Production of monoclonal antibodies to the prion protein and their characterization

Antibodies to the prion protein (PrP), particularly, monoclonal antibodies, are necessary tools in the diagnostics and study of prion diseases and potential means of their immunotherapy. For the production of monoclonal antibodies, BALB/c mice were immunized by a recombinant bovine PrP. Three stable hybridomas producing antibodies of IgM class were prepared. The antibodies were bound to PrP in a solid-phase enzyme immunoassay and immunoblotting. The epitope mapping accomplished with the use of synthetic peptides showed that an epitope located in region 25–36 of PrP corresponds to one antibody, and epitopes located in region 222–229, to the other two. The antibodies to fragment 222–229 purified by affinity chromatography recognized with a high specificity conglomerates of a pathogenic prion in the brain tissue of cows suffering from spongiform encephalopathy. Thus, in nontransgenic mice, PrP-specific monoclonal antibodies were produced, useful in studies and diagnostics of prion diseases.     

Structure prediction for peptides capable of inducing antibody formation in mice

A simple method for the sequence prediction of peptides capable of the in vivo stimulation of antibody production in mice without conjugation with protein carriers was proposed on the basis of literature data on the structure of T-helper epitopes active in vivo. According to this approach, a potentially active peptide should contain a nine-membered sequence with a hydrophobic amino acid residue in the first position and a positively charged residue in the ninth position. The efficiency of this approach was confirmed by the presence of such sequences in the previously described synthetic peptides with immune activities, by the application of this approach to the choice of immunogenic fragments within the sequences of various proteins that exhibited further the specific activity, and by the construction of immunogenic peptides on the basis of inactive natural sequences.     

Immunization with a synthetic fragment 155–164 of neurotrophin receptor p75 prevents memory loss and decreases beta-amyloid level in mice with experimentally induced Alzheimer’s disease

Neurotoxic beta-amyloid peptide plays an important role in the pathology of Alzheimer’s disease. In aggregated form it binds to several proteins on the surface of the brain cells leading to their death. p75 receptor involved in supporting of cell balance is one of the targets for toxic beta-amyloid. We proposed that induction of antibodies against potential binding sites of p75 with beta-amyloid can be a promising approach towards development of new anti-Alzheimer’s disease treatment. Four potentially immunoactive fragments of p75 were chosen and chemically synthesized. Investigation of immunoprotective effect of the peptide fragments carried out in mice with experimentally induced form of Alzheimer’s disease helped to reveal two fragments effectively preserving murine memory from impairment. Results obtained by ELISA biochemical analysis showed that only immunization with fragment p75 155–164 led to significant decrease in beta-amyloid level in the brain of the experimental mice. Thus, immunization with both fragments of p75 receptor pro-vides a new insight into anti-Alzheimer’s disease drug design.     

Antibodies to synthetic fragment 95–123 of the prion protein protect neurons and astrocytes from beta-amyloid toxicity

Molecular mechanisms of β-amyloid toxic effect on brain cells during Alzheimer’s disease is associated with oxidative stress, intracellular Ca²⁺ increase in neurons and astrocytes and activation of reactive oxygen species production. Prion protein is known to be involved in beta-amyloid toxicity. Here we investigated an effect of affinity purified antibodies to synthetic fragment 95–123 of the prion protein (PrP-(95–123)) on β-amyloid induced cell death, Ca²⁺-signal, reactive oxygen species production and caspase 3 activation. We have shown that antibodies to PrP-(95–123) are able to protect neurons and astrocytes from beta-amyloid induced cell death with no effect on the intracellular Ca²⁺ concentration altered by beta-amyloid treatment. However, the antibodies significantly reduced β-amyloid induced reactive oxygen species production in astrocytes and decreased caspase 3 activation in neurons and astrocytes. Thus, induction of antibodies to PrP-(95–123) of the prion protein provides a new approach to anti-Alzheimer’s disease vaccine design.     

Synthesis and biological properties of polysaccharide-peptide conjugates as potential antigens for a vaccine against meningococci of serogroups A and B

A new approach to the development of a vaccine against meningococci of serogroups A and B was proposed. It involves the synthesis of conjugates of high-molecular capsule polysaccharides of the serogroup A meningococcus (PsA) with earlier synthesized protective fragments of membrane proteins from serogroup B meningococci. The conjugates were synthesized using a method that consists of the generation of aldehyde groups by oxidizing free vicinal hydroxyl groups of PsA and subsequent reaction of these groups with amino groups of the peptide. The reaction proceeds with the intermediate formation of the Schiff base, which is reduced to the stable secondary amine. The main parameters of the reaction were optimized in the synthesis of a PsA conjugate with a model peptide and methods of their characterization were developed. The reproducibility and efficiency of the synthetic procedure were demonstrated by the example of synthesis of PsA conjugates with fragments of protein PorA from the outer membrane of the serogroup B meningococcus. It was shown that, when administered without adjuvant, a conjugate of PsA with a protective peptide, which represents an exposed conserved fragment 306–332 of protein PorA, stimulates the formation of antibodies to the peptide and polysaccharide moieties of the molecule and is also capable of decreasing the degree of bacteremia in animals infected with serogroup A and serogroup B meningococci. The approach can be applied to the development of a complex vaccine for serogroup A and serogroup B meningococci.     

Antibodies to synthetic peptides for the detection of survivin in tumor tissues

Survivin, an endogenous protein, is a promising marker for the cancer diagnosis. The aim of the present work was to obtain antibodies specific to survivin and capable of detecting this protein in tumor tissues. Four peptides corresponding to fragments (1–22), (54–74), (80–88)–(153–165), and (118–144) of the survivin-2B sequence were selected and synthesized. Rabbits were immunized with the synthetic peptides. It has been shown that all peptides in a free state, without conjugation with a high-molecular-weight carrier, stimulate the production of antibodies capable of binding with recombinant survivin. Antipeptide antibodies were isolated from sera and their performance in the immunohistochemical detection of survivin in human tumor tissues was studied. It was shown that only antibodies to the (80–88)–(153–165) peptide bind to the survivin present in breast and bladder tumors. The ability of antibodies to this peptide to detect survivin in tumor tissue lysates was demonstrated by immunoblotting. The part of the sequence targeted by the antibodies against the (80–88)–(153–165) peptide was localized using truncated peptide fragments.     

Synthetic peptide designs based on immunoactive fragments of the VP1 protein of the foot-and-mouth disease virus strain A22

Peptide constructs consisting of 44-53 aa were synthesized on the basis of sequences 135-159, 170-190 and 197-213 of VP1 from the foot-and-mouth disease A22 strain. Immunogenic and protective properties of the peptide constructs were studied in guinea pigs and mice of three lines. The constructs were shown to induce higher levels of antibodies and exhibit higher protective effects than the separate peptides. The most active among the peptides studied was the construct involving the VP1 fragments 135-160 and 170-190: it protected pigs from the experimental infection by the foot-and-mouth disease virus.     

New approaches to the immunotherapy of Alzheimer’s disease with the synthetic fragments of α7 subunit of the acetylcholine receptor

The effect of immunization with the synthetic fragments of the α7 subunit of the acetylcholine nicotine receptor on the spatial memory of mice subjected to olfactory bulbectomy, which causes the development of the neurodegenerative disease of Alzheimer’s type, was studied. NMRI mice were immunized with the KLH conjugates of two peptide fragments of the N-terminal fragment of the α7 subunit extracellular fragment, subjected to olfactory bulbectomy to cause the development of the neurodegenerative disease of Alzheimer’s type, and then the state of the spatial memory was evaluated. It was shown that 20% of bulbectomized mice immunized with N-terminal 1–23 fragment exhibited good spatial memory after training. Immunization with the peptide construct (159–167)-(179–188) consisting of two hydrophilic exposed regions of α7-subunit induced good spatial memory in 50% of bulbectomized mice, while in the control group, which received only KLH, none of animals were learned. Thus, the development of immunotherapy with peptide (159–167)-(179–188) seems to be a promising approach to prophylaxis and treatment of Alzheimer’s disease.     

Antitumor immunotherapy with the use of synthetic fragments of survivin

The endogenous protein survivin is present in tumor cells and inhibits apoptosis. The influence of vaccination of mice by survivin fragments on growth of various types of tumors was studied in order to examine the possibility of creation of an antitumor vaccinating agent on its basis. Two peptides corresponding to the 118–144 and (80–88)-(153–165) sequences of survivin 2B were chosen and synthesized on the basis of literature data and theoretical calculations. Their ability to stimulate antibody production in mice of the C57BL/6J line (b-haplotype) and in BDF1 hybrids (b × d-haplotype) was investigated. Both peptides were shown to stimulate production of antibodies that bound the recombinant survivin in the BDF1 mice. Immunization of BDF1 and C57BL/6J mice with the recombinant survivin resulted in formation of antibodies that reacted with 118–144 peptide. The effect of preventive vaccination with the peptides and the recombinant protein on dynamics of growth of several species of tumors was studied. Vaccination with the (80–88)-(153–165) peptide was found to cause an antitumor effect in BDF1 mice suffered from sarcoma S-37. Thus, creation of antitumor agent on the basis of this peptide is a promising area of further studies.     

A synthetic fragment 60–70 of the receptor for advanced glycation end products exhibits a therapeutic effect in an animal model of Alzheimer’s disease

Six synthetic peptides overlapping a fragment 60–76 of the receptor for advanced glycation end products (RAGE) were studied on a protective effect on spatial memory of animals in the experimental model of Alzheimer’s disease. It was shown that only a peptide corresponding to the fragment 60–70 of RAGE exhibits a therapeutic activity. Intranasal administration of this peptide into bulbectomized mice, which develop neurodegenerative features of the Alzheimer type, completely protects animal memory. Thus, it was found that the N-terminal region (60–70) within the peptide sequence 60–76 of RAGE is responsible for the revealed protective effect. The synthetic peptide RAGE-(60–70) could be the basis for the development of a new drug for the treatment of Alzheimer’s disease.