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Li W, Wang S, Jin Y, Mu X, Guo Z, Qiao S, Jiang S, Liu Q, Cui X. The role of the hepatitis B virus genome and its integration in the hepatocellular carcinoma. Front Microbiol 2024; 15:1469016. [PMID: 39309526 PMCID: PMC11412822 DOI: 10.3389/fmicb.2024.1469016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2024] [Accepted: 08/19/2024] [Indexed: 09/25/2024] Open
Abstract
The integration of Hepatitis B Virus (HBV) is now known to be closely associated with the occurrence of liver cancer and can impact the functionality of liver cells through multiple dimensions. However, despite the detailed understanding of the characteristics of HBV integration and the mechanisms involved, the subsequent effects on cellular function are still poorly understood in current research. This study first systematically discusses the relationship between HBV integration and the occurrence of liver cancer, and then analyzes the status of the viral genome produced by HBV replication, highlighting the close relationship and structure between double-stranded linear (DSL)-HBV DNA and the occurrence of viral integration. The integration of DSL-HBV DNA leads to a certain preference for HBV integration itself. Additionally, exploration of HBV integration hotspots reveals obvious hotspot areas of HBV integration on the human genome. Virus integration in these hotspot areas is often associated with the occurrence and development of liver cancer, and it has been determined that HBV integration can promote the occurrence of cancer by inducing genome instability and other aspects. Furthermore, a comprehensive study of viral integration explored the mechanisms of viral integration and the internal integration mode, discovering that HBV integration may form extrachromosomal DNA (ecDNA), which exists outside the chromosome and can integrate into the chromosome under certain conditions. The prospect of HBV integration as a biomarker was also probed, with the expectation that combining HBV integration research with CRISPR technology will vigorously promote the progress of HBV integration research in the future. In summary, exploring the characteristics and mechanisms in HBV integration holds significant importance for an in-depth comprehension of viral integration.
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Affiliation(s)
- Weiyang Li
- Jining Medical University, Jining, China
- School of Biological Science, Jining Medical University, Rizhao, China
| | - Suhao Wang
- School of Biological Science, Jining Medical University, Rizhao, China
| | - Yani Jin
- School of Biological Science, Jining Medical University, Rizhao, China
| | - Xiao Mu
- School of Biological Science, Jining Medical University, Rizhao, China
| | - Zhenzhen Guo
- Jining First People's Hospital, Shandong First Medical University, Jining, China
| | - Sen Qiao
- Jining First People's Hospital, Shandong First Medical University, Jining, China
| | - Shulong Jiang
- Jining First People's Hospital, Shandong First Medical University, Jining, China
| | - Qingbin Liu
- Jining First People's Hospital, Shandong First Medical University, Jining, China
- Clinical Medical Laboratory Center, Jining First People's Hospital, Shandong First Medical University, Jining, China
| | - Xiaofang Cui
- Jining Medical University, Jining, China
- School of Biological Science, Jining Medical University, Rizhao, China
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Tan WS, Ho KL. Phage display creates innovative applications to combat hepatitis B virus. World J Gastroenterol 2014; 20:11650-11670. [PMID: 25206271 PMCID: PMC4155357 DOI: 10.3748/wjg.v20.i33.11650] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/25/2013] [Accepted: 05/05/2014] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20th century has reduced significantly the rate of the viral infection. However, currently there is no effective treatment for chronic HBV carriers. Newly emerging vaccine escape mutants and drug resistant strains have complicated the viral eradication program. The entire world is now facing a new threat of HBV and human immunodeficiency virus co-infection. Could phage display provide solutions to these life-threatening problems? This article reviews critically and comprehensively the innovative and potential applications of phage display in the development of vaccines, therapeutic agents, diagnostic reagents, as well as gene and drug delivery systems to combat HBV. The application of phage display in epitope mapping of HBV antigens is also discussed in detail. Although this review mainly focuses on HBV, the innovative applications of phage display could also be extended to other infectious diseases.
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Kalaycioglu AT, Russell PH, Howard CR. The characterization of the neutralizing bovine viral diarrhea virus monoclonal antibodies and antigenic diversity of E2 glycoprotein. J Vet Med Sci 2012; 74:1117-20. [PMID: 22673562 DOI: 10.1292/jvms.11-0187] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Bovine viral diarrhea virus (BVDV) is associated with a range of economically important diseases of cattle including reproductive disorders and an acute fatal hemorrhagic disease. Neutralizing antibodies that bind to the E2 glycoprotein are important predictors of vaccinal immunity. Neutralization tests using the NADL strain of BVDV and five anti-E2 monoclonal antibodies showed one, Wb163, neutralized the NADL strain of BVDV in an unexpected manner. Its titer was 10,000 compared to <35 as reported previously. The present stock of NADL differed from that of the earlier study in that the amino acid at position 79 of E2 was Valine instead of Glutamic acid. MAb Wb163 may, however, recognize a less important neutralizing epitope than another mAb Wb166, because it was less cross reactive than mAb Wb166, had a neutralizing titer 50-fold lower than Wb166 and was of lower relative affinity than Wb166. Variations in the amino terminus of E2 will be discussed in the context of vaccinal immunity.
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Chen SM, Kung CM, Yang WJ, Wang HL. Efficacy of the nationwide hepatitis B infant vaccination program in Taiwan. J Clin Virol 2011; 52:11-6. [PMID: 21767983 DOI: 10.1016/j.jcv.2011.06.012] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2011] [Revised: 06/20/2011] [Accepted: 06/24/2011] [Indexed: 12/17/2022]
Abstract
BACKGROUND Taiwan launched a nationwide infant vaccination program for hepatitis B (HB) in 1984. OBJECTIVES This study evaluated the seroprevalence of hepatitis B virus (HBV) and the incidence of high alanine aminotransferase (ALT) level among young adults prior to, during, and since the introduction of the nationwide HBV vaccination program. STUDY DESIGN Researchers recruited 101,584 freshmen (male:female=1.114:1; mean age, 18.5±0.5 years) from 21 universities between 1995 and 2009 (birth cohorts 1977-1991) in Taiwan, testing for serum hepatitis B surface antigens (HBsAg), hepatitis e antigens (HBeAg), antibodies against HBsAg (anti-HBs), and liver function tests, including ALT and aspartate aminotransferase (AST). RESULTS The results showed that the prevalence of HBsAg decreased significantly from 14.3% in 1995 to 1.1% in 2009 and the seroprevalence of HBeAg decreased significantly from 5.9% in 1995 to 0.3% in 2009. Seroconversion to anti-HBs maintained a steady rate above 50% between 1995 and 2007, but declined considerably to 36.6% and 36.4% in 2008 and 2009, respectively. Subject with HBeAg seropositivity was in 43.94% of HBV carriers. Double seronegativity for HBsAg and anti-HBs was observed in 2007 (47.8%), 2008 (62.3%), and 2009 (62.5%). High ALT level was observed in 5.74% of the subjects, particular among HBV-carriers (16.5% of HBV carrier vs. 5.0% of non-HBV carrier; ORs, 3.733; 95% CIs, 3.463-4.023, p<0.0001). Subjects with high ALT level were significantly positively associated with HBeAg (10.5% of HBeAg seropositive vs. 1.9% of HBeAg seronegative; ORs, 6.195; 95%CI, 5.629-6.818; p<0.0001). Male subjects were more easily infected by HBV than female subjects were (HBsAg, ORs, 1.355, 95% CI, 1.283-1.431; HBeAg, ORs, 1.324, 95% CI, 1.218-1.439, p<0.0001), and significantly more male subjects had high ALT levels than female subjects did (ORs, 4.087; 95% CI, 3.819-4.375, p<0.0001). CONCLUSIONS The mass vaccination program successfully reduced the HBV carrier rate and prevalence of chronic hepatitis B in Taiwan. However, the low percentage of anti-HBV in 2008 and 2009 remains unresolved.
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Affiliation(s)
- Szu-Ming Chen
- Department of Medical Laboratory Science and Biotechnology, College of Biomedical Science and Technology, Yuanpei University, No. 306, Rd. Yuanpei, Hsinchu (300), Taiwan
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Kalaycioglu AT, Russell PH, Howard CR. Peptide mimics of hapten DNP: the effect of affinity of anti-DNP monoclonal antibodies for the selection of phage-displayed mimotopes. J Immunol Methods 2011; 366:36-42. [PMID: 21262229 DOI: 10.1016/j.jim.2011.01.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2010] [Revised: 12/17/2010] [Accepted: 01/10/2011] [Indexed: 11/18/2022]
Abstract
Biopanning of two linear (6- and 15-mer) and two constrained (10- and 17-mer) phage-displayed peptide libraries with two anti-DNP monoclonal antibodies (mAbs) selected seven unique peptide sequences using only the low affinity anti-DNP monoclonal antibody. The selected peptides contained two of 6, one of 10, two of 15 and two of 17 amino acids in length. They were all rich in hydrophobic residues. Both 15-mer peptides had antigenic regions of eight amino acids as revealed by a spot scan assay. Two of the 17-mer and one of the 10-mer peptides displayed on phage competed with free DNP for the low affinity anti-DNP mAb. These findings highlight (i) the selective power of phage displayed peptide libraries to identify peptides that mimic the shape of a small hapten molecule such as DNP, (ii) the possible preferential bias of phage libraries towards low affinity antibodies, (iii) the importance of using a panel of phage libraries for selecting peptide mimics.
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Affiliation(s)
- Atila T Kalaycioglu
- Department of Pathology and Infectious Diseases, Royal Veterinary College, University of London, Royal College Street, London NW1 0TU, UK.
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El-Attar LMR, Partidos CD, Howard CR. A peptide mimotope of hepatitis C virus E2 protein is immunogenic in mice and block human anti-HCV sera. J Med Virol 2010; 82:1655-65. [PMID: 20827761 DOI: 10.1002/jmv.21857] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Conformational B-cell epitopes on the HCV E2 protein recognized by human antibodies were characterized by the use of a peptide mimotope named K1. K1 was identified by two HCV anti-E2 monoclonal antibodies (mAbs) following selection and purification of phage clones containing a 15-mer random peptide insert. Murine antisera to the mimotope K1 recognized the E2 protein. Five of eight human sera from patients who had cleared HCV recognized the K1 mimotope. Binding to E2 in four individuals with the capacity to block E2-CD81 interaction was inhibited by the mimotope K1. The results demonstrate that anti-E2 antibodies in sera from patients who have cleared HCV infection are directed against a conformational B-cell epitope on E2 that can be mimicked with linear synthetic peptides. These findings could have implications for vaccine design by employing linear mimotopes to direct B-cell responses against those specific E2 epitopes that may correlate with immunity.
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Affiliation(s)
- L M R El-Attar
- Department of Pathology and Infectious Diseases, Royal Veterinary College, London, UK.
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Gous N, Bhimma R, Kew M, Kramvis A. Retrospective characterization of the S open reading frame of HBV isolated from children with membranous nephropathy treated with interferon-alpha2b. Antivir Ther 2010; 15:61-9. [PMID: 20167992 DOI: 10.3851/imp1487] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
BACKGROUND A causal relationship exists between HBV infection and membranous nephropathy. The association is especially close in Black children in sub-Saharan Africa. Interferon-alpha2b is commonly used to treat this condition, but is effective in only 30-40% of patients. The reason for the poor response is unknown. The objective of this study was to determine if mutations in the surface gene of HBV isolated from Black children with HBV-associated membranous nephropathy before, during and after interferon treatment, have any effect on treatment response and vice versa. METHODS HBV DNA was extracted from a responder, a reverter and a non-responder before and after initiation of 16 weeks of interferon-alpha2b treatment. The preS1/preS2/S region was amplified, cloned and sequenced. RESULTS The preS2 region was the most variable in the reverter and the non-responder, and the S region was the most variable in the non-responder. Phylogenetic analysis showed that the viral population dynamics between the responder and the reverter/non-responder strains differed as a result of mutations in the surface gene. CONCLUSIONS The presence of mutations in the S region of HBV could be used as predictive markers to differentiate interferon-alpha2b responders from non-responders provided that detailed analysis of further genomes confirms our findings.
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Affiliation(s)
- Natasha Gous
- Hepatitis Virus Diversity Research Programme (formerly MRC/CANSA/University Molecular Hepatology Research Unit), Department of Internal Medicine, University of the Witwatersrand, Johannesburg, South Africa
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Chang DK, Lin CT, Wu CH, Wu HC. A novel peptide enhances therapeutic efficacy of liposomal anti-cancer drugs in mice models of human lung cancer. PLoS One 2009; 4:e4171. [PMID: 19137069 PMCID: PMC2614347 DOI: 10.1371/journal.pone.0004171] [Citation(s) in RCA: 89] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2008] [Accepted: 12/02/2008] [Indexed: 12/02/2022] Open
Abstract
Lung cancer is the leading cause of cancer-related mortality worldwide. The lack of tumor specificity remains a major drawback for effective chemotherapies and results in dose-limiting toxicities. However, a ligand-mediated drug delivery system should be able to render chemotherapy more specific to tumor cells and less toxic to normal tissues. In this study, we isolated a novel peptide ligand from a phage-displayed peptide library that bound to non-small cell lung cancer (NSCLC) cell lines. The targeting phage bound to several NSCLC cell lines but not to normal cells. Both the targeting phage and the synthetic peptide recognized the surgical specimens of NSCLC with a positive rate of 75% (27 of 36 specimens). In severe combined immunodeficiency (SCID) mice bearing NSCLC xenografts, the targeting phage specifically bound to tumor masses. The tumor homing ability of the targeting phage was inhibited by the cognate synthetic peptide, but not by a control or a WTY-mutated peptide. When the targeting peptide was coupled to liposomes carrying doxorubicin or vinorelbine, the therapeutic index of the chemotherapeutic agents and the survival rates of mice with human lung cancer xenografts markedly increased. Furthermore, the targeting liposomes increased drug accumulation in tumor tissues by 5.7-fold compared with free drugs and enhanced cancer cell apoptosis resulting from a higher concentration of bioavailable doxorubicin. The current study suggests that this tumor-specific peptide may be used to create chemotherapies specifically targeting tumor cells in the treatment of NSCLC and to design targeted gene transfer vectors or it may be used one in the diagnosis of this malignancy.
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Affiliation(s)
- De-Kuan Chang
- Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan
- Institute of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Chin-Tarng Lin
- Institute of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Chien-Hsun Wu
- Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan
| | - Han-Chung Wu
- Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan
- Institute of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan
- * E-mail:
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Lee YJ, Choi HJ, Lim JS, Earm JH, Lee BH, Kim IS, Frøkiær J, Nielsen S, Kwon TH. A novel method of ligand peptidomics to identify peptide ligands binding to AQP2-expressing plasma membranes and intracellular vesicles of rat kidney. Am J Physiol Renal Physiol 2008; 295:F300-9. [DOI: 10.1152/ajprenal.00006.2008] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Aquaporin-2 (AQP2), the vasopressin-regulated water channel in collecting duct principal cells, plays a key role in the regulation of body water balance. We aimed to isolate high-affinity peptide ligands that bind to immunoisolated AQP2-expressing plasma membrane (PM) or intracellular vesicle (ICV) preparations from rat kidney by the in vitro phage display technique. Immunoblotting revealed that AQP2 was exclusively expressed in the immunoisolated AQP2 membrane fractions (PM and ICV), compared with the nonimmunoisolated or preimmune IgG pulldown rat kidney samples. Moreover, AQP1 or H+-ATPase (B1 subunit) expression was minimal in the immunoisolated AQP2 membrane fractions, indicating the specificity of AQP2 membrane isolation. A phage peptide library based on T7 415-1b phage vector displaying CX7C was constructed. After three rounds of biopanning, seven phage clones of high frequency were selected, which showed high affinity to the AQP2-containing PM or ICV fractions compared with a nonrecombinant T7 insertless phage clone. In contrast, these phage clones showed lower affinity to H+-ATPase-containing fractions. Fluorescein-conjugated peptide labeling was associated with intracellular compartment and PM of primary cultured inner medullary collecting duct cells, relative to absent or very weak labeling with fluorescein-conjugated control peptide. Library analyses demonstrated proteins that had motifs homologous to the peptide ligands, albeit with a high probability of a random match due to short peptide sequences. In summary, we applied the in vitro phage display technique to identify high-affinity peptide ligands to AQP2-expressing membranes. Library analyses identified proteins having homologous motifs, which need to be examined for involvement in AQP2 trafficking and regulation.
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Kalaycioglu AT, Russell PH, Howard CR. Selection of mimotopes of Bovine Viral Diarrhoea Virus using a solid-phase peptide library. Vaccine 2007; 25:7081-6. [PMID: 17825961 DOI: 10.1016/j.vaccine.2007.07.066] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2007] [Revised: 07/18/2007] [Accepted: 07/28/2007] [Indexed: 11/22/2022]
Abstract
Bovine Viral Diarrhoea Virus (BVDV) is an important pathogen of cattle, causing important economical losses world-wide. In this study, an 8-mer solid-phase peptide library was screened with a neutralising monoclonal antibody 157 to generate mimotopes mimicking a conformational neutralising epitope of BVDV E2 protein. Two sequences selected 157A1 LFEQYYYF and 157A2 LYRFGEFD that did not show a high structural or sequence similarity with BVDV E2 glycoprotein reacted specifically with monoclonal antibody 157 when presented as solid-phase peptides in a SPOT scan assay. These results indicate that combinatorial peptide libraries can be used to identify potential mimotopes of conformational epitopes.
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Affiliation(s)
- Atila T Kalaycioglu
- Department of Pathology and Infectious Diseases, Royal Veterinary College, Royal College Street, London NW1 OTU, UK.
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Chen YC, Huang HN, Lin CT, Chen YF, King CC, Wu HC. Generation and characterization of monoclonal antibodies against dengue virus type 1 for epitope mapping and serological detection by epitope-based peptide antigens. CLINICAL AND VACCINE IMMUNOLOGY : CVI 2007; 14:404-11. [PMID: 17287314 PMCID: PMC1865613 DOI: 10.1128/cvi.00249-06] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Dengue virus (DEN), the pathogen behind dengue hemorrhagic fever, remains a public health problem in Asia and South America. In this study, monoclonal antibodies (MAbs) against DEN serotype 1 (DEN-1) were generated by fusing NSI/1-Ag4-1 mouse myeloma cells with lymphocytes from BALB/c mice immunized with DEN-1. Twelve MAbs were found to react specifically to the DENs by enzyme-linked immunosorbent assay, immunofluorescence analysis, and immunoblotting analysis. Five MAbs, namely, DA4-7, DA6-7, DA9-5, DA10-2, and DA11-13, were found to react with envelope proteins of DEN-1. Two serotype-specific MAbs of DEN-1, DA6-7 and DA11-13, were further shown to neutralize DEN-1 infection by a plaque reduction neutralization test. The neutralizing epitopes of these MAbs were further identified from a random peptide library displayed on phage. Immunopositive phage clones reacted specifically with these MAbs and did not react with normal mouse serum. Epitope-based peptide antigens were proved able to detect antibodies in serum samples collected from DEN-1-infected patients but not in those taken from DEN-2-infected patients or healthy controls. We believe that these MAbs and neutralizing epitopes will provide information that will lead to the development of DEN-1 serotype-specific diagnostic reagents and vaccines.
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Affiliation(s)
- Yun-Ching Chen
- Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan
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Szardenings M. Phage display of random peptide libraries: applications, limits, and potential. J Recept Signal Transduct Res 2004; 23:307-49. [PMID: 14753295 DOI: 10.1081/rrs-120026973] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
The identification of ligands from large biological libraries by phage display has now been used for almost 15 years. Most of the successful reports on high-affinity ligand identification originated from work with different antibody libraries. In contrast, the progress of applying phage display to random peptide libraries was relatively slow. However, in the last few years several improvements have led to an increasing number of published peptide ligands identified by phage display from such libraries and which exhibited good biological activity and high affinity. This review summarizes the current state and the technical progress of the application of random peptide libraries using filamentous phage for ligand identification.
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Affiliation(s)
- Michael Szardenings
- Institute of Biochemistry and Biotechnology, Technical University of Braunschweig, Braunschweig, Germany.
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Wu HC, Jung MY, Chiu CY, Chao TT, Lai SC, Jan JT, Shaio MF. Identification of a dengue virus type 2 (DEN-2) serotype-specific B-cell epitope and detection of DEN-2-immunized animal serum samples using an epitope-based peptide antigen. J Gen Virol 2003; 84:2771-2779. [PMID: 13679612 DOI: 10.1099/vir.0.19228-0] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
In this study, a serotype-specific monoclonal antibody (mAb), D2 16-1 (Ab4), against dengue virus type 2 (DEN-2) was generated. The specificity of Ab4, which recognized DEN-2 non-structural protein 1, was determined by ELISA, immunofluorescence and immunoblotting analyses. The serotype-specific B-cell epitope of Ab4 was identified further from a random phage-displayed peptide library; selected phage clones reacted specifically with Ab4 and did not react with other mAbs. Immunopositive phage clones displayed a consensus motif, His–Arg/Lys–Leu/Ile, and a synthetic peptide corresponding to the phage-displayed peptide bound specifically to Ab4. The His and Arg residues in this epitope were found to be crucial for peptide binding to Ab4 and binding activity decreased dramatically when these residues were changed to Leu. The epitope-based synthetic peptide not only identified serum samples from DEN-2-immunized mice and rabbits by ELISA but also differentiated clearly between serum samples from DEN-2- and Japanese encephalitis virus-immunized mice. This mAb and its epitope-based peptide antigen will be useful for serologic diagnosis of DEN-2 infection. Furthermore, DEN-2 epitope identification makes it feasible to dissect antibody responses to DEN and to address the role of antibodies in the pathogenesis of primary and secondary DEN-2 infections.
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Affiliation(s)
- Han-Chung Wu
- Graduate Institute of Oral Biology, College of Medicine, National Taiwan University, Taipei, Taiwan 100, Republic of China
| | - Mei-Ying Jung
- Graduate Institute of Oral Biology, College of Medicine, National Taiwan University, Taipei, Taiwan 100, Republic of China
| | - Chien-Yu Chiu
- Graduate Institute of Oral Biology, College of Medicine, National Taiwan University, Taipei, Taiwan 100, Republic of China
| | - Ting-Ting Chao
- Graduate Institute of Oral Biology, College of Medicine, National Taiwan University, Taipei, Taiwan 100, Republic of China
| | - Szu-Chia Lai
- Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan 100, Republic of China
| | - Jia-Tsrong Jan
- Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan 100, Republic of China
| | - Men-Fang Shaio
- Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan 100, Republic of China
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Yamada T, Iwabuki H, Kanno T, Tanaka H, Kawai T, Fukuda H, Kondo A, Seno M, Tanizawa K, Kuroda S. Physicochemical and immunological characterization of hepatitis B virus envelope particles exclusively consisting of the entire L (pre-S1 + pre-S2 + S) protein. Vaccine 2001; 19:3154-63. [PMID: 11312011 DOI: 10.1016/s0264-410x(01)00017-2] [Citation(s) in RCA: 64] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The hepatitis B virus (HBV) envelope (env) protein is composed of three regions; the 108- or 119-residue pre-S1 region involved in the direct interaction with hepatocytes, the 55-residue pre-S2 region associated with the polymerized albumin-mediated interaction, and the major 226-residue S protein region. Thus, to improve the immunogenic potency of conventional HB vaccines, development of a new vaccine containing the entire pre-S1 region in addition to pre-S2 and S is desired. We previously reported the efficient production of the HBV env L (pre-S1 + pre-S2 + S) protein in the recombinant yeast cells [J Biol Chem 267 (1992) 1953]. In this study, the HBV env L protein produced as nano-particles in yeast has been purified and characterized. By equilibrium sedimentation, an average molecular weight of L particle was estimated to be approximately 6.4 x 10(6), indicating that about 110 molecules of L proteins are assembled into an L particle. By atomic force microscopy in a moist atmosphere, the L particles were observed as large spherical particles with a diameter of 50-500 nm. The L particles were stable on short-time heating at a high temperature and long-time storage at a low temperature but rather unstable on repeated freezing and thawing and treatment with dithiothreitol. When immunized in mice, L particles elicited efficiently and simultaneously the anti-S, anti-pre-S2, and anti-pre-S1 antibodies. The ED(50) values in mice for the anti-S and anti-pre-S2 antibodies were similar to those elicited by the M (pre-S2 + S) particles. Furthermore, the anti-pre-S1 rabbit antibodies were found to recognize various segments of the pre-S1 region, including the pre-S1 (21-47) segment. These results show the high ability of L particles to induce all antibodies against HBV env proteins, hence promising the future application of L particles for the next generation HB vaccine.
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Affiliation(s)
- T Yamada
- Department of Structural Molecular Biology, Institute of Scientific and Industrial Research (SANKEN), Osaka University, 8-1 Mihogaoka, 567-0047, Ibaraki, Japan
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Wu HC, Huang YL, Chao TT, Jan JT, Huang JL, Chiang HY, King CC, Shaio MF. Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients. J Clin Microbiol 2001; 39:977-82. [PMID: 11230414 PMCID: PMC87860 DOI: 10.1128/jcm.39.3.977-982.2001] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Using a serotype-specific monoclonal antibody (MAb) of dengue virus type 1 (DEN-1), 15F3-1, we identified the B-cell epitope of DEN-1 from a random peptide library displayed on phage. Fourteen immunopositive phage clones that bound specifically to MAb 15F3-1 were selected. These phage-borne peptides had a consensus motif of HxYaWb (a = S/T, b = K/H/R) that mimicked the sequence HKYSWK, which corresponded to amino acid residues 111 to 116 of the nonstructural protein 1 (NS1) of DEN-1. Among the four synthetic peptides corresponding to amino acid residues 110 to 117 of the NS1 of DEN-1, -2, -3, and -4, only one peptide, EHKYSWKS (P14M) of DEN-1, was found to bind to 15F3-1 specifically. Furthermore, P14M was shown to inhibit the binding of phage particles to 15F3-1 in a competitive inhibition assay. Histidine(111) (His(111)) was crucial to the binding of P14M to 15F3-1, since its binding activity dramatically reduced when it changed to leucine(111) (Leu(111)). This epitope-based peptide demonstrated its clinical diagnostic potential when it reacted with a high degree of specificity with serum samples obtained from both DEN-1-infected rabbits and patients. Based on these observations, our DEN-1 epitope-based serologic test could be useful in laboratory viral diagnosis and in understanding the pathogenesis of DEN-1.
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Affiliation(s)
- H C Wu
- Institute of Preventive Medicine, National Defense Medical Center, P.O. Box 90048-700, San-Hsia, Taipei, Taiwan, Republic of China.
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16
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D'Mello F, Howard CR. An improved selection procedure for the screening of phage display peptide libraries. J Immunol Methods 2001; 247:191-203. [PMID: 11150550 DOI: 10.1016/s0022-1759(00)00318-5] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Peptide sequences that bind to a wide range of ligands such as monoclonal antibodies, receptors and carbohydrates have been successfully identified after screening phage display peptide libraries. However, these procedures tend to select mainly medium to low affinity-binding clones. A modified screening procedure has been developed in order to improve the efficiency of this process such that high avidity/affinity binding clones are preferentially selected. Three different solid phase binding surfaces were evaluated for the attachment of antibody during the screening procedure and a stepwise decrease in the pH of the elution buffer introduced during the final round of biopanning. The monoclonal antibody MA 18/7 was used to screen a 15-mer peptide library. This antibody is well-characterised and its binding site has been mapped to residues 28-37 of the pre-S1 protein of the hepatitis B virus. The antibody was either biotinylated and attached to polystyrene plates via a streptavidin-biotin 'bridge', or bound directly to 1/4 in. polystyrene beads, or to 11 microm latex beads. A significant enrichment of binding clones was observed when the monoclonal antibody was attached directly to polystyrene or latex beads as compared to the biotinylated antibody. All mimotopes identified after biopanning with the antibody attached to the polystyrene beads possessed a central core motif, identical or similar to the sequence DPAF contained within the epitope binding site of MA 18/7 on the native pre-S molecule. However, this motif was only observed in 30% of clones isolated after biopanning using the 11 microm latex beads and in 2% of clones isolated after biopanning on the streptavidin-coated plates. Immunoblotting with the monoclonal antibody MA 18/7 confirmed binding to clones containing the DPAF sequence or a similar motif. A stepwise reduction in the pH of the elution buffer in the final round of biopanning resulted in the removal of clones that possessed low affinity binding motifs, thereby increasing the percentage of clones containing high affinity binding motifs in the final elution step at pH 2.0. Thus, the combined use of polystyrene beads and a stepwise decrease in the pH of the elution buffer in the final round of biopanning resulted in the elimination of non-binding clones and an increase in the efficiency in isolating high affinity binding clones.
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Affiliation(s)
- F D'Mello
- Royal Veterinary College, Department of Pathology and Infectious Diseases, Royal College Street, London NW1 OTU, UK
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17
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Abstract
Theoretically it seems highly unlikely that relatively small peptides could mimic functionally discontinuous epitopes of antigens. Nevertheless various recent reports show this to be the case. Peptide mimics of protein-, polysaccharide- and DNA-epitopes have been shown to be able to replace the native epitope. Moreover, some of them are able to induce, when used in a vaccine, antibodies with the same activity as that of the antibody used as a template. These mimics, called mimotopes, can be used in vaccines and diagnostics and can be developed more or less systematically using solely antibodies and random, semi-random and dedicated peptide arrays or libraries. Furthermore, the mimotope concept which seems to have proven itself for antibody and antigen interaction can be applied equally well to many receptor ligand interactions and thus may form a new generic approach to the development of drugs. Ltd.
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Affiliation(s)
- R H Meloen
- Pepscan Systems BV, Lelystad, The Netherlands
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18
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Heiskanen T, Lundkvist A, Soliymani R, Koivunen E, Vaheri A, Lankinen H. Phage-displayed peptides mimicking the discontinuous neutralization sites of puumala Hantavirus envelope glycoproteins. Virology 1999; 262:321-32. [PMID: 10502511 DOI: 10.1006/viro.1999.9930] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We selected peptide ligands mimicking the surface structure of discontinuous binding sites of Puumala hantavirus-neutralizing monoclonal antibodies from a random 18-amino acid peptide library containing a disulfide bridge in a fixed position and displayed on a filamentous phage. The varying of selection conditions, either by shortening of the association time or by competitive elution with antigen, was crucial for the selection of peptide inserts that could be aligned with the primary sequences of the envelope glycoproteins G1 and G2. Correspondingly, when the envelope glycoprotein sequences were synthesized as overlapping peptides as spots on membrane, the same site in primary structure was found as with phage display, which corroborates the use of the two methods in mapping of conformational epitopes. Also, epitopes reactive with early-phase sera from Puumala virus infection were defined with the pepspot assay in the amino-terminal region of G1. Similarities of the selected phage clones to a monoclonal antibody-escape mutant site and to a linear early-phase epitope were found.
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Affiliation(s)
- T Heiskanen
- Department of Virology, University of Helsinki, Helsinki, FIN-00014, Finland.
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19
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Tang Y, Beuerlein G, Pecht G, Chilton T, Huse WD, Watkins JD. Use of a peptide mimotope to guide the humanization of MRK-16, an anti-P-glycoprotein monoclonal antibody. J Biol Chem 1999; 274:27371-8. [PMID: 10488067 DOI: 10.1074/jbc.274.39.27371] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
A mimotope-guided strategy for engineering antibodies directed against orphan targets or antigens that are difficult to purify was developed and used to humanize the murine MRK-16 monoclonal antibody (mAb). MRK-16 recognizes a conformational epitope of a 170-kDa membrane protein, termed P-glycoprotein (P-gp). Elevated expression of P-gp on tumor cells is associated with resistance to cytotoxic drugs, a major obstacle in chemotherapy. Murine MRK-16 was used to enrich and screen a phage-displayed peptide library to identify reactive mimotopes. One peptide, termed ALR1, was enriched to a greater extent than others and subsequently was expressed as a fusion protein with glutathione S-transferase. ALR1 fusion protein bound MRK-16 specifically and inhibited binding of MRK-16 to cells expressing elevated levels of P-gp. To humanize MRK-16, the murine complementarity determining regions were grafted onto homologous human heavy and light chain variable region frameworks. Framework residues that differed between the murine MRK-16 and the homologous human templates were analyzed and subsequently, five framework positions potentially important for maintaining the specificity and affinity of MRK-16 were identified. A combinatorial library consisting of 32 variants encoding all possible combinations of murine and human residues at the five differing framework positions was expressed in a phage system. In the absence of purified P-gp, ALR1 fusion protein was used as surrogate antigen to screen the antibody library to identify the framework combination that most preserved the binding activity of the mAb. On the basis of the initial screening against the mimotope four antibody variants were selected for further characterization. The binding affinity of these variants for the ALR1 fusion protein correlated with their binding to cells expressing elevated levels of P-gp. Thus, peptide mimotopes which can be identified for virtually any antibody including those that recognize conformational or carbohydrate epitopes, can serve as antigen templates for antibody engineering.
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Affiliation(s)
- Y Tang
- Ixsys, Inc., San Diego, California 92121, USA
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20
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D'Mello F, Kairo SK, Howard CR, Partidos CD. Mapping the specificity of an anti-feline immunodeficiency virus monoclonal antibody using a filamentous phage displayed peptide library. Vaccine 1999; 18:371-5. [PMID: 10506664 DOI: 10.1016/s0264-410x(99)00208-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Abstract
Recent developments in peptide technology enable the use of random peptide libraries for identifying linear amino acid sequences (mimotopes) which can mimic conformational epitopes without necessarily exhibiting amino acid sequence homology with the native linear sequence. In this study a 15-mer random peptide library displayed on the surface of a filamentous phage has been used to characterise the conformational epitopes recognised by a monoclonal antibody raised against the envelope protein gp120 of feline immunodeficiency virus (FIV). Three mimotopes were identified that reacted with the selecting antibody in an immunoblot assay. Sequence analysis revealed that, whereas the three mimotopes had several amino acids in common, there was no significant homology with the primary amino acid sequence of gp120 although some amino acids were shared between the variable region (V3) and the three mimotopes. Petide mimtopes of complex retroviral glycoproteins may have potential uses as novel vaccines and for the serological diagnosis of FIV.
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Affiliation(s)
- F D'Mello
- Department of Pathology and Infectious Diseases, Royal Veterinary College, Royal College Street, London, UK
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21
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Küttner G, Kramer A, Schmidtke G, Giessmann E, Dong L, Roggenbuck D, Scholz C, Seifert M, Stigler RD, Schneider-Mergener J, Porstmann T, Höhne W. Characterization of neutralizing anti-pre-S1 and anti-pre-S2 (HBV) monoclonal antibodies and their fragments. Mol Immunol 1999; 36:669-83. [PMID: 10509818 DOI: 10.1016/s0161-5890(99)00074-7] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Single-chain Fv fragments (scFv) were generated from two murine monoclonal antibodies directed to the neutralizing epitopes of the pre-S1 and pre-S2 region of hepatitis B virus, respectively, using different assembly cloning strategies. The scFv fragments were solubly expressed in E. coli. Dissociation constants were in the nanomolar range for all forms (whole IgG antibodies, Fab fragment and scFv fragments). The epitopes of both antibodies were mapped using solid phase peptide synthesis on continuous cellulose membranes and turned out to be linear determinants. The minimal epitope for the anti-pre-S2 antibody 1F6 was identified to be DPRVRGLYF (amino acid 133-141 of the pre-S region). For the anti-pre-S1 antibody MA 18/7 the minimal epitope proved to be the hexamer LDPAFR (amino acid 30-35 of the pre-S region). Complete substitutional analyses as well as truncation experiments revealed key residues for these antibody-antigen interactions. On the basis of those results we used computer-assisted modeling techniques to suggest models for both antibody-peptide interactions providing insight into the structural basis of these molecular recognitions.
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Affiliation(s)
- G Küttner
- Institut für Biochemie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Germany.
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22
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Sunyach C, Rollier C, Robaczewska M, Borel C, Barraud L, Kay A, Trépo C, Will H, Cova L. Residues critical for duck hepatitis B virus neutralization are involved in host cell interaction. J Virol 1999; 73:2569-75. [PMID: 10074101 PMCID: PMC104011 DOI: 10.1128/jvi.73.4.2569-2575.1999] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
To date, no detailed analysis of the neutralization properties of duck hepatitis B virus (DHBV) has been reported, and it is not clear whether any of the known neutralization epitopes correspond to the viral receptor binding site or to sequences involved in the cell entry pathway. We demonstrate here that antibodies directed against two overlapping peptides (amino acids 83 to 97 and 93 to 107), covering the sequences of most DHBV pre-S neutralizing epitopes, both inhibit virus binding to primary duck hepatocytes and neutralize virus infectivity. An extensive mutagenesis of the motif 88WTP90, which is the shortest sequence of the epitope recognized by the virus-neutralizing monoclonal antibody (MAb) 900 was performed in order to define the amino acids involved in these interactions. Single point mutations within this epitope affected neither virus replication nor infectivity but abolished virus neutralization by MAb 900 completely. Interestingly, mutants with two and three consecutive residue replacements (SIP and SIH) within this epitope retained replication competence but were no longer infectious. The loss of infectivity of SIH and SIP mutant particles was associated with significantly reduced binding to primary duck hepatocytes and could be rescued by trans complementation with wild-type pre-S protein. Taken together, these results indicate that each amino acid of the DHBV pre-S sequence 88WTP90 is critical for recognition by the neutralizing MAb 900 and that replacement of the first two or all three residues strongly reduces virus interaction with hepatocytes and abrogates infectivity. These data imply that the motif 88WTP90 contains key residues which are critical for interaction with both the neutralizing MAb and the host cell.
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Affiliation(s)
- C Sunyach
- Unité de Recherche sur les Virus des Hépatites, les Rétrovirus Humains, et les Pathologies Associées, Institut National de la Santé et de la Recherche Médicale 271, 69424 Lyon Cedex 03, France
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23
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Borisova G, Borschukova O, Skrastina D, Dislers A, Ose V, Pumpens P, Grens E. Behavior of a short preS1 epitope on the surface of hepatitis B core particles. Biol Chem 1999; 380:315-24. [PMID: 10223334 DOI: 10.1515/bc.1999.043] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
The major immunodominant region of hepatitis B core particles is widely recognized as the most prospective target for the insertion of foreign epitopes, ensuring their maximal antigenicity and immunogenicity. This region was mapped around amino acid residues 79-81, which were shown by electron cryo-microscopy to be located on the tips of the spikes protruding from the surface of hepatitis B core shells. Here we tried to expose a model sequence, the short immunodominant hepatitis B preS1 epitope 31-DPAFR-35, onto the tip of the spike, with simultaneous deletion of varying stretches from the major immunodominant region of the HBc molecule. Accessibility to the monoclonal anti-preS1 antibody MA18/7 and specific immunogenicity of the preS1 epitope depended on the location and length of the deletion. While chimeras with deletions within the stretch 79-88 presented the preS1 epitope on their surface and demonstrated remarkable preS1 immunogenicity, the corresponding chimeras without any deletion or with a more prolonged deletion (79-93) were unable to provide such presentation and possessed a lower specific preS1 immunogenicity. Deletion of the stretch 79-81 was sufficient to avoid the intrinsic HBc immunogenicity of the core particles, although chimeras with deleted major immunodominant region retained their property to be recognized by human polyclonal or hyperimmune anti-HBc antibodies.
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Affiliation(s)
- G Borisova
- Biomedical Research and Study Centre, University of Latvia, Riga
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24
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Chirinos-Rojas CL, Steward MW, Partidos CD. A phage-displayed mimotope inhibits tumour necrosis factor-alpha-induced cytotoxicity more effectively than the free mimotope. Immunol Suppl 1999; 96:109-13. [PMID: 10233684 PMCID: PMC2326710 DOI: 10.1046/j.1365-2567.1999.00660.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
A phage-displayed peptide library was screened by direct interaction with human tumour necrosis factor-alpha (TNF-alpha) to identify novel antagonistic molecules of its biological activities. After several rounds of affinity selection, a phage displaying a mimotope sequence was shown to strongly inhibit, in a dose-dependent fashion, both mouse and human TNF-alpha-mediated cytotoxicity in L929 cells. The identified mimotope did not bear any sequence homology to the primary structures of the extracellular domains of either the 55 000 MW or the 75 000 MW TNF-alpha receptors, suggesting that it represents or mimics a conformational epitope involved with binding to TNF-alpha. The free 15-mer mimotope weakly inhibited TNF-alpha-induced cytotoxicity in vitro, and it did not bind to TNF-alpha as assessed by surface plasmon resonance, demonstrating the importance of mimotope presentation for its biological activities. In conclusion, this study highlights the potential of random combinatorial peptide libraries for the identification of novel inhibitors, which may serve as important tools in research that could lead to the development of TNF-alpha antagonists with therapeutic potential.
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Affiliation(s)
- C L Chirinos-Rojas
- Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK
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25
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Chirinos-Rojas CL, Steward MW, Partidos CD. A Peptidomimetic Antagonist of TNF-α-Mediated Cytotoxicity Identified from a Phage-Displayed Random Peptide Library. THE JOURNAL OF IMMUNOLOGY 1998. [DOI: 10.4049/jimmunol.161.10.5621] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Abstract
Phage-displayed peptide libraries represent a vast collection of peptide sequences that can be used to identify novel therapeutic molecules. In this report, a 15-mer phage-displayed peptide library was used to identify potential TNF-α antagonists. After direct interaction of recombinant human TNF-α with the library, four randomly selected phage clones were shown to inhibit in a dose-dependent fashion both mouse and human TNF-α-induced cytotoxicity in vitro. DNA sequencing of the positive clones revealed a common amino acid sequence that does not bear any structural similarity to the known primary structures of the extracellular domains of either 55-kDa or 75-kDa TNF receptors. This sequence was synthesized, and the peptidomimotope was shown i) to bind to the recombinant human TNF-α using surface plasmon resonance (biosensor) technology and ii) to inhibit both recombinant mouse and human TNF-α-induced cytotoxicity in vitro in a dose-dependent fashion.
These findings highlight the potential of phage-displayed random peptide libraries for the identification of novel low molecular antagonistic molecules that can block the biologic activities of TNF-α.
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Affiliation(s)
- Carlos L. Chirinos-Rojas
- *Department of Infections and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; and
| | - Michael W. Steward
- *Department of Infections and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; and
| | - Charalambos D. Partidos
- †Department of Pathology and Infectious Diseases, Royal Veterinary College, London, United Kingdom
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