WO2015037992A1 - Anticorps spécifiques du parechovirus - Google Patents

Anticorps spécifiques du parechovirus Download PDF

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WO2015037992A1
WO2015037992A1 PCT/NL2014/050624 NL2014050624W WO2015037992A1 WO 2015037992 A1 WO2015037992 A1 WO 2015037992A1 NL 2014050624 W NL2014050624 W NL 2014050624W WO 2015037992 A1 WO2015037992 A1 WO 2015037992A1
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sequence
amino acid
antibody
hpevl
hpev
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PCT/NL2014/050624
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Adrianus Quirinus Bakker
Tim Beaumont
Brenda Mirande WESTERHUIS
Klazine Christine WOLTHERS
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Aimm Therapeutics B.V.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1009—Picornaviridae, e.g. hepatitis A virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to the fields of biology, immunology and medicine.
  • 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.
  • 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.
  • 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
  • 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).
  • CRE cis- acting replication element
  • 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).
  • RGD arginine-glycine-aspartic acid
  • 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).
  • CAV9 Coxsackievirus A9
  • FMDV foot-and-mouth disease virus
  • characterized genotypes are lacking the RGD motif.
  • Parechovirus 1 Parechovirus 1
  • HPeVl Parechovirus 1
  • enterovirus infection 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.
  • HPeV3 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).
  • CSF cerebrospinal fluid
  • 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.
  • IVIg intravenous immunoglobulin
  • the present invention for the first time discloses fully human monoclonal antibodies that are specific for human Parechovirus. Antibodies directed against multiple antibodies
  • 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”.
  • antibody refers to an antigen binding protein comprising at least a heavy chain variable region (Vh) that binds to a target epitope.
  • Vh heavy chain variable region
  • 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.
  • 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.
  • an antibody referred to herein as being specific for HPeV is also specific for said other virus comprising the same epitope.
  • 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).
  • Enteroviruses such as human Enteroviruses and Echovirus
  • adenovirus yellow fever virus
  • Kaposi's sarcoma- associated herpesvirus (KSHV) and FMDV foot-and-mouth disease virus.
  • 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.
  • binding 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.
  • Parechovirus subtype refers to genetically different Parechoviruses.
  • Parechovirus subtypes are HPeVl, HPeV2, HPeV3, HPeV4, HPeV5, HPeV6, HPeV7, HPeV8, HPeV9, HPeVIO, HPeVll, HPeV12, HPeV13, HPeV14, HPeV15 and HPeV16.
  • HPeV strain 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).
  • 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 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.
  • HPeVl a viral protein of at least one HPeV subtype.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HPeV3 which is associated with severe life-threatening disease
  • HPeV subtypes which are usually associated with mild disease.
  • 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.
  • 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.
  • 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.
  • kits 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.
  • 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.
  • 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).
  • tTG tissue transglutaminase
  • 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.
  • antibody AM28 does not recognize isolated VP proteins and linear peptides, but binds to a conformational epitope.
  • 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.
  • RGD motif 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 sub
  • 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 .
  • antibody AM 18 specifically recognizes the VPl protein.
  • AM 18 binds an epitope comprising the RGD motif.
  • 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 also specifically binds to other viruses comprising the same epitope or amino acid sequence.
  • RGD amino acid sequence
  • examples of such other viruses comprising an RGD domain are Coxsackievirus, in particular Coxsackievirus A9, other viruses comprising an RGD domain.
  • Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma-associated herpesvirus (KSHV), and FMDV.
  • a virus other than HPeV which comprises a viral protein comprising an RGD motif.
  • virus other than HPeV is preferably selected from the group consisting of Coxsackievirus, in particular Coxsackievirus A9, other viruses.
  • Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma-associated herpesvirus (KSHV) and FMDV.
  • Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma-associated herpesvirus (KSHV) and FMDV.
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • FMDV Kaposi's sarcoma-associated herpesvirus
  • an antibody or functional part or equivalent according to the invention that specifically binds to the same epitope as antibody AM 18.
  • an antibody or functional part or equivalent according to the invention that competes with antibody AM 18 for specific binding to HPeV VP1.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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,
  • 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.
  • 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.
  • 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.
  • AM18 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:
  • l-5 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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%.
  • AT12-015 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%.
  • AT12-017 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 are 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.
  • 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%.
  • AT12-018 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%.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • conservative amino acid substitution examples 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.
  • an antibody or functional part or equivalent 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.
  • a nucleic acid molecule encoding a CDR sequence according to the invention is mutated, for instance using random - or site-directed - mutagenesis.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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, .
  • 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,
  • KD dissociation constant
  • 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.
  • 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.
  • 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.
  • 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.
  • a nucleic acid molecule or nucleic acid sequence of the invention preferably comprises a chain of nucleotides, more preferably DNA and/or RNA.
  • 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.
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • ribozyme a nucleic acid sequence
  • 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.
  • 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 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.
  • said nucleic acid molecule comprises at least one CDR encoding sequence.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • said producer cell line is suitable for producing antibodies for use in humans.
  • said producer cell line is preferably free of pathogenic agents such as pathogenic micro-organisms.
  • antibodies consisting of human sequences are generated using a nucleic acid molecule or vector according to the invention.
  • 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 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.
  • an antiviral agent refers to any substance that reduces or blocks the function, or growth, of a virus and/or causes destruction of a virus.
  • a moiety that is coupled to an antibody according to the invention is an antimicrobial peptide.
  • antimicrobial peptide 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.
  • 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.
  • CRAMP cathelicidin-related antimicrobial peptide
  • 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.
  • 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.
  • 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.
  • LPETGG sortase recognition site
  • Another example is a thioether linkage, whereby an antibody according to the invention is coupled to another moiety via such thioether linkage.
  • 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
  • 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.
  • scFv single chain variable fragments
  • Fab fragments bispecific Fab fragments
  • F(ab')2 fragments bispecific F(ab')2 fragments.
  • 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.
  • 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.
  • 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.
  • two antibodies according to the invention are coupled to each other.
  • sortase catalized transamidation which is described herein before and in detail in WO 2010/087994.
  • 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.
  • both said antibodies are preferably engineered as described herein before and in detail in WO 2010/087994.
  • 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.
  • 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,
  • determining whether an individual is suffering from a Parechovirus infection and/or from a disorder caused by a Parechovirus infection Preferably it is determined whether an individual is suffering from a Parechovirus infection and/or from a disorder caused by a Parechovirus infection.
  • a method for determining whether an individual is suffering from a Parechovirus infection and/or from a disorder caused by a Parechovirus infection comprising:
  • 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.
  • said individual is a human.
  • 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.
  • 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.
  • 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.
  • antibody AT12-015 comprising a heavy chain sequence of SEQ ID NO:33 and a light chain sequence of
  • 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.
  • 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
  • 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.
  • such sample is a faeces sample, a saliva sample or swab, or a alveolar sample, lavage or swab.
  • a sample is from an individual, preferably a human, that is suspected of suffering from a Parechovirus infection.
  • HPeV3 which is associated with severe life -threatening disease
  • 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.
  • 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.
  • 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.
  • 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 are 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.
  • 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 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 viruses comprising an RGD domain.
  • Enteroviruses such as human Enteroviruses and Echovirus, adenovirus, yellow fever virus, Kaposi's sarcoma-associated herpesvirus (KSHV) and FMDV.
  • 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).
  • ELISA enzyme-linked immunosorbent assays
  • RIA radio-immuno assays
  • the antibody can be labelled directly and immune complexes of antibody and antigen can be detected via the label.
  • labels which can be used include enzymes, fluorescent compounds, radioisotopes, chemiluminescent compounds and bioluminescent compounds.
  • 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.
  • an antibody according to the invention for use as a medicament and/or prophylactic agent.
  • 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.
  • a nucleic acid molecule according to the invention When 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 may also specifically bind to other viruses comprising the same epitope or amino acid sequence.
  • 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.
  • 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.
  • 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.
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • FMDV Kaposi's sarcoma-associated herpesvirus
  • 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.
  • 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.
  • suitable carriers for instance comprise a solution, like for example saline, keyhole limpet haemocyanin (KLH), serum albumin (e.g. BSA or RSA) and ovalbumin.
  • KLH keyhole limpet haemocyanin
  • serum albumin e.g. BSA or RSA
  • ovalbumin 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.
  • 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.
  • a liquid carrier such as fatty oil.
  • 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.
  • an RGD motif preferably selected from the group consisting of Coxsackievirus, in particular Coxsackievirus A9, other Enteroviruses such as human Enteroviruses and Echovirus, a
  • “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.
  • 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.
  • 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.
  • FIG. 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 ⁇ g/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 .
  • FIG. 4 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 ⁇ g/ml antibody is injected on a SPR chip coupled with a peptide library containing the entire sequence of HPeVl-VPl protein.
  • AM18 captured on a spot coated with 5.0 ⁇ g/ml biotin-anti-IgG. Curve fits (1: 1 binding model) are shown as grey lines.
  • 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.
  • FIG. 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).
  • FIG. 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.
  • 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.
  • 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
  • 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).
  • 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.
  • 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).
  • PFA paraformaldehyde
  • 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 ⁇ g/ml) (Riemer) and ampicillin (0.1 ⁇ g/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.
  • FCS Fetal Calf Serum
  • 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).
  • Black clear round bottom 96 wells plate (Greiner) with Vero or HT29 cells line were inoculated with a virus solution in EMEM 8% FCS.
  • CPE cytopathic effect
  • PFA paraformaldehyde
  • 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.
  • 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.
  • the supernatant (20 ⁇ ) 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 ⁇ elution buffer and reverse transcribed as previously described. 5 ⁇ 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).
  • 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 StarTM (DE3) One Shot® cells.
  • Single bacterial colony was inoculated in LB medium supplemented with 100 ⁇ g/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).
  • IPTG isopropyl ⁇ -d-thiogalactopyranoside
  • ELISA plates were coated with 200ng HPeV purified virus, or the HPeV capsid proteins VPO, VPl and VP3 in ⁇ 50mM NaHCo3 overnight at room temperature (RT).
  • the plate was washed 3x with PBS-tweenO.05%.
  • To avoid unspecific binding wells were blocked with 300 ⁇ 1 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%.
  • 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.
  • 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.
  • SPR surface plasmon resonance
  • the peptide library used in this experiment consists of 86 12-mer peptides, containing the entire sequence of the Parechol-VPl protein.
  • 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 are 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
  • 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).
  • 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 ⁇ g/ml) followed by a 2.0 ⁇ g/ml VPl-solution. Kinetic constants were fitted to the binding curves, using a 1: 1 binding model.
  • the Fab fragments from AM28 were produced using a PierceTM 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.
  • 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 ⁇ g/ml) and ampicillin (0.1 ⁇ g/ml), supplemented with 8% heat-inactivated Fetal Calf Serum (FCS).
  • EMEM Eagle's Minimum Essential Medium
  • FCS heat-inactivated Fetal Calf Serum
  • HT29 cells were cultured -90% confluent in a T175 flask before being infected with HPeVl-Harris at a MOI 0.1.
  • 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).
  • 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
  • PDB id lqqp and lbbt
  • Poliovirus 1 Poliovirus 1
  • PB id Poliovirus 1
  • PB id Poliovirus 1
  • PB id Poliovirus 1
  • PB id Poliovirus 1
  • PB id Poliovirus 1
  • PB id Poliovirus 1
  • PB id Poliovirus 1
  • PB id Poliovirus 1
  • PB id lpov
  • PB id Bovine enterovirus
  • PB id Theiler murine encephalomyelitis virus
  • PB id Poliovirus 1 mahoney strain
  • PB id Poliovirus 1 mahoney strain
  • VP1 Triatoma virus
  • PDB id Triatoma virus
  • 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).
  • Poliovirus 1 Poliovirus 1
  • PDB id Human enterovirus 71
  • PDB id Human enterovirus 71
  • PB id 3vbh
  • Equine rhinitis A virus PB id: 2xbo
  • Coxsackievirus A9 PB id: ld4m
  • Human rhinovirus 16 PB 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.
  • 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).
  • PDB ID: 3NAP triatoma virus
  • PB ID: 1D3I human rhinovirus 14
  • PB ID: 1B35 cricket paralysis virus
  • PB ID: 4EJR rabbit hemorrhagic disease virus
  • echovirus 7 (PDB ID: 1M11) and bovine enterovirus (PDB ID: lBEV).
  • 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).
  • 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.
  • 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
  • HPeV5 GenBank ID: AF055846
  • GenBank IDs for HPeVl are JX441355, JX575746, S45208, EF051629, FJ840477, GQ 183035, GQ 183034, HQ696574, HQ696572, HQ696570, HQ696573,
  • the alignment was visualized with Jalview (Hadfield et al. 1997).
  • 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.
  • reaction volumes were set up per well in a 96-well PCR plate and each reaction contained 2.5 ⁇ of 200X Sybr Safe DNA gel stain (Invitrogen, also binds RNA) and the protein sample which was one of the following HPeVl (10 ⁇ of lmg/ml stock), AM18 (10 ⁇ of 2 mg/ml stock), AM28 (10 ⁇ of 2 mg/ml stock), HPeVl-AM18 complex (20 ⁇ ) or HPeVl-AM28 complex (20 ⁇ ).
  • the total volume was made up to 25 ⁇ 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).
  • 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.
  • 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.
  • HPeV3 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 ⁇ 2-018). 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).
  • 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.
  • 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.
  • 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.
  • HPeVl did not cause a shift in the mobility of the pentamers relative to pentamers in the absence of antibody.
  • 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
  • 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 ⁇ -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 ⁇ -barrels and long helices of the models.
  • Table 3 Mapping conformational epitopes for AM28 and linear epitopes from peptide scanning of sera on the capsid protein amino acid sequence.
  • Pandurangan AP Shakeel S, Butcher S, Topf M. Combined approaches to flexible fitting and assessment in virus capsids undergoing conformational change. Journal of Structural Biology. 2014 185: 427-439.
  • Neutralizing antibodies can initiate genome release from human enterovirus 71. Proc Natl Acad Sci U S A 111: 2134-2139.

Abstract

L'invention concerne des anticorps isolés, synthétiques ou recombinés et des parties fonctionnelles de ceux-ci, spécifiques du parechovirus, ainsi que des séquences d'acides nucléiques codant ces anticorps. L'invention concerne également l'utilisation de ces anticorps dans le diagnostic d'une infection à parechovirus et un médicament et/ou un agent prophylactique servant à traiter ou atténuer au moins partiellement les symptômes d'une infection à parechovirus.
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