ITFI20120122A1 - MONOCLONAL ANTIBODIES THAT CAN TIE THE E2 VIRAL PROTEIN PREPARATION AND USE. - Google Patents

Description

Monoclonal antibodies capable of binding the viral protein E2 their preparation and use.

FIELD OF THE INVENTION

The present invention relates to the field of monoclonal antibodies

State of the art

Hepatitis C infections represent one of the major health emergencies of the 21st century. In fact, it is estimated that around 170 million individuals worldwide are infected with HCV; in addition, chronic patients have a high probability of developing cirrhosis and hepatocellular cancer; HCV infections are also the leading cause of liver transplantation in the US.

HCV is made up of multiple genotypes, and a certain heterogeneity is also present between the different subgroups of genotypes. This leads to high drug resistance and an obvious rush to use combined therapies.

Current treatments for HCV infections are based on interferon-α, either alone or in combination with generic anti-virals such as ribavirin, but these treatments are only effective in 20% and 40% of patients, respectively.

HCV is an enveloped virus with a positive strand RNA genome of 9.5 kbases. A highly conserved non-coding region of 341 base pairs is located at the 5 'end of the viral genome while downstream of this region there is an Open Reading Frame (ORF) which encodes a polypeptide of about 3010 amino acids. During the maturation of the virus, this polypeptide is processed by a pool of viral and endogenous proteases generating 10 mature proteins. Among these are the two 2 envelope glycoproteins anchored to the virus membrane, called E1 (or gp35) and E2 (or gp72), containing 5 or 6 and 11 N-glycosylation sites, respectively.

The proteins of the envelope E1 and E2 are responsible for the binding and entry of the virus into the host cells, they therefore represent targets of choice of drugs that block the virus from the early stages of infection. However, due to the high replication rate in vivo and the highly error-prone nature of the RNA polymerase, HCV exhibits a very high genetic variability that is particularly evident in the regions encoding the envelope proteins; in particular, two hypervariable regions were found at the N-terminus of the E2 protein, known as HVR1 and HVR2, which are among the most involved in binding with receptors on host cells.

It has been reported that a region immediately downstream of HVR1 contains epitopes whose antibodies very effectively inhibit the binding of the virus to the CD81 receptor [18]. In particular, a linear epitope corresponding to residues 412-423 and defined by the monoclonal antibody AP33 inhibits the interaction between CD81 and a variety of E2 genotypes, E1-E2 dimers and virus-like particles.

Patents WO 2006/100449 and the most recent 20110002926, published on January 6, 2011, report that the monoclonal antibody AP33 and the humanized variant, bind and can neutralize the six genotypes of HCV, therefore the linear epitope 412-423 it is clearly a target for anti-HCV ligands and an immunogen for the generation of anti-HCV polyclonal and monoclonal antibodies [Owsianka A, Tarr AW, Juttla VS, Lavillette D, Bartosch B, Cosset FL, Ball JK, Patel AH. Monoclonal Antibody AP33 Defines a Broadly Neutralizing Epitope on the Hepatitis C Virus E2 Envelope Glycoprotein. 2005, J. Virai. , 79 (17): 11095-11104],

However, these patents describe monoclonals that recognize a linear epitope of the E2 protein, while it has been hypothesized and reported that the 412-423 region or a slightly shorter region, could be found, in the protein structure, in a loop region [Yagnik AT , Lahm A, Meola A, Roccasecca RM, Ercole BB, Nicosia A, Tramontano A. A model for the hepatitis C virus envelope glycoprotein E2. 2000, Proteins, 15; 40 (3): 355-6],

It is therefore evident, from the above, how interesting it would be to develop antibodies against cyclic variants of this epitope for the optimization of the binding, neutralization and specificity properties of the antibodies, despite its ability to cross-react with different genotypes.

Summary of the invention

The invention refers to monoclonal antibodies of murine or human origin capable of binding and neutralizing the entry into the cells of the HCV virus to varying degrees and the antigens used to generate the antibodies as above.

Brief description of the Figures

Figure 1 (A-C) shows the ELISA binding assays between the synthetic peptides corresponding to the cyclic epitope (Peptide I) and the corresponding linear epitope (linear Peptide II) to the Mabs I - VII, respectively.

Figure 2 (A - G) shows the Sensorgrams related to the binding of Peptide I to Mabs I - VII.

Figure 3 shows the Biacore Sensorgrams related to the binding of linear Peptide II to monoclonal antibodies I - VII, immobilized on biochips.

Figure 4 (A-G) shows the Biacore sensorgrams relating to the binding of the cyclic peptides: III Muti, IV Mut2, V Mut3, III Muti, IV Mut2, V Mut3 and VI Mut4 respectively with: the monoclonal II (Mab II), the monoclonal III (Mab III) and monoclonal VI (Mab VI).

Figure 5 (A and B) shows the results of the western blot analysis of the recognition of E2 of genotype 1a and 3 by the mixture of monoclonal antibodies MAb Ι-MAbV II.

Figure 6 (A and B) shows the neutralizing activity of the developed monoclonal antibodies and the AP33 control.

Figure 7 shows the neutralizing activity of the developed monoclonal antibodies and the AP33 control.

Figure 8 shows the neutralizing activity of MAb II, MAb VI and MAb VII tested in single, in binary combination with each other and all together.

DETAILED DESCRIPTION OF THE INVENTION

The present invention allows to meet the aforementioned needs by making available monoclonal antibodies against epitopes derived from the E2 protein of the hepatitis C virus (HCV) and their use in therapeutic and diagnostic applications. The monoclonal antibodies according to the invention are therefore able to bind the viral protein E2 and neutralize HCV infections in mammals.

The antibodies according to the invention are of murine or human origin and are characterized by the fact that at least one of the chains (light or heavy) of the complementary determining region (CDR) has sequence (1) (heavy chain): AELVRPGGSVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIQPSDSETRLN QKFKDKYSTLTGDSLTWDQGTLTDQGTLTDQLTWDQGTWDQGTWG ) (light chain): [SEQ. ID. No 1]

PASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESG VPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRS [SEQ. ID. No 2] and at the same time, the other chain has an identity percentage between 50% and 100% with the respective sequences 1 or 2.

The amino acid sequence is reported according to the single letter notation, known to those skilled in the art, and refers to amino acids in the L configuration.

In particular, the monoclonal antibody that has sequence 1 as a heavy chain and sequence 2 as a light chain in the CDR is defined below as Mab II.

Other examples of human or murine monoclonal antibodies included in the present invention as characterized by having the following light and heavy chains as defined above, are:

Mab V having

heavy chain:

AELVRPGGSVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIQPSDSETRLN QKFKDKATLTLDRSSSTVYMQLSSPTSDDSAVYYCARTGRGDAWLTYWGQG

[SEQ. ID. No 3]

light chain:

QKFMSTSVGDRVSITCKASQNVRTAVAWYQQKPGQSPKALIYLASNRHTGVPD RFTGSGSGTDFTLTISNVQSEDLAAYFCQQYLNYPRTRSEGGPSWN [SEQ. ID. No 4]

Mab VI having:

heavy chain:

AELVKPGASVKLSCTASGFNIKDTYMHWIKQRPEQGLEWIGRIAYADGNIKIDPKF QGKATITTVTSSNTAYLQLSSLTSEDTAVYYCAYYGLLFAYWGQG [SEQ. ID. No 5]

light chain:

PASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESG VPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRS [SEQ. ID. No. 6] Mab VI. The following Tables I and II show the percentages of identity between the light (table I) and heavy (table II) chains of the monoclonal antibodies Mab II, Mab V and Mab VI.

The percentages of identity with the AP33 monoclonal antibody are also reported as a comparison.

TABLE I

The V VI AP33

On - 55.6 99.0 71.2

V - - 52.6 53.8

VI - - - 71.2

AP33 -. . .

Table II

The V VI AP33

Il - 100 56.7 38.2

V - - 56.7 38.2

VI - 41.2

AP33

These antibodies are mainly characterized by their ability to specifically bind the E2 protein of the HCV virus.

The properties of the monoclonal antibodies called Mab II, V and VI and characterized by having the previously reported sequences in the CDR, are described in detail in examples 1-3.

The corresponding nucleotide sequences of the amino acid sequences characterizing the above antibodies are shown below:

Nucleotide sequence 2p - heavy chain of Mab II [SEQ. ID. No. 64] GCTGAGCTGGTGAGGCCTGGAGGTTCAGTGAAGCTGTCCTGCAAGGCGTCT GGCTACTCCTTCACCACCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGAC AAG G C CTT G AGT G G ATT G G C AT G ATT C AGC GTT C C G AT AGT G AAACT AG GTT AAATCAGAAGTTCAAGGACAAGGCCACATTGACTTTAGACAGATCCTCCAGCA CAGTCTACATGCAACTCAGTAGTCCGACATCTGATGACTCTGCGGTCTATTAC TGTGCAAGAACGGGCAGGGGGGACGCTTGGTTAACTTACTGGGGCCAAGG

Nucleotide sequence 2I - Mab II light chain [SEQ. ID No. 65] CCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGG CCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAG AAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATC TGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCT CAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAC ATT AG G G AG CTT AC AC GTT C G GAG

Nucleotide sequence 5a - heavy chain of Mab V [SEQ. ID. No. 66] GCAGAACTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTG GCTTCAACATTAAAGACACCTATATGCACTGGATAAAGCAGAGGCCTGAACA GGGCCTGGAGTGGATTGGAAGGATTGCTTATGCGGATGGTAATATTAAAATT GACCCGAAGTTCCAGGGCAAGGCCACTATAACAACAGTCACATCTTCCAACA CAGCTTACCTGCAGCTCAGCAGCCTGACATCAGAGGACACTGCCGTCTATTA CTGTGCTTATTACGGCCTCTTATTTGCTTACTGGGGCCAAGGGACTCTGGTT

Nucleotide sequence 5b - Mab V light chain [SEQ. ID. No. 67] CCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGG CCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAG AAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATC TGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCT CAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAC ATTAGGGAGCTTACNCGTTCGGANG

Nucleotide sequence 6a - heavy chain of Mab VI [SEQ. ID. No. 68] GCAGAACTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTG GCTTCAACATTAAAGACACCTATATGCACTGGATAAAGCAGAGGCCTGAACA GGGCCTGGAGTGGATTGGAAGGATTGCTTATGCGGATGGTAATATTAAAATT GACCCGAAGTTCCAGGGCAAGGCCACTATAACAACAGTCACATCTTCCAACA CAGCTTACCTGCAGCTCAGCAGCCTGACATCAGAGGACACTGCCGTCTATTA CTGTGCTTATTACGGCCTCTTATTTGCTTACTGGGGCCAAGGGACTCTGGTT

Nucleotide sequence 6b - Mab VI light chain [SEQ. ID. No. 69] CCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGG CCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAG AAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATC TGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCT CAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAC ATTAGGGAGCTTACNCGTTCGGANG

According to a further embodiment, the invention also includes the aforementioned sequences suitably modified in order to obtain more stable conjugates in biological media or less immunogenic or more soluble or less prone to aggregation.

Such conjugates can be obtained through well-known chemical or enzymatic reactions using suitable substituents and whose action on the molecule is well known.

Preferably, the derivatives or conjugates of the antibodies object of the present invention are functional derivatives, meaning by this derivatives chemically or enzymatically modified which, in functional assays, retain the same biological function - or a similar biological function - of the unmodified products, on the other hand, exhibiting one or more of the properties just described.

For example, functional derivatives of the described antibodies can be antibodies of identical sequence, but carrying modifications introduced by chemical or enzymatic reactions of: amidation, glycosylation, carbamoylation, acylation (for example acetylation), sulfation, phosphorylation, lipidization, pegylation, biotinylation, fluoresceination.

Other functional derivatives with biological properties similar or identical to those of the antibodies object of the present invention, can be originated by applying the method of preparation of "single chains", abbreviated ScFv, in which the polypeptide chains of the original antibodies are assembled in a single chain in in order to obtain two immunoglobulin domains that reproduce the CH-4 and

CL-4 fused together, or divalent and trivalent ScFv or tetravalent ScFv.

Canonical, divalent, trivalent and tetravalent ScFvs can be prepared as fusion products with other proteins using known methods. Any protein or polypeptide can be used as a fusion partner, but preferred fusion partners must not have undesirable biological effects in vivo.

These fusion partners can be introduced both at the C-terminus and at the N-terminus of the ScFvs and optionally a short polypeptide segment can be introduced between the polypeptide sequences of the ScFvs and the fusion partners. These segments can optionally be recognition sequences for endogenous proteases; in this way, the ScFvs can be removed in vitro or in vivo from their fusion partner. Examples of such segments include, but are not limited to: D-D-D-D-Y (SEQ. ID NO. 75), G-P-R, V-P-R, A-G-G and H-P-F-H-L (SEQ. ID NO. 76), which can be hydrolyzed by enterokinase, thrombin, ubiquitin cleaving enzyme and renin, respectively (see U.S. Patent No. 6,410,707).

A chemically or enzymatically modified functional derivative can be a pegylated derivative of the monoclonal antibodies or related ScFvs. The pegylated compounds can provide additional advantages, such as, for example, better solubility, greater stability and longer persistence in circulation and reduced immunogenicity (see U.S. Patent No. 4,179,337).

Other useful modifications include, but are not limited to: ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. A polymer to be used for derivatization can have any molecular weight and can be linear or branched. Polymers of different molecular weight can be used on the basis of the desired therapeutic profile, for example the duration in circulation or the release time, or any other property that modifies their biological properties. The choice of the polymer and its characteristics may also depend on the ease of use, the degree or lack of immunogenicity or other known effects on the therapeutic properties of monoclonals or ScFvs. For example, the PEG can have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

Preferably, the monoclonal antibodies object of the present invention are human or humanized monoclonal antibodies.

Also included by the present invention are fragments of the antibodies as described above which retain the same antigen binding properties (functional antibody fragments).

Examples of such functional fragments are Fabs and (Fab) 2 or polypeptides corresponding to isolated complementarity determining regions (CDR), not considered in the context of an lg-like domain, which consider the same or a similar ability to bind the antigens, i.e. the whole E2 protein or its fragments or its homodimers or heterodimers of the E1 and E2 proteins. Included in this example are synthetic polypeptides or the same ones obtained with the recombinant DNA technique, of sequence [SEQ. ID. No 1 - SEQ. ID. No 6] ·

These sequences, in the context of whole antibodies, ScFvs, Fabs, (Fab) 2s or other functional derivatives obtained by chemical or enzymatic method, can be conveniently modified, using molecular biology techniques, by replacing some residues with homologous residues with similar physico-chemical properties. For example, glutamine residues can be replaced by asparagine residues and vice versa, glutamic residues with aspartic residues and vice versa, lysine residues with arginine or histidine residues and vice versa, serine residues with threonine residues and vice versa, aromatic residues with other aromatic residues. Similarly, some residues can be removed (in a maximum number of 3) or inserted in some points (in a maximum number of 3) so as not to alter the structure of the antibody in the vicinity of the CDR, or in order to alter it to obtain variants a greater affinity.

An integral part of this first aspect of the invention are monoclonal antibodies or their fragments or functional derivatives whose CDR sequences, in the context of whole antibodies, ScFv, Fab, (Fab) 2 or other functional derivatives obtained by chemical or enzymatic, are identical for at least 50% to the aforementioned sequences [SEQ. ID. No 1 - SEQ. ID. No 6],

Also an integral part of this first aspect of the invention are all the compositions containing the antibodies object of this invention or their fragments or functional derivatives, alone or in binary or ternary mixtures, or in general multiple, able to bind and neutralize in various measures the entry of the virus, or of experimental artefacts that reproduce its activity, in model cell systems such as, for example, those described in EP1551960A2.

Alternatively, such compositions thus described are used to neutralize the action of the virus also in recently described animal models of the disease. Preferably, such compositions can contain at least one antibody object of this invention or a derivative or functional fragment thereof and at least a carrier or an adjuvant or a diluent. Furthermore, such compositions can contain at least one monoclonal antibody or functional fragments thereof, possibly chemically or enzymatically modified as described.

The antibodies and / or their fragments, modified or not, and their preparations just described, can be used in many applications related to the prevention and treatment of hepatitis C virus infections.

Some of the applications are:

Passive immunization of healthy or FICV infected mammals Prevention of recurrence of FICV infections in non-FICV infected livers transplanted to patients with chronic FICV infections

Prevention of FICV infections in non-FICV infected mammals.

Prevention of FICV infections in non-FICV infected mammals after accidental contact with FICV infected needles

Prevention of transmission of FICV infections during pregnancy and / or birth from FICV infected mothers to unborn children.

Treatment of HCV infections in infected mammals.

In each of the applications listed above, the treatments with neutralizing antibodies or their fragments or functional derivatives or compositions described in this invention can be further combined with any other known drug or therapy, endowed with therapeutic properties against ITHCV; it is understood that this combination can take place in different ways, for example: Simultaneous administration.

Administration of pre-made mixtures.

Administration of known anti-HCV drugs or therapies and subsequent administration of molecules or compositions object of the present invention. Administration of molecules or compositions object of the present invention and subsequent administration of known anti-HCV drugs or therapies.

It goes without saying that mammals include humans.

Furthermore, the described monoclonal antibodies or their fragments or functional derivatives as described above can be used in analytical methods to identify new compounds capable of neutralizing HCV infection.

These methods include the following steps:

- setting up the conditions for an interaction assay between the neutralizing antibodies or their derivatives or functional fragments described in this invention and the E2 protein or its variants or fragments.

- add the compound for which you want to evaluate the potential neutralizing properties of the HCV activity, before, after or simultaneously with the contact between the antibodies or their fragments or functional derivatives object of the present invention and the E2 protein or its variants or fragments

- measure the binding of antibodies or their functional fragments or derivatives with the E2 protein or its fragments or variants

- determine in step (iii) if the compound added in step (ii) is able to interfere with the binding of antibodies or their fragments or functional derivatives with the E2 protein.

- confirm the neutralizing activity of the selected molecules in a HCV neutralization assay.

Obviously the antibodies and / or their fragments or functional derivatives as described above can be used as positive controls in the previously described method for determining the neutralizing properties of new compounds.

Alternatively, compositions containing at least one antibody or a functional fragment or derivative thereof and at least one carrier or adjuvant or diluent can be used.

Furthermore, the anti-HCV monoclonal antibodies or their fragments or functional derivatives, can be used in diagnostic kits to identify and quantify the E2 protein and its variants in biological samples.

The anti-HCV monoclonal antibodies, and their functional fragments or derivatives as described above can be prepared and purified with known techniques.

A method of preparation and purification of Mab II, V and VI is shown in example 2.

In particular, this method includes the following steps:

(i) obtaining crude preparations of antibodies, or their functional fragments by culturing the corresponding hybridomas, recombinant expression or by chemical synthesis;

(ii) possible obtaining of their functional derivatives by chemical or enzymatic reactions;

(iii) purification of these antibodies, their functional fragments or their derivatives obtained in (i) and (ii) as described in example 2.

A further aspect of the present invention are the antigens used to generate the antibodies as described above.

The main epitope of these antigens is represented by the amino acid sequence that corresponds to the 412 - 422 region of the E2 protein (QLINTNGSWHI) [SEQ.ID. 77] or its variants in which two other residues are added at the two ends that allow to generate cyclic structures having the same number of atoms or a number of atoms equal to:

N - i or N + i, with N = number of atoms present in the structure as defined above (41 atoms) in which said added residues are two cysteines and i varies from 1 to 10 and in which at the C-terminus and at the N - two lysine residues are possibly inserted at the end.

An example of said residues are two cysteines, one on each side, joined by a disulfide bridge so as to generate a cyclic structure.

The possible addition of the two lysines serves to increase the solubility of the molecule and above all to promote conjugation to carrier proteins, such as KLH, used to stimulate the antibody response in the immunization phase of animals.

An example of an epitope (hereinafter Epitope 1) according to the invention therefore has a sequence:

KKCQLINTNGSWHIC [SEQ. ID. No. 7]

The amino acid sequence is reported according to the single letter notation, known to those skilled in the art, and refers to amino acids in the L configuration.

This sequence can be reduced by removing amino acids from the N-terminus and / or from the C-terminus in order to obtain new epitopes of smaller size, down to a minimum of 5 amino acids.

This aspect, in relation to Epitope 1, refers to the sequence included within the two cysteine residues.

Other epitopes obtained by replacing the sequence included between the two cysteines with one of the following amino acid sequences corresponding to different genetic isolates of the same virus are an integral part of this fourth aspect of the present invention:

QLINTNGSWH [SEQ. ID. No. 8]

QLINTDGSWH [SEQ. ID. No. 9]

QLVNTNGSWH [SEQ. ID. No. 10]

QLMNTNGSWH [SEQ. ID. No. 11]

QLISTNGSWH [SEQ. ID. No. 12]

QLINSNGSWH [SEQ. ID. No. 13]

QLINTSGSWH [SEQ. ID. No. 14]

ELINTNGSWH [SEQ. ID. No. 15]

RLINTNGSWH [SEQ. ID. No. 16]

HLINTNGSWH [SEQ. ID. No. 17] KLINTNGSWH [SEQ. ID. No. 18] YLINTNGSWH [SEQ. ID. No. 19] SLINTNGSWH [SEQ. ID. No. 20] NLINTNGSWH [SEQ. ID. No. 21] GLINTNGSWH [SEQ. ID. No. 22] QLINTGGSWH [SEQ. ID. No. 23] QLINTNGGWH [SEQ. ID. No. 24] QLTNTNGSWH [SEQ. ID. No. 25] QLMNTNGSWH [SEQ. ID. No. 26] QLIDTNGSWH [SEQ. ID. No. 27] QLIYTNGSWH [SEQ. ID. No. 28] QLIRTNGSWH [SEQ. ID. No. 29] QLIHTNGSWH [SEQ. ID. No. 30] QLIKTNGSWH [SEQ. ID. No. 31] QLIYTNGSWH [SEQ. ID. No. 32] QLINANGSWH [SEQ. ID. No. 33] QLINTDGSWH [SEQ. ID. No. 34] ELINSNGSWH [SEQ. ID. No. 35] ELVNTSGSWH [SEQ. ID. No. 36] HLVNSNGSWH [SEQ. ID. No. 37] NLVNTNGSWH [SEQ. ID. No. 38] KLINSNGSWH [SEQ. ID. No. 39] RLINSNGSWH [SEQ. ID. No. 40] RLVNTNGSWH [SEQ. ID. No. 41] SLINSNGSWH [SEQ. ID. No. 42] NLIKTNGSWH [SEQ. ID. No. 43] HLVNSNGSWH [SEQ. ID. No. 44] QFVNTNGSWH [SEQ. ID. No. 45] QLVKTNGSWH [SEQ. ID. No. 46] QLVNKNGSWH [SEQ. ID. No. 47] QLVNANGSWH [SEQ. ID. No. 48] QLVNTSGSWH [SEQ. ID. No. 49] QLVNTNGRWH [SEQ. ID. No. 50] QLMYTNGSWH [SEQ. ID. No. 51] QLVNSNGSWH [SEQ. ID. No. 52] QLVNKNGSWH [SEQ. ID. No. 53] QLVNRNGSWH [SEQ. ID. No. 54] QLVNTDGSWH [SEQ. ID. No. 55] QLINSNXSWH [SEQ. ID. No. 56] WHERE x =

Glycine or Serine

QLVNASGSWH [SEQ. ID. No. 57] QLVKTEGNWH [SEQ. ID. No. 58] QLVHANGSWH [SEQ. ID. No. 59] QLVNSHGSWH [SEQ. ID. No. 60] QLVNSNGSRH [SEQ. ID. No. 61] QLVHSNGSWH [SEQ. ID. No. 62] QLIKNGSSWH [SEQ. ID. No. 63]

As mentioned above, the variants of Epitope 1 as indicated above, can also be prepared and used by omitting the two lysines present at N-terminus. An integral part of this aspect of the present invention are epitopes in which the amino acids of Epitope 1 can be in configuration D. Alternatively, the same peptide molecules can be generated in the retro or retro-reverse form, since it is known that antibodies against a peptide relative, they are also able to recognize the variants with amino acids in the D configuration, and the retro and retro-inverse variants.

Some embodiment examples of the present invention are reported below

EXAMPLE 1:

Design and preparation of cyclic and linear immunogens for the preparation of conformational anti-E2 Mabs.

The surface antigens were designed on the basis of a known epitope of the HCV E2 protein. This epitope corresponds to region 412-419, sequence QLINTNGS (SEQ. ID NO. 78) [Pei Zhang, Charles G. Wu, Kathleen Mihalik, Maria Luisa Virata-Theimer, Mei-ying W. Yu, Harvey J. Alter, and Stephen M. Feinstone.

Hepatitis C virus epitope-specific neutralizing antibodies in Igs prepared from human plasma ". PNAS, May 15, 2007, vol. 104, no. 20, 8449-8454], against which antibodies with high neutralizing capacity have already been prepared and developed.

To obtain even more specific and possibly higher affinity antibodies, a cyclic structure containing this amino acid region and reproducing an exposed and folded portion of the protein was devised and designed. Based on a theoretical model of E2 described in reference [Yagnik AT, Lahm A, Meda A, Roccasecca RM, Ercole BB, Nicosia A, Tramontano A. A model for the hepatitis C virus envelope glycoprotein E2. 2000, Proteins, 15; 40 (3): 355-6.].

Considering the variability of the antibody response, it is possible to isolate clones that selectively recognize different conformations of the peptide, therefore, by testing multiple clones, first as peptide binders and subsequently in neutralization assays, it is possible to identify neutralizing agents of HCV infection that selectively recognize a three-dimensional structure of the protein and that they are therefore more specific, selective and related.

In order to study the binding site of antibodies to the antigen, epitope 1, of sequence [SEQ. ID. No. 7], its methylated variant and 4 mutants as indicated in Table III.

TABLE III

Chemical synthesis of antigens

The syntheses were performed using a SYRO I synthesizer purchased from Multisyntech (Germany). The reagents for the solid phase peptide synthesis (resin, amino acids protected with Fmoc and condensing agents), were purchased from the companies Inbios (Naples), GL Biochem (Shanghai, China) and NovaBiochem (Laufelfingen, Switzerland). The solvents for the synthesis, dimethylformamide (DMF), dichloromethane (DCM), isopropanol (Iso-Pro) and ether (Et20) were purchased from the company DelChimica (Naples) and from Sigma-Aldrich (Milan). Piperidine (PIP), used to remove the Fmoc group, trifluoroacetic acid (TFA) and triisopropylsilane (TIS) used for the detachment of peptides from the resin, were purchased from the Sigma-Aldrich company (Milan). Acetonitrile (ACN) used for HPLC analyzes and purifications was purchased from Sigma-Aldrich (Milan). The analytical and preparative reverse phase columns for the analysis and purification of peptides were purchased from the company Phenomenex (Bologna). The preparatory chromatographic systems are produced by Shimadzu (Milan). The LC-MS system for the characterization of crude and purified peptides is a ThermoFisher product (Milan).

The peptides were prepared by chemical synthesis by means of a solid phase technique on a RINK AMIDE resin which allows to have amidated peptides at the C-terminus. The syntheses were performed on a scale of 50 μmol for each peptide. The condensation reactions between the amino acids were performed for 25 minutes at room temperature, using HATU and DIPEA (HATU: 2- (1 H-7-Azabenzotriazol-1 -yl) - 1, 1, 3,3-tetramethyl uronium hexafluorophosphate Methanaminium; DIPEA: Ν, Ν-Diisopropylethylamine) using an amino acid excess of 10 (500 μmol). Deprotection reactions of the Fmoc group were carried out for 10 minutes at room temperature, using 30% solutions of PIP in DMF. The resins obtained at the end of the synthesis were washed manually with DMF (3 times, 1 mL), with DCM (3 times, 1 mL), with Iso-Pro (3 times, 1 mL) and with Et20 (5 times, 1 mL) mL). The resins were dried under vacuum and treated with a mixture of TFA: H20: TIS (3 mL for approximately 100 mg of resin) for 5 hours at room temperature. The products obtained in the acid solutions were precipitated by addition of cold ethyl ether, separated by centrifugation and freeze-dried from a 50% solution of H20 / ACN. The crude products were characterized by LC-MS in terms of purity and molecular weight (ESTIMATED AVERAGE PURITY ABOUT 65%).

The theoretical and experimental weights are shown in the following Table IV.

Table IV

Conjugation to KLH and BSA by glutaraldehyde

Conjugation to KLH (Keyhole Limpet Hemocyanin) was carried out only with Peptide I, reported in Tables III and IV.

For conjugation:

0.5 mg of peptide was dissolved in 0.5 mL of 50 mM phosphate buffer, pH 7.0. The solution was mixed with a solution of KLH (product SIGMA H7017, Milan), 1.5 mg in 0.5 mL of phosphate buffer, pH 7.0 and finally 0.5 mL of 0.2% glutaraldehyde solution in water (Sigma-Aldrich, Milan). The mixture was incubated for 2 hours at room temperature, then 1 mL of 1 M glyein solution in phosphate buffer was added, allowing it to react for another 1 hour at room temperature. The solution was finally dialyzed twice against 50 mM phosphate buffer pH 7.0 and finally lyophilized.

In parallel, under the same conditions, the conjugation of Peptide I to BSA was performed (Albumin Serica Bovina, Sigma-Aldrich, Milan).

Preparation of methylated linear Peptide II (see Table IV).

The starting linear peptide was methylated in solution on the thiol groups of the cysteines to have a stable molecule in solution during the testing phase of the monoclonals and at the same time, modified as little as possible.

The methylation reaction was carried out on 3 mg of crude peptide (about 1.7 pmoles, about 3.4 pmoles of SH groups) dissolved in 500 µL of ACN containing 1% DIPEA. 100 µL of a solution of Methyl Iodide (CH3I, Sigma-Aldrich, Milan) in ACN at 10% v / v (10 mg, about 170 pmoles) was added to the solution and left to react for 1 minute at room temperature. The reaction was stopped by cooling on ice and then adding 100 µL of H20 / TFA 9: 1.

After evaporating the solvents, the product was taken up in 2 mL of H20 / ACN 9: 1, 0.1% TFA and was purified by HPLC on a reverse phase column, obtaining after purification about 1.5 mg of purified product. The methylation occurred was verified by LC-MS analysis of an aliquot of purified sample.

EXAMPLE 2:

Immunization of mice and generation of monoclonal antibodies.

Immunization

For the production of monoclonal antibodies, Peptide I conjugated with KLH was used as the antigen. The TiterMax Gold Adjuvant (Sigma) was used as an adjuvant, which guarantees optimal activation of innate immunity, an essential event to obtain a better production of antibodies. Particular attention was paid to mixing the adjuvant with the peptide to create a perfect emulsion. For this purpose 500 μl of adjuvant were loaded into a syringe and 500 μΙ_ of antigen at a concentration of 4 mg / mL in PBS buffer (50 mM phosphate, 150 mM NaCl), were loaded into a second syringe. Using a three-way connection, the adjuvant was mixed with the antigen, making sure that the aqueous phase (antigen) entered the oily phase (adjuvant) and not vice versa. The mixing operation continued until the emulsion became homogeneous. The final emulsion thus obtained was used to immunize mice of the Balb / c strain obtained by the Jackson Lab. 3 female mice of 5 weeks of age were used. Each mouse was immunized with 50 µL of emulsion (containing 100 µg of immunogen) subcutaneously, injecting 25 µL at two different sites (time (T) = 0). The day before the immunization, about 250 pL of blood were taken from the caudal vein in order to have the pre-immune serum available for each mouse, used in all subsequent phases as a control. Thirty days (T = 30) from the first immunization, the mice were again immunized with 50 μL of antigen-antibody emulsion (containing 100 μg of immunogen) subcutaneously, injecting 25 μL in two different sites. On day 40, the mice were again immunized with 100 µg of immunogen emulsified with the adjuvant, subcutaneously. After another 20 days (T = 60) a new blood sample was taken from the tail vein and the concentration of antibodies directed against Peptide I was determined by ELISA. The two mice showing the highest antibody titer were sacrificed and splenectomized.

WISE ELISA

In the ELISA assays, Peptide I or linear Peptide II, conjugated with BSA, were used as antigen. This allows to measure the antibodies actually produced against Peptide I, avoiding the interference of anti-KLH antibodies.

For this purpose, solutions of Peptide I (100 μl_), conjugated to BSA and dissolved in PBS, at a concentration of 1 μg / mL, were incubated in a 96-well ELISA plate (Immunoplate, NUNC). After one night at 4 ° C, the plates were washed three times with 200 µL of a 1% PBS-Tween solution (Fluka, Milan), to remove excess reagents. The wells were saturated with BSA, incubating with 200 µL of a solution of BSA (Sigma-Aldrich, Milan) at 3% (weight / volume) in PBS, for two hours at room temperature. The plates were washed three times with 200 µL of 1% PBS-Tween. Solutions, in scalar dilutions, of the immune serum and the pre-immune serum of each mouse were dispensed into the wells. The dilutions (1: 100; 1: 1000; 1: 5000; 1: 10000; 1: 100000) were prepared by diluting the serum in PBS. The plates were incubated for 1 hour in a humid chamber at room temperature. At the end of the incubation the plates were washed three times with 200 µL of 1% PBS-Tween, and a solution containing a secondary antibody conjugated with peroxidase that specifically recognizes mouse immunoglobulin G was added (Biorad, Milan) at a dilution of 1: 1000. After incubating the antibody for one hour at room temperature in a humid chamber, the plates were washed three times with 200 µL of 1% PBS-Tween. The detection was performed by adding 100 µL of an ABTS peroxidase substrate solution (2,2'-azino-bis acid (3-ethylbenzothiazolin-6-sulfonic, Sigma-Aldrich, Milan) and reading the absorbance at 415 nm. The antibody titer was defined sufficient for values of the immune serum OD / pre-immune serum OD ratio greater than 3 for dilutions of at least 1: 10000. To determine the presence of antibodies directed against Peptide I in the supernatant of the hybridomas, it was followed the same procedure, but using 50 µL of hybridoma culture medium instead of animal serum Culture media containing antibodies capable of binding only Peptide I and not linear Peptide II were evaluated positive.

FUSION OF MYELOMA CELLS-SPLENOCYTES.

The two mice that showed the best antibody titer were sacrificed by cervical dislocation. The spleen was taken and placed in a 50 mL Falcon tube containing 10 mL of RPMI-GM medium (the composition of which is shown in the section below), preheated to 37 ° C. The spleen was transferred to a 100 mm plate containing 10 mL of medium and was disrupted using a cell sieve (BD-Falcon, Milan). The solution containing the splenocytes was centrifuged at 800 rpm for 5 minutes at room temperature. At the end of the centrifugation the supernatant was removed and the cells were resuspended in 5 mL of RPMI-GM medium and counted. To carry out the count, the following dilutions were prepared:

0.1 mL of splenocytes 0.9 mL of PBS (dilution 1:10);

0.1 mL of splenocytes diluted 1:10 0.9 mL of PBS (dilution 1: 100).

For the counting of the cells, 10 μL of the different dilutions of splenocytes were taken and 10 μL of the 0.4% Tripan blue dye (Sigma-Aldrich, Milan) was added to exclude dead cells from the count.

A number of myeloma cells equal to 1/5 of the splenocytes resuspended in 10 mL of RPMI-GM was used to fuse the splenocytes and SP2 / 0 myeloma cells (ATCC). Myeloma cells were added to the splenocytes, then 15 mL of RPMI-GM (final volume 30 mL) was added. The cells were centrifuged at 600 rpm for 5 minutes, at room temperature. The supernatant was removed and the cell pellet was resuspended in 1 mL of RPMI-GM containing PEG 1300-1600 (Hybri-Max, Sigma-Aldrich, Milan) and 75 µL of DMSO (Sigma-Aldrich, Milan). The cell pellet was gently disrupted with the tip of a pipette and 9 mL of RPMI-GM added with 10% FBS (Foetal Bovine Serum, Sigma-Aldrich, Milan) was then slowly added, taking care to continuously mix the suspension. cell phone. The cells were centrifuged at 500 rpm for 7 minutes at room temperature and the pellet was resuspended in 200 mL of RPMI-HAT selection medium (Sigma-Aldrich, Milan). The cells were plated in 20 96-well plates and left in the incubator for at least 10 days.

After approximately two weeks the cell culture medium was tested for the presence of antibodies directed against Peptide I, by means of ELISA assays, using linear Peptide II as a control. Following this screening, 30 clones were isolated which gave a signal ratio between Peptide 1 / Peptide II linear of at least 5. By way of example, the values obtained for some clones are reported in Table V.

Table V

The 30 selected clones were isolated and sub-cloned. Following this step, 8 clones lost productivity and the remaining 22 were subjected to a stabilization process.

CULTURE MEDIA USED FOR THE PREPARATION OF MONOCLONAL ANTIBODIES

Medium for the maintenance of myeloma cells:

RPMI 1640 (Sigma-Aldrich, Milan).

Foetal Bovine Serum (FBS) 10% (Sigma-Aldrich, Milan).

Penicillin-streptomycin-glutamine (PSG) 1% (Sigma-Aldrich, Milan).

RPMI-GM

RPMI 1640 (Sigma-Aldrich, Milan)

Non-essential amino acids (NEAA) 1% (Sigma-Aldrich, Milan)

Medium for myeloma / splenocyte fusion

RPMI 1640 (Sigma-Aldrich, Milan)

FBS 15%

Hypoxanthine-aminoipterine-thymine (HAT) 2%

PSG 1%

Hybridoma enhnacing supplement (HES) 10%

Medium for the selection of RPMI-HAT clones

RPMI 1640 (Sigma-Aldrich, Milan)

FBS 15%

HAT 2%

PSG 1%

STABILIZATION OF CLONES

To stabilize the clones, three sequential cycles of limiting dilution cloning were performed. The clones were diluted to 3 cells per mL and 20 mL of this dilution was plated in two 96-well plates (100 µL / well = 0.3 cells / well). The cells were left in the incubator for two weeks and then re-analyzed by ELISA assays for their ability to produce anti-Peptide I antibodies. The supernatants with high antibody titer were isolated and re-cloned by limit dilution.

This procedure was repeated three times, always testing the productivity of the single clones by means of ELISA. Following this procedure, 7 clones were selected and expanded and named:

E2-I, E2-II, E2-III, E2-IV, E2-V, E2-VI, E2-VII or simply antibodies or Mab l-VII.

SCREENING AND SELECTION OF CLONES

The antibody-producing clones capable of recognizing only the cyclic antigen, Peptide I, were preselected, identified by ELISA assays in which both Peptide I and linear Peptide II were tested. The data obtained are reported in the following Figures 1A-C, in which the monoclonals were tested with a series of controls. In Figure 1A, ELISA data of dose-response binding to unconjugated Peptide I immobilized on the plate at a concentration of 0.5 pg / mL and increasing concentrations of unpurified monoclonal antibodies II, III, IV and V, over the concentration range between 0 and 7 nM. Unconjugated linear Peptide II and a BSA conjugate were also immobilized as a control.

The latter reagent serves to exclude any possible recognition of monoclonals with glutaraldehyde. As can be seen in Figure 1 A, the 4 antibodies bind Peptide I with a dose-response trend, with saturation already reached at a concentration of about 1.3 nM, while no binding is observed with the linear variant, nor with the conjugated BSA.

In Figure 1B, on the other hand, a dose-response ELISA assay is shown with immobilized Peptide I at a concentration of 0.5 pg / mL and increasing concentrations of all 7 antibodies I-VII, not purified at concentrations between 0 and about 3.3 nM. Also in this case a dose-response trend of the signal is observed, with saturation reached at a concentration of about 1.7 nM. Also in this assay the control with linear Peptide II is reported, with which no binding is observed. Finally, Figure 1C shows the result obtained from a dose-response ELISA assay again performed with 0.5 pg / mL of immobilized peptide and increasing concentrations of purified Mab l-VII (0 - 3.5 nML

As shown, a dose-response trend is still observed with saturation reached at the concentration still about 1.7 nM. From the analysis of these curves, KD values were extrapolated, determined as the antibody concentration that produces a signal equal to half of the saturation signal.

The values are shown in the following Table VI.

SPR CHARACTERIZATION OF SELECTED CLONES AND COMPARATIVE ANALYSIS OF MUTED ANTIGENS

The SPR (Surface Plasmon Resonance) characterization was carried out using a Biacore 3000 instrument (GE Healthcare, Milan) and CM5 type biochip from the same company.

The 7 selected antibodies were covalently immobilized on the surface of the biochip using an EDC / NHS chemistry [Ethyl-3- (3-dimethylaminopropyl) carbodiimide / (N-Hydroxy-succinimide, GE Healthcare, Milan], following the manufacturer's instructions [GE Healthcare, Milan], Immobilization was performed with antibody solutions at a concentration of 5.0 pg / mL, obtaining for all an immobilization level between 6500 and 8000 RU (Resonance Units). As a negative control it was immobilized on a channel of reference an uncorrelated IgG (purified human polyclonal Ig) at the same concentration and therefore with the same level of immobilization. The binding assays were carried out at a flow of 30 pL / min for 2 minutes in HBS, by injecting Peptide I solutions at concentrations increasing between 6.25 nM and 5 pM, value beyond which the curves reach saturation). Regeneration was performed with 50 mM NaOH, injected for 10 sec.

The KDs were calculated using the BiaEvaluation software, vers. 4.1 (GE Healthcare, Milan), implemented in the instrument software. The calculation was performed by determining the Kone and koffe for each single experiment, obtaining the KD from the Koff / kon ratio. The values were then averaged for all concentrations. Figures 2A-G show the sensorgrams obtained after processing and subtracting the reference curves. In the Insets, the graphs of RUmax vs concentration are also shown, attesting that the link is dose-response and saturable.

Table VII, on the other hand, shows the KDs determined by the SPR analysis compared to the KDs relating to the same antibodies determined by the ELISA method. A certain discrepancy is observed between the values obtained with the two methods. However, it is believed that values determined by the SPR technique are much more accurate.

Table VII

To verify the specificity of the monoclonals for the cyclic variant, SPR binding experiments with linear Peptide II at a concentration of 1.0 μΜ were performed on the monoclonal antibodies Mab l-VII. The curves obtained are shown in Figure 3 and, as evident, no recognition between the linear Peptide II and the monoclonals can be observed.

To further evaluate the specificity of antibody recognition with Peptide I, binding experiments were performed between monoclonal antibodies II and VI and the Peptides III-VI reported in Table III. The experiments were conducted under the same conditions used for the analysis of Peptide I and linear Peptide II (flow 30 uL / min, temperature 25 ° C). The peptides were evaluated at variable concentrations in the various experiments (shown in the figures and related legends), but between 125 nM and 5000 nM.

Example sensorgrams referring to the binding of Peptides III, IV and V with Mab II and Mab VI, are reported in Figures 4A-G, while in Tables Villa and Vlllb the KD values determined for all peptides are reported.

Villa table

Table Vlllb

The binding data obtained suggest that:

- the monoclonal antibody II and does not bind the cyclic antigen in the vicinity of the NTN amino acids, but recognizes residues in the vicinity of the disulfide bridge, in particular the QLI and GSWHI residues (SEQ ID NO. 81).

- antibody VII, recognizes the WHI amino acids and in part also GS with high specificity.

The analysis of the mutants makes it possible to exclude that there is a non-specific recognition of the portions of the molecule close to the disulfide bridge or to the two N-terminal lysines.

EXAMPLE 3:

EVALUATION OF RECOGNITION OF HCV PROTEIN E2 AND NEUTRALIZING ACTIVITY NEUTRALIZATION TEST

Cell lines and propagation

Cell line 293T, human embryonic kidney epithelial cells transformed with Simian Virus 40 (SV40) T antigen, was maintained in DMEM Hi-Glucose medium (Dulbecco's Modified Eagle's Medium Hi Glucose, Sigma-Aldrich, Milan) added with FBS (Gibco, Invitrogen, Carlsbad, CA) 10% inactivated at 56 ° C for 30 minutes, L-glutamine 2 mM (Sigma-Aldrich), penicillin 1000U / mL (Sigma-Aldrich), streptomycin (Sigma-Aldrich) 100 pg / mL, 1% non-essential amino acids (Sigma-Aldrich).

The HuH-7 cell line, human hepato-carcinoma cells susceptible to entry of HCV pseudoparticles, were cultured in DMEM medium (Dulbecco's Modified Eagle's Medium, Sigma-Aldrich) supplemented with FBS (EuroClone Ltd., Torquay, UK ) at 10%, inactivated at 56 ° C for 30 minutes, 2mM L-glutamine (Sigma-Aldrich), penicillin 1000U / mL (Sigma-Aldrich), streptomycin (Sigma-Aldrich) 100 pg / mL, 1% non-essential amino acids (Sigma-Aldrich).

Cells were cultured in T75 flasks (BD Falcon, Bedford, MA, USA) in a cell culture incubator (Sanyo Incubatoi ", Japan) at 37 ° C and 5% C02.

When confluent, the cells were washed with 4 mL of PBS and treated with Trypsin / EDTA (Lonza, Walkersville, MD, USA) for 5 minutes at 37 ° C. The trypsin was then inactivated with 5 mL of complete medium and the cells were centrifuged for 5 minutes at 400g. Once the supernatant was discarded, the cells were resuspended in 10 mL of complete medium and counted in a Burker chamber. 3x10 <6> 293T or 1.5x10 <6> HuH-7 cells were then seeded in T75.

PLASMIDES FOR HCV AND VSV-G CONTROL PSEUDOPARTICLE PRODUCTION

FlV-derived constructs produced at the Retrovirus Center of the University of Pisa were used to produce particles with vector derived from feline immunodeficiency virus (FIV), pseudotyped with E1-E2 and expressing GFP as a reporter gene. The constructs are based on a split component system in which three constructs are used: packaging, called pAenvI (deriving from p34TF10, a molecular clone of FIV containing the entire genome of the Petaluma isolate); vector, indicated with the acronym LAW-GFP and carrier GFP as reporter gene ;, and three envelope constructs used in an alternative way. The first, pE1E2gen1a, containing the sequences coding for the surface proteins E1 and E2 of HCV genotype 1 a, the second, pE1E2gen3a, containing the sequences coding for E1 and E2 of genotype 3a, and the third called pVSV-G encoding the protein G of vesicular stomatitis virus (VSV-G). Plasmid constructs expressing E1 / E2 of genotype 1a and 3a were produced starting from HCV clinical isolates using plasma from peripheral blood as a source. Cloning of E1 / E2 from plasma extracted HCV RNA was performed as described by Cosset et al. (Bartosch et al., J. Exp. Med. 197: 633-642, 2003). The FlV-derived viral particles pseudotyped with E1-E2 of genotype 1a or 3a were named FIV-E1E2 / 1a and FIV-E1E2 / 3a while those pseudotyped with the G protein, FIV-VSV.

The plasmids were transformed into MAX Efficiency Stbl Cells (Invitrogen, Milan) bacterial cells by thermal shock at 42 ° C for 45 seconds. The transformed cells were then incubated on ice for 2 minutes, added with SOC medium and incubated for 1 hour under shaking at 37 ° C. The cells were then pelleted at 3000 rpm for 5 minutes, resuspended in 100 µl of Luria Broth (LB, tryptone 10 g / l, yeast extract 5 g / l, NaCI 10 g / l, [Sigma-Aldrich]) and seeded with a sterile loop in Petri dishes containing 1.5% LB agar (Sigma-Aldrich) and 50 pg / mL ampulla (Sigma-Aldrich, Milan). The plates were incubated overnight at 37 ° C. A polymase chain reaction was performed on the colonies the following day. A positive colony for each vector was grown overnight in 250 mL of LB (Sigma-Aldrich) and 50 pg / mL ampulla (Sigma-Aldrich). The following day, an aliquot of 800 µl was taken from each vector to obtain a stock by adding glycerol (150 µl) and stored at -80 ° C. Bacterial growth was used to perform a Maxi plasmid DNA extraction (Pure Yield Plasmid Maxiprep System, Promega Corporation, Madison, Wl, USA). The extracted DNA was subsequently quantified by spectrophotometric reading at a wavelength of 260 nm and an aliquot was run on 1% agarose gel to verify its integrity.

PRODUCTION AND FUNCTIONAL ANALYSIS OF HCV AND VSV-G CONTROL PSEUDOPARTICLES.

The pseudoparticles were produced in HEK293T cells. Twenty-four hours prior to transfection, 3x1 0 <6> cells were seeded in 10 cm Petri dishes (BD Biosciences, Milan). The following day the cells were transfected with 20µg of LAW-GFP vector construct, 10µg of pAenvl packaging construct and 5µg of envelope construct (pE1 E2gen1a, pE1 E2gen3a or VSV-G). For transfection, 10 μΜ polyethyleneimine polymer (PEI, Sigma-Aldrich) was used; briefly, the DNA was mixed with a 150 mM NaCl solution up to a volume of 700 µl; in the same way 100 µL of PEI was brought to 700 µL with 150 mM NaCl. The two solutions were mixed and incubated at room temperature for 15 minutes after which the new solution was gently aliquoted on the cells. Before proceeding to transfection, the medium present on the cells was replaced with 5 mL of DMEM Hi-Glucose containing 2 mM L-glutamine, while 6 hours after transfection the medium was replaced with 10 ml_ of complete Hi-GIu DMEM.

The supernatant containing the carrier particles was collected 48 hours after transfection, clarified at 1800 RPM for 10 minutes at room temperature and then stored in aliquots at -80 ° C until its use.

To verify the transduction efficacy of the produced pseudoparticles, 2x10 <4> HuH-7 were sown in 48-well multiwells. The following day, each well of cells was transduced with 200 µl of vector in order to test the three different pseudoparticle preparations. Six hours after transduction, the medium containing the pseudoparticles was replaced with 200 μΙ_ of complete DMEM. 48 hours after transduction, the wells were washed with 200 μΙ_ of PBS, treated with 100 μΙ_ of trypsin / EDTA (Lonza, Walkersville, MD, USA) and incubated for 5 minutes at 37 ° C. The cells were then spiked with 150 μΙ of complete DMEM and each well was collected in a tube from FACS (Sarsted, Verona). The reading of the samples for the evaluation of the fluorescence was carried out with a FACScan model flow cytometer (BD Biosciences) and the analysis of the samples with the CelIQuest software (BD Biosciences). The efficiency of the test was evaluated as a percentage of GFP-positive cells, compared to a negative control of non-transduced cells.

HCV ANTI-PROTEIN E2 ANTIBODIES

The antibodies were stored at -20 ° C. If necessary, they were thawed and two aliquots were prepared for each antibody, using PBS as diluent, corresponding to 10 pg / mL and 100 pg / mL dilutions. Aliquots were frozen at -20 ° C until used.

Western blot analysis for recognition of HCV genotype 1a and 3 anti-E2 protein

To assess the effective binding of the anti-HCV antibodies to the E2 protein, a western blot was carried out on transfected cells and on the pseudotyped vectors generated by them. HEK293T cells transfected, as described above, with LAW-GFP constructs, pAenvl packaging and envelope (pE1E2gen1a, pE1E2gen3a or VSV-G), were lysed 2 days after transfection and the supernatant containing the vector particles collected for western analysis blot. Cell lysis took place by cold incubation for 1 hour under gentle agitation in the following lysis buffer: 50 mM Tris pH 7.5, 150 mM NaCI, 1% Triton, 1 mM EDTA, 0.1% protease inhibitor, 5 mg / mL MG123 proteasome inhibitor [Merck, Milan, Italy]). After clarification of the cell debris by centrifugation at 6000g for 15 minutes at 4 ° C, the cell lysate was frozen in aliquots at -20 ° C until use. The supernatant (10 ml_) was ultracentrifuged at 70,000 g at 4 ° C for 2 hours (Beckman L-70 ultracentrifuge) and resuspended in 50 μl of lysis buffer (10 mM Tris, 2 mM EDTA, 0.15 mM NaCI, 0.5% Nonidet P-40). The cell lysate at concentrations of 60 and 600 µg of total proteins and the virus resuspended in the lysis buffer were added with 20 µl of the dye SDS sample buffer 2X (0.5 M Tris HCI pH 6.8, 10% glycerol, 10% SDS, 0.05 % β-mercaptoethanol, 0.05% bromine phenol blue). The samples thus prepared were boiled for 7 minutes and half were loaded on a 10% acrylamide / bis-acrylamide gel consisting of a stacking gel (30% Acrilamide / Bis Solution 29: 1 [Bio-Rad Italy, Milan]; 0.5 mM Tris HCI pH 6.8, 10% SDS, 10% APS, 10% TEMED) and a resolving gel (30% Acrylamide / Bis Solution 29: 1, 1.5 mM Tris HCI pH 8.8, 10% SDS, 10% APS, 10 % TEMED). The run was performed in 1X running buffer (25 mM Tris, 0.1% SDS, 1.44% gliin, pH 9.0) at a potential difference of 80-100 volts for 1-2 hours.

The transfer of cellular and viral proteins onto nitrocellulose membranes (GE Healthcare) was carried out in the presence of the transfer buffer (0.3% Tris, 1.44% gliin, 20% methanol) at a potential difference of 100 volts for 90 minutes.

Subsequently, the blocking was carried out by incubating the membranes with 1x PBS, 0.05% Tween-20 (PBS-T), 3% skim-milk at 3%, at room temperature, overnight under gentle agitation. The following day, after 3 washes with PBS-T / skim-milk, the membranes were hybridized with the mixture of anti-E2 antibodies used in the same proportions and diluted 1: 500 in PBS-T / skim-milk at 1% . After 90 minutes, 3 washes with PBS-T were carried out to remove the unbound antibody and the secondary anti-mouse antibody conjugated with the enzyme peroxidase diluted 1: 1000 in PBS-T / skim-milk at 0.5 was added. %. The detection of the bands was performed with the Horseradish Peroxidase Conjugate Substrate Kit (Bio-Rad). The monoclonal antibody AP33, provided by Dr. Arvind Patel (MRC Virology Unit, Institute of Virology, University of Glasgow), directed against an epitope of Linear and highly conserved E2 among the various HCV genotypes [Owsianka, A., A. W. Tarr, V. S. Juttla, D. Lavillette, B. Bartosch, F.-L. Cosset, J. K. Ball, and A. H. Patel. 2005. Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein. J. Virol. 79: 11095-11104],

Neutralization test

Each antibody was tested at two different concentrations 0.5 pg / mL and 5 pg / mL. In addition, three wells were seeded for each antibody tested to perform a triplicate test. As a positive control, the monoclonal antibody AP33 was used which recognizes a linear epitope in E2 partially overlapping with the epitope present in Peptide I. For a more direct comparison of the neutralizing activity, also AP33 was used at the concentration of 0, 5 pg / mL and 5 pg / mL.

The following tests were then carried out in the assay:

- the cells only (Kc) to determine a possible fluorescence background (in triplicate)

- cells transduced with virus only (Kv) to evaluate the transduction efficiency (in triplicate)

- cells incubated with the virus and AP33 (0.5 pg / mL and 5.0 pg / mL

to test the efficiency of neutralization (in triplicate).

- cells incubated with the virus and each antibody (0.5 pg / mL and 5.0 pg / mL, to evaluate the efficiency of neutralization (in triplicate).

Each test was carried out using both the particles pseudotyped with VSV-G (as a positive control for cell transduction), and with E1E2 of genotype 1a and genotype 3a to evaluate the neutralization efficacy of the antibodies tested against these two different genotypes.

For the neutralization assay 2x10 <4> HuH-7 were seeded in 48-well multiwells. One multiwell was seeded for each vector preparation used.

The day following cell seeding, the neutralization assay was set up: for each well of seeded cells, 200 μl_ of each vector (FIV-E1E2 / 1a, FIV-E1E2 / 3a, FIV-E1E2 / VSV-G) are were incubated in eppendorf tubes with 10 µl of prediluted antibody at 10 g / ml_ and 100 g / ml_ dilutions to obtain samples in which the antibodies have a concentration of 0.5 μg / mL and 5 μg / mL, respectively. After one hour of incubation of the samples at 37 ° C, the medium was removed from the cells and this was replaced with the mixture of pseudoparticles and antibodies. The multiwells were then incubated at 37 ° C and 5% CO2 for 6 hours.

After this time, the mixture on the cells was removed and replaced with 500 µL of complete DMEM medium.

After 48 hours the wells were washed with 200 µL of PBS, treated with 100 µL of trypsin / EDTA (Lonza, Walkersville, MD, USA) and incubated for 5 minutes at 37 ° C. The cells were then spiked with 150 µl of complete DMEM and each well was collected in a FACS (Sarsted) tube.

Reading of the samples for fluorescence assessment was performed with a model FACScan flow cytometer (BD Biosciences) and analysis of the samples with CelIQuest software (BD Biosciences) to evaluate the decrease in the number of fluorescent cells pre-incubated with the samples of antibodies compared to controls (Kc and Kv). The neutralizing capacity of the sample was expressed as a percentage of entry reduction. The 40% reduction in infectivity is considered as a cut-off for neutralizing activity.

Monoclonal antibodies, purified and resuspended in Tris-Glycine buffer, at pH 7.0, were tested for their ability to reduce infectivity against E1 / E2 pseudotyped particles of HCV genotype 1a and 3a to which more than 70% of the HCV isolates belong. circulating in Italy, Europe and North America. In all tests, pseudotyped particles with VSV-G using a phospholipid ubiquitously present in cytoplasmic membranes as a receptor were used as a negative control. Any significant reduction in infectivity against VSV-G pseudotyped particles would imply that the anti-E2 monoclonal antibodies actually bind in different portions of the particle or in any case prevent the infection process for mechanisms not dependent on the blocking of the Interaction between the HCV E2 protein and cell receptors (non-specific neutralization). The AP33 monoclonal antibody was used as a positive control in the various neutralization tests, capable of neutralizing a broad spectrum of HCV isolates from different genotypes and in a high range of concentrations [Owsianka, A., A. W. Tarr, V. S. Juttla, D. Lavillette, B. Bartosch, F.-L. Cosset, J. K. Ball, and A. H. Patel. 2005. Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein. J. Virol. 79: 11095-11104],

ANALYSIS OF THE RECOGNITION OF HCV E2 OF GENOTYPE 1a AND 3 WITH WESTERN BLOT

The analysis was conducted on transfected and lysed cells and purified pseudotyped virions as described above. Figure 5A shows the analysis of the expression of E2 in the transfected cells. Not knowing the expression level of the protein, due to the high glycosylation and the presence of multiple forms (precursors and variously glycosylated finished product), the cell lysate transfected with the carrier, packaging and E1 / E2 constructs of genotypes la and 3, it was seeded at a concentration of 600 and 60 μg of total protein and tested with AP33 and the MAbl-MAbVII mixture. As can be seen in Figure 5A and despite the fact that E2 appears rather blurred as a result, probably, of the different patterns of glycosylation [Owsianka, A., A. W. Tarr, V. S. Juttla, D. Lavillette, B. Bartosch, F.-L. Cosset, J. K. Ball, and A. H. Patel. 2005. Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein. J. Virol. 79: 11095-11104] the anti-E2 MAbl-MAbVII monoclonal antibody mixture recognizes the genotype 1a and 3 protein practically as AP33 the monoclonal antibody which, as mentioned above, recognizes a highly conserved linear epitope of E2 among the genotypes of HCV. Similarly, the MAbl-MAbVII mixture specifically recognizes genotype 1a and 3 E2 glycoproteins on pseudotyped viral particles generated from transfected and purified cells. The heavier weight band is glycoprotein E1 / E2 present as immature form which is split into E1 and E2 during the maturation process, the final phase of the viral replication cycle which occurs at an extracellular level not or partially glycosylated. Finally, it is important to underline that the monoclonal mixture does not recognize VSV-G at all, which has dimensions of just over 50 KDa (Figure 5B). This result therefore demonstrates that the developed anti-E2 monoclonal antibodies specifically recognize genotype 1a and 3 HCV glycoprotein.

ANALYSIS OF THE NEUTRALIZING ACTIVITY OF MONOCLONAL ANTI-E2 ANTIBODIES

The tests were repeated four times at different times and with different viral preparations and gave very similar results. In general, all monoclonal antibodies produced have neutralizing activity against pseudotyped particles with VSV-G zero or less than 10%. The neutralizing activity shown by all antibodies is therefore highly specific. The activity also decreases, as expected, in proportion to the decrease in the concentration of antibodies and with scaling very similar to that of the positive control AP33. Figure 6A shows the neutralization data, from one of the experiments conducted, for all 7 monoclonal antibodies, compared to the AP33 antibody, at concentrations of 0.5 pg / mL and 5.0 pg / mL, while in Figure 6B shows the data obtained using pseudotyped particles with control VSV-G.

As for the anti-E2, the antibodies showing the greatest neutralizing activity are MAb II, MAb VI and MAbVII which reduce the infectivity of the pseudotyped particles with E1 / E2 of genotype 1a by about 40 and 30% using 5.0 respectively. and 0.5 pg / mL of antibody in the assay. This neutralizing activity is completely superimposable to that shown by AP33 and is considered clinically significant [Keck ZY, Machida K, Lai MM, Ball JK, Patel AH, Foung SK. Therapeutic control of hepatitis C virus: the role of neutralizing monoclonal antibodies Curr Top Microbiol Immunol. 2008; 317: 1-38, Stamataki Z, Grove J, Balie P, McKeating JA. Hepatitis C virus entry and neutralization. Clin Liver Dis 2008; 12: 693-712], The other antibodies instead have more modest activities that are around 30% at a concentration of 5.0 pg / mL. Anti-E2 antibodies were then tested against genotype 3a (Figure 7), using pseudotypic E1 / E2 pseudoparticles cloned from an isolate of this genotype. MAb I, MAb III, MAb IV and MAb V show modest neutralizing activity also towards this genotype. As for the MAb II, MAb VI and MAb VII, the first and the last maintain the good performances also towards this genotype which instead is not neutralized by the MAb VI, probably following an amino acidic difference of the genotype 3a compared to the Peptide I used for the production of antibodies. Based on these results, MAb II, MAb VI and MAb VII were used in binary combination with each other and all together. For a better comparison with what could occur in vivo, if they were used in the therapeutic setting, the total concentration of antibodies present in the assay remained 0.5 and 5.0 μg / mL. So in the case of the combination between two antibodies the concentration of the single MAb is halved. Instead, it is reduced to a third in the combination of the three antibodies put together. Regardless of the association used, no increase in neutralizing activity was observed compared to the same antibodies tested individually (Figure 8). Indeed, the combination of the three antibodies significantly reduces their effectiveness, thus suggesting a possible competition or steric hindrance between the antibodies for binding to the epitope.

Claims (19)

Claims 1. Monoclonal antibodies of murine or human origin characterized by the fact that at least one of the chains, light or heavy, of their complementary determining region CDR has a sequence (heavy chain): AELVRPGGSVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIQPSDSETRLN QKFKDKATLTLDRSSSTVYMQLSSPTSDDSAVYYCARTGRGDAWLTYWGQG [SEQ. ID. No 1] o sequence (light chain): PASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESG VPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRS [SEQ. ID. No 2] and at the same time, the other chain has an identity percentage between 50% and 100% with the respective sequences [SEQ. ID. No 1] and [SEQ. ID. No 2] or their functional fragments. 2. Monoclonal antibodies according to claim 1 characterized in that the heavy and light chains in the CDR have the following sequences: a) AELVRPGGSVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIQPSDSETRLN QKFKDKATLTLDRSSSTVYMQLSSPTSDDSAVYYCARTGRGDAWLTYWGQG [SEQ. ID. No 1] and PASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESG VPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRS [SEQ. ID. No 2] b) AELVRPGGSVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIQPSDSETRLN QKFKDKATLTLDRSSSTVYMQLSSPTSDDSAVYYCARTGRGDAWLTYWGQG [SEQ. ID. No 3] and QKFMSTSVGDRVSITCKASQNVRTAVAWYQQKPGQSPKALIYLASNRHTGVPD RFTGSGSGTDFTLTISNVQSEDLAAYFCQQYLNYPRTRSEGGPSWN [SEQ. ID. No 4] c) AELVKPGASVKLSCTASGFNIKDTYMHWIKQRPEQGLEWIGRIAYADGNIKIDPKF QGKATITTVTSSNTAYLQLSSLTSEDTAVYYCAYYGLLFAYWGQG [SEQ. ID. No 5] and PASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESG VPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRS [SEQ. ID. No 6] 3. Antibodies according to claim 2 wherein the corresponding nucleotide sequences of the amino acid sequences are: a) [SEQ. ID. 64] GCTGAGCTGGTGAGGCCTGGAGGTTCAGTGAAGCTGTCCTGCAAGGCGTCT GGCTACTCCTTCACCACCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGAC AAGGCCTTGAGTGGATTGGCATGATTCAGCGTTCCGATAGTGAAACTAGGTT AAATCAGAAGTTCAAGGACAAGGCCACATTGACTTTAGACAGATCCTCCAGCA CAGTCT ACATGCAACT CAGT AGT CCG ACAT CTG ATG ACT CTGCGGTCT ATT AC TGTGCAAGAACGGGCAGGGGGGACGCTTGGTTAACTTACTGGGGCCAAGG And [SEQ. ID. 65] CCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGG CCAGCAAAAGT GTCAGT ACAT CTGGCT AT AGTT AT AT GC ACT GG AACCAACAG AAACCAGG AC AGCCACCCAG ACTCCT CAT CT AT CTTGT AT CCAACCT AG AAT C TGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCT CAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAC ATT AGGG AGCTT ACACGTT CGGAG b) [SEQ. ID. 66] GCAGAACTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTG GCTT CAACATT AAAG ACACCT AT ATGCACTGG AT AAAGCAG AGGCCT G AACA GGGCCTGGAGTGGATTGGAAGGATTGCTTATGCGGATGGTAATATTAAAATT GACCCGAAGTTCCAGGGCAAGGCCACTATAACAACAGTCACATCTTCCAACA C AG CTT ACCTG C AG CT C AGC AG CCT G AC AT C AG AG G AC ACT G CCGTCT ATT A CTG TG CTT ATT ACGGCCTCTTATTTGCTTACTGGGGCCAAGGGACTCTGGTT And [SEQ. ID. 67] CCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGG CCAGCAAAAGT GTCAGT AC AT CTGGCT AT AGTT AT AT GC ACT GG AACCAACAG AAACCAGG AC AGCCACCCAG ACTCCT CAT CT AT CTTGT AT CCAACCT AG AAT C TGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCT CAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAC ATTAGGGAGCTTACNCGTTCGGANG c) [SEQ. ID. 68] GCAGAACTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTG GCTT CAACATT AAAG ACACCT AT ATGCACTGG AT AAAGCAG AGGCCT G AACA GGGCCTGGAGTGGATTGGAAGGATTGCTTATGCGGATGGTAATATTAAAATT GACCCGAAGTTCCAGGGCAAGGCCACTATAACAACAGTCACATCTTCCAACA C AG CTT ACCTG C AG CT C AGC AG CCT G AC AT C AG AG G AC ACT G CCGTCT ATT A CTG TG CTT ATT ACGGCCTCTTATTTGCTTACTGGGGCCAAGGGACTCTGGTT e [SEQ. ID. 69] CCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGG CCAGCAAAAGT GTCAGT AC AT CTGGCT AT AGTT AT AT GC ACT GG AACCAACAG AAACCAGG AC AGCCACCCAG ACTCCT CAT CT AT CTTGT AT CCAACCT AG AAT C TGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCT CAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAC ATTAGGGAGCTTACNCGTTCGGANG 4. Antibodies according to claim 1 - 3 wherein said sequences are suitably chemically or enzymatically modified by chemical or enzymatic reactions of: amidation, glycosylation, carbamoylation, acylation (for example acetylation), sulfation, phosphorylation, lipidization, pegylation, biotinylation. 5. Anticopres according to claims 1 - 3 wherein the polypeptide chains of the original antibodies are assembled in a single chain so as to obtain two immunoglobulin domains which reproduce the CH-4 and CL-4 domains fused together, or divalent and trivalent ScFv or tetravalent ScFv. 6. Antibodies according to claim 5 in which said canonical, divalent, trivalent and tetravalent ScFvs are fusion products with other proteins introduced both at the C-terminus and at the N-terminus of the ScFvs possibly inserting between the polypeptide sequences of the ScFvs and partners of fusion a polypeptide segment. 7. Antibodies according to claim 6 wherein said polypeptide segments are recognition sequences for endogenous proteases. 8. Antibodies according to claim 7 wherein said recognition sequences for endogenous proteases are selected from: D-D-D-D-Y (SEQ ID NO.75), G-P-R, V-P-R, A-G-G and H-P-F-H-L (SEQ ID NO. 76). 9. Use of antibodies according to claims 1 - 7 in compositions capable of binding and neutralizing to varying degrees the entry into the cells of the FICV virus. 10 Use according to claim 9 for passive immunization of healthy or FICV infected mammals, prevention of recurrence of FICV infections in non-FICV-infected livers transplanted in patients with chronic FICV infections, prevention of FICV infections in non-FICV infected with FICV, treatment of FICV infections in infected mammals possibly in combination with other drugs with therapeutic properties against FHCV. 11. Use of antibodies according to claim 10 in analytical methods to identify new compounds capable of neutralizing FICV infection or as positive controls in methods for determining the neutralizing properties of new compounds. 12. Compositions containing at least one antibody or a functional fragment or derivative thereof and at least one carrier or adjuvant or diluent. 13. Diagnostic kits for identifying and quantifying the E2 protein and its variants in biological samples comprising antibodies according to claims 1 - 7. 14. Epitope consisting of the sequence of amino acids that corresponds to the 412 - 422 region of the E2 protein (QLINTNGSWFII (SEQ ID NO. 78)) or to its variants in which two other residues are added to the two ends which allow to generate cyclic structures having the same number of atoms or a number of atoms equal to: N-i or N + i, with N = 41, or number of atoms present in the structure in which said residues are two cysteines and i varies from 1 to 10 and in which the C- two lysine residues are eventually added to the terminal and to the N-terminal. 15. Epitope according to claim 14 wherein said residues are two cysteines, one on each side, joined by a disulfuric bridge so as to generate a cyclic structure. 16. Epitope according to claim 15 having sequence: KKCQLINTNGSWHIC [SEQ. ID. No. 7] 17. Epitope according to claim 14 wherein said included sequence is a known sequence corresponding to the different genetic isolates of the same virus. 18. Epitopes according to claim 17 wherein the amino acids are either in D, retro or retro-reverse configuration. 19. Use of the epitopes according to claims 13 - 17 for the production of the antibodies according to claims 1 - 8.