ANTIBODIES AND PEPTIDES TO TREAT HCMV RELATED DISEASES

Abstract

The present invention relates to the treatment of HCMV relates diseases. The inventors conducted a study to find an essential domain of pUL56 for its interaction with pUL89 which is important in the effect of the CMV. Sequences alignments allowed them to predict one sequence in C-terminal of pUL56 potentially necessary for interaction with pUL89. BAC mutagenesis and AlphaLISA technologies using purified proteins allowed to validate that the short sequence .sub.671WMVVKYMGFF.sub.680 (SEQ ID NO: 1) in C-terminal of pUL56 is involved in interaction with pUL89. Knowing this important information, antibodies directed against this sequence or peptides derived from this sequence could be useful to invalidate the interaction of pUL56 to pUL89 and thus to treat HCMV related diseases. Thus, the present invention relates to an isolated anti-pUL56 antibody, binding to the SEQ ID NO:1 or a peptide comprising the amino acids sequence: WMVVKYMGFF (SEQ ID NO: 1) or a function-conservative variant thereof for use in the treatment of HCMV related diseases.

Claims

1. An isolated anti-pUL56 antibody, wherein said antibody binds to an epitope of the pUL56 protein comprising residues 671 to 680 of the amino acid sequence SEQ ID NO:2, or an antigen binding fragment thereof.

2. The isolated anti-pUL56 antibody of claim 1, wherein said antibody is a chimeric or a humanized antibody.

3. The isolated anti-pUL56 antibody of claim 1 wherein the antigen binding fragment is selected from the group consisting of a Fab, a F(ab)2, a single domain antibody, a ScFv, a Sc(Fv)2, a diabody, a triabody, a tetrabody, a unibody, a minibody, a maxibody, and a small modular immunopharmaceutical (SMIP).

4. (canceled)

5. A nucleic acid molecule encoding an antibody according to claim 1, or an antigen binding fragment thereof.

6. An isolated peptide comprising the amino acid sequence of formula (I):
W-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-F-Xaa8, wherein: Xaa1, Xaa2, Xaa3, Xaa4, Xaa6 and Xaa7 are selected from the group consisting of: Alanine (A), Arginine (R), Asparagine (N), Aspartic acid (D), Cysteine (C), Glutamic acid (E), Glutamine (Q), Glycine (G), Histidine (H), Isoleucine (I), Leucine (L), Lysine (K), Methionine (M), Phenylalanine (F), Proline (P), Serine (S), Threonine (T), Tryptophan (W), Tyrosine (Y), Valine (V), allyl glycine (AllylGly), norleucine, norvaline, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2-naphtylalanine (2-Nal), ornithine (Orn) and pentafluorophenylalanine; and Xaa5 and Xaa8 are Phenylalanine (F) or Tyrosine (Y).

7. The isolated peptide according to claim 6 wherein the isolated peptide comprises the amino acid sequence WMVVKYMGFF (SEQ ID NO: 1) or a function-conservative variant thereof.

8. The isolated peptide according to claim 6 wherein the isolated peptide comprises the amino acid sequence: TABLE-US-00002 (SEQIDNO:3) WMVVKFMGFF, (SEQIDNO:4) WMVVKFMGFY, (SEQIDNO:16) SRKTDWTVSKFRGFYDFSTI or (SEQIDNO:5) WMVVKYMGFY.

9. The isolated peptide according to claim 7 wherein the function-conservative variant differs from SEQ ID NO: 1 by 1, 2 or 3 amino acids.

10. The isolated peptide according to claim 7 wherein the function-conservative variant has at least 95% identity to SEQ ID NO: 1.

11-12. (canceled)

13. A therapeutic composition comprising an isolated anti-pUL56 antibody, wherein said antibody binds to an epitope of the pUL56 protein comprising residues 671 to 680 of the amino acid sequence SEQ ID NO:2; or an isolated peptide comprising the amino acid sequence of formula (I): W-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-F-Xaa8, wherein: Xaa1, Xaa2, Xaa3, Xaa4, Xaa6 and Xaa7 are selected from the group consisting of: Alanine (A), Arginine (R), Asparagine (N), Aspartic acid (D), Cysteine (C), Glutamic acid (E), Glutamine (Q), Glycine (G), Histidine (H), Isoleucine (I), Leucine (L), Lysine (K), Methionine (M), Phenylalanine (F), Proline (P), Serine (S), Threonine (T), Tryptophan (W), Tyrosine (Y), Valine (V), allyl glycine (AllylGly), norleucine, norvaline, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2-naphtylalanine (2-Nal), ornithine (Orn) and pentafluorophenylalanine; and Xaa5 and Xaa8 are Phenylalanine (F) or Tyrosine (Y).

14. A method for treating a HCMV related diseases in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an isolated anti-pUL56 antibody, wherein said antibody binds to an epitope of the pUL56 protein comprising residues 671 to 680 of the amino acid sequence SEQ ID NO:2; or an isolated peptide comprising the amino acid sequence of formula (I): W-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-F-Xaa8, wherein: Xaa1, Xaa2, Xaa3, Xaa4, Xaa6 and Xaa7 are selected from the group consisting of: Alanine (A), Arginine (R), Asparagine (N), Aspartic acid (D), Cysteine (C), Glutamic acid (E), Glutamine (Q), Glycine (G), Histidine (H), Isoleucine (I), Leucine (L), Lysine (K), Methionine (M), Phenylalanine (F), Proline (P), Serine (S), Threonine (T), Tryptophan (W), Tyrosine (Y), Valine (V), allyl glycine (AllylGly), norleucine, norvaline, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2-naphtylalanine (2-Nal), ornithine (Orn) and pentafluorophenylalanine; and Xaa5 and Xaa8 are Phenylalanine (F) or Tyrosine (Y).

Description

FIGURES

[0133] FIG. 1: A. Structure of HCMV terminase subunit pUL56 according to Champier et al., 2008 with a putative leucine zipper pattern annotated as pUL56-LZ. B. Sequences alignment of conserved regions from 21 herpesviruses. Sequence numbering is consistent with that of AD169 residues. Key residues are highlighted as white letters on a black background. SSP: Secondary structure prediction of pUL56-LZ (h= helix).

[0134] FIG. 2: Determination of pUL56 binding domains for the interaction with pUL89 Alpha assay results. The Alpha assay for the binding of full-length pUL89 (His-pUL89, 1.5E+03 nM) was performed with 5E+02 nM wild-type pUL56 (HA-pUL56) or a deletion mutant of pUL56 (HA-pUL56 Del W671-F680). As a negative control, proteins were used alone and a reaction was performed without proteins (mock). Two measures for each reaction were performed in duplicate.

EXAMPLE

Material & Methods

Identification of Conserved Patterns and Secondary Structure Prediction

[0135] The pUL56 amino acid sequence of reference strain AD169 (Chee et al., 1990) was aligned with the sequences of 21 homologous proteins from other herpesviruses, as described in supplementary table 1. Alignments were performed with Clustal Omega () multiple sequence alignment (MSA) tool provided by the EMBL-EBI bioinformatics web and programmatic tools framework (Sievers et al., 2011) (McWilliam et al., 2013) (Li et al., 2015). Secondary structure prediction was carried by Phyre2 web portal (Kelley et al., 2015).

Cells and Bacterial Strains

[0136] Human fibroblasts MRC-5 (Biomerieux, France) were cultivated at 37 C. in 5% CO.sub.2 and grown in minimal essential medium (MEM) containing 10% fetal bovine serum with antibiotics. HEK293 (ATCC CRL-1573) were cultivated at 37 C. in 5% CO.sub.2 and grown in minimal essential medium (MEM) containing 10% fetal bovine serum with antibiotics. E. coli strain DH5 and Stellar (Clontech, USA) were used for cloning procedures. E. coli strain GS1783 was used for BAC mutagenesis (Borst et al., 1999).

BAC Mutagenesis and Reconstitution of Mutant Viruses

[0137] Conserved domains were deleted by en passant mutagenesis, a two-step markerless Red recombination system for BAC mutagenesis in E. coli strain GS1783. UL56 point mutations were introduced into an EGFP-expressing HCMV-BAC (Borst et al., 1999) to generate several mutants (primers used for mutagenesis are described in Table I). Presence of mutations in UL56 gene of each virus was confirmed by sequencing prior to transfection. The HCMV-BAC contains an enhanced green fluorescent protein (EGFP) gene in the unique short region and was derived from parental strain pHB5, a BAC-cloned genome of the CMV laboratory strain AD169 (Borst et al., 1999). The impact of different mutations on viral growth was assessed using transfection of mutated HCMV-BAC into human fibroblasts MRC-5 using liposomal reagent Transfast (Promega, USA) following manufacturer's instructions (Hantz et al., 2013).

Library Construction and Whole-Genome DNA Sequencing

[0138] After HCMV-BAC preparation, amplicons were purified using magnetic beads (Agencourt AMPure XP) and fragmented using the Ion Xpress Plus DNA Fragment Library Preparation kit (Life Technologies). Barcodes adapters were ligated to fragment ends and 250 bp fragments were collected. The library was PCR amplified, then sequenced on the Ion Proton with the Ion Sequencing kit (Life Technologies). Bases callings were performed with Torrent Suite Software version 5.0.2. Mutations were obtained using Torrent Variant Caller using Somatic variant frequency and AD169_ATCC as reference. Mutations were then filtered against reference (Wild-type HCMV-BAC) using vcftools version 0.1.13.

Viral Immediate Early and Late Protein Expression

[0139] A transfection of mutated HCMV-BAC into human fibroblasts MRC-5 using liposomal reagent Transfast (Promega, USA) was performed. Cells were fixed at 5 days post transfection, and immunostaining was performed for viral immediate early (anti-IE1 antibody; Argene, France) and late (anti-gB antibody; Abcam, United Kingdom) proteins in transfected cells.

Plasmids Construction for Alpha Analysis

[0140] For protein production, the SC784 expression plasmid encoding full-length amino-terminal 3xHA-tagged pUL56 and driven by an upstream HCMV major immediate early promoter was cloned in vector pGEM3z. In-Fusion (Clontech, USA) kit was used following manufacturer's instructions to clone several UL56 mutants from source HCMV-BAC in SC784 plasmid. ORF encoding pUL89 is composed of two exons separated by an intron. Both exons were generated by assembling PCR from AD169 genotype and cloned into pCI-neo (Promega, USA) with His tag to obtain pCI-neo His-pUL89. Transformations were performed in DH5 cells. The nucleotide sequence of all constructs generated was verified by Sanger sequencing prior to use.

Transfection and Proteins Purification

[0141] HEK293 were transfected with the appropriate expression vectors by use liposomal reagent Transfast following manufacturer's instructions, washed and lysed 48 h later with CelLytic M (Sigma-Aldrich, USA). Lysates were cleared by centrifugation.

[0142] For purification of HA-tagged pUL56, the cell-free reaction was performed with Anti-HA Immunoprecipitation Kit according to the manufacturer's protocol (Sigma-Aldrich, USA).

[0143] For purification of His-tagged pUL89, the cell-free reaction was performed with Ni resin (Clontech, USA).

[0144] All proteins were concentrated approximately 5-fold using Pall centrifugal filters (Pall, USA), and protein concentration was determined by the Bradford method using bovine serum albumin (Sigma-Aldrich, USA) as standard protein

Western Blotting

[0145] SDS-PAGE was performed under reducing conditions on Mini-PROTEAN TGX Stain-Free gels (BioRad, USA). Proteins were then transferred onto a Trans-Blot Turbo PVDF Western blotting membrane (BioRad, USA). Antibody dilutions were 1:1,000 for the mouse anti-HA antibody (catalogue number: 2367, Cell Signaling, USA), mouse anti-His antibody (catalogue number: 2366, Cell Signaling, USA) and secondary anti-mouse horseradish peroxidase (HRP)-linked antibody (catalogue number: 7076, Cell Signaling, USA). Signals were visualized using the Substrat HRP Immobilon Western (Merck Millipore, USA) and a ChemiDoc imager (BioRad, USA).

Protein/Protein Interaction Analysis by Alpha

[0146] Alpha (Amplified Luminescent Proximity Homogeneous Assay) experiments were conducted according to the manufacturer's protocol (PerkinElmer, USA). Five L of transfected MRC-5 lysate with HCMV-BAC is first disposed in wells of a 96-well AlphaPlate. The final concentration of each proteins was optimized to obtain the best value of interaction. Ten L of each purified protein were combined (to give a final assay concentration of 500 nM of 3xHA-pUL56 and 1.5 M of 6xHis-pUL89). Ten L and 15 L of 10 mg/mL of donor beads and acceptor beads, respectively, were added and incubated for 1 hour. Plates were read on a PerkinElmer EnVision plate reader using an excitation wavelength of 680 nm and emission detection was set at 615 nm.

Results

A Putative Conserved Protein Interface in pUL56 Subunit

[0147] Selection of a potent pUL56 fragment for pUL89 interaction was supported by three hints. First, based on the sequences alignment of pUL56 with 20 herpesviruses homologues (FIG. 2), the peptide .sub.671WMVVKYMGFF.sub.680 (pUL56(671-680) seems to be broadly conserved in betaherpesviruses proteins, which supported a major role either in function or structure of pUL56. Secondly, its secondary structure is predicted as an alpha helix. Previous studies demonstrated that the peptide pUL89(580-600) implicated in the pUL56-pUL89 interface (Thoma et al., 2006) adopts an alpha helix secondary structure (Couvreux et al., 2010; Nadal et al., 2010). Moreover, wide protein-protein interfaces analyses revealed a preferential interaction of an helix of one protein with one of its counterpart (Eilers et al., 2002; Ansari and Helms, 2005). Thirdly, pUL56(671-680) is within the C-terminal part previously described to be sufficient for interaction with pUL89 (Hwang and Bogner, 2002). Interestingly, this motif belongs to the pUL56 region carrying the ATP binding site. As a parallel, pUL89(580-600) is enclosed into the endonuclease domain of pUL89 (Nadal et al., 2010). Both activities, ATPase of pUL56 on the one hand and nuclease of pUL89 on the other hand are dependent on the association between the two terminase subunits (Hwang and Bogner, 2002; Scheffczik et al., 2002). Taken together, these observations make pUL56(671-680) a good candidate to interact with pUL89.

A Deletion or Targeted Mutations of .SUB.671.WMVVKYMGFF.SUB.680 .(SEQ ID NO:1) pUL56 Domain Affects Viral Replication in MRC-5 Cells

[0148] To evaluate the importance of the pUL56 predicted domain for viral replication, we produced by en passant mutagenesis recombinant EGFP-virus with complete deletion of UL56(671-680) or point mutations in this sequence. Analyse of HCMV genome confirmed that UL56 have no gene on the other strand (Bradley et al., 2009). Thus, mutations in the virus are silent on the other strand and thus cannot impact the function of another gene expressed from the other strand. To ensure that no other mutations that could have a negative impact on viral replication was introduced in the BAC backbone during the manipulations, we performed NGS sequencing on both the mutant and the original BAC. The deletion was found in 100% of the mutant BAC sequences whereas other SNPs were located in genes non essential for viral replication and represent less than 30% of the sequences both in the original BAC and in the mutant.

[0149] Unlike the wild-type HCMV-BAC, eleven days after the transfection of human fibroblasts (MRC-5 cells), we observed no foci of cytopathic effect for the mutant which has a deletion of .sub.671WMVVKYMGFF.sub.680 sequence (data not shown). This deletion dramatically impaired viral replication and propagation in cell-culture. In the same way, recombinant EGFP-viruses with a single or a combination of mutations among W671A, Y676A, F679A and F680A do not produce progeny virion as well. These residues were selected for mutagenesis because they are perfectly or for the less highly conserved (i.e. replaced by another aromatic amino acid) among all the 20 herpesviruses homologues of pUL56 (data not shown). To check if these deletion or mutations may disrupt another step of the viral replication, immunostaining assays were performed to detect proteins produced at immediate early and late stages of viral cycle (IE and late proteins). Expression of immediate early (IEA) and late (gB) viral genes were detected indicating that mutations have no impact on viral gene expression (data not shown). Therefore W671, Y676, F679 and F680 within pUL56(671-580) are critical amino acids for viral replication.

pUL56(671-680) is Necessary for pUL89 Association

[0150] HEK293 were transfected with SC784 and pCI-neo His-89 expression plasmids and protein-protein interactions were carried out by the Alpha assay. This technology represents a powerful method to highlight protein-protein interactions (Ullman et al., 1994) (Waller et al., 2010). Since we have no virion production for mutant viruses, we chose to study in vitro biochemical interactions after protein overexpression in HEK cells which allow introduction of tags (HA and His) for the Alpha assay.

[0151] Alpha assay needs both acceptor and donor beads. For this study, HA-coated Donor beads and 6xHis-coated Acceptor were used. A singlet of oxygen diffuses from Donor bead to the Acceptor bead, resulting in light production at 615 nm. In the absence of a specific biological interaction between proteins, singlet molecules produced by the Donor bead cannot be detected beyond 200 nm from the Acceptor bead (data not shown). First step consisted in verifying the interaction between pUL56-WT and pUL89-WT as a valuable positive control. Alpha assays with 3xHA-pUL56 and 6xHis-pUL89 results in the production of over 9,000 relative light units (RLU), over two-fold more than negative controls (3xHA-pUL56 or His-pUL89) (FIG. 2). pUL56 depleted of its W671-F680 fragment was in turn soaked with pUL89-WT and their affinity assessed by Alpha analysis. The lack of pUL56(671-680) decreased the interaction signal by 50% which is significant in this assay. These data strongly suggest that .sub.671WMVVKYMGFF.sub.680 is necessary for interaction with pUL89.

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