Screening and identification of mimotopes of LPS conservative epitope from random phage display peptide library

Objective: To screen and identify the mimotopes of lipopolysaccharide (LPS) epitope. Methods: A random phage display dodecapeptide library was screened with monoclonal antibody 2B4 specifically against LPS conservative epitope. The positive clones were identified by phage EUSA and competitive inhibition assay with either S. typhimurium T861 LPS or E. coli Olll:B4 LPS. Results: After 3 rounds of biopanning, the clones bound with monoclonal antibody 2B4 were well enriched with the positive rate of 80%. The bindings between 12 positive phage clones and screening antibody were inhibited by both kinds of LPS. The positive peptide sequences were deduced from the corresponding DNA sequences. There were some identical sequences among them: PPQWFFSQPQL (5/12, 41. 7%), LPQYFW NTATTA (3/12, 25%), FPQNHWNVPWAT(2/12, 16. 6% ),HSQSFWNAPLAM and AHPWTHGYFPPL (l/12, 8. 3% ). Conclusion: The peptides screened with 2B4 antibody are mimotopes of LPS conservative epitope.

Isolation of mimotopes to the anti-human VEGF antibody Bevacizumab by mRNA display using random peptide libraries and the vaccination of a rabbit

An mRNA display system using synthetic DNA cod-ing for random 10- and 20-amino-acid peptide li-braries was employed to isolate mimotopes that could substitute for the anti-human VEGF antibody Bevacizumab. After six rounds of affinity selection, three clones that bound to the antibody were isolated. Their random-peptide portions were chemically syn-thesized, and further characterized. All of the pep-tides showed clear specific binding to the antibody. Two of them were further conjugated with Keyhole limpet hemocyanin (KLH) to immunize a rabbit. Af-ter five immunizations biweekly, antibodies to the peptides were purified with a column conjugated with the peptides. The purified antibodies reacted specifically to the antibody’s original antigen, human VEGF. mRNA displays could be useful for the isola-tion of mimotopes for vaccines to substitute for ther-apeutic antibodies.

Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes

Hepatitis C(HCV) genome is highly variable,particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene.The variability of HCV genome has been a major obstacle for de-veloping HCV vaccines.Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes,we synthesized an minigene of HCV-derived multi-epitope peptide an-tigen(CMEP) ,which contains 9 B-cell HVR1 mimotopes in E2,2 conserved CTL epitopes in C,1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3.This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP.The immunogenic properties of CEMP were characterized by HCV infected patients' sera,and found that the reactivity frequency reached 75%.The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%.Meanwhile,we constructed an HCV DNA vaccine candidate,plasmid pVAX1.0-st-CMEP carrying the recombinant gene(st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene.Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody,which was of the same cross reactivity as the fusion protein GST-CMEP.Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes,and would be of the value as a candidate for the development of HCV vaccines.

Screening and identification of mimotopes of the major shrimp allergen tropomyosin using one-bead-one- compound peptide libraries

The one-bead-one-compound （OBOC） combinatorial peptide library is a powerful tool to identify ligand and receptor interactions. Here, we applied the OBOC library technology to identify mimotopes specific to the immunoglobulin E （IgE） epitopes of the major shellfish allergen tropomyosin. OBOC peptide libraries with 8-12 amino acid residues were screened with serum samples from patients with shellfish allergy for IgE mimotopes of tropomyosin. Twenty-five mimotopes were identified from the screening and their binding reactivity to tropomyosin-specific IgE was confirmed by peptide ELISA. These mimotopes could be divided into seven clusters based on sequence homology, and epitope mapping by EpiSearch of the clustered mimotopes was performed to characterize and confirm the validity of mimotopes. Five out of six of the predicted epitopes were found to overlap with previously identified epitopes of tropomyosin. To further confirm the mimicry potential of mimotopes, BALB/c mice were immunized with mimotopes conjugated to keyhole limpet hemocyanin and assayed for their capacity to induce tropomyosin-specific antibodies. BALB/c mice that received mimotope immunization were found to have an elevated level of tropomyosin-specific immunoglobulin G, but not mice that received an irrelevant mimotope. This study pioneers the successful application of the OBOC libraries using whole sera to screen and identify multiple shrimp allergen mimotopes and validates their mimicry potential using in vitro, in vivo, and in silico methods.

A mimotope of Pre-S_2 region of surface antigen of viral hepatitis B screened by phage display

To acquire the phage-displayed mimotopes which mimic the specificity of hepatitis B virus surface antigen (HBsAg), a random peptide library expressing linear peptide with 12 amino asids in length were used to screen with the serum from a hepatitis B virus infected patient in the recovery phase. After 3 rounds of biopanning, the positive phages were confirmed by competitive ELISA using HBsAg/P33. Two phagotopes were identified and one of them was confirmed as mimotope by competition experiment. Based on the mimotpe, a multiple antigenic peptide with four branches was synthesized by solid phase peptide synthesis. The antiginicity and specificity of the synthesized antigen was tested in BALB/c mice compared with the native epitope-based antigen. The results showed that the mimotope-based antigen could evoke higher titer of antibodies with the same specificity of the epitope-based antigen. Those findings indicate mimotopes can be used in antigen and vaccine design.

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag

AIM: Using a monoclonal antibody against gastric cancer antigen named MGbl to screen a phage-displayed random peptide library fused with coat protein plII in order to get some information on mimotopes.lV^37BODS: Through affinity enrichment and EUSA screening,positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced)By blocking test, specificity of the mimic phage epitopes was identified.RESULTS: There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGbl-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced.According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGbl-Ab to gastric cancer cell KATO-Ⅲ. The percentage of blocking was from (21.0±1.6) %to (39.0±2.7) %.CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.

Mimotope peptide modified pompon mum-like magnetic microparticles for precise recognition,capture and biotransformation analysis of rituximab in biological fluids

Due to low immobilized ligand density,limited binding capacity,and severe interference from serum proteins,developing ideal peptide-based biomaterials for precise recognition and in vivo analysis of biopharmaceuticals remains a huge challenge.In this study,mimotope peptide modified pompon mum-like biomimetic magnetic microparticles(MMPs,3.8μm)that mimic the specific functionalities of CD20 on malignant B cells were developed for the first time.Benefit from the numerous ligand binding sites(Ni^(2+))on the pompon mum-like MMPs,these novel materials achieved≥10 times higher peptide ligand densities(>2300 mg/g)and antibody binding capacities(1380 mg/g)compared to previous reported biomaterials.Leveraging the high specificity of the mimotope peptide,rituximab can be precisely recognized and enriched from cell culture media or serum samples.We also established an LC-MS/MS method using the MMPs for tracking rituximab biotransformation in patient serum.Intriguingly,deamidation of Asn55 and Asn33,as well as oxidation of Met81 and Met34 were observed at the key complementarity determining regions of rituximab,which could potentially influence antibody function and require careful monitoring.Overall,these versatile biomimetic MMPs demonstrate superior recognition and enrichment capabilities for target antibodies,offering interesting possibilities for biotransformation analysis of biopharmaceuticals in patient serum.