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However, to our knowledge, no method has been widely accepted. Anti-idiotypic vaccines would therefore represent a novel approach for prion disease prevention and treatment. Results of passive vaccination and cell-culture studies indicate that only monoclonal antibodies that bind to the membrane PrP C are efficient in blocking prion propagation and pathogenesis [ 32 — 35 ]; at the same time, it has been shown that monoclonal antibodies that recognized both PrP C and PrP Sc , blocked PrP Sc accumulation in vitro even more efficiently than monoclonal antibodies specific for PrP C only [ 36 ].

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However, the cross-linking of PrP C in vivo caused extensive neuronal damage [ 37 ] and in our opinion this finding needs to be noted when potent PrP C -specific antibodies are to be induced or introduced for treatment or prophylaxis purposes. Therefore an approach to exclusively target PrP Sc would avoid all of the problems regarding the binding of antibodies to PrP C and the resultant cessation of normal PrP C function.

To directly target the pathological isoform in the development of an anti-idiotypic vaccine, a PrP Sc -specific antibody is a prerequisite.

What is an Anti-Idiotypic Antibody?

However, only a few PrP Sc -specific antibodies have been reported [ 23 , 24 , 38 — 40 ], and due to their individual characteristics, not all of them are suitable for this purpose. The V5B2 monoclonal antibody that was previously described by our group is a potent PrP Sc -specific antibody that reacts with the native PrP Sc deposits in immunohistochemistry [ 24 ].

This thus represents an appropriate candidate to preserve the information of the native PrP Sc -specific epitope in its antibody-combining site. In the present study, the main ways of producing Ab2 antibodies against the PrP Sc -specific antibody have been studied in two experimental models: in xenogeneic and syngeneic systems. Mammalian proteins are highly immunogenic in chickens. As expected for xenogeneic immunization, the antibody titre in the antiserum was high.

Although the immune response was expected against the whole immunoglobulin molecule, anti-idiotypic response was surprisingly potent, with the ability to totally block the binding of V5B2 to the P1 peptide or PrP Sc. However, despite promising results after testing the immune sera and affinity purified Ab2 IgY antibodies, we were not able to isolate stable chicken hybridoma cell lines that retained the antibody production. The main reason appears to be that to date chicken hybridoma technology has been poorly studied in comparison to mouse hybridoma technology; the system therefore still needs to be optimized for use as a routine experimental procedure.

Whole IgG V5B2 was the original and the least chemically treated molecule used for immunization. It is a relatively large molecule, predominantly composed of the self constant region, with only a minor part being immunogenic in a syngeneic experimental system. Although the immune tolerance to the whole constant part of the immunoglobulin molecule was expected, we also wanted to assess whether the use of the smaller fragment Fab V5B2, lacking the constant Fc-region, potentiates the immune response to idiotopes.

In addition to the whole V5B2 and Fab V5B2, the Fab V5B2, covalently coupled to the highly immunogenic carrier molecule KLH, was used for immunization, to maximize the specific helper T-cell response and to potentiate the immune response against idiotopes on V5B2. Nevertheless, a prominent immune response was achieved with all three antigens. Surprisingly, even though the immune response against the whole IgG V5B2 was relatively strong, the antisera capacity to inhibit the V5B2 binding to the P1 peptide was much lower in comparison to the inhibitory capacity of the antisera obtained by immunization with the smaller fragment, Fab V5B2.

This could contribute to the lower immune response and lower inhibitory capacity of the obtained sera. It is also possible that the immune response was directed predominantly against the glycan moieties of the constant region in case of the whole IgG, as the glycosylation profile of monoclonal antibodies is affected by the cell culture conditions and can differ from the normal antibody glycosylation observed in vivo [ 41 ].

The resulting hybridoma cell lines produced predominantly IgG Ab2 antibodies, in contrast to other Ab2 antibody studies, where the antibodies were mainly of the IgM class [ 4 , 5 , 42 , 43 ]. The main objective in the present study was to select Ab2 antibodies that recognize private idiotopes. Therefore, the selection process included Fab fragments of monoclonal antibodies with the same specificities for the peptide antigen, but with different specificities for the prion protein isoforms.

Indeed, the majority of the antibodies used in the negative selection process Table 1 were directed against the P1 peptide derived from the C-terminal amino-acid sequence of the human prion protein, which can adopt different conformations in solution, and therefore, which can elicit antibodies of different specificities when injected into wild-type mice [ 23 ].

The comparison of the variable region amino-acid sequences of both the light and heavy chains, between antibodies that had been used in the negative selection process and our antigen, Fab V5B2, revealed considerable analogy. The V H and V L amino-acid sequences of these antibodies differ only slightly, and most importantly, the differences between these antibodies are predominantly restricted to the CDR regions Table 2. Nevertheless, all of the Ab2 monoclonal antibodies selected still distinguished between the Fab fragments of these antibodies, which indicated a very precise selectivity of our Ab2 monoclonal antibodies for private idiotopes, most probably restricted to the CDR regions of V5B2.

Based on the selection process described and the affinities of the Ab2 antibodies for Fab V5B2 as well as their inhibition potential, two Ab2 monoclonal antibodies, 5D12 and 4F8, were selected for further characterization.

Since no functional test is available to determine the presence of Ab2 antibodies that bind to the antigen-combining site of a PrP Sc -specific antibody, at first an ELISA-based inhibition assay was designed to characterize the Ab2 antibody response. Ab2 antibodies in chicken and mouse polyclonal immune sera and Ab2 murine monoclonal antibodies Figs.

This indicated that in agreement with idiotypic network theory, immunization with Fab V5B2 induced Ab2 antibodies, which bear the functional epitope for Ab1 that competes with the original antigen, the P1 peptide, and in turn have to be similar to the P1 peptide structure. The reasons for the stronger inhibition of the polyclonal immune sera in comparison to monoclonal antibodies can be accounted for by the higher antibody concentration, a wider variety of Ab2 antibody specificities, and consequently also a wider variety of target epitopes present in polyclonal preparations.

In addition to the ELISA-based inhibition assay, the competitive immunohistochemistry assay was set up to show that Ab2 antibodies inhibit not only binding of V5B2 to the P1 peptide but also to the original target molecule, PrP Sc. The inhibition of binding was concentration dependent, which indicates the specificity of the effects seen.

What is an Anti-idiotypic antibody?

The results of the competitive immunohistochemical studies, together with the results of the competitive ELISA, strongly indicate that the selected Ab2 antibodies bind into the V5B2 binding site, thereby hindering the binding of V5B2 to the P1 peptide, and most importantly to the original target, the PrP Sc molecule. In addition, the structural model of the 5DV5B2 complex, obtained by molecular docking, also strongly supports the close interaction of both antibody-combining regions, since the majority of the interacting residues are located in the CDR regions Figs.

Based on previous studies, Ab2 antibodies can resemble the amino-acid sequences of nominal antigens in their variable regions, especially when the nominal antigen is a peptide [ 44 — 46 ]. The P1 peptide motif could be preserved in a single CDR sequence or in a conformational stretch across different CDR loops of a single chain, as well as across different loops of both the heavy and light chains. The latter, i.

The comparison of the 5D12 binding interface to the P1 peptide reveals structural similarities to its C-terminal.

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This is especially important in the light of our recent unpublished data, which indicate that the C-terminal residues are crucial for the V5B2 binding to the P1 peptide. It is therefore possible that the 5D12 binding surface mimics the crucial part of the P1 peptide and the epitope, unique to the PrP Sc molecule. Nevertheless, the computer modelling data must be interpreted cautiously; still, the obtained docking model is in agreement with the observed experimental data. The proposed model also achieved an outstanding ranking score using both the surface complementarity and the global energy criteria.

With all of the data collected, we have shown that it is possible to induce a strong Ab2 antibody immune response against a PrP Sc -specific monoclonal antibody in both the xenogeneic and the syngeneic experimental systems. Among the three different antigens tested, the Fab fragment was the most successful form for the immunization of mice.

In addition, we have demonstrated that it is possible to select Ab2 monoclonal antibodies specific for private idiotopes using a two-step selection process. In our case, the availability of related antibodies that predominantly differ in CDR regions only, enabled the critical selection step in the negative selection process. According to the convincing results of the competition studies and the computer modelling data, 5D12 is a highly plausible candidate for being an internal image antibody, mimicking the PrP Sc -specific epitope.

All supernatants from murine hybridoma cells were purified by gravity-flow affinity chromatography on Protein G Sepharose Amersham using 0. Five chickens were immunized intramusculary every 3 weeks with the whole V5B2 monoclonal antibody as antigen. After the 4 th immunization, the titres of anti-V5B2 antibodies were estimated by ELISA and the two chickens with the highest antibody titres were selected.

They received two more boosters of the Fab fragment of V5B2.

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  6. Three days after the last immunization booster, these chickens were sacrificed, and their blood was collected for the preparation of the polyclonal anti-serum and their spleens were removed for monoclonal antibody production. The chicken hybridoma cells were produced as described previously [ 49 , 50 ]. Fab V5B2 was covalently immobilized to agarose beads Pierce according to the manufacturer protocol. Blood serum from the chicken with the most potent immune response, collected after immunization, was applied to the column with the immobilized Fab V5B2.

    After extensive washing the bound fraction was eluted using 0. Each mouse was immunized subcutaneously with 0. On day 52, the mice were bled from the tail vein and the immune sera were collected. The mice were sacrificed on day 61, and their spleens were removed. The presence of specific antibodies was determined in supernatants after 10 to 14 days by indirect ELISA. Selected hybridomas were cultured until stable cell lines were established, and then subcloned by limiting dilution, and frozen in liquid nitrogen.

    The next day, the plates were washed three times with washing buffer sodium phosphate buffer, containing mM NaCl, 0.


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    The plates were washed again and then incubated with the following primary antibodies: polyclonal immune sera, cell culture supernatant, or purified monoclonal antibodies, all diluted in blocking buffer, for 1. After incubation with the 2,2'-azino-bis 3-ethylbenzothiazolinesulfonic acid ABTS, Sigma substrate in citrate-phosphate buffer, pH 4. The 5D12 and 4F8 clones were chosen for further experiments, as they consistently produced the highest titre antibodies, as determined by indirect ELISA.

    The 6C5 murine IgG1 monoclonal antibody, specific for antigen B of the human polysaccharide blood group system AB0, served as an isotype negative control monoclonal antibody throughout the study. The affinity constants Kaff of the Ab2 monoclonal antibodies were determined in a non-competitive immunoassay [ 51 ]. The incubations with the secondary antibody and the ABTS substrate were performed as described above.