unique molecular and biological properties.

Like other Picornaviruses, Parechoviruses are small non-enveloped particles containing a monocistronic RNA genome with positive polarity which is packed in a protein capsid consisting of 60 copies of each of the structural VP proteins.

Most Picornaviruses share essentially the same genome organisation. The structural capsid proteins (1A, IB, 1C andlD, commonly known as VP4, VP2, VP3 and VP lrespectively) are encoded towards the N-terminal end of the polyprotein and the non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D) are encoded downstream thereof. Human parechoviruses together with kobuviruses have a different

organisation of their structural proteins compared to other Picornaviruses. The structural protein VP0, which is normally a precursor for VP4 and VP2, is not cleaved, resulting in only 3 structural proteins (VP0, VP3 and VP1). This VP0 maturation of human Parechovirus is thought to be critical for capsid stability and infectivity. The HPeV capsid is composed of 60 copies of each of the capsid proteins. Figure 6 shows the organization of the capsid of HPeV and human Enterovirus, as well as the genomic organization of the structural and non-structural proteins. The function of the different capsid proteins is not extensively studied. It has been shown that the VP0 protein contains an SLS that may function as a cis- acting replication element (CRE). The CRE has been shown to be critical for RNA replication in several other Picornaviruses and to consist of an RNA stem-loop, generally, but not

exclusively, located in the non-structural region. Secondly, sequence analyses of

HPeVl and HPeV2 showed the highly conserved arginine-glycine-aspartic acid (RGD) motif near the C-terminus of VP1 (amino acids 222-224 of HPeVl VP1 protein). This motif has been found to be functional in binding to host cell integrins, and virus neutralization in other Picornaviruses, like for instance Coxsackievirus A9 (CAV9), echovirus 9 and FMDV (foot-and-mouth disease virus). For HPeVl this motif has been shown to be essential receptor binding and entry. HPeV3, and some newly

characterized genotypes are lacking the RGD motif.

Members of the genus Parechovirus are common pathogens with a worldwide distribution and studies have shown that Parechovirus 1 (HPeVl) infections occur early in life and that the number of seropositive children increases rapidly after one year of age. At the age of 5, almost all children have seroconverted suggesting that the incidence of HPeVl infection is extremely high. Determining incidence of HPeVl infection is difficult since HPeVl infection, in most cases, only causes asymptomatic or mild infections of the gastrointestinal or respiratory tract. When compared to, for instance, enterovirus infections, the involvement of the central nervous system is less frequent and severe disease is rare, although cases of encephalitis and paralysis have been reported for HPeVl infection. Myocarditis, necrotizing enterocolitis and hemolytic uremic syndrome have also been associated with HPeVl infection.

In contrast to HPeVl, HPeV3 can cause more serious illness. HPeV3 has been isolated first from stool specimens of a young Japanese child with transient paralysis. From Canada, three additional cases of neonatal sepsis associated with HPeV3 were reported in infants 7-27 days old. All children were hospitalized with high fever, erythematous rash, and tachypnea for a median of 5 days. HPeV3 has subsequently been shown to be specifically associated with sepsis and fever in young infants, in particular infants up to 3 months of age and infants with neonatal encephalitis with white matter injury. HPeV3 is the most common HPeV recovered from cerebrospinal fluid (CSF).

HPeV6 was originally isolated from the cerebrospinal fluid of a 1 year old child with Reye syndrome. Subsequently it has been recovered from stool and respiratory tract samples of children.

HPeVl, HPeV3 and HPeV6 infections are most frequently detected.

HPeVl infections are very common. By contrast, HPeV2 infections seem more rare, and are associated with the same clinical symptoms as HPeVl. HPeV3 has a biannual circulation pattern and is currently mainly recovered from patients in the summer of even years. Circulation patterns of HPeV4, 5 and 7- 14 have not yet been determined. HPeV infections can be severe and even life-threatening. Hence, reliable diagnosis methods and treatment is urgently needed. To date, therapy against human parechovims infection is not available. One case report is described about a twin with neonatal sepsis and hepatitis infected with HPeV3, whereby one child received IVIg (intravenous immunoglobulin) and subsequently recovered. However, high antibody titers in IVIg against the specific HPeV serotype are needed for protection. Recently, the development of polyclonal rabbit antibodies directed against His-tagged human Parechovirus VP1 protein has been reported (Yu et at. 2012). However, monoclonal human antibodies have not yet been described.

Serotyping for diagnostic purposes has long been performed with specific antisera directed to the different HPeV subtypes. Currently, genotyping is used as an alternative method for diagnosis of Parechovirus infection. However, United States Food and Drug Administration (FDA) - approved HPeV PCR kits are not yet available.

Based on the above, it is apparent that there is a need for human antibodies specific for Parechovirus, useful for diagnosis, prevention and treatment of Parechovirus infection.

It is an object of the present invention to provide antibodies specific for human Parechovirus, and functional parts or equivalents of such antibodies. The present invention for the first time discloses fully human monoclonal antibodies that are specific for human Parechovirus. Antibodies directed against multiple

Parechovirus subtypes, as well as subtype specific antibodies are provided. Further, antibodies are provided that are capable of neutralizing Parechovirus. The present invention further provides isolated nucleic acids encoding such antibodies, host cells transformed with nucleic acids, and pharmaceutical compositions comprising human Parechovirus specific antibodies or isolated nucleic acids encoding such antibodies. Accordingly, the invention provides an isolated, synthetic and/or recombinant antibody or functional part or equivalent thereof specific for human Parechovirus (HPeV). Isolated, synthetic and/or recombinant antibodies or functional parts or equivalents thereof thereof according to the present invention are herein also referred to as "antibodies according to the invention".

The term "antibody" as used herein, refers to an antigen binding protein comprising at least a heavy chain variable region (Vh) that binds to a target epitope.

A "functional part of an antibody" is defined herein as a part that has at least one shared property with said antibody in kind, not necessarily in amount. Said functional part is capable of binding the same antigen as said antibody, albeit not necessarily to the same extent. Functional parts of an antibody that retain binding capacity include a Fab fragment or a F(ab')2 fragment, a single domain antibody, a single chain antibody, a nanobody, an unibody, a single chain variable fragment (scFv). A functional part of an antibody is also produced by altering an antibody such that at least an antigen-binding property of the resulting compound is essentially the same in kind, not necessarily in amount. This is done in many ways, for instance through conservative amino acid substitution, whereby an amino acid residue is substituted by another residue with generally similar properties (size, hydrophobicity, etc), such that the overall functioning of the antibody is essentially not affected.

A "functional equivalent of an antibody" is defined herein as an artificial binding compound, comprising at least one CDR sequence of an antibody.

As used herein "specific for" and "capable of specifically binding" are used herein interchangeably and refer to the interaction between an antibody and its epitope, indicating that said antibody preferentially binds to said epitope over other antigens or amino acid sequences. Thus, although the antibody may non-specifically bind to other antigens or amino acid sequences, the binding affinity of said antibody for its epitope is significantly higher than the non-specific binding affinity of said antibody for any other antigen or amino acid sequence. "Binding affinity" refers to the strength of the total sum of the noncovalent interactions between a single binding site of an antibody and its epitope. An antibody that interacts with a particular epitope of HPeV can also be specific for a virus other than HPeV if said epitope of HPeV is present in said other virus. In that case an antibody referred to herein as being specific for HPeV is also specific for said other virus comprising the same epitope. For instance, antibody AM 18 described herein interacts with an epitope comprising an RGD (Arg-Gly-Asp) domain in viral protein 1 (VP1) of HPeV. Because the same epitope is present in several other viruses, AM 18 is specific for HPeV and for said other viruses comprising the epitope of HPeV comprising an RGD domain. Examples of other viruses comprising an RGD domain are Coxsackievirus, in particular

Coxsackievirus A9, other Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma- associated herpesvirus (KSHV) and FMDV (foot-and-mouth disease virus). Hence, an antibody or functional part or equivalent according to the invention which specifically binds to an epitope of VP1 comprising an amino acid sequence RGD (Arg-Gly-Asp), may also specifically bind other viruses comprising the same epitope or amino acid sequence.

The term "specifically binding" as used herein refers to the process of a non-covalent interaction between an antibody according to the invention and an epitope, for instance an epitope of a viral protein of HPeV.

A "Parechovirus subtype" as used herein refers to genetically different Parechoviruses. examples of Parechovirus subtypes are HPeVl, HPeV2, HPeV3, HPeV4, HPeV5, HPeV6, HPeV7, HPeV8, HPeV9, HPeVIO, HPeVll, HPeV12, HPeV13, HPeV14, HPeV15 and HPeV16.

An "HPeV strain" as used herein refers to different HPeV of the same subtype, for example HPeVl strains Harris, 20550163, 251949 and 452252 and HPeV3 strains 150237, A308-99 and 1930.

"Neutralizing activity" as used herein is defined as the inhibition or reduction of a Parechovirus' capacity of infecting a host cell. Neutralizing activity of an antibody can be measured by any method known in the art, for instance by measuring the ability of the antibody to lower the titer of infectious virus in vitro in cultured cells. One of such methods is detailed in the Examples of this application and involves the inhibition of Parechovirus infection of cultured cells by monoclonal antibodies. In this method, Parechovirus is mixed with B-cell supernatants or antibody and after 1 hour of incubation added to, for instance, HT29 cells (HPeVl) or Vero cells (HPeV3). After infection, for instance at day 7 post infection, the cytophatic effect on the cells can be measured for instance directly by e.g. detection of cell rounding or by the detection of the number of virus copies. Potent antibodies will prevent or reduce Parechovirus infection and number of virus copies in the target cell. Neutralizing activity can be quantified by measurement of the IC50. "IC50" is a term well known in the art and refers to the concentration of Parechovirus neutralizing antibody necessary to inhibit or reduce Parechovirus infectivity of host cells by half. The lower the value of IC50 of an antibody, the stronger the neutralizing activity of the antibody, and the greater its potential as a therapeutic agent.

The percentage of identity of an amino acid or nucleic acid sequence, or the term "% sequence identity", is defined herein as the percentage of residues in a candidate amino acid or nucleic acid sequence that is identical with the residues in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art, for example "Align 2".

Antibodies according to the invention are specific for Parechovirus, preferably for Parechovirus viral proteins. Parechovirus is a viral genus consisting of two species, human Parechovirus (HPeV) and Ljungan virus. Preferred antibodies of the invention are specific for human Parechovirus (HPeV) because infection with HPeV can be severe and even life-threatening in humans, in particular in young children. Antibodies provided by the invention are capable of specifically binding at least one Parechovirus subtype, preferably a viral protein of at least one HPeV subtype. Currently, 16 different HPeV subtypes have been identified, designated HPeVl to HPeV16. Preferred antibodies of the invention have broad subtype specificity, meaning that the antibody has cross-binding activity, i.e. is capable of binding more than one Parechovirus, preferably HPeV, subtype. Such antibody can be used to bind and/or neutralize more than one HPeV subtype. Preferably, said antibody is specific for at least two HPeV subtypes, more preferably for at least three HPeV subtypes, more preferably for at least four specific subtypes, or for at least five specific subtypes. Preferably an antibody according to the invention is specific for one or more HPeV selected from the group consisting of HPeVl, HPeV2, HPeV4, HPeV5 and HPeV6, preferably for at least two HPeV selected from said group. A particularly preferred antibody of the invention is specific for at least two HPeV subtypes selected from the group consisting of HPeVl, HPeV2, HPeV3 and HPeV6, because these HPeV subtypes are most prevalent.

Further antibodies according to the invention are specific for one HPeV subtype, preferably for a viral protein thereof. Such antibodies are also referred to as subtype-specific antibodies. For instance, antibodies of the invention are specific for HPeVl, HPeV2, HPeV3, HPeV4, HPeV5 or HPeV6. A preferred subtype specific antibody of the invention is specific for HPeVl, HPeV3 or HPeV6 or for a viral protein of said HPeV subtypes because these subtypes are most frequently detected. HPeV3 infection has been associated with severe, life-threatening disease, including sepsis, meningitis and encephalitis, whereas infection with any of the other HPeV subtypes usually causes mild symptoms or is asymptomatic. It is thus particularly important that HPeV3 infections can be readily detected. Particularly preferred are thus HPeV3 subtype specific antibodies both for diagnosis of HPeV3 infection, and treatment of HPeV3 infection, either prophylactic or curative.

Because of differences in disease severity, it is of great importance to be able to discriminate between HPeV3, which is associated with severe life-threatening disease, and other HPeV subtypes, which are usually associated with mild disease. Now that an HPeV3 specific antibody has been provided that allows for specifically detecting the presence of HPeV3 in a sample it can be determined whether an individual, preferably a human, is suffering from HPeV3 infection. Therefore, an

HPeV3 specific antibody useful in diagnosis is preferable subtype-specific. Said HPev3 specific antibody is preferably not capable of specifically binding other HPeV subtypes such as HPeVl, HPeV2, HPeV4, HPeV5 and HPeV6.

An antibody according to the invention having broad subtype specificity is preferably specific for at least two HPeV subtypes other than HPeV3. For instance, such antibody is specific for at least two subtypes selected from the group consisting of HPeVl, HPeV2, HPeV4, HPeV5, HPeV6, HPeV7, HPeV8, HPeV9, HPeVIO, HPeVl 1, HPeV12, HPeV13, HPeV14, HPeV15 and HPeV16. Preferably, said antibody is specific for at least two subtypes selected from HPeVl, HPeV2 and HPeV6, but not for HPeV3. More preferably, said antibody is specific for at least HPeVl, HPeV2 and HPeV6, but not for HPeV3.

Particularly useful is a combination of a HPeV3 subtype specific antibody and an antibody having a broad subtype specificity. Using such combination allows for discriminating between the presence of or infection with HPeV3 on the one hand or the presence of or infection with another HPeV subtype on the other hand. Provided is therefor a kit of parts comprising a subtype specific antibody or functional part or equivalent thereof according to the invention specific for HPeV3 and a broad subtype specific antibody or functional part or equivalent thereof according to the invention specific for at least two subtypes selected from the group consisting of HPeVl, HPeV2, HPeV4, HPeV5, HPeV6, HPeV7, HPeV8, HPeV9, HPeVIO, HPeVl 1, HPeV12,

HPeV13, HPeV14, HPeV15 and HPeV16, preferably at least three subtypes selected from said group. Preferably said broad subtype specific antibody is specific for at least two subtypes of HPeVl, HPeV2 and HPeV6, more preferably for HPeVl, HPeV2 and HPeV6.

Antibodies of the invention are specific for human Parechovirus, preferably a HPeV viral protein. The term "viral protein" as used herein refers to any protein of a Parechovirus, for instance the viral capsid, structural proteins VPO, VP1 and VP3, and non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C and 3D. A preferred antibody according to the invention is specific for the HPeV viral capsid. Preferably said antibody specific for the viral capsid recognizes and binds to one of the structural proteins that form the viral capsid, i.e. VPO, VP1 or VP3, or to intact viral capsid composed of proteins VPO, VP1 and VP3. A subtype specific antibody according to the invention, preferably specific for HPeVl, HPeV2, HPeV3, HPeV4, HPeV4 or HPeV6, is preferably specific for the VP1 protein, because the VP1 protein of HPeV is the most immunogenic protein. Preferred antibodies of the invention are specific for a conformational epitope of a HPeV viral protein or viral capsid. A "conformational epitope" or "conformation dependent epitope" is herein defined as an epitope which is formed by the amino acid sequence and the three-dimensional shape of an antigen (e.g. as a result of folding and/or interactions between individual amino acids). The amino acids making up the epitope can be relatively few in number and can be spread along the length of the molecule. Such epitope is brought into the correct conformation via folding of the antigen. Posttranslational modification by the cells in which a

Parechovirus replicates may contribute to formation of a conformational epitope. Non- limiting examples of such posttranslational modifications are phosphorylation, palmitoylation through a thioether linkage, acylation, sumoylation and cross-linking by tissue transglutaminase (tTG). In general, antibodies recognizing conformational epitopes have broader specificity for multiple HPeV strains and/or subtypes because conformational epitopes are more conserved. Such antibodies may therefore offer broader therapeutic application for ameliorating or preventing HPeV infection than antibodies able to bind only linear epitopes. Antibody AM28 is such antibody recognizing a conformational epitope. As is demonstrated in the Examples and Figures 4A, 4C, 5B and 12 antibody AM28 does not recognize isolated VP proteins and linear peptides, but binds to a conformational epitope. Hence, antibody AM28 having a sequence as depicted in table 1, and variant antibodies thereof are preferred antibodies according to the invention. Such variant antibodies have at least 70%, preferably at least 80%, more preferably at least 85%, sequence identity with the heavy and light chain CDR's of antibody AM28, more preferably with the heavy and light chain sequences of antibody AM28.

Other preferred antibodies according to the invention are specific for an epitope in the VP1 protein comprising an RGD motif. The term "RGD motif or "RGD domain" refers to the amino acid sequence Arg-Gly-Asp. This motif is located near the C-terminus of VP1, at amino acid positions of VP1 corresponding to amino acid positions 222-224 of the VP- 1 protein of HPeVl, which also corresponds to amino acid positions 764-766 of the HPeVl sequence depicted in Figure 7 (the VP-1 sequence starting at position 543 of the HPeVl sequence depicted in Figure 7). This epitope is present in VP1 of all HPeV subtypes except HPeV3. In HPeVl, the RGD motif is located at amino acid positions 222 to 224 of the VP- 1 protein. "Located at amino acid positions corresponding to amino acid positions 222 to 224 of the VP-1 protein of HPeVl" means that in HPeV subtypes other than HPeVl, the amino acid positions of the RGD motif may vary, but they correspond to amino acids 222 to 224 of HPeVl VP1, which also corresponds to amino acid positions 764-766 of the HPeVl sequence depicted in Figure 7. Hence, an antibody capable of interacting with this epitope is preferably an antibody having broad subtype specificity against at least two, more preferably at least three, HPeV subtypes other than HPeV3. Said epitope of VP1 preferably comprises also an amino acid sequence VTSSR, located at amino acid positions 215 to 219 of the VP1 protein of HPeVl, which also corresponds to amino acid positions 757-761 of the HPeVl sequence as depicted in Figure 7. As detailed in the Examples, residues VTSSR located N-terminal to the RGD likely increase the binding specificity of antibodies of the invention, preferably AM 18, to the protein, most probably by increasing the accessibility of the RGD motif. Said epitope of VP1 further preferably comprises an amino acid sequence VTSSRALRGDMA

(ValThrSerSerArgAlaLeuArgGlyAspMetAla) of VPl, located at amino acid positions of VP1 corresponding to amino acid positions 215 to 226 of the VP- 1 protein of HPeVl, which also corresponds to amino acid positions 757-768 of the HPeVl sequence as depicted in Figure 7. A preferred antibody or functional part or equivalent thereof according to the invention therefore specifically binds to an epitope of VPl comprising an amino acid sequence Arg-Gly-Asp, preferably wherein said epitope is located at amino acid positions of VPl corresponding to amino acid positions 222 to 224 of the VP-1 protein of HPeVl, and corresponding to amino acid positions 764-766 of the HPeVl sequence as depicted in Figure 7. Preferably, said antibody or functional part or equivalent specifically binds said epitope of VPl of at least one HPeV subtype selected from the group consisting of HPeVl, HPeV2, HPeV4, HPeV5, HPeV6, HPeV7, HPeV8, HPeV9, HPeVlO, HPeVl 1, HPeV12, HPeV13, HPeV14, HPeV15 and HPeV16, preferably of at least two HPeV subtypes selected from said group, more preferably of at least three HPeV subtypes selected from said group. More preferably, said at least one, preferably at least two, more preferably at least three HPeV subtypes are selected from the group consisting of HPeVl, HPeV2, HPeV4, HPeV5 and HPeV6. A preferred antibody or functional part or equivalent thereof according to the invention therefore specifically binds an epitope of VPl comprising an amino acid sequence VTSSRALRGDMA (ValThrSerSerArgAlaLeuArgGlyAspMetAla) of VPl, located at amino acid positions of VPl corresponding to amino acid positions 215 to 226 of the VP-1 protein of HPeVl, which also corresponds to amino acid positions 757-768 of the HPeVl sequence as depicted in Figure 7. A particularly preferred antibody or functional part or equivalent binds an epitope consisting of an amino acid sequence RGD, or of amino acids sequences RGD and VTSSR, or of an amino acid sequence VTSSRALRGDMA . As is demonstrated in the Examples and Figures 3 and 4, antibody AM 18 specifically recognizes the VPl protein. In particular AM 18 binds an epitope comprising the RGD motif. Hence, antibody AM18 having a sequence as depicted in table 1, and variant antibodies thereof are preferred antibodies according to the invention. Such variant antibodies have at least 70%, preferably at least 80%, more preferably at least 85%, sequence identity with the heavy and light chain CDR's of antibody AM 18, more preferably with the heavy and light chain sequences of antibody AM 18. An antibody or functional part or equivalent according to the invention which specifically binds to an epitope of VPl comprising an amino acid sequence RGD (Arg-Gly-Asp), also specifically binds to other viruses comprising the same epitope or amino acid sequence. Examples of such other viruses comprising an RGD domain are Coxsackievirus, in particular Coxsackievirus A9, other

Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma-associated herpesvirus (KSHV), and FMDV. Hence, such preferred antibody or functional part or equivalent according to the invention is further specific for a virus other than HPeV, which comprises a viral protein comprising an RGD motif. Such virus other than HPeV is preferably selected from the group consisting of Coxsackievirus, in particular Coxsackievirus A9, other

Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma-associated herpesvirus (KSHV) and FMDV. Also provided is an antibody or functional part or equivalent according to the invention that specifically binds to the same epitope as antibody AM 18. Further provided is an antibody or functional part or equivalent according to the invention that competes with antibody AM 18 for specific binding to HPeV VP1.

Other preferred antibodies according to the invention are specific for an epitope of HPeV comprising a groove formed by VPO and VP3. Said HPeV is preferably HPeVl and/or HPeV2. Said groove is preferably formed by HPeVl VPO and HPeVl VP3 and/or by HPeV2 VPO and HPeV2 VP3. Said groove is further preferably formed by VPO and VP3 from different pentamers. As described in the Examples, modelling of the HPeVl VPO and VP3 proteins indicates that amino acids in the following loops in HPeVl are involved this epitope: amino acid sequence

PLSIPTGSANQ of VPO, amino acid sequence FFPNATT of VP3 and amino acid sequence ATTAPQSIVH of VP3, more specifically amino acid sequences HEWTPSWA, HQDKP and PLSIPTGSANQ VD of VPO and amino acid sequences

MADSTTPSENHG, ATTAPQSIVH and FFPNATTDST of VP3 . A model of the groove formed by HPeV VPO and VP3 is shown in Figures 10 and 12B, D and E. This epitope is conserved over HPeVl strains. Amino acid sequence PLSIPTGSANQ of VPO corresponds to amino acid positions 250 to 260 of the HPeVl sequence as depicted in Figure 7. Amino acid sequence HEWTPSWA of VPO corresponds to amino acid positions 123 to 150 of the HPeVl sequence as depicted in Figure 7. Amino acid sequence HQDKP of VPO corresponds to amino acid positions 148 to 152 of the HPeVl sequence as depicted in Figure 7. Amino acid sequence PLSIPTGSANQ VD of VPO corresponds to amino acid positions 250 to 262 of the HPeVl sequence as depicted in Figure 7. Amino acid sequence FFPNATT of VP3 corresponds to amino acid positions 448 to 454 of the HPeVl sequence as depicted in Figure 7. Amino acid sequence ATTAPQSIVH of VP3 corresponds to amino acid positions 393 to 402 of the HPeVl sequence as depicted in Figure 7. Amino acid sequence MADSTTPSENHG of VP3 corresponds to amino acid positions 371 to 382 of the HPeVl sequence as depicted in Figure 7. Amino acid sequence FFPNATTDST of VP3 corresponds to amino acid positions 448 to 457 of the HPeVl sequence as depicted in Figure 7. Provided is therefore an antibody or functional part or equivalent according to the invention which specifically binds to an epitope of HPeV comprising a groove formed by VPO and VP3. Said HPeV is preferably HPeVl and/or HPeV2. Said groove is preferably formed by HPeVl VPO and HPeVl VP3 and/or by HPeV2 VPO and HPeV2 VP3. Said epitope comprises at least one amino acid from an amino acid sequence corresponding to amino acid sequence of PLSIPTGSANQ of HPeVl VPO, at least one amino acid from an amino acid sequence corresponding to the amino acid sequence FFPNATT of HPeVl VP3 and at least one amino acid from an amino acid sequence corresponding to the amino acid sequence ATTAPQSIVH of HPeVl VP3. Hence, said epitope comprises at least one amino acid from an amino acid sequence corresponding to amino acid positions 250 to 260 of the HPeVl sequence as depicted in Figure 7, at least one amino acid from an amino acid sequence corresponding to amino acid positions 448 to 454 of the HPeVl sequence as depicted in Figure 7 and at least one amino acid from an amino acid sequence corresponding to amino acid positions 393 to 402 of the HPeVl sequence as depicted in Figure 7. More preferably, said epitope comprises at least one amino acid from an amino acid sequence corresponding to amino acid sequence of HEWTPSWA of HPeVl VPO, at least one amino acid from an amino acid sequence corresponding to amino acid sequence of HQDKP of HPeVl VPO, at least one amino acid from an amino acid sequence corresponding to amino acid sequence of PLSIPTGSANQVD of HPeVl VPO, at least one amino acid from an amino acid sequence corresponding to the amino acid sequence MADSTTPSENHG of HPeVl VP3, at least one amino acid from an amino acid sequence corresponding to the amino acid sequence ATTAPQSIVH of HPeVl VP3 and at least one amino acid from an amino acid sequence corresponding to the amino acid sequence FFPNATTDST of HPeVl VP3. Hence, said epitope comprises at least one amino acid from an amino acid sequence corresponding to amino acid positions 123 to 150 of the HPeVl sequence as depicted in Figure 7, at least one amino acid from an amino acid sequence corresponding to amino acid positions 148 to 152 of the HPeVl sequence as depicted in Figure 7, at least one amino acid from an amino acid sequence corresponding to amino acid positions 250 to 262 of the HPeVl sequence as depicted in Figure 7, at least one amino acid from an amino acid sequence corresponding to amino acid positions 393 to 402 of the HPeVl sequence as depicted in Figure 7, at least one amino acid from an amino acid sequence corresponding to amino acid positions 371 to 382 of the HPeVl sequence as depicted in Figure 7 and at least one amino acid from an amino acid sequence corresponding to amino acid positions 448 to 457 of the HPeVl sequence as depicted in Figure 7. Located at amino acid positions

corresponding to amino acid positions 123 to 150, 148 to 152, 250 to 260, 250 to 262, 371 to 382, 448 to 454, 448 to 457 or 393 to 402 of the HPeVl sequence as depicted in Figure 7 means that in HPeV subtypes other than HPeVl or in different strains of the HPeV subtype, the amino acid positions of the epitope may vary, but they correspond to the indicated amino acids of HPeVl as depicted in Figure 7. More preferably said epitope comprises at least two amino acids from each of said amino acid sequences in VPO and VP3, more preferably at least three amino acids, more preferably at least four amino acids, more preferably at least five amino acids of each of said amino acid sequences in VPO and VP3. More preferably said epitope comprises the amino acid sequences PLSIPTGSANQ of VPO, FFPNATT of VP3 and ATTAPQSIVH of VP3. Even more preferably said epitope comprises the amino acid sequences HEWTPSWA, HQDKP and PLSIPTGSANQ VD of VPO and amino acid sequences

MADSTTPSENHG, ATTAPQSIVH and FFPNATTDST of VP3. Preferably, said antibody or functional part or equivalent specifically binds said epitope of HPeV of at least one HPeV subtype selected from the group consisting of HPeVl, HPeV2, HPeV4, HPeV5, HPeV6, HPeV7, HPeV8, HPeV9, HPeVIO, HPeVl 1, HPeV12, HPeV13,

HPeV14, HPeV15 and HPeV16, more preferably of at least HPeVl and/or HPeV2. Hence, said epitope preferably comprises at least one, more preferably at least two, more preferably at least three, more preferably at least four, more preferably at least five, more preferably all, amino acids of the amino acid sequences PLSIPTGSANQ of HPeVl VPO, FFPNATT of HPeVl VP 3 and ATTAPQSIVH of HPeVl VP3 or of the amino acid sequences PLSIPSGSSNQ of HPeV2 VPO, FFPNSST of HPeV2 VP3 and ANSDPQAIVH of HPeV2 VP3. A particularly preferred antibody according to the invention specifically binds to an epitope of HPeV consisting of a groove formed by VPO and VP3. As is demonstrated in the Examples, antibody AM28 specifically recognizes an epitope of HPeV comprising a groove formed by VPl and VP3. Antibody AM28 having a sequence as depicted in table 1, and variant antibodies thereof are preferred antibodies according to the invention. Such variant antibodies have at least 70%, preferably at least 80%, more preferably at least 85%, sequence identity with the heavy and light chain CDR's of antibody AM28, more preferably with the heavy and light chain sequences of antibody AM28. Particularly preferred antibodies or functional parts or equivalents thereof specific for an epitope of HPeV comprising a groove formed by VPl and VP3 comprises at least the heavy chain and light chain CDRs of antibody AM28. Also provided is an antibody or functional part or equivalent according to the invention that specifically binds to the same epitope as antibody AM28. Further provided is an antibody or functional part or equivalent according to the invention that competes with antibody AM28 for specific binding to HPeV, preferably HPeVl and/or HPeV2. Said antibody preferably specifically binds to an epitope of HPeV comprising a groove formed by VPO and VP3. More preferably said antibody specifically binds to an epitope comprising at least one amino acid from an amino acid sequence corresponding to amino acid positions 250 to 260 of the HPeVl sequence as depicted in Figure 7, at least one amino acid from an amino acid sequence corresponding to amino acid positions 448 to 454 of the HPeVl sequence as depicted in Figure 7 and at least one amino acid from an amino acid sequence corresponding to amino acid positions 393 to 402 of the HPeVl sequence as depicted in Figure 7, most preferably at least one amino acid from the amino acid sequence PLSIPTGSANQ of HPeVl VPO, at least one amino acid from the amino acid sequence FFPNATT of HPeVl VP3 and at least one amino acid from the amino acid sequence ATTAPQSIVH of HPeVl VP3.

An HPeV specific antibody according to the invention preferably exhibits neutralizing activity against Parechovirus, preferably against human PeV (HPeV), more preferably against multiple HPeVs. Such neutralizing antibody is useful in the prophylactic or therapeutic treatment of a Parechovirus infection. An antibody according to the invention may have Parechovirus neutralizing activity in the presence of complement, whereby a chain of events leading to complement-mediated cell lysis is initiated, or have complement-independent neutralizing activity, which is independent from another biological system. The presence of neutralizing activity independent of complement is for instance determined by testing neutralizing activity of an antibody both in the presence and absence of added complement. If the neutralizing activity is higher in the presence of complement, the antibody exhibits complement-dependent neutralizing activity. If the neutralizing activity if comparable in the presence and absence of complement, the neutralizing activity is complement independent.

Preferred HPeV antibodies according to the invention are AM18, AM28, AT12-015, AT12-017 and AT12-018, because these antibodies have been demonstrated to have particularly desired binding and/or neutralizing characteristics. AM18, AM28, AT12-015, AT12-017 and AT12-018 have heavy chain sequences of SEQ ID NO's: 31, 32, 33, 34 and 35 as depicted in table 1, respectively, and light chain sequences of SEQ ID NO's: 36, 37, 38, 39 and 40 as depicted in table 1, respectively. The heavy and light chain CDR sequences of these preferred antibodies are also depicted in table 1.

SEQ ID NO's: 1, 6 and 11 are the heavy chain CDR1, CDR2 and CDR3 sequences of AM18 respectively. SEQ ID NO's: 16, 21 and 26 are the light chain CDR1, CDR2 and CDR3 sequences of this antibody, respectively.

SEQ ID NO's: 2, 7 and 121 are the heavy chain CDR1, CDR2 and CDR3 sequences of AM28 respectively. SEQ ID NO's: 17, 22 and 27 are the light chain CDR1, CDR2 and CDR3 sequences of this antibody, respectively.

SEQ ID NO's: 3, 8 and 13 are the heavy chain CDR1, CDR2 and CDR3 sequences of AT12-015 respectively. SEQ ID NO's: 18, 23 and 28 are the light chain CDR1, CDR2 and CDR3 sequences of this antibody, respectively.

SEQ ID NO's: 4, 9 and 14 are the heavy chain CDR1, CDR2 and CDR3 sequences of AT12-017 respectively. SEQ ID NO's: 19, 24 and 29 are the light chain CDR1, CDR2 and CDR3 sequences of this antibody, respectively.

SEQ ID NO's: 5, 10 and 15 are the heavy chain CDR1, CDR2 and CDR3 sequences of AT12-018 respectively. SEQ ID NO's: 20, 25 and 30 are the light chain CDR1, CDR2 and CDR3 sequences of this antibody, respectively.

The terms "AM18", "AM28", "AT12-015", "AT12-017" and "AT12-018" as used herein encompass all antibodies and functional equivalents having the indicated heavy chain and light chain sequences, for instance isolated and/or purified antibodies or recombinantly produced antibodies.

The invention thus provides an antibody or functional part or equivalent thereof according to the invention comprising:

- a heavy chain CDRl sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's: l-5, and/or

- a heavy chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's:6-10, and/or

- a heavy chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's: l l-15, and/or

- a light chain CDRl sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's: 16-20, and/or

- a light chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's:21-25, and/or

- a light chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's:26-30. Preferably, said antibody or functional part or equivalent comprises heavy chain CDRl, CDR2 and/or CDR3 sequences and/or light chain CDRl, CDR2 and/or CDR3 sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical to these sequences.

Preferably, an antibody according to the invention comprises a heavy chain sequence and/or a light chain sequence, or a sequence which has at least 70% sequence identity thereto, as depicted in table 1. Also provided is therefore an antibody or functional part or equivalent thereof according to the invention, having a heavy chain sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's:31-35 and/or having a light chain sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's:36-40, or sequences that are at least at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical to any one of these heavy chain or light chain sequences. The higher the identity, the more closely an antibody resembles an antibody depicted in table 1.

Antibody AM18 is a preferred antibody because it is capable of specifically binding multiple HPeV subtypes. As shown in the Examples, AM18 is capable of binding at least HPeVl, HPeV2, HPeV4, HPeV5 and HPeV6. Further, AM18 is capable of binding multiple strains of a single subtype: for subtype HPeVl, AM18 has been demonstrated to bind both HPeVl-Harris and HPeVlB. Thus, AM18 is a broad subtype specific antibody. AM18 does not recognize or bind HPeV3. AM18 is thus particularly suitable to detect the presence of a HPeV other than HPeV3. It can suitable be used in combination with a HPeV3 subtype specific antibody such as AT12-015, AT12-017 or AT12-018 to discriminate between severe and life-threatening disease causing HPeV3 and other HPeV subtypes that generally cause mild disease. Antibody AM18 is further preferred because it is capable of neutralizing HPeVl, HPeV2, HPeV4, HPeV5 and HPeV6, with a particularly high neutralizing activity for HPeVl and HPeV2 as shown in the Examples. AM18 is thus particularly suitable for use in treatment or prevention of HPeVl, HPeV2, HPeV4, HPeV5 or HPeV6 infection or a disorder caused by such infection. AM 18 is further preferred because it specifically recognizes the VPl protein of HPeV, which is the immunogenic protein of HPeV. More in particular, AM18 specifically binds to an epitope in VPl comprising an RGD motif, which epitope in particular further comprises an amino acid sequence VTSSR. Such RGD motif is also present in several other viruses, such as

Coxsackievirus, in particular Coxsackievirus A9, other Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma- associated herpesvirus (KSHV) and FMDV. Therefore, AM 18 is also useful in the detection of such other viruses comprising an RGD motif, and/or in the treatment and/or prevention of infection by such other viruses, or a disorder caused by such infection. AM 18 is further preferred because it has a high affinity for HPeVl VP1 with a KD of 11.5 pM. A preferred antibody or functional part or equivalent thereof of the invention thus has heavy chain CDRl, CDR2 and CDR3 sequences and light chain CDRl, CDR2 and CDR3 sequences of antibody AM18, comprising the sequence of

SEQ ID NO: l, SEQ ID NO:6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO:21 and SEQ ID NO:26, or sequences that are at least 70% identical thereto, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%. Said antibody or functional part or equivalent thereof preferably has the entire heavy chain and light chain sequences of antibody AM 18, comprising the sequence of SEQ ID NO:31 and SEQ ID NO:36, or sequences that are at least 70% identical thereto, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%.

Another preferred antibody is AM28. AM28 is capable of specifically binding multiple HPeV subtypes. As shown in the Examples, AM28 is capable of binding at least HPeVl and HPeV2. Further, AM28 is capable of binding multiple strains of a single subtype: for subtype HPeVl, AM28 has been demonstrated to bind both HPeVl-Harris and HPeVlB. AM28 does not recognize or bind HPeV3, HPeV4, HPeV5 and HPeV6. AM28 is thus particularly suitable to detect the presence of a HPeVl and HPeV2. It can suitable be used in combination with a HPeV3 subtype specific antibody such as AT12-015, AT12-017 or AT12-018 to discriminate between severe and life -threatening disease causing HPeV3 and other HPeV subtypes that generally cause mild disease. Antibody AM28 is further preferred because it is capable of neutralizing HPeVl and HPeV2 with a high neutralizing activity as shown in the Examples. AM28 is thus particularly suitable for use in treatment or prevention of HPeVl or HPeV2 infection or a disorder caused by such infection. AM28 is further preferred because it specifically recognizes a conformational epitope in HPeV viral protein. Conformational epitopes are generally conserved, which indicates that AM28 offers broad therapeutic application for ameliorating or preventing HPeV infection. More in particular, AM28 specifically binds to an epitope of HPeV that comprises a groove formed by VP0 and VP3, involving at least part of the amino acid sequences PLSIPTGSANQ of VPO, FFPNATT of VP3 and ATTAPQSIVH of VP3, in particular involving at least part of the amino acid sequences HEWTPSWA, HQDKP and PLSIPTGSANQ VD of VPO and amino acid sequences MADSTTPSENHG, ATTAPQSIVH and FFPNATTDST of VP3. A preferred antibody or functional part or equivalent thereof of the invention thus has heavy chain CDRl, CDR2 and CDR3 sequences and light chain CDRl, CDR2 and CDR3 sequences of antibody AM28, comprising the sequence of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO:22 and SEQ ID NO:27, or sequences that are at least 70% identical thereto, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%. Said antibody or functional part or equivalent thereof preferably has the entire heavy chain and light chain sequences of antibody AM28, comprising the sequence of SEQ ID NO:32 and SEQ ID NO:37, or sequences that are at least 70% identical thereto, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%.

Another preferred antibody is AT 12-015 because it is a HPeV3 subtype specific antibody. AT12-015 does not recognize or bind HPeVl, HPeV2, HPeV4, HPeV5 and HPeV6. AT12-015 is thus particularly suitable to detect the presence of a HPeV3. It can suitable be used in combination with an antibody specific for HPeV subtypes other than HPeV3 such as AM18 or AM28 to discriminate between severe and life -threatening disease causing HPeV3 and other HPeV subtypes that generally cause mild disease. A preferred antibody or functional part or equivalent thereof of the invention thus has heavy chain CDRl, CDR2 and CDR3 sequences and light chain CDRl, CDR2 and CDR3 sequences of antibody AT12-015, comprising the sequence of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO:23 and SEQ ID NO:28, or sequences that are at least 70% identical thereto, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%. Said antibody or functional part or equivalent thereof preferably has the entire heavy chain and light chain sequences of antibody AT 12- 015, comprising the sequence of SEQ ID NO:33 and SEQ ID NO:38, or sequences that are at least 70% identical thereto, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%.

Another preferred antibody is AT12-017 because it is a HPeV3 subtype specific antibody. AT12-017 does not recognize or bind HPeVl, HPeV2, HPeV4,

HPeV5 and HPeV6. AT12-017 is thus particularly suitable to detect the presence of a HPeV3. It can suitable be used in combination with an antibody specific for HPeV subtypes other than HPeV3 such as AM18 or AM28 to discriminate between severe and life -threatening disease causing HPeV3 and other HPeV subtypes that generally cause mild disease. A preferred antibody or functional part or equivalent thereof of the invention thus has heavy chain CDRl, CDR2 and CDR3 sequences and light chain CDRl, CDR2 and CDR3 sequences of antibody AT12-017, comprising the sequence of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO:24 and SEQ ID NO:29, or sequences that are at least 70% identical thereto, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%. Said antibody or functional part or equivalent thereof preferably has the entire heavy chain and light chain sequences of antibody AT12- 017, comprising the sequence of SEQ ID NO:34 and SEQ ID NO:39, or sequences that are at least 70% identical thereto, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%.

Another preferred antibody is AT 12-018 because it is a HPeV3 subtype specific antibody. AT12-018 does not recognize or bind HPeVl, HPeV2, HPeV4, HPeV5 and HPeV6. AT12-018 is thus particularly suitable to detect the presence of a HPeV3. It can suitable be used in combination with an antibody specific for HPeV subtypes other than HPeV3 such as AM18 or AM28 to discriminate between severe and life -threatening disease causing HPeV3 and other HPeV subtypes that generally cause mild disease. A preferred antibody or functional part or equivalent thereof of the invention thus has heavy chain CDRl, CDR2 and CDR3 sequences and light chain CDRl, CDR2 and CDR3 sequences of antibody AT12-018, comprising the sequence of SEQ ID NO:5, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO:20, SEQ ID NO:25 and SEQ ID NO:30, or sequences that are at least 70% identical thereto, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%. Said antibody or functional part or equivalent thereof preferably has the entire heavy chain and light chain sequences of antibody AT 12- 018, comprising the sequence of SEQ ID NO:35 and SEQ ID NO:40, or sequences that are at least 70% identical thereto, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%. As is well known by the skilled person, a heavy chain of an antibody is the larger of the two types of chains making up an immunoglobulin molecule. A heavy chain comprises constant domains and a variable domain, which variable domain is involved in antigen binding. A light chain of an antibody is the smaller of the two types of chains making up an immunoglobulin molecule. A light chain also comprises a constant domain and a variable domain. The variable domain is often, together with the variable domain of the heavy chain, involved in antigen binding. Complementary- determining regions (CDRs) are the hypervariable regions present in heavy chain variable domains and light chain variable domains. The CDRs of a heavy chain and the connected light chain of an antibody together form the antigen-binding site.

Based on the antibodies depicted in table 1, it is possible to produce an antibody or functional part or equivalent thereof comprising at least one CDR sequence of an antibody variable domain depicted in table 1 which is specific for HPeV. Provided is therefor an isolated, recombinant and/or synthetic antibody or functional part or equivalent thereof comprising at least one CDR sequence of an antibody variable region depicted in table 1. Preferably, antibodies are provided which comprises at least two CDR's, more preferably at least three CDR's, of the same antibody indicated in table 1. Hence, preferably at least two or three CDR's of AM18, AM28, AT12-015, AT12-017 and AT12-018, are jointly present in one antibody or functional part or equivalent according to the invention. Preferably, an antibody according to the invention comprises all three heavy chain CDR's and all three light chain CDR's of the same antibody indicated in table 1. Optionally, said at least one CDR sequence is optimized, preferably in order to improve binding efficacy or stability. This is for instance done by mutagenesis experiments where after the stability and/or binding efficacy of the resulting compounds are preferably tested and an improved HPeV neutralizing antibody is selected. A skilled person is well capable of generating variants comprising at least one altered CDR sequence according to the invention. For instance, conservative amino acid substitution is applied. Examples of conservative amino acid substitution include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, and the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine. It is also possible to alter at least one CDR sequence depicted in table 1 in order to generate a variant antibody, or a functional part thereof, with at least one altered property as compared to the original antibody such as for instance an improved binding affinity, selectivity and/or stability. Preferably, an antibody or functional part or equivalent is provided comprising a CDR sequence which is at least 70% identical to a CDR sequence as depicted in table 1, so that the favourable binding and/or neutralizing characteristics of a Parechovirus specific antibody according to the invention are maintained or even improved. Variant antibodies or functional parts thereof comprising an amino acid sequence which is at least 70% identical to a CDR sequence as depicted in table 1 are therefore also within the scope of the present invention. Various methods are available in the art for altering an amino acid sequence. For instance, a heavy chain or light chain sequence with a desired CDR sequence is artificially synthesized. Preferably, a nucleic acid molecule encoding a CDR sequence according to the invention is mutated, for instance using random - or site-directed - mutagenesis.

Besides optimizing CDR sequences in order to improve binding efficacy or stability, at least one sequence in at least one of the framework regions can be optimized. This is preferably done in order to improve binding efficacy or stability. Framework sequences are for instance optimized by mutating a nucleic acid molecule encoding such framework sequence where after the characteristics of the resulting antibody - or functional part - are preferably tested. This way, it is possible to obtain improved antibodies or functional parts. In a preferred embodiment, human germline sequences are used for framework regions in antibodies according to the invention. The use of human germline sequences minimizes the risk of immunogenicity of said antibodies, because these sequences are less likely to contain somatic alterations which are unique to individuals from which the framework regions are derived, and may cause an immunogenic response when applied to another human individual.

An antibody or functional part or equivalent thereof according to the invention preferably comprises a human variable region. More preferably, said antibody or part or equivalent comprises a human constant region and a human variable region. Most preferably, said antibody or part or equivalent is a human antibody or part or equivalent. The use of human HPeV specific antibodies is advantageous over the use of non-human antibodies. The in vivo use of non-human antibodies for the diagnosis and treatment of human diseases is hampered by a number of factors. In particular, the human body may recognize non-human antibodies as foreign, which can elicit a immunogenic response against the non- human antibodies, which may also result in rapid clearance of the antibodies from the circulation. A human antibody diminishes the chance of side-effects due to an immunological reaction against non-human antibodies when administered to a human individual and results in a prolonged period in the circulation because of reduced clearance when compared to non-human antibodies. In another embodiment an antibody according to the invention is a humanized antibody. Humanized antibodies are made by incorporating non-human hypervariable domains into human antibodies and therefore immunogenic properties are diminished as compared to fully non-human antibodies. In another embodiment an antibody according to the invention is a chimeric antibody. In a chimeric antibody, sequences of interest, such as for instance a binding site of interest, are included into an antibody according to the invention.

Further, antibodies or functional parts or equivalents thereof according to the invention preferably are monoclonal antibodies or parts or equivalents thereof. Human serum globulin preparations, such as IVlg, may contain polyclonal antibodies against one or more subtypes of Par echo virus. However, the content of these antibodies is very low. Due to this low content the neutralizing activity against Parechovirus is relatively low. A large amount of serum preparation is necessary to achieve the desired neutralizing activity, if this activity is achieved at all. A monoclonal antibody is an antibody consisting of a single molecular species, and a titer can be obtained that is significantly higher than that of antibodies present in an antiserum. In addition, monoclonal antibodies can be produced in large quantities by monoclonal antibody-producing cells or recombinant DNA technology.

Preferred antibodies according to the invention have a high binding affinity for the Parechoviral protein, preferably for VPO, VP3 and/or VP1.

Measurement of the affinity constant and specificity of binding between antigen and antibody is preferred in determining the efficacy of prophylactic, therapeutic, diagnostic and research methods using antibodies of the invention. "Binding affinity" generally refers to the strength of the total sum of the noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity can generally be represented by the equilibrium dissociation constant (KD), which is calculated as the k_a to ka ratio, . see, e.g., Chen et al. 1999. Affinity can be measured by common methods known in the art, such as for instance a surface plasmon resonance (SPR) assay such as BiaCore or IBIS-iSPR instrument at IBIS Technologies BV (Hengelo, the Netherlands) or solution phase assays, such as Kinexa. Preferably an antibody according to the invention has a binding affinity for an epitope on VPO, VP3 and/or VP1, preferably for an epitope comprising part of VPO and part of VP3 or an epitope on VP1,

characterized by a dissociation constant (KD) of at most 100 nM, more preferably at most 50 nM, more preferably at most 25 nM, more preferably at most 10 nM, more preferably at most 5 nM, more preferably at most 2 nM, more preferably at most 1 nM, more preferably at most 0.5 nM, more preferably at most 0.3 nM, more preferably at most 0.1 nM. Particularly preferred antibodies of the invention recognizing the VP1 protein have a binding affinity for an epitope on VPlcharacterized by a dissociation constant (KD) of at most 100 pM, more preferably at most 50 pM, more preferably at most 25 pM, more preferably at most 15 pM. The invention further provides an isolated, synthetic or recombinant nucleic acid molecule with a length of at least 15 nucleotides, or a functional equivalent thereof, encoding at least one CDR sequence of an antibody, functional part or functional equivalent thereof according to the invention. An "isolated, synthetic or recombinant nucleic acid molecule with a length of at least 15

nucleotides, or a functional equivalent thereof, encoding at least one CDR sequence of an antibody according to the invention" is herein also referred to as "a nucleic acid molecule according to the invention". Preferably a nucleic acid molecule according to the invention has a length of at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 75 nucleotides. A nucleic acid molecule according to the invention is for instance isolated from a B-cell which is capable of producing an antibody according to the invention or produced recombinantly. Preferably, a nucleic acid molecule according to the invention encodes an antibody according to the invention, preferably an antibody comprising a heavy chain and/or a light chain of antibodies AM18, AM28, AT12-015, AT12-017 or AT12-018 as depicted in table 1. Nucleic acid sequences encoding heavy chain and light chain CDR's of antibodies AM18, AM28, AT12-015, AT12-017 and AT12-018 are depicted in table 1. However, nucleic acid molecules encoding a heavy or a light chain CDR of an antibody according to the invention comprising nucleic acid sequences which differ from the CDR nucleic acid sequences depicted in table 1 but comprising nucleic acid codons encoding the amino acid sequence of said heavy chain or light chain CDR are also encompassed by the invention. A preferred nucleic acid molecule according to the invention comprises a nucleic acid sequence encoding at least one CDR sequence of an antibody, functional part or functional equivalent thereof according to the invention which is specific for an epitope of VP1 comprising an amino acid sequence Arg-Gly-Asp, preferably wherein said epitope is located at amino acid positions of VP1 corresponding to amino acid positions 222 to 224 of the VP- 1 protein of HPeVl, and corresponding to amino acid positions 764-766 of the HPeVl sequence as depicted in Figure 7. Nucleic acid molecules encoding a heavy or light chain CDR of an antibody depicted in table 1 which has been altered, for instance by conservative amino acid substitution, are also encompassed by the invention, as long as the resulting CDR has at least 70% sequence identity with a CDR depicted in table 1.

As used herein, a nucleic acid molecule or nucleic acid sequence of the invention preferably comprises a chain of nucleotides, more preferably DNA and/or RNA. In other embodiments a nucleic acid molecule or nucleic acid sequence of the invention comprises other kinds of nucleic acid structures such as for instance a DNA/RNA helix, peptide nucleic acid (PNA), locked nucleic acid (LNA) and/or a ribozyme. Such other nucleic acid structures are referred to as functional equivalents of a nucleic acid sequence. The term "functional equivalent of a nucleic acid molecule" also encompasses a chain comprising non-natural nucleotides, modified nucleotides and/or non-nucleotide building blocks which exhibit the same function as natural nucleotides.

Preferably, a nucleic acid molecule according to the invention comprises:

- a heavy chain CDR1 sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:41- 45, and/or

- a heavy chain CDR2 sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:46- 50, and/or

- a heavy chain CDR3 sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:51- 55, and/or

- a light chain CDR1 sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:56- 60, and/or

- a light chain CDR2 sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:61- 65, and/or

- a light chain CDR3 sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:66- 70. A nucleic acid molecule according to the invention preferably comprises a sequence which has at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to said CDR sequences. Preferably, said nucleic acid molecule comprises at least one CDR encoding sequence.

Further provided is a nucleic acid molecule or functional equivalent thereof comprising a sequence which has at least 70% sequence identity, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to at least one nucleic acid sequence selected from SEQ ID NO's:41-70, said nucleic acid molecule or functional equivalent having at least 15 nucleotides.

A nucleic acid molecule according to the present invention preferably encodes an amino acid sequence which has at least 70% sequence identity to the amino acid sequence of a heavy chain and/or a light chain of antibodies AM18, AM28, AT12-015, AT12-017 and AT12-018 as depicted in table 1. Thus, a preferred nucleic acid molecule according to the invention comprises a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO's:71- 75 and/or a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO's:76-80. More preferably, a nucleic acid molecule according to the invention comprises a heavy chain encoding sequence and a light chain encoding sequence which resemble the heavy and the light chain encoding sequences of the same antibody depicted in table 1. Thus, one preferred nucleic acid molecule according to the invention comprises a heavy chain encoding sequence of antibody AM18, comprising the sequence of SEQ ID NO:71 and a light chain encoding sequence of antibody AM18, comprising the sequence of SEQ ID NO:76 or sequences that are at least 70%, preferably at least 75%, more preferably at least

80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical thereto.

Another preferred nucleic acid molecule according to the invention comprises a heavy chain encoding sequence of antibody AM28, comprising the sequence of SEQ ID NO:72 and a light chain encoding sequence of antibody AM28, comprising the sequence of SEQ ID NO:77, or sequences that are at least 70%, %, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical thereto.

Another preferred nucleic acid molecule according to the invention comprises a heavy chain encoding sequence of antibody AT 12-015, comprising the sequence of SEQ ID NO:73 and a light chain encoding sequence of antibody AT12-015, comprising the sequence of SEQ ID NO:78, or sequences that are at least 70%, %, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical thereto.

Another preferred nucleic acid molecule according to the invention comprises a heavy chain encoding sequence of antibody AT12-017, comprising the sequence of SEQ ID NO:74 and a light chain encoding sequence of antibody AT12-017, comprising the sequence of SEQ ID NO:79, or sequences that are at least 70%, %, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical thereto.

Another preferred nucleic acid molecule according to the invention comprises a heavy chain encoding sequence of antibody AT 12-018, comprising the sequence of SEQ ID NO:75 and a light chain encoding sequence of antibody AT12-018, comprising the sequence of SEQ ID NO:80, or sequences that are at least 70%, %, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical thereto.

The invention further provides a vector comprising a nucleic acid molecule according to the invention. As used herein "a vector comprising a nucleic acid molecule according to the invention" is also referred to as "a vector according to the invention". Methods for constructing a vector comprising a nucleic acid molecule or a nucleic acid sequence of a nucleic acid molecule according to the invention are well known in the art. Non-limiting examples of vectors suitable for generating a vector of the invention are retroviral and lentiviral vectors. A vector according to the invention is suitable for a variety of applications. For instance, a vector according to the invention can be used for in vitro expression of a nucleic acid molecule of interest in a cell, i.e. for the generation of antibodies, functional parts or functional equivalents according to the invention. Further, a vector of the invention comprising the nucleic acid sequence of a therapeutically beneficial nucleic acid molecule is suitable for prophylactic or therapeutic applications against HPeV. Administration of such vector to an individual, preferably a human, in need thereof results in expression of said prophylactic or therapeutic nucleic acid molecule in vivo resulting in treatment or prophylaxis against HPeV.

A nucleic acid molecule or vector according to the invention is particularly useful for generating antibodies or functional parts, or immunoglobulin chains or functional equivalents, which are specific for human Parechovirus, preferably HPeV viral protein. This is for instance done by introducing such nucleic acid molecule or vector into a cell so that the cell's nucleic acid translation machinery will produce the encoded antibodies or functional parts, immunoglobulin chains or functional equivalents. In one embodiment, a nucleic acid molecule or vector according to the invention is expressed in so called producer cells, such as for instance cells of a Chinese hamster ovary (CHO), NSO (a mouse myeloma) or 293(T) cell line, some of which are adapted to commercial antibody production. Proliferation of such producer cells results in a producer cell line capable of producing antibodies according to the invention. Preferably, said producer cell line is suitable for producing antibodies for use in humans. Hence, said producer cell line is preferably free of pathogenic agents such as pathogenic micro-organisms. Preferably, antibodies consisting of human sequences are generated using a nucleic acid molecule or vector according to the invention. Also provided is therefore an isolated or recombinant cell comprising a nucleic acid molecule or a vector according to the invention. Such isolated or recombinant cell is herein also referred to as an "antibody producing cell" and is defined herein as a cell which is capable of producing and/or secreting antibodies or functional equivalents thereof, and/or which is capable of developing into a cell which is capable of producing and/or secreting antibodies or functional equivalents thereof. A method for producing an antibody according to the invention is also provided, said method comprising providing a cell, preferably an antibody producing cell, with a nucleic acid molecule or a vector according to the invention, and allowing said cell to translate a nucleic acid sequence of said nucleic acid molecule or vector, thereby producing antibodies, functional parts or functional equivalents according to the invention. A method according to the invention preferably further comprises a step of harvesting, purifying and/or isolating the antibodies, functional parts or functional equivalents. Obtained antibodies, functional parts or functional equivalents according to the invention are preferably used in diagnosis or in human prophylactic or therapeutic therapy, optionally after additional purifying, isolation or processing steps.

An antibody according to the invention may be coupled to another moiety to form an antibody- drug conjugate. An antibody according to the invention is for instance coupled to an antiviral agent, such as acyclovir, penciclovar, lamivudine, ribavirin, zanamivir, laninamivir, peramivir, idoxuridine, oseltamivir, amantadine, remantidine, maxamine, peramivir, or thymalfasin. The term "antiviral agent" as used herein refers to any substance that reduces or blocks the function, or growth, of a virus and/or causes destruction of a virus. Alternatively, a moiety that is coupled to an antibody according to the invention is an antimicrobial peptide. The term

"antimicrobial peptide" as used herein refers to small amphipathic peptides of variable length (typically 6 to 100 amino acids), sequence and structure with activity against microorganisms such as for instance bacteria, protozoa, yeast, fungi and/or viruses. Antimicrobial peptides usually act through relatively non-specific mechanisms resulting in memhranolytic activity but several antimicrobial peptides can also stimulate the innate immune response. Said antimicrobial peptide preferably has anti-viral activity. Non-limiting examples of suitable antimicrobial peptides are magainins, PGLa, cathelicidins (such as LL-37 or derivatives thereof, and

cathelicidin-related antimicrobial peptide (CRAMP)), alamethicin, mellitin and cecropin, hydramacin- 1, pexiganan, MSI-78, MSI-843, MSI-594, polyphemusin, human antimicrobial peptide, defensins, protegrins and indolicidin. Another example of a moiety that is coupled to an antibody according to the invention is an

immunomodulatory molecule such as an CD3 antibody. Such CD3 antibody is capable of binding T cells and, if coupled to an antibody according to the invention, targeting T cells to Parechovirus infected cells.

Said other moiety is preferably coupled to an antibody according to the invention via a linker such as for instance an acid-labile hydrazone linker, or via a peptide linker like citruline-valine, or through a thioether linkage, or by sortase catalized transamidation, which is described in detail in WO 2010/087994. Sortase catalized transamidation involves engineering of a sortase recognition site (LPETGG) on the heavy chain of an antibody, preferably on the C-terminal part of the heavy chain, and on the moiety to be coupled to said antibody. The antibody and the moiety further typically contain a GGGGS sequence and a tag for purification purposes, such as a HIS tag. Subsequently sortase mediated transamidation is performed followed by click chemistry linkage. In a sortase catalized transaminidation, "click chemistry linkage" typically involves chemical coupling of, for instance, an alkyne-containing reagent and, for instance, an azide-containing reagent which are added by sortase through addition of glycines to the sortase motif on the heavy chain of the antibody and to a sortase motif on the moiety (such as a protein, peptide or antibody) to be coupled to the antibody. In one embodiment, the invention therefore provides an antibody according to the invention wherein a sortase recognition site (LPETGG) is engineered on the heavy chain of the antibody, preferably on the C-terminal part of the heavy chain, the antibody preferably further containing a GGGGS sequence and a purification tag, such as a HIS tag.

Another example is a thioether linkage, whereby an antibody according to the invention is coupled to another moiety via such thioether linkage. In such case, one or more cysteines are preferably incorporated into an antibody according to the invention. Cysteines contain a thiol group and, therefore, incorporation of one or more cysteines into an antibody according to the invention, or replacement of one or more amino acids by one or more cysteines of an antibody according to the invention, enable coupling of said antibody to another moiety. Said one or more cysteines are preferably introduced into an antibody at a position where it does not significantly influence folding of said antibody, and does not significantly alter antigen binding properties or effector function of said antibody. The invention therefore also provides an antibody according to the invention wherein at least one amino acid other than cysteine has been replaced by a cysteine. Said at least one amino acid other than cysteine is preferably located in a part of said antibody not involved in epitope binding.

The invention further provides an HPeV bispecific antibody with specificity for at least two different HPeV subtypes, preferably at least three different HPeV subtypes, more preferably at least four different HPeV subtypes. An "HPeV bispecific antibody" as used herein is defined as an antibody capable of

simultaneously binding at least two different HPeV subtypes, such as two, three or four subtypes, and is also referred to as an "HPeV bispecific antibody according to the invention" or a "bispecific antibody according to the invention". The term "HPeV bispecific antibody" also encompasses functional parts of such bispecific antibody which have retained their capability of binding at least two different HPeV subtypes simultaneously, such as bispecific single chain variable fragments (scFv), bispecific Fab fragments and bispecific F(ab')2 fragments. Also provided is a pharmaceutical composition comprising an HPeV bispecific antibody according to the invention.

A bispecific antibody according to the invention preferably comprises two non-identical heavy chain-light chain combinations, thus having two antigen-binding regions which recognize two different HPeV subtypes, preferably viral proteins thereof. For instance, an HPeV bispecific antibody comprises a heavy and light chain of an antibody according to the invention as depicted in table 1 and a heavy and light chain of another antibody according to the invention as depicted in table 1. Bispecific single chain variable fragments (scFv), bispecific Fab fragments and bispecific F(ab')2 fragments comprise for instance a scFv or Fab or F(ab')2 fragment of an antibody according to the invention and a scFv or Fab or F(ab')2 fragment of another antibody according to the invention. In a preferred embodiment, a bispecific antibody according to the invention comprises a heavy and light chain of two antibodies selected from the group consisting of AM 18, AM28, AT12-015, AT12-017 and AT12-018 as depicted in table 1, or a scFv or Fab fragment thereof.

Alternatively, two antibodies according to the invention are coupled to each other. This is preferably done by sortase catalized transamidation, which is described herein before and in detail in WO 2010/087994. For this purpose, sortase catalized transamidation involves engineering of a sortase recognition site (LPETGG) on the heavy chains of both antibodies to be coupled, preferably on the C-terminal part of the heavy chains. The antibodies further typically contain a GGGGS sequence and a purification tag, such as a HIS tag. Thus, if two antibodies according to the invention are coupled, both said antibodies are preferably engineered as described herein before and in detail in WO 2010/087994. Subsequently sortase mediated transamidation is preferably performed followed by click chemistry linkage to couple both antibodies via their heavy chains. As herein explained before, "click chemistry linkage" involves chemical coupling of, for instance, an alkyne -containing reagent and, for instance, an azide-containing reagent which are added by sortase through addition of glycines to the sortase motif on the heavy chain of a first antibody and to the heavy chain of a second antibody that is to be coupled to the first antibody. In a preferred embodiment, two antibodies according to the invention are coupled to each other by sortase catalized transamidation, whereby said two antibodies are preferably selected from the group consisting of AM18, AM28, AT12-015, AT12-017 and AT12- 018 as depicted in Table 1. Antibodies according to the invention are capable of binding and/or neutralizing human Parechoviruses. Antibodies according to the invention are therefore particularly suitable for use in diagnosis. Antibodies according to the invention are further particularly suitable as a medicament or prophylactic agent. The invention therefore provides an antibody or functional part or equivalent according to invention for use in diagnosis, preferably of a HPeV infection.

The invention further provides a method for determining whether a HPeV is present in a sample comprising: - contacting said sample with an antibody or functional part or equivalent according to the invention,

- allowing said antibody or functional part or equivalent to bind HPeV, preferably a HPeV viral protein, if present, and

- determining whether HPeV is bound to said antibody or functional part or equivalent, thereby determining whether a HPeV is present.

Preferably it is determined whether an individual is suffering from a Parechovirus infection and/or from a disorder caused by a Parechovirus infection. Provided is therefore a method for determining whether an individual is suffering from a Parechovirus infection and/or from a disorder caused by a Parechovirus infection comprising:

- contacting a sample from said individual with an antibody according to the invention,

- allowing said antibody to bind said Parechovirus, if present, and

- determining whether Parechovirus is bound to said antibody thereby determining whether said individual is suffering from a Parechovirus infection and/or from a disorder caused by a Parechovirus infection. Preferably said individual is a human.

Preferred antibodies for use in diagnosis are antibodies AM18, AM28, AT12-015, AT12-017 and AT12-018, which have heavy and light chain sequences as depicted in Table lor a functional part or equivalent thereof. Provided is thus antibody AM18, comprising a heavy chain sequence of SEQ ID NO:31 and a light chain sequence of SEQ ID NO:36, for use in diagnosis. Also provided is antibody AM28, comprising a heavy chain sequence of SEQ ID NO:32 and a light chain sequence of SEQ ID NO:37, for use in diagnosis. Also provided is antibody AT12-015, comprising a heavy chain sequence of SEQ ID NO:33 and a light chain sequence of

SEQ ID NO:38, for use in diagnosis. Also provided is antibody AT12-017, comprising a heavy chain sequence of SEQ ID NO:34 and a light chain sequence of SEQ ID NO:39, for use in diagnosis. Also provided is antibody AT 12-018, comprising a heavy chain sequence of SEQ ID NO:35 and a light chain sequence of SEQ ID NO:40, for use in diagnosis.

Parechovirus, preferably human HPeV, may be detected using the antibodies of the invention when present in any type of sample. Any sample containing a detectable amount of Parechovirus, preferably HPeV, can be used. Non- limiting examples of a sample are urine, saliva, mouth or throat swabs, alveolar samples, lavage or swabs, nasopharyngeal aspirate sample or swabs, blood, serum or the like, cerebrospinal fluid, tissues and feces. In a preferred embodiment, such sample is a faeces sample, a saliva sample or swab, or a alveolar sample, lavage or swab. Preferably, a sample is from an individual, preferably a human, that is suspected of suffering from a Parechovirus infection.

As detailed herein above, it is of great importance to be able to discriminate between HPeV3, which is associated with severe life -threatening disease, and other HPeV subtypes, which are usually associated with mild disease. The invention provides both antibodies that are specific for HPeV3, e.g. antibodies AM18 and AM18, and antibodies that have broad subtype specificity for at least two HPeV subtypes other than HPeV3, e.g. antibodies AT12-015, AT12-017 and AT12-018. Particularly useful is a combination of such HPeV3 subtype specific antibody and such antibody having a broad subtype specificity that does not recognize HPeV3. Using such combination allows for discriminating between the presence of or infection with HPeV3 or the presence of or infection with another HPeV subtype on the other hand. Provided is therefore a kit of parts comprising an antibody or functional part or equivalent that is specific for one or more HPeV subtypes selected from the group consisting of HPeVl, HPeVlb, HPeV2, HPeV4, HPeV5, HPeV6, HPeV7, HPeV8, HPeV9, HPeVlO, HPeVll, HPeV12, HPeV13, HPeV14, HPeV15 and HPeV16, preferably for at least two HPeV selected from said group, and an antibody or functional part or equivalent specific for HPeV3. Further provided is the use of at least two antibodies according to the invention for diagnosis, at least one that is specific for one or more HPeV subtypes selected from the group consisting of HPeVl, HPeVlb, HPeV2, HPeV4, HPeV5, HPeV6, HPeV7, HPeV8, HPeV9, HPeVlO, HPeVll, HPeV12, HPeV13, HPeV14, HPeV15 and HPeV16, preferably for at least two HPeV selected from said group, and the other antibody having HPeV3 specificity. Said first antibody preferably has broad HPeV subtype specificity, and is specific for at least two subtypes selected from the group consisting of HPeVl, HPeV2, HPeV4, HPeV5, HPeV6, HPeV7, HPeV8, HPeV9, HPeVlO, HPeVll, HPeV12, HPeV13, HPeV14,

HPeV15 and HPeV16. Preferably, said antibody is specific for at least two subtypes of HPeVl, HPeV2 and HPeV6, more preferably all three, but not for HPeV3. The use of such combination of at least two antibodies enables determining whether an HPeV infection is present in an individual, and, more importantly, it enables discrimination between HPeV3, which may lead to severe, even life -threatening disease, and any other HPeV subtype, which are generally associated with mild disease. A preferred combination of antibodies for use in diagnosis is one antibodies selected from AM18 and AM28 and one antibody selected from AT12-015, AT12-017 and AT12-018, having heavy and light chain sequences as depicted in table 1.

As described herein before an antibody or functional part or equivalent according to the invention which specifically binds to an epitope of VP1 comprising an amino acid sequence RGD (Arg-Gly-Asp), may also specifically bind to other viruses comprising the same epitope or amino acid sequence. Antibody that interacts with a particular epitope of HPeV can also be specific for another virus if said epitope of HPeV is also present in said other virus. Examples of such other viruses comprising an RGD domain are Coxsackievirus, in particular Coxsackievirus A9, other

Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma-associated herpesvirus (KSHV) and FMDV. Hence, also provided is an antibody or functional part or equivalent according to the invention for use in diagnosis of an infection by a virus comprising an viral protein comprising an RGD motif, preferably selected from the group consisting of Coxsackievirus, in particular Coxsackievirus A9, other Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma-associated herpesvirus (KSHV), and FMDV.

Diagnosis using an antibody of the invention can be performed by conventional methods known in the art, such as enzyme-linked immunosorbent assays (ELISA) or radio-immuno assays (RIA). For such methods, the antibody can be labelled directly and immune complexes of antibody and antigen can be detected via the label. Examples of labels which can be used include enzymes, fluorescent compounds, radioisotopes, chemiluminescent compounds and bioluminescent compounds. Alternatively, the antibody is unlabelled and the antibody - antigen complex can be detected with an labelled antibody, for instance an enzyme-conjugated antibody, directed against the antibody of the invention.

Kits of parts for performing diagnosis using antibodies according to the invention are also provided. Such kit of part comprises at least one antibody according to the invention, and one or more of the following compounds or parts: an antigen immobilizing material, a labelled antibody, such as an enzyme-conjugated antibody, against the antibody of the invention, an appropriate substrate, a suitable buffer for dilution and washing, and instructions for carrying out a diagnostic test. An antibody according to the invention for use as a medicament or prophylactic agent preferably consist of human sequences, in order to reduce the chance of adverse side effects when human individuals are treated. Such human sequences can be isolated from a human or synthetically or recombinantly produced based on the sequence of human antibodies. Provided is an antibody according to the invention for use as a medicament and/or prophylactic agent. Also provided is a nucleic acid molecule or functional equivalent thereof according to the invention or a vector according to the invention comprising such nucleic acid molecule for use as a medicament and/or prophylactic agent. When a nucleic acid molecule according to the invention is administered, it will be translated in situ by the host's machinery into an antibody according to the invention. Produced antibodies according to the invention are capable of preventing and/or counteracting a Parechovirus infection. Preferred antibodies for use as a medicament or prophylactic agent are antibodies AM 18, AM28, AT12-015, AT12-017 and AT12-018, which have heavy and light chain sequences as depicted in Table lor a functional part or equivalent thereof. Provided is thus antibody AM18, comprising a heavy chain sequence of SEQ ID NO:31 and a light chain sequence of SEQ ID NO:36, for use as a medicament and/or prophylactic agent. Also provided is antibody AM28, comprising a heavy chain sequence of SEQ ID NO:32 and a light chain sequence of SEQ ID NO:37, for use as a medicament and/or prophylactic agent. Also provided is antibody AT12-015, comprising a heavy chain sequence of SEQ ID NO:33 and a light chain sequence of SEQ ID NO:38, for use as a medicament and/or prophylactic agent. Also provided is antibody AT12-017, comprising a heavy chain sequence of SEQ ID NO:34 and a light chain sequence of SEQ ID NO:39, for use as a medicament and/or prophylactic agent. Also provided is antibody AT12-018, comprising a heavy chain sequence of SEQ ID NO:35 and a light chain sequence of SEQ ID NO:40, for use as a medicament and/or prophylactic agent.

Also provided is an antibody or functional part or equivalent, or a nucleic acid molecule, or a vector according to the invention for use as a medicament and/or prophylactic agent. An antibody according to the invention, or a nucleic acid molecule or functional equivalent thereof according to the invention is preferably used for at least in part treating and/or preventing a Parechovirus infection, preferably a human Parechovirus infection. As used herein "at least in part treating a Parechovirus infection" includes counteracting a Parechovirus infection, alleviating symptoms resulting from a Parechovirus infection and/or counteracting inflammation resulting from a Parechovirus infection. Provided is therefore an antibody or functional part or equivalent according to the invention, or a nucleic acid molecule or according to the invention, or a vector according to the invention for use in a method for the treatment or prevention of a HPeV infection and/or a disorder caused by a HPeV infection..

Examples of symptoms resulting from a Parechovirus infection include, but are not limited to, inflammation, fever, diarrhea, sepsis, meningitis, encephalitis and paralysis. Also provided is therefore an antibody according to the invention, or a nucleic acid molecule or functional equivalent thereof according to the invention, or a vector according to the invention, for use in a method of at least in part treating and/or preventing a disorder caused by Parechovirus infection wherein said disorder is selected from the group consisting of inflammation, fever, diarrhea, sepsis, meningitis, encephalitis and paralysis. Further provided is a use of an antibody or functional part or functional equivalent or a nucleic acid molecule according to the invention or a vector according to the invention for the preparation of a medicament and/or prophylactic agent for at least in part treating and/or preventing a

Parechovirus infection and/or a disorder caused by a Parechovirus infection.

Preferably, said disorder is selected from the group consisting of inflammation, fever, diarrhea, sepsis, meningitis, encephalitis and paralysis. Preferred antibodies are antibodies AM18, AM28, AT12-015, AT12-017 and AT12-018, which have heavy chain and light chain sequences as depicted in table 1.

As described herein before an antibody or functional part or equivalent according to the invention which specifically binds to an epitope of VPl comprising an amino acid sequence RGD (Arg-Gly-Asp), may also specifically bind to other viruses comprising the same epitope or amino acid sequence. Examples of such other viruses comprising an RGD domain are Coxsackievirus, in particular Coxsackievirus A9, other Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma-associated herpesvirus (KSHV) and FMDV. Hence, also provided is an antibody or functional part or equivalent according to the invention for use in a method for the treatment and/or prevention of an infection by a virus comprising a viral protein comprising an RGD motif, preferably selected from the group consisting of Coxsackievirus, in particular Coxsackievirus A9, other

Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma-associated herpesvirus (KSHV) and FMDV, and/or a disorder caused by such virus.

The invention further provides a pharmaceutical composition comprising an antibody according to the invention, and/or a bispecific antibody according to the invention, and a pharmaceutical acceptable carrier, diluent and/or excipient. Also provided is a pharmaceutical composition comprising a nucleic acid molecule according to the invention, or a vector according to the invention comprising such nucleic acid molecule, and a pharmaceutical acceptable carrier, diluent and/or excipient. Examples of suitable carriers for instance comprise a solution, like for example saline, keyhole limpet haemocyanin (KLH), serum albumin (e.g. BSA or RSA) and ovalbumin. A pharmaceutical composition according to the invention is preferably suitable for human use. A "pharmaceutical composition comprising an antibody or functional part or equivalent, or a nucleic acid molecule, or a vector according to the invention and a pharmaceutically acceptable carrier, diluent and/or excipient" is herein also referred to as a pharmaceutical composition according to the invention.

A pharmaceutical composition according to the invention may further comprise an adjuvant. Examples of adjuvants which can be incorporated in tablets, capsules and the like are a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as microcrystalline cellulose; a disintegrating agent such as corn starch, pre gelatinized starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; a flavoring agent such as peppermint, oil of wintergreen or cherry. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as fatty oil. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propyl parabens as preservatives, and a flavoring such as cherry or orange flavor.

The invention further provides a method for at least in part treating and/or preventing a Parechovirus infection, preferably a human Parechovirus infection, and/or a disorder caused by such infection, comprising administering to an individual in need thereof a therapeutically effective amount of an antibody according to the invention, and/or a bispecific antibody according to the invention, and/or a nucleic acid molecule according to the invention, and/or a vector according to the invention, and/or a pharmaceutical composition according to the invention. Also provided is a method for at least in part treating and/or preventing an infection by a virus comprising a viral protein comprising an RGD motif, preferably selected from the group consisting of Coxsackievirus, in particular Coxsackievirus A9, other Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma-associated herpesvirus (KSHV) and FMDV, and/or a disorder caused by such virus, comprising administering to an individual in need thereof a therapeutically effective amount of an antibody according to the invention, and/or a bispecific antibody according to the invention, and/or a nucleic acid molecule according to the invention, and/or a vector according to the invention, and/or a pharmaceutical composition according to the invention. As used herein, an

"individual" is a human or an animal, preferably an animal that can be infected by Parechovirus, such as rodents, humans and other mammals. In a preferred embodiment of the invention said individual is a human.

In order to at least in part treat or prevent a Parechovirus infection and/or a disorder caused by such infection, an antibody, a nucleic acid molecule, a vector, and/or a pharmaceutical composition according to the invention is preferably administered to an individual before infection has taken place. Alternatively, an antibody, a nucleic acid molecule, a vector, and/or a pharmaceutical composition according to the invention is administered when an individual is already infected. In that case, a Parechovirus infection is counteracted, symptoms resulting from a Parechovirus infection are alleviated and/or inflammation resulting from a

Parechovirus infection is counteracted. Said antibody or functional equivalent is particularly suitable for administered to individuals with an increased risk of complications, such as hospitalized individuals, for instance infants, individuals with compromised immunity and/or elderly people. An antibody, a nucleic acid molecule, a vector, and/or a pharmaceutical composition according to the invention is preferably administered via one or more injections.

The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.

Brief description of the drawings

Figure 1. Neutralization of anti-HPeVl-Harris, HPeVlB, HPeV2, HPeV4, HPeV5 and HPeV6 by AM 18 and AM28. Virus was inoculated for 1 hr with purified antibodies before being added to confluent HT29 cells. The antibody workstock (400 μg/ml) was diluted 16-times, followed by a 2-step dilution series. When the presence of CPE was detected, cells were harvested and HPeV replication was determined by real time PCR ³. Figure 2. Neutralization of HPeV3 strains by AT12-015, AT12-017 and ΑΊΊ2-018. In all assays virus was mixed for 1 hr with purified recombinant protein before being added to confluent target cells. At day 7 after infection cells were harvested and presence of virus was detected by real time PCR (read out viral copies/PCR). The antibody workstock was 250 μg/ml and diluted 16-times, followed by a 2-step dilution series. Indicated in (A) is neutralization of strains HPeV3- 150237, -1595 and - 1930 by AT12-015, AT12-017 and AT12-018, in (B) is indicated the neutralization of HPeV3 strains 150237 and 1930 on Vero, MBG and lower panel Caco2 cells by AT12-015. (C) shows neutralization of all antibodies on several more recent clinical isolates. Figure 3. Antibody Immunofluorescence assay on HPeV infected cells. (A) Binding of AM18 and AM28 to HPeVl, HPeV2, HPeV3 and HPeV4 infected HT29 cells. After infection the target cells were fixed and incubated with the indicated antibodies and DAPI to stain nuclei (shown in grey). Antibody binding was detected with goat anti- human IgG-Alexa488 on the Operetta (shown in light grey), staining are shown as an overlay. An identical detection method was used to determine binding of AT 12-015 (upper panel) and AT12-017 (lower panel) to HPeV3 infected Vero cells (Figure3B) and Figure 3C of AT12-018. The left panel shows the overlay of DAPI and Alexa488 staining; the left panel the virus binding (Alexa488 staining) only. Figure 4. (A) ELISA antibody binding to plates directly coated with purified virus particles or recombinant capsid proteins and to 12 aa peptides of VP1 for AM 18 (B) and AM28 (C). Figure 5. (A) Binding of AM18 to HPeVl-VPl peptides. 3.0 μg/ml antibody is injected on a SPR chip coupled with a peptide library containing the entire sequence of HPeVl-VPl protein. (B) Binding of AM18 to HPeVl-VPl 3.0 μg/ml antibody is injected on a SPR chip; subsequently 2.0 μg/ml HPeVl-VPl is injected. Shown is the response to HPeVl-VPl (peptide) injection, on a spot coated with 5.0 μg/ml biotin-anti-IgG. (C) Binding of a concentration range of AM18 to HPeVl-VPl. A concentration series (2.0 - 20 μg/ml) of antibody is injected on a SPR chip; subsequently 2.0 μg/ml HPeVl-VPl is injected. Shown is the response to HPeVl-VPl (protein) injection (black lines), binding to

AM18 captured on a spot coated with 5.0 μg/ml biotin-anti-IgG. Curve fits (1: 1 binding model) are shown as grey lines.

Figure 6. Organization of the capsid of HPeV and human Enterovirus, as well as the genomic organization of the structural and non-structural proteins (Source: SIB Swiss Institute of Bioinformatics, 2008).

Figure 7. Amino acid sequence of HPeVl VP0-VP3-VP1 as described in Ghazi et al., J. Gen Virol. 1998;79 ( Pt ll):2641-2650.

Figure 8. (A) Raw micrograph of HPeVl in complex with AM28 Fab. Bar, 50 nm. (B) Central cross-section of HPeVl-AM28 Fab complex with two-fold (21), five-fold (51) and three-fold (31) symmetry axes marked. Scalebar 15 nm. (C) Three-dimensional reconstruction of the HPeVl capsid with 60 Fab molecules bound, seen at high radius (arrows indicate two such molecules sticking out from the surface of the capsid.

Figure 9. Overlay of the integrin-bound form of HPeVl with the AM28-HPeVl complex. Arrow indicates one of 60 equivalent positions where the integrin is bound. Figure 10. 3D reconstruction of HPeV in grey with atomic models for VP0 (left hand, molecule derived from echovirus levl VP2), VP3 (right hand molecule, derived from echovirus levl VP3) and a Fab molecule fitted in to the cleft formed by VPO and VP3 from different pentamers (bottom picture).

Figure 11. Comparison of nucleotide sequences in 22 published HPeVl strains over the regions coding for the loops of VPO and VP3 (VPO PLSIPTGSANQ, VP3 FFPNATT and VP 3 ATTAPQSIVH).

Figure 12. Epitopes on HPeVl for AM28. (A) Homology models of VP1 (dark grey), VPO (light grey) and VP3 (medium grey) built using I-TASSER. (B) Final fits of VP1, VPO and VP 3 homology models into an asymmetric unit of HPeVl (EMD- 1690). (C) Superimposing asymmetric units of echovirus 1 (PDB ID: 1EV1), poliovirus 1 (PDB ID: 1POV), enterovirus 71 (PDB ID: 3VBF) and foot mouth disease virus (PDB ID: 1QQP) on final fits of HPeVl VP1, VPO and VP3. (D) Mapping epitopes for AM28 on HPeVl surface by fitting AM28 Fab variable region homology model into the Fab density seen in HPeVl-AM28 Fab reconstruction and superimposing VP1, VPO and VP3 fits for HPeVl (EMD- 1690) into the HPeVl-AM28-Fab reconstruction (mesh). AM28 variable heavy chain and variable light chain are indicated. (E) Roadmap showing the density of AM28 Fab (line contour, radius 155-156A) and the epitopes HEWTPSWA (VPO), HQDKP (VPO), PLSIPTGSANQ VD (VPO), MADSTTPSENHG (VP3), ATTAPQSIVH (VP3) and FFPNATTDST (VP 3). Epitopes having the same color are identical, e.g. all black epitopes are epitope MADSTTPSENHG (VP 3). An asymmetric unit is marked by straight black lines. (F) Distance between the symmtery-related Fab shown as wire. Figure 13. Conservation of epitopes. Amino acid sequences of HPeVl-5 used for neutralization were aligned against complete genome sequences for HPeVl-6. The sequence annotation on the left hand side is 'virus genotype/GenBank ID'. The epitopes are marked in black on the HPeVl-Harris strain (GenBank ID: L02971) that was used as the basis for the HPeVl homology modelling. The alignment is coloured according to percent sequence identity, from a scale of white (no identity) to dark grey (full identity). The conservation panel below the alignment gives the numerical values for the conservation based on the BLOSUM 62 score of the alignment and

physicochemical conservation (Livingstone and Barton 1993) where * is 100% identity. The arrowheads indicate irrelevant regions of the sequence that have been hidden in the final representation for simplicity (1- 120, 160-246, 270-372, 413-453). The figure was made with Jalview (Waterhouse et al. 2009). Figure 14. Thermofluor assay. Plot of temperature (x-axis) versus first derivative of fluorescence (y-axis) showing the change in Tm when HPeVl is bound to antibodies AM18 or AM28 compared to HPeVl alone. Arrows indicate the Tm for each sample. AM18 and AM28 were used as the negative control for the RNA binding dye.

Examples

Material & Methods Generation of monoclonal HPeV specific antibodies

The AM18 and AM28 antibodies were obtained using a direct virus neutralization assay of HPeVl virus pre-incubated with supernatant of cultures with different B cell densities (from healthy donors, generated as described in Kwakkenbos MJ et al. 2010 and Kwakkenbos MJ et al. 2012). In brief, CD27+ memory B cells were isolated from peripheral blood by FACS sorting. Cells were stimulated for 36 hrs with CD40L and interleukin (IL)21 and subsequently transduced with a retrovirus containing the Bcl-6 and Bcl-xL transgenes together with the marker gene GFP. Transduced B cells can be maintained for prolonged periods of time and harbor a Germinal Center phenotype, which is characterized by expression of cell surface immunoglobulin (the B Cell Receptor -BCR-) and secretion of soluble immunoglobulin. Antibodies present in the supernatant can for example be tested for binding or functionally. Here we tested the HPeVl neutralizing capacity of antibodies in B cell supernatants by co-incubation of supernatants with virus for 1 hr before they were added to HT29 cells. Infection was determined by cell rounding. Cultures that did not show cell rounding were single cell subcloned to obtain monoclonal B cell cultures. The antibody heavy and light chain genes were recovered from these B cell clones and expressed as recombinant protein in 293T cells. IgGl antibodies were subsequently purified using HiTrap Protein A or G columns on an AKTA instrument (GE) . The HPeV3 specific antibodies (AT12-015, AT12-017 en AT12-018) were obtained from supernatants of B cell cultures that were screened for binding to HPeV3 infected monolayers of Vero cells.. Before, infected Vero cells were incubated with B cell derived supernatants (from HPeV3 infected patients, generated as described in Kwakkenbos MJ et al. 2010 and Kwakkenbos MJ et al. 2012) and anti-human IgG- Alexa488 secondary antibody they were fixed with 4% paraformaldehyde (PFA).

Fluorescent binding of the secondary antibody to human IgG binding to the HPeV3 infected cells was detected using the Operetta instrument (PerkinElmer). Cell lines

The Human colon carcinoma (HT29) and African green monkey kidney (Vero) cell line were used for HPeV virus culture. The cells were maintained in Eagle's Minimum Essential Medium (EMEM) (Lonza) supplemented with L-glutamic acid (0.2X) (Gibco), non-essential amino acid (IX) (Gibco), streptomycin (0.1 μg/ml) (Riemer) and ampicillin (0.1 μg/ml). The medium was supplemented with 8% heat-inactivated Fetal Calf Serum (FCS) (Sigma). Only when the cell lines were infected with virus the medium contained 2% FCS. In addition human colon adenocarcinoma (Caco2) and buffalo green monkey kidney (BGM, kindly provided by Dr. van Kuppeveld, St.

Radboud University, Nijmegen) were used.

Virus strains and virus cultivation

The following HPeV strains were used: HPeVIA Harris, HPeV2-751312 and the HPeV3 strains HPeV3- 150237, HPeV3 A308-99, HPEV3 1930, HPeV3 1595, HPEV3 2736, and HPeV3 1825. The A308-99 Japanese patient strain was a kind gift from Dr. Shimizu, National Institute of Infectious Diseases, Tokyo, Japan (Ito et al., 2004). HPeV4-251176, HPeV5-552322 and HPeV6-550389 (Benschop et al., 2006, 2008, 2010). HPeVl-Harris and the HPeV2-751312 strains were provided by the Dutch National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands. HPeVl, 2, 4, 5 and 6 were cultured in the HT29 cell line, HPeV3 in the Vero cell line and the virus working stocks were stored in aliquots at -80°C. The virus concentration was determined by the median tissue culture infective dose (TCID50) and calculated by the Reed and Muench method (Reed L.J. & Muench H., 1938). The following HPeV3 strains were used, HPeV3 A308-99, HPEV3 1930, HPeV3 1595, HPEV3 2736, and HPeV3 1825. The A308-99 Japanese patient strain was a kind gift from Dr. Shimizu, National Institute of Infectious Diseases, Tokyo, Japan (Ito et al., 2004).

Immunofluorescence assay

Black clear round bottom 96 wells plate (Greiner) with Vero or HT29 cells line were inoculated with a virus solution in EMEM 8% FCS. When a cytopathic effect (CPE) of 2+ (25-50% of cells infected) was observed, the infected cells were fixed with 4% paraformaldehyde (PFA)/PBS for 15 minutes. The PFA was removed and the cells were washed consecutively 3 times with PBS, 25mM NH4CI/PBS, and PBS. The cells were placed in 0.1% triton/PBS for 10 minutes and washed three times with PBS. To avoid unspecific binding wells were blocked with 1% BSA/PBS for 30 minutes. The blocking buffer was removed and the cells were incubated with B cell derived culture supernatants, control antibodies or purified, recombinant HPeV specific monoclonal antibodies, for lhr at 37°C. As control Abs the following Abs were used: polyclonal antiHPeVl -Harris, kindly provided by the RIVM, Bilthoven, the Netherlands, and the polyclonal antiHPeV3-A308-99, kindly provided by dr. Shimizu from the National institute of Infectious Diseases, Tokyo, Japan. Plates were washed consecutively 3 times with PBS, 0.1% Tween/PBS, and PBS. 15 μg/ml of either goat anti-human Alexa Fluor 488 (Life Technologies), anti-rabbit or anti-guinea pig secondary goat antibody - fluorescein isothiocyanate (FITC) labelled IgG (Jackson Immuno Research) was used as secondary antibody staining and to stain nuclei 0.5 μg/ml 4',6-diamidino-2- phenylindole (DAPI) (Sigma) was added and incubated for lhr 37°C. Plates were washed 3 times with PBS. The fluorescence was conserved in Glycerol/PBS and examined with a fluorescence microscope (Leica) or the Operetta automated fluorescent microscope (PE).

Neutralization assay

Neutralization assays were performed as described in Westerhuis et al. 2012. B-cell supernatants or antibodies (Abs) were mixed with the different HPeVl and HPeV3 virus suspension containing 100 ΤΟΙΏ50/50μ1. Mixtures were incubated at 37°C for lhr, and were used to inoculate HT29 cells (HPeVl, 2, 4, 5, and 6) and Vero cells (HPeV3) on a 96-wells plate (200 μΐ). In addition, neutralization of Coxsackie A9 (CAV9) by AM18 and AM28 was determined. Virus, cell and Ab controls were included as positive and negative control. The cells were examined for the

appearance of CPE every 24hrs for 7 days. For the purified Abs an end point neutralization was performed at day seven the virus copies/PCR were measured with real time PCR.

Virus replication

The supernatant (20 μΐ) was extracted by automatic extraction using the total nucleic acid isolation kit with the MagnaPure LC instrument® (Roche Diagnostics). The RNA was eluted in 50 μΐ elution buffer and reverse transcribed as previously described. 5 μΐ of cDNA was used for real-time PCR using the LC480® (Roche Diagnostics) (Benschop et al., 2008; Benschop et al. 2010) . The virus copies per PCR were calculated with a standard curve as previously described (Benschop et al., 2008; Benschop et al. 2010).

Expression and purification of HPeV capsid proteins

The HPeVl-Harris, HPeV3, and HPeV4 strain was used to generate the capsid proteins VPO, VP3 and VPl sequences. The fragments were cloned into the expression vector Petl02 with his-tag and expressed in Escherichia coli BL21 Star™ (DE3) One Shot® cells. Single bacterial colony was inoculated in LB medium supplemented with 100 μg/ml and propagated at 37°C with 220rpm speed on a shaker incubator till the culture reached logarithmic growth phase (at OD600 0.6-0.7). The culture was then induced by addition of isopropyl β-d-thiogalactopyranoside (IPTG), and the proteins were purified by immobilized Ni ion affinity chromatography (Invitrogen) in accordance with the manufacturer's instructions (Invitrogen)

ELISA

ELISA plates were coated with 200ng HPeV purified virus, or the HPeV capsid proteins VPO, VPl and VP3 in ΙΟΟμΙ 50mM NaHCo3 overnight at room temperature (RT). The plate was washed 3x with PBS-tweenO.05%. To avoid unspecific binding wells were blocked with 300μ1 blocking buffer (5% skimmed dried milk powder) for 1 hr. The purified antibodies were diluted in 1% BSA-PBS and incubated for 1 hr at RT. After washing 3X with PBS-tweenO.05% the plate was incubated with horseradish peroxidase -conjugated secondary IgG diluted 1:4000 in PBS-tweenO.05%. Next the plate was washed 3X with PBS and incubated with ΙΟΟμΙ 3,3 ,5,5 - tetramethylbenzidine (TMB) substrate solution. The colour reaction was stopped after 10 minutes with stopping solution (H2SO4). The OD-value is measured and calculated at 450-625Nm in the spectrophotometric reader. Peptide ELISA

Streptavidin coated ELISA plates were blocked with 2% BSA/PBS for 2 hours at room temperature. The biotin- labelled HPeVl overlapping peptides (12 aa in length with 3 aa overlap) were diluted (1:500) in 1% BSA in PBS and bound to the plate for 1 hr at room temperature. The plate is washed 3 times with PBS/tween-0.1% and incubated with the primary aHPeVl Abs AM18 or AM28 for 1 hr at RT. After washing 3 times with PBS/tween-0.1%, the plate was incubated with horseradish peroxidase- conjugated secondary anti-human IgG diluted 1:4000 in PBS-tween0.1% the plate was washed 3X with PBS and incubated with ΙΟΟμΙ 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate solution. The colour reaction was stopped after 10 minutes with stopping solution (H2SO4). The OD-value is measured and calculated at 450-625Nm in the spectrophotometric reader. SPR Epitope Mapping of AM18

The epitope of antibody AM18 is mapped with surface plasmon resonance (SPR), by measuring binding of AM18 to a peptide library that is spotted on an SPR chip. The peptide library used in this experiment consists of 86 12-mer peptides, containing the entire sequence of the Parechol-VPl protein.

SPR

SPR is performed on an IBIS Mx96 SPR instrument (IBIS Technologies). Biotinylated peptides are coupled on a streptavidin-coated gel type gold SPR chip (G- strep, Ssens Technologies) using a Continuous Flow Microfluidic Spotter (Wasatch Microfluidics). Peptides are 100x-diluted in spotting buffer (PBS + 0.05% sodium azide + 0.03% Tween20) and coupled to the chip for 45 min (at room temperature). Results were analyzed using SprintX software (IBIS).

Peptide Library

Peptide library is synthesized by Henk Hilkmann (NKI, Amsterdam). The library consists of 86 12-mer peptides, containing the entire sequence of the Parechol---VPl protein. Between consecutive peptides, there is an overlap of three amino acids (peptide 1 consists of amino acids 1-12, peptide 2 consists of amino acids 10-21 etc.). To facilitate coupling to streptavidin, each peptide is biotinylated at the N- terminus. Antibodies and Antigens:

AM18, AM28 and D25 (described in WO 2008/147196); anti-IgG: AffiniPure Goat anti-human IgG, F y-fragment specific (Jackson); anti-IgG is biotinylated using a biotin-XX microscale antibody labeling kit (Invitrogen).

Measurements

The activity of the antibody (Figure 5B) is measured with an IgG capture assay, in which the antibody is first immobilized on an anti-IgG-coated spot. After capturing the antibodies, Parecho antigen (HPeVl-VPl) is injected over the chip. To measure antibody affinity (Figure 5C), first a concentration series of antibody is injected (2.0 - 20 μg/ml) followed by a 2.0 μg/ml VPl-solution. Kinetic constants were fitted to the binding curves, using a 1: 1 binding model.

Epitope mapping (Figure 5A) is done with AM 18, AM28 and D25. The antibodies are injected on an SPR chip, to which the Parecho peptide library was coupled. Based on the ELISA- mapping results, we chose to include peptides 44-86 in the SPR

experiment (since the first 43 peptides were negative in the ELISA experiment).

Identification of epitopes on HPeVl capsids by labeling of the virus with AM28

Fab fragments

The Fab fragments from AM28 were produced using a Pierce™ Fab Micro Preparation Kit according to the manufacturer's protocol. The Fab was mixed with HPeVl at a molar ratio of 5: 1 in lxTNM for 30 minutes at room temperature. Virus culture and purification

HT29 cell line was used for culturing the HPeVl Harris strain. The cells were maintained in Eagle's Minimum Essential Medium (EMEM) supplemented with L- glutamic acid (0.2X), non essential amino acid (IX), streptomycin (0.1 μg/ml) and ampicillin (0.1 μg/ml), supplemented with 8% heat-inactivated Fetal Calf Serum (FCS). HT29 cells were cultured -90% confluent in a T175 flask before being infected with HPeVl-Harris at a MOI 0.1. After appearance of CPE (cytopathic effect) grade 4+ (75-100% infection) the cells and spent media were freeze thawed twice (-80°C), and centrifuged at 4000rpm for 15 minutes before the supernatant was filtered. After filtration of virus through 0.22 μηι filter, it was centrifuged at 32000rpm, for 2h at 4°C in the ultracentrifuge (rotor SW32Ti, Beckman). The pellet was dissolved in lxTNM buffer (lOmM Tris-HCl, pH 7.5, 150mM NaCL, ImM MgC12) and virus was purified on a cesium chloride step gradient made of 40% (w/v) bottom layer, 15% (w/v) top layer by centrifuging at 32000rpm, for 16hrs at 4°C (rotor SW41Ti, Beckman). The fraction containing the virus was harvested and exchanged with TNM buffer and concentrated with lOOkDa cutoff filter (Millipore).

Imaging, three-dimensional reconstruction, homology modelling

Aliquots of the Fab-virus mixture were vitrified on Quantifoil R2/2 holey carbon nickel grids in a home-built guillotine by plunging into liquid ethane maintained in a liquid nitrogen bath. After vitrification, the grids were stored in liquid nitrogen until use. The grids were examined in a FEI F20 transmission electron microscope at 200 kEV using a Gatan 626 cryostage. The images were recorded on a Gatan Ultrascan 4000 under low dose conditions at a magnification of 69000 x with sampling size of 2.17A per pixel.

The contrast transfer function of each micrograph was estimated using CTFFIND3 and images containing drift or astigmatism were discarded (Shakeel et al. 2013). Particles were picked using the program ETHAN (Pandurangan et al. 2014) with box size of 401 pixels and inspected by eye in the program suite EMAN (Wildenbeest et al. 2010). The previous reconstruction of HPeVl from Seitsonen et al. 2010 (EMDB ID: 1690) was used as a starting model to initiate full orientation and origin

determinations of the Fab labelled set of images using AUT03DEM (Yan et al. 2007). The final reconstructions calculated to the Nyquist frequency were used to estimate the B-factors with EM-Bfactor, and then the reconstructions were truncated to the resolution indicated by the Fourier shell correlation analysis with a threshold criterion of 0.5 (Shakeel et al 2013, Wang et al 2012, Ren et al 2013). The HPeVl- AM28 Fab density map was deposited in the Protein Databank in Europe.

Homology modelling and fitting of models into cryoEM maps The structures of the three HPeVl capsid proteins were predicted by multiple- template comparative modeling using the I-TASSER server (Roy et al. 2010; Zhang 2007). In a first analysis, the template structures for VP0 were Foot mouth disease virus

(PDB id: lqqp and lbbt), Poliovirus 1 (PDB id: lpov), Equine rhinitis A virus (PDB id: 2wff), Bovine enterovirus (PDB id: lbev), Theiler murine encephalomyelitis virus (PDB id: ltmf), Poliovirus 1 mahoney strain (PDB id: lhxs) . For VP1 they were Triatoma virus (PDB id: 3nap), Coxsackievirus A9 (PDB id: ld4m), Human rhinovirus 16 (PDB id: laym), Human rhinovirus 14 (PDB id: ld3i), Mycobacterium smegmatis biosynthetic protein (PDB id: 2grv), Theiler murine encephalomyelitis virus (PDB id: 1TME). For VP3, they were Poliovirus 1 (PDB id: lpov and lal2), Human enterovirus 71 (PDB id: 3vbh), Equine rhinitis A virus (PDB id: 2xbo), Coxsackievirus A9 (PDB id: ld4m), Human rhinovirus 16 (PDB id: laym). The resulting models along with the Echovirus 1 atomic model (PDB id: levl) were flexibly-fitted into the previously published model of HPEV1 with and without integrin bound (Seitsonen et al, 2010) (EMDB ID: 1689 and 1690 respectively). The Fab -virus complex, HPeVl with and without integrin bound and the two flexibly fitted models were compared to identify the footprint of the antibody on the virus. The sequences contributing to three loops on HPeVl Harris strain were compared with the equivalent sequences of HPeV2.

In a second analysis, template structures for VP0 were foot and mouth disease virus (PDB ID: 1QQP, 1FMD and 1BBT), poliovirus 1 (PDB ID: lPOV), bovine enterovirus (PDB ID: lBEV) and Seneca Valley virus-001 (PDB ID: 3CJI). For VP1, they were triatoma virus (PDB ID: 3NAP), human rhinovirus 14 (PDB ID: 1D3I), cricket paralysis virus (PDB ID: 1B35), rabbit hemorrhagic disease virus (PDB ID: 4EJR), echovirus 7 (PDB ID: 1M11) and bovine enterovirus (PDB ID: lBEV). For VP3, they were human enterovirus 71 (PDB ID: 3VBF), Seneca Valley virus-001 (PDB ID: 3CJI), human rhinovirus 16 (PDB ID: 1AYM) and poliovirus Mahoney strain (PDB ID:

1HXS). An atomic model of echovirus 1 capsid (PDB ID: 1EV1) (Basavappa et al.

1994) was placed into an 8.5 A resolution HPeVl map (EMD-1690) (Seitsonen et al. 2010). The fitting was done using a modification of a protocol described elsewhere (Smyth et al. 1995). The homology models were aligned with the echovirus 1 capsid, before being rigidly fitted into the HPeVl map (EMD- 1690) (Seitsonen et al. 2010) using the 'fit in map' feature in UCSF-Chimera (Venkataraman et al. 2008). The N- terminus of VP0, VP3 and VP1 were truncated to avoid inter-subunit clashes. Using the 'zoning' feature in UCSF-Chimera (Venkataraman et al. 2008), the HPeVl capsid map was zoned to an asymmetric unit with a radius of 6 A using the truncated VP0- VP3-VP1 rigidly-fitted model. RIBFIND based rigid bodies were identified for the truncated VP0-VP3-VP1 model (Squires et al. 2013) and the model was flexibly fitted into the asymmetric unit using one iteration in FlexEM (Kolatkar et al. 1999) followed by iMODfit based flexible fitting using the default settings (Tate et al. 1999). The resulting homology model of the complete HPeVl capsid was then placed directly into the Fab-labelled reconstruction to identify the probable binding sites. The variable regions of AM28 Fab were modelled using the WAM webserver (Wang et al. 2013) and manually fitted into the corresponding Fab density in the HPeVl-AM28 Fab reconstruction and the fit was optimized by 'fit in map' feature in UCSF-Chimera. All the visualization was carried out in UCSF-Chimera (Venkataraman et al. 2008).

Confirmation of the macromolecular assembly recognized by AM28

Pentamers of HPeVl were incubated with AM28 and loaded on to a native gel. The mobility of the pentamers alone and with AM28 was compared.

Sequence alignment

The PI amino acid sequences of HPeVl (GenBank ID: L02971, GQ183023, GQ183022, GQ183021, GQ183020, GQ183019, GQ183018, GQ183025, GQ183024), HPeV2 (GenBank ID: NC_001897), HPeV3 (GenBank ID: GQ 183026), HPeV4 (GenBank ID: DQ315670) and HPeV5 (GenBank ID: AF055846) used for AM18 and AM28 neutralization assays were aligned using Clustal Omega (He et al. 2002) with additional HPeV strains for which the complete genome sequences were available in GenBank (GenBank IDs for HPeVl are JX441355, JX575746, S45208, EF051629, FJ840477, GQ 183035, GQ 183034, HQ696574, HQ696572, HQ696570, HQ696573,

HQ696571, FM178558; for HPeV3 are GQ183027, GQ183028, GQ183029; for HPeV4 are AB433629, AM235750; for HPeV5 is AM235749; for HPeV6 are EU077518, AB252583). The alignment was visualized with Jalview (Hadfield et al. 1997).

Thermofluor assay

In order to test the capsid stability in the presence of antibodies, AM18 and AM28 mAb were mixed with HPeVl virions (so capsids containing RNA) at a molar ratio of 66: 1 and incubated at room temperature for 30 min. Dye-accessibility to the RNA increasing with heat was detected with a fluorescent dye. The reaction volumes were set up per well in a 96-well PCR plate and each reaction contained 2.5 μΐ of 200X Sybr Safe DNA gel stain (Invitrogen, also binds RNA) and the protein sample which was one of the following HPeVl (10 μΐ of lmg/ml stock), AM18 (10 μΐ of 2 mg/ml stock), AM28 (10 μΐ of 2 mg/ml stock), HPeVl-AM18 complex (20 μΐ) or HPeVl-AM28 complex (20 μΐ). The total volume was made up to 25 μΐ for each reaction volume using IX TNM buffer. The assay was run from 25°C to 95°C with readout every 0.33 s in an Mx3005P qPCR instrument (Agilent Technologies). The Sybr Safe DNA gel stain dye was excited at 492 nm and emission was read at 516 nm (Miller et al. 2001).

Results Identification and generation of HPeV specific antibodies

Human memory IgG+ B cells were obtained from three healthy donors and were frozen at -150°C in 96 well plates at 50 cells /well after transduction with Bcl-6 and Bcl-xL. In parallel, supernatants containing antibodies were kept and frozen and tested for direct neutralization of HPeVl in a HT29 cell rounding assay at the appropriate time. From the cultures that showed reduced cell round, single cell cultures were generated to retrieve the original monoclonal B cell. From clones that showed repetitive neutralization of HPeVl, RNA was isolated to retrieve the antibody heavy and light chain sequences. These sequences were used to generate recombinant protein from 293T cells.

Using this procedure clone AM18 and AM28 were discovered. Both antibodies efficiently neutralize HPeVl and HPeV2 at concentrations ranging from 100 to 200 ng/ml on both Vero and (Figure 1). AM 18, did broadly neutralize HPeV since it also neutralized HPeV strains 4, 5 and 6, it did not neutralize HPeV3. In addition, AM18 neutralized Coxsackie A9 at relatively high concentration, while AM28 did not.

In addition we screened the human B cell repertoire of two healthy adult donors who had a documented HPeV3 infected several months in advance. Since HPeV3 cannot be propagated easily in the lab a direct neutralization assay using B cell supernatant was not feasible. Therefore development of HPeV3 specific antibodies was performed by detecting antibody binding to a monolayer of infected Vero cells. Positive cultures were single cell sorted to generate monoclonal B cells. Using this method we could generate three HPeV3 strain specific antibodies (AT12-015, AT12-017 and ΑΊΊ2-018). Although these antibodies are highly specific for HPeV3 we could only detect a reduction of 20 to 30% in viral titer with AT12-015 on both Vero and BGM cells (Figure 2A). In another experiment this neutralization was determined more carefully in Vero cells (upper panel), BMG cells (middle panel) and Caco2 cells (lower panel) with AT12-015 and HPeV3 strains 150237 and 1930 (Figure 2B). A trend towards in vitro neutralization of HPeV3 is observed. To test neutralization of several more common (currently circulating) viruses AT12-015 was tested on strains HPeV3 1930 (ctrl) and HPeV3-1595, -2736 and -1825 (Figure 2C). Again AT12-015 could reduce virus replication in an HPeV3 infection model, especially of isolate HPeV3- 1825, which was reduced by almost ½.

To determine antibody specificity we tested binding to PFA fixed monolayers of Vero and HT29 cells infected with different HPeV strains (Figure 3A to C). Except for HPeV3, AM 18 binds to HPeVl, -2 and -4 while AM28 binds to HPeVl and -2 and not HPeV3 and HPeV4 (Figure 3A); data that corresponds with the neutralization data. FITC staining is also clearly observed for the AT12-015, -017 and -018 antibodies that recognize two different strains of HPeV3 (Figure 3B and 3C).

ELISA and SPR Epitope Mapping

To determine the direct target of the different antibodies to HPeV we tested all antibodies in ELISA, using total virus lysate or recombinant expressed proteins (VP0, VP1 and VP3) as depicted in Figure 4A. Besides AM18, which showed high binding to lysates of purified virus samples of HPeVl, -2, -4, -5 and -6 and the VP1 protein of HPeVl and -4 all other antibodies did not react in ELISA assays (not shown). This strongly suggests that the epitope recognized by the antibodies is a non-linear structure (e.g. conformation dependent) in one of the surface proteins of HPeV. In addition, 12-mer peptides spanning the whole VPl protein of HPeVl were used to determine the specific binding region of AM 18. Both by ELISA (Figure 4B) and by SPR (Figure 5A) we found that the peptide that contained a RGD motif and several surrounding residues was specifically recognized (peptide number 85 sequence VTSSRALRGDMA) and to a lesser extent to the second peptide containing the RGD motif (peptide number 86; ALRGDMANLTNQ). The AM28 antibody showed no binding to the linear overlapping peptides in the ELISA (Figure 4C), strongly suggesting that the epitope recognized by it is a non-linear, conformational- dependent epitope, hence we progressed with three-dimensional epitope mapping on the intact virions.

SPR analysis could also confirm that AM18 is specific for HPeVl VPl (Figure 5B) and that AM28 and antibody D25 (negative control) do not bind to HPeVl VPl in the SPR assay. Binding to peptide number 86 (sequence ALRGDMANLTNQ), which contains the RDG motif, by AM18 was only found by ELISA while SPR signals (OD) were very weak. Likely, this is due to immobilization: in SPR the peptides are coupled to the streptavidin-coated chip via the biotin moiety that is attached to the N-terminus of the peptide; in ELISA, however, the peptides are randomly coated on a plate. The site- directed linking used for SPR would obscure the N-terminal amino acids in peptide 86 (these contain the AM18-epitope), causing the loss of binding signal. Then, this result also suggests that the amino acids directly N-terminal to the RGD-motif (VTSSRAL) contribute to binding of AM 18.

SPR analysis further demonstrated that AM 18 has high affinity for HPeVl VPl (Figure 5C); with a KD of 11.5 pM (Table 4).

Identification of epitopes on HPeVl capsids by labeling of the virus with AM28

The HPeVl AM28-labelled reconstruction showed additional density adjacent to the two-fold axis of symmetry corresponding to the Fab. in order to approximate the binding site, a homology model of the HPeVl cap si d was generated and compared to the reconstruction. The reconstruction statistics of HPeVl-AM28 Fab are summarized in Table 2. This showed that the antibody recognizes a conformational epitope which has contributions from both VPO and VPS. Thus the AM28 antibody binds to HPeVl capsids, either side of the two fold axes of symmetry (figure 8). The AM28 Fab does not overlap with the integrin binding site which is the binding site for AM 18 (Figures 8 and 9).

The AM28 antibody footprint sits across two protomers from adjacent pentamers in the capsid recognizing both VPO and VP3. The addition of AM28 to pentamers of

HPeVl did not cause a shift in the mobility of the pentamers relative to pentamers in the absence of antibody. Hence the conformation recognized by AM28 requires two adjacent protomers from two different pentamers, one contributing VPO, the other contributing VP3. Modelling of the HPeVl VPO and VP3 proteins indicates that amino acids in the following loops in HPeVl are involved in the footprint: VPO

PLSIPTGSANQ, VP3 FFPNATT and VP3 ATTAPQSIVH (Figure 10).

Comparison of HPeVl, HPeV2 and HPeV4 sequences for these three regions indicates that there is some sequence conservation between HPeVl and HPeV2 not seen in HPeV4 in these loop regions. The conservation at the nucleic acid level was compared for 22 HPeVl sequences available in the public databases (Figure 11).

In order to further approximate the AM28 binding site, a homology model of the HPeVl capsid was generated (Figure 12A) and subsequently superposed on the 20A resolution HPeVl-AM28 Fab reconstruction in a second analysis. The highest confidence model was obtained for VP3 with an I-TASSER based confidence score (C- score) of -0.38 followed by VPO with a C-score of - 1.60. In contrast, the VP1 had a C- score of only -3.77. The typically C-score ranges from -5 to 2 with high score means better confidence in the quality of modelling. In general, a C-score of - 1.5 means more than 90% of the quality predictions are correct, thus, the VP1 model was only used to constrain the fitting of VPO and VP3 in the asymmetric unit (Figure 12B-C). All the models had the characteristic eight-stranded β-barrels found in all picornaviruses capsid proteins (Figure 12C). Since the termini in picornaviruses are least conserved in the 3D conformation within the capsids and prediction was unreliable, we truncated the termini of the homology models. The placement of the individual capsid proteins within the capsid shell was improved using flexible fitting, resulting in improved fitting of the β-barrels and long helices of the models. In addition a model of the AM28 Fab variable region was also generated and fitted into the Fab density in the HPeVl-AM28 Fab reconstruction. This showed that the antibody recognizes a conformational epitope which has contributions from both VPO and VP3 (Figure 12D). Modelling and fitting of the HPeVl VPO, VP1 and VP3 proteins indicated that amino acids in the following loops in HPeVl were involved in the footprint: βΒ-βϋ (VPO), aA- βΌ (VPO), βΙ-βΗ (VPO), αΖ-βΒ (VP3), βΒ-βϋ (VP3) and βΕ-αΒ (VP3) (Figure 12D and E and summarized in Table 3 as underlined amino acids). These identified antigenic regions are distinct from linear epitopes of VPO and VP3 that have been described previously by peptide scanning (Table 3, amino acids indicated in italic). The fitting of the Fab variable region was unambiguous, with a cross correlation value of 0.88 compared to the 0.84 if the molecule was rotated by 180°. The distance between the two Fab molecules across the two-fold symmetry axis was on average about ~53A (Figure 12F). We compared amino acid sequence alignments of the six newly identified VPO and VP3 antigenic regions from different HPeVl isolates with those of HPeV2-6. They were well conserved in HPeVl, moderately conserved in HPeV2 and poorly conserved in HPeV3-6 (Figure 13) which explains why mAb AM28 cross-binds and cross- neutralizes HPeV2, but no reactivity was detected against HPeV3-6.

Mode of neutralization

In order to understand the mechanism of neutralization, we performed a capsid thermal stability assay using an RNA binding dye, which has previously been used to explain the mode of action of EV71 neutralizing antibodies (Plevka et al. 2014). The Tm of the HPeVl, HPeVl-AM18 and HPeVl-AM28 were 53°C, 54°C and 56°C respectively (Figure 14). The 3°C shift in Tm for HPeVl-AM28 compared to HPeVl alone indicates that the bivalent binding of AM28 across neighbouring pentamers stabilizes the capsid, inhibiting RNA release from the capsid. In contrast, a shift of 1°C in Tm for HPeVl-AM18 suggests that increasing the capsid stability is not the primary reason for neutralization by AM 18.

Table 1. Preferred HPeV specific antibodies according to the invention (CDR numbering according to Kabat et al. 1991)

30 AT12-018 Light chain CDR3 QQYNTYF

31 AM18 Heavy chain QVQFMQSGAEVKKPGASVKVSCKASGDTFSNFAVHWVRQAPGQRPEWMGWVNAGDSNLKYSQRFQGRLI ITRDTFAST

VYLDLSSLTSKDTAVYYCAGGNFGGNSATFDYWGQGTLVTVSS

32 AM28 Heavy chain EVQLLDSGGGLVQPGGSLRLSCAASGFPFSDYAMSWIRQAPGKGLEWVSPI SGSGGRAYYADSVKGRFTI SRDNSEGT

VYLQMNSLSPDDTALYYCAKDGSGSHYNNNYFDSWGQGTLVTVSS

33 AT12-015 Heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI SGGGDSRYYADSVKGRFTI SRDNSKNT

LYLQMNSLGAEDTALYYCAKRLGRVAEYYFDYWGQGTLVTVSP

34 AT12-017 Heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHEMNWVRQAPGKGLEWVSHITSGGNS IKYADSVKGRFTI SRDNAKSS

LYLQMNSLRAEDTAVYYCARILLGQQWLPTYYYYGMDVWGQGTTVTVSS

35 AT12-018 Heavy chain QEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSTSGTGAGTYYADSVKGRFTI SRDNSKNT

LYLQMNSLRAEDTAVYYCAKRLGRVAEYYFDYWGQGTLVTVSS

36 AM18 Light chain EIVLTQSPGTLSLSPGERATLSCRASQSVSDNYLAWYQQKPGQAPRLLIYGASHKSTGIPDRFSGSGSGTDFTLTI SR

LEPEDFAVYYCQQYGGSPLTFGGGTKVEIKRTVAA

37 AM28 Light chain EIVMTQSPATLSVSPGERATLSCRASQYVHSTLAWYQQKPGQAPRLLIYYASTRATGIPDRFSGSGSGTEFTLTI SSL

QSEDFAVYYCQQYNNWPYTFGQGTKLEIERTVAA

38 AT12-015 Light chain DIQMTQSPSTLSASVGDRVTITCRTSQSISNWLAWYQQKPGKAPKLLIYQASTLENGVPSRFTGSGSGTEFSLTISSL

QPDDFATYYCQQYNNYMALTFGGGTKVEIKRTVAA

39 AT12-017 Light chain DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSL

QPDDFATYYCQEYNNYPMCSFGQGTKLEIKRTVAA

40 AT12-018 Light chain DIQMTQSPSTLSASVGDRVTITCRASQSINNWLAWYQQKPGKAPKLLIYRASYLESGVPSRFSGSGSGTEFTLTISSL

QPDDFATYYCQQYNTYFALTFGGGTKVEIKRTVAA

41 AM18 Heavy chain CDR1 aac ttt get gtt cat

42 AM28 Heavy chain CDR1 gac tat gec atg age

43 AT12-015 Heavy chain CDR1 agt tat gec atg age

44 AT12-017 Heavy chain CDR1 agt cat gaa atg aac

45 AT12-018 Heavy chain CDR1 agt tat ggc atg agt

46 AM18 Heavy chain CDR2 tgg gtc aac get ggc gat agt aat eta aaa tat tea cag aga ttc cag ggc

47 AM28 Heavy chain CDR2 cct att agt gga agt ggt ggc agg gca tac tac gca gac tec gtg aag ggc egg

48 AT12-015 Heavy chain CDR2 get att agt ggt ggt ggt gat age aga tac tac gca gac tec gtg aag ggc

49 AT12-017 Heavy chain CDR2 cac att act agt ggt ggt aat tec ata aag tac gca gac tct gtg aag ggc

50 AT12-018 Heavy chain CDR2 act agt ggt act ggt get ggc aca tac tac gca gac tec gtg aag ggc

51 AM18 Heavy chain CDR3 gga aac ttc ggt ggt aac tec gca act ttt gac tac

52 AM28 Heavy chain CDR3 gat ggt teg ggg agt cac tac aat aac aac tac ttt gac tec

53 AT12-015 Heavy chain CDR3 agg ttg ggg cga gtg get gag tac tac ttt gac tac

54 AT12-017 Heavy chain CDR3 att etc ttg gga cag cag tgg ctg cca act tac tac tac tac ggt atg gac gtc

55 AT12-018 Heavy chain CDR3 aga ttg ggg cga gtg get gag tac tac ttt gac tac

56 AM18 Light chain CDR1 agg gec agt cag agt gtt age gac aac tac tta gee

57 AM28 Light chain CDR1 agg gec agt cag tat gtt cac age acc tta gec

58 AT12-015 Light chain CDR1 egg acc agt cag agt att agt aac tgg ttg gec

59 AT12-017 Light chain CDR1 egg gec agt cag agt att agt agt tgg ttg gec

60 AT12-018 Light chain CDR1 egg gee agt cag agt att aat aac tgg ttg gec

61 AM18 Light chain CDR2 ggt gca tec cac aag tec act

62 AM28 Light chain CDR2 tat gca tec acc agg gec act

63 AT12-015 Light chain CDR2 cag gcg tct act tta gaa aat

64 AT12-017 Light chain CDR2 aag gcg tct act tta gaa agt

65 AT12-018 Light chain CDR2 agg gcg tct tat tta gaa agt

66 AM18 Light chain CDR3 cag cag tat ggt ggc tea ccg

67 AM28 Light chain CDR3 cag cag tat aat aac tgg ccg

68 AT12-015 Light chain CDR3 caa cag tat aat aat tat atg

69 AT12-017 Light chain CDR3 caa gaa tat aat aat tac ccc

70 AT12-018 Light chain CDR3 caa caa tat aat act tat ttc

71 AM18 Heavy chain cag gtc cag ttt atg cag tct ggg get gag gtg aag aag cct ggg gec tea gtg aag gtt tec tgc aag gec tct ggt gac acc ttc agt aac ttt get gtt cat tgg gtg cgc cag gee ccc gga caa agg ccg gag tgg atg gga tgg gtc aac get ggc gat agt aat eta aaa tat tea cag aga ttc cag ggc aga etc att att acc agg gac act ttc gcg age aca gtc tac ctg gac ctg age age ctg aca tct aaa gac acg get gtc tat tac tgt gcg ggt gga aac ttc ggt ggt aac tec gca act ttt gac tac tgg ggc cag gga acc ctg gtc act gtc tec tea

72 AM28 Heavy chain gag gtg cag ctg ttg gac tct ggg gga ggc ttg gta cag cct ggg ggg tec ctg aga etc tec tgc gcg gec tct gga ttc ccc ttt age gac tat gec atg age tgg ate cgc cag get cca gga aag ggg ctg gag tgg gtc tea cct att agt gga agt ggt ggc agg gca tac tac gca gac tec gtg aag ggc egg ttc acc ate tec aga gac aat tec gag ggc acg gtg tat ctg caa atg aac age ctg age ccc gac gac acg gee eta tat tac tgt gcg aag gat ggt teg ggg agt cac tac aat aac aac tac ttt gac tec tgg ggc cag gga acc ctg gtc acc gtc tec tea

73 AT12-015 Heavy chain gag gtg cag ttg ttg gag tct ggg gga ggc ttg gta cag cct ggg ggg tec ctg aga etc tec tgt gca gec tct gga ttc acc ttt age agt tat gec atg age tgg gtc cgc cag get cca ggg aag ggc ctg gag tgg gtc tea get att agt ggt ggt ggt gat age aga tac tac gca gac tec gtg aag ggc egg ttc acc ate tec aga gac aat teg aag aac aca etc tat eta caa atg aac age ctg gga gec gag gac acg gee ctg tat tac tgt gcg aaa agg ttg ggg cga gtg get gag tac tac ttt gac tac tgg ggc cag gga

acc ctg gtc acc gtc tec cca

74 AT12-017 Heavy chain gag gtg cag ctg gtg gag tct ggg gga ggc ttg gta cag cct gga ggg tec ctg aga etc tec tgt gca gcc tct gga ttc acc ttc agt agt cat gaa atg aac tgg gtc cgc cag get cca ggg aag ggg ctg gag tgg gtt tea cac att act agt ggt ggt aat tec ata aag tac gca gac tct gtg aag ggc cga ttc acc ate tec aga gac aac gcc aag age tea ctg tat ctg caa atg aac age ctg aga gcc gag gac acg get gtt tat tac tgt gcg aga att etc ttg gga cag cag tgg ctg cca act tac tac tac tac ggt atg gac gtc tgg ggc caa ggg acc acg gtc act gtc tec tea

75 AT12-018 Heavy chain gag gtg cag ttg ttg gag tct ggg gga ggc ttg gta cag ccg ggg ggg tec ctg aga etc tec tgt gca gcc tct gga ttc acc ttt age agt tat ggc atg agt tgg gtc cgc cag get cca ggg aag ggg ctg gag tgg gtc tea act agt ggt act ggt get ggc aca tac tac gca gac tec gtg aag ggc egg ttc acc ate tec aga gac aat tec aag aac aca ctg tat ctg caa atg aac age ctg aga gcc gag gac acg gcc gtc tat tat tgt gcg aaa aga ttg ggg cga gtg get gag tac tac ttt gac tac tgg ggc cag gga acc ctg gtc acc gtc tec tea

76 AM18 Light chain gaa att gtg ttg acg cag tct cca ggc acc ctg tct ttg tct cca ggg gaa aga gcc acc etc tec tgc agg gcc agt cag agt gtt age gac aac tac tta gcc tgg tac cag cag aaa cct ggc cag get ccc egg etc ctt ate tat ggt gca tec cac aag tec act ggc ate cca gac agg ttc age ggc agt ggg tct ggg aca gac ttc act etc acc ate age aga ctg gag cct gaa gat ttt gca gtg tat tac tgt cag cag tat ggt ggc tea ccg etc act ttc ggc gga ggg acc aag gtg gag ate aaa cga act gtg get gca

77 AM28 Light chain gaa ata gtg atg acg cag tct cca gcc acc ctg tct gtg tct cct ggg gaa aga gcc acc etc tec tgc agg gcc agt cag tat gtt cac age acc tta gcc tgg tac cag cag aaa cct ggc cag get ccc agg etc etc ate tat tat gca tec acc agg gcc act ggt ate cca gac agg ttc agt ggc agt ggg tct ggg aca gag ttc act etc acc ate age age ctg cag tct gaa gat ttt gca gtt tat tac tgt cag cag tat aat aac tgg ccg tac act ttt ggc cag ggg acc aag ctg gag ate gag cga act gtg get gca

78 AT12-015 Light chain gac ate cag atg acc cag tct cct tec acc ctg tct gca tct gta gga gac aga gtc acc ate act tgc egg acc agt cag agt att agt aac tgg ttg gcc tgg tat cag cag aaa cca ggg aaa gcc cct aaa etc ctg ate tat cag gcg tct act tta gaa aat ggg gtc cca tea agg ttc acc ggc agt gga tct ggg aca gaa ttc agt etc acc ate age age ctg cag cct gat gat ttt gca act tat tac tgc caa cag tat aat aat tat atg gcg etc act ttc ggc gga ggg acc aag gtg gag ate aaa cga act gtg get gca

79 AT12-017 Light chain gac ate cag atg acc cag tct cct tec acc ctg tct gca tct gta gga gac aga gtc acc ate act tgc egg gcc agt cag agt att agt agt tgg ttg gcc tgg tat cag cag aaa cca ggg aaa gcc cct aaa etc ctg ate tat aag gcg tct act tta gaa agt ggg gtc cca tea agg ttc age ggc agt gga tct ggg aca gaa ttc act etc acc ate age

age ctg cag cct gat gat ttt gca act tat tac tgc caa gaa tat aat aat tac ccc atg tgc agt ttt ggc cag ggg acc aag ctg gag ate aaa cga act gtg get gca

80 AT12-018 Light chain gac ate cag atg acc cag tct cct tec acc ctg tct gca tct gta gga gac aga gtc acc ate act tgc egg gcc agt cag agt att aat aac tgg ttg gcc tgg tat cag cag aaa cca ggg aaa gcc cct aag etc ctg ate tat agg gcg tct tat tta gaa agt ggg gtc cca tct agg ttc age ggc agt gga tct ggg aca gaa ttc act etc acc ate age age ctg cag cct gat gat ttt gca act tat tac tgc caa caa tat aat act tat ttc gcg etc act ttc ggc gga ggg acc aag gtg gag ate aaa cga act gtg get gca

Table 2. Statistics of the HPeVl-AM28 Fab reconstruction.

Table 3. Mapping conformational epitopes for AM28 and linear epitopes from peptide scanning of sera on the capsid protein amino acid sequence.

Capsid Amino acid sequence*

protein

VPO METIKSIADMATGWSSVDSTINAVNEKVESVGNEIGGNLLTKVA

(Genbank DDASNILGPNCFATTAEPENKNWQATTTVNTT LT ff¾AP /»f id: L02971, PFSPDFSNS I⁾NFHSMAYDITTGDKNPSKLWLETiIIi:W rpSW AR amino GYQITHVELPKVFWD! -] :QpK PAYGQSRYFAAVRCGFHFQVQVNV acids 1- NQGTAGSALWYEPKPWTYDSKLEFGAFTNLPHVLMNLAETTQ 289) ADLCIPYVADTNYVKTDSSDLGQLKVYVWT Pi .A iPTGS ANQ V DVT

VP3 APNGKEXNWKKJMTMSTKYEWTRr^{^}^^

(Genbank TGAQSVALVGERAFYDPRTAGSKSRFDDLVKIAQLFSVM ADSTTl ' id: L02971, SENH GVDAKGYFKWSATIAPQSIVIIRNI LRLFPNLNVFVNSY amino SYFRGSLVLRLS ASTFNRGRLRMGFFPNATTDSTSTLDNAIYT acids 290- ICDIGA/} APAAA:PP¾A¾71! M/iKTNGHPIGLFQIEVLNRLTYNSS 542) SPSEVYCIVQGKMGQDARFFCPTGSWTFQ Table 4. Kinetic Constants of AM 18 binding to VPl.

k_a in 10⁴ sec^"1*M^"1, kd in 10 ⁵ sec ¹, KD in pM. Data is shown as mean ± SD, from at least two independent measurements

Antibody: ka. kd: KD:

AM 18 20.8 (± 1.8) 0.23 (± 0.05) 11.5 (± 3.3)

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Claims

1. An isolated, synthetic and/or recombinant antibody or functional part or equivalent thereof specific for human Parechovirus (HPeV) .

2. An antibody or functional part or equivalent according to claim 1 which is a monoclonal antibody.

3. An antibody or functional part or equivalent according to claim 1 which is specific for one or more HPeV subtypes selected from the group consisting of HPeVl, HPeVlb, HPeV2, HPeV4, HPeV5, HPeV6, HPeV7, HPeV8, HPeV9, HPeVlO, HPeVl 1, HPeV12, HPeV13, HPeV14, HPeV15 and HPeV16, preferably for at least two HPeV selected from said group.

4. An antibody or functional part or equivalent according to claim 1 which is specific for HPeV3.

5. An antibody or functional part or equivalent according to any one of claims 1-3 which specifically binds to an epitope of VPl comprising an amino acid sequence Arg- Gly-Asp, preferably wherein said epitope is located at amino acid positions of VPl corresponding to amino acid positions 222 to 224 of HPeVl VPl and corresponding to amino acid positions 764-766 of the HPeVl sequence as depicted in Figure 7.

6. An antibody or functional part or equivalent according to any one of claims 1-5, comprising:

- a heavy chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's: l-5, and/or

- a heavy chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's: l l-15, and/or - a light chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's: 16-20, and/or - a light chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's:21-25, and/or

- a light chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO's:26-30.

7. An antibody or functional part or equivalent according to any one of claims 1-6, having a heavy chain sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:31- 35 and/or having a light chain sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:36-40.

8. An isolated, synthetic and/or recombinant nucleic acid molecule comprising a nucleic acid sequence with a length of at least 15 nucleotides, or a functional equivalent thereof, encoding at least one CDR sequence of an antibody or functional part or equivalent according to any one of claims 1-7.

9. A nucleic acid molecule or functional equivalent according to claim 8, comprising:

- a heavy chain CDR1 sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 41- 45, and/or

- a heavy chain CDR2 sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 46- 50, and/or

- a heavy chain CDR3 sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51- 55, and/or

- a light chain CDR1 sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 56- 60, and/or

- a light chain CDR2 sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 61- 65, and/or - a light chain CDR3 sequence comprising a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 66- 70.

10. A vector comprising a nucleic acid molecule or functional equivalent according to any one of claims 8-9.

11. A pharmaceutical composition comprising an antibody or functional part or equivalent according to any one of claims 1-7, or a nucleic acid molecule or functional equivalent according to any one of claims 8-9, or a vector according to claim 10, and a pharmaceutically acceptable carrier, diluent and/or excipient.

12. A kit of parts comprising an antibody or functional part or equivalent according to claim 3 and an antibody or functional part or equivalent according to claim 4.

13. An antibody or functional part or equivalent according to any one of claims 1-7 for use in diagnosis, preferably of a HPeV infection.

14. A method for determining whether a HPeV is present in a sample comprising:

- contacting said sample with an antibody or functional part or equivalent according to any one of claims 1-7,

- allowing said antibody or functional part or equivalent to bind HPeV, if present, and

15. An antibody or functional part or equivalent according to any one of claims 1-7, or a nucleic acid molecule or functional equivalent according to claims 8-9, or a vector according to claim 10 for use as a medicament and/or prophylactic agent, preferably for use in a method for the treatment or prevention of a HPeV infection and/or a disorder caused by a HPeV infection, preferably wherein said disorder is selected from the group consisting of inflammation, fever, diarrhea, sepsis, meningitis, encephalitis and paralysis.

16. An antibody or functional part or equivalent according to any one of claims 1-3 that specifically binds to an epitope of HPeV comprising a groove formed by VPO and VP3.

17. An antibody or functional part or equivalent according to claim 16 wherein said epitope comprises at least one amino acid from an amino acid sequence corresponding to amino acid positions 250 to 260 of the HPeVl sequence as depicted in Figure 7, at least one amino acid from an amino acid sequence corresponding to amino acid positions 448 to 454 of the HPeVl sequence as depicted in Figure 7 and at least one amino acid from an amino acid sequence corresponding to amino acid positions 393 to 402 of the HPeVl sequence as depicted in Figure 7, preferably said epitope comprises at least one amino acid from an amino acid sequence corresponding to amino acid positions 123 to 150 of the HPeVl sequence as depicted in Figure 7, at least one amino acid from an amino acid sequence corresponding to amino acid positions 148 to 152 of the HPeVl sequence as depicted in Figure 7, at least one amino acid from an amino acid sequence corresponding to amino acid positions 250 to 262 of the HPeVl sequence as depicted in Figure 7, at least one amino acid from an amino acid sequence corresponding to amino acid positions 393 to 402 of the HPeVl sequence as depicted in Figure 7, at least one amino acid from an amino acid sequence corresponding to amino acid positions 371 to 382 of the HPeVl sequence as depicted in Figure 7 and at least one amino acid from an amino acid sequence corresponding to amino acid positions 448 to 457 of the HPeVl sequence as depicted in Figure 7.

18. An antibody or functional part or equivalent according to claim 16 or 17 wherein said epitope comprises at least one amino acid from the amino acid sequence PLSIPTGSANQ of HPeVl VPO, at least one amino acid from the amino acid sequence FFPNATT of HPeVl VP3 and at least one amino acid from the amino acid sequence ATTAPQSIVH ₀f HPeVl VP3, preferably said epitope comprises at least one amino acid from an amino acid sequence corresponding to amino acid sequence of

HEWTPSWA of HPeVl VPO, at least one amino acid from an amino acid sequence corresponding to amino acid sequence of HQDKP of HPeVl VPO, at least one amino acid from an amino acid sequence corresponding to amino acid sequence of PLSIPTGSANQVD of HPeVl VPO, at least one amino acid from an amino acid sequence corresponding to the amino acid sequence MADSTTPSENHG of HPeVl VP3, at least one amino acid from an amino acid sequence corresponding to the amino acid sequence ATTAPQSIVH of HPeVl VP3 and at least one amino acid from an amino acid sequence corresponding to the amino acid sequence FFPNATTDST of HPeVl VP3.