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In Silico Screening of Aptamers Configuration against Hepatitis B Surface Antigen. Adv Bioinformatics 2019; 2019:6912914. [PMID: 31346332 PMCID: PMC6617924 DOI: 10.1155/2019/6912914] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2018] [Revised: 04/20/2019] [Accepted: 04/30/2019] [Indexed: 01/05/2023] Open
Abstract
Aptamer has been long studied as a substitute of antibodies for many purposes. However, due to the exceeded length of the aptamers obtained in vitro, difficulties arise in its manipulation during its molecular conjugation on the matrix surfaces. Current study focuses on computational improvement for aptamers screening of hepatitis B surface antigen (HBsAg) through optimization of the length sequences obtained from SELEX. Three original aptamers with affinity against HBsAg were truncated into five short hairpin structured aptamers and their affinity against HBsAg was thoroughly studied by molecular docking, molecular dynamics (MD) simulation, and Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) method. The result shows that truncated aptamers binding on HBsAg “a” determinant region are stabilized by the dynamic H-bond formation between the active binding residues and nucleotides. Amino acids residues with the highest hydrogen bonds hydrogen bond interactions with all five aptamers were determined as the active binding residues and further characterized. The computational prediction of complexes binding will include validations through experimental assays in future studies. Current study will improve the current in vitro aptamers by minimizing the aptamer length for its easy manipulation.
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Temporal trend of hepatitis B surface mutations in the post-immunization period: 9 years of surveillance (2005-2013) in eastern China. Sci Rep 2017; 7:6669. [PMID: 28751727 PMCID: PMC5532365 DOI: 10.1038/s41598-017-07085-z] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2017] [Accepted: 06/22/2017] [Indexed: 01/21/2023] Open
Abstract
Limited information is available about the temporal trend in the prevalence and evolution of hepatitis B virus (HBV) S-gene mutations in the post-immunization era in China. From 2005 to 2013, 1077 hepatitis B cases under 15 years of age reported through Chinese National Notifiable Disease Reporting System (NNDRS) were successfully sequenced of S-gene in Shandong province, China. A total of 97 (9.01%) cases had amino acid (aa) substitution in the “α” determinant of HBsAg. The yearly prevalence from 2005 to 2013 maintained at a relatively stable level, and showed no significant change (P > 0.05). Multivariate logistic regression analysis demonstrated that the prevalence of “α” mutations was independently associated with the maternal HBsAg status (P < 0.05), and not with surveillance year and hepatitis B vaccination (P > 0.05). The hottest mutation position was aa126 (I126S/N and T126A, 29.63%), and aa 145 (G145R/A, 25.93%). Mutated residue 126 tended to occur less frequent, while that of residue 145 was more frequent with increasing year. Our data showed that there was no increase in the frequency of HBV “α” mutations over time during the post-immunization period. However, long-term vaccination might enhance the change of HBV mutational pattern, and G145 mutation was becoming dominant.
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MEILANI MEILANI, UTSUMI TAKAKO, JUNIASTUTI JUNIASTUTI, AMIN MOCHAMAD, SOETJIPTO SOETJIPTO, HAYASHI YOSHITAKE, INGE LUSIDA MARIA. High Prevalence of Occult Hepatitis B Infection (OBI) and its Molecular Characteristics among Pregnant Women in Surabaya, Indonesia. MICROBIOLOGY INDONESIA 2016. [DOI: 10.5454/mi.10.1.1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
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Modification of Asparagine-Linked Glycan Density for the Design of Hepatitis B Virus Virus-Like Particles with Enhanced Immunogenicity. J Virol 2015; 89:11312-22. [PMID: 26339047 DOI: 10.1128/jvi.01123-15] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2015] [Accepted: 08/20/2015] [Indexed: 01/20/2023] Open
Abstract
UNLABELLED The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic components of the HBV vaccine and are platforms for the delivery of foreign antigenic sequences. To investigate structure-immunogenicity relationships for the design of improved immunization vectors, we have generated biochemically modified virus-like particles (VLPs) exhibiting glycoengineered HBsAgS. For the generation of hypoglycosylated VLPs, the wild-type (WT) HBsAgS N146 glycosylation site was converted to N146Q; for constructing hyperglycosylated VLPs, potential glycosylation sites were introduced in the HBsAgS external loop region at positions T116 and G130 in addition to the WT site. The introduced T116N and G130N sites were utilized as glycosylation anchors resulting in the formation of hyperglycosylated VLPs. Mass spectroscopic analyses showed that the hyperglycosylated VLPs carry the same types of glycans as WT VLPs, with minor variations regarding the degree of fucosylation, bisecting N-acetylglucosamines, and sialylation. Antigenic fingerprints for the WT and hypo- and hyperglycosylated VLPs using a panel of 19 anti-HBsAgS monoclonal antibodies revealed that 15 antibodies retained their ability to bind to the different VLP glyco-analogues, suggesting that the additional N-glycans did not shield extensively for the HBsAgS-specific antigenicity. Immunization studies with the different VLPs showed a strong correlation between N-glycan abundance and antibody titers. The T116N VLPs induced earlier and longer-lasting antibody responses than did the hypoglycosylated and WT VLPs. The ability of nonnative VLPs to promote immune responses possibly due to differences in their glycosylation-related interaction with cells of the innate immune system illustrates pathways for the design of immunogens for superior preventive applications. IMPORTANCE The use of biochemically modified, nonnative immunogens represents an attractive strategy for the generation of modulated or enhanced immune responses possibly due to differences in their interaction with immune cells. We have generated virus-like particles (VLPs) composed of hepatitis B virus envelope proteins (HBsAgS) with additional N-glycosylation sites. Hyperglycosylated VLPs were synthesized and characterized, and the results demonstrated that they carry the same types of glycans as wild-type VLPs. Comparative immunization studies demonstrated that the VLPs with the highest N-glycan density induce earlier and longer-lasting antibody immune responses than do wild-type or hypoglycosylated VLPs, possibly allowing reduced numbers of vaccine injections. The ability to modulate the immunogenicity of an immunogen will provide opportunities to develop optimized vaccines and VLP delivery platforms for foreign antigenic sequences, possibly in synergy with the use of suitable adjuvanting compounds.
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Characterization of C69R variant HBsAg: effect on binding to anti-HBs and the structure of virus-like particles. Arch Virol 2015; 160:2427-33. [DOI: 10.1007/s00705-015-2515-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2014] [Accepted: 06/26/2015] [Indexed: 12/11/2022]
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Ma Q, Wang Y. Comprehensive analysis of the prevalence of hepatitis B virus escape mutations in the major hydrophilic region of surface antigen. J Med Virol 2012; 84:198-206. [PMID: 22170538 DOI: 10.1002/jmv.23183] [Citation(s) in RCA: 75] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Escape mutations in the major hydrophilic region (MHR) of hepatitis B surface antigen (HBsAg) are reported widely worldwide; these mutations lead to diagnostic problems, emergence of vaccine-escape mutants, and hepatitis B immunoglobulin (HBIG) therapy failure. However, the prevalence of these mutations in different genotypes remains to be studied systematically. In the current study, 11,221 non-redundant hepatitis B virus (HBV) sequences of 8 genotypes (from A to H), obtained from the National Center for Biotechnology Information (NCBI), were analyzed to determine the prevalence of HBsAg escape mutations that were previously described. Eight important mutations associated with diagnostic failure, P120T, T126S, Q129H, G130N, S143L, D144A, and G145A/R, were prevalent in one or more genotypes, with the frequency of no less than 1%. With regard to escape variants that evade vaccine or immunoglobulin therapy, mutations were located mainly at positions 120, 126, 129, 130, 133, 134, 137, 140, 143, 144, and 145. The majority of such mutations showed genotypic heterogeneity, indicating the different distribution of the escape mutations. Most of the escape mutations clustered in the "a" determinant, indicating that this region was more likely to be affected by immune selection or antiviral therapy than other regions. Understanding the prevalence and heterogeneity of escape mutations could provide useful guidance for the improvement of diagnostic assays, design of new vaccines, and prevention of failure of HBIG therapy.
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Affiliation(s)
- Qiang Ma
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China
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Shanmugam A, Suriano R, Chaudhuri D, Rajoria S, George A, Mittelman A, Tiwari RK. Identification of PSA peptide mimotopes using phage display peptide library. Peptides 2011; 32:1097-102. [PMID: 21539876 DOI: 10.1016/j.peptides.2011.04.018] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/13/2010] [Revised: 04/15/2011] [Accepted: 04/16/2011] [Indexed: 11/21/2022]
Abstract
Prostate cancer (PCa) is one of the most common types of cancer in men in the United States and is the second leading cause of cancer related death in men. Clinically, secreted prostate specific antigen (PSA) has gained recognition because of its proteolytic activity being directly linked to PCa cell proliferation leading to disease initiation and progression. Using phage display technology, we identified four distinct cyclical peptides. These peptides apart from differences in their amino acid sequence, elicited minimal cross reactive antibody responses against each other. One of the four peptides analyzed produced an antibody response that recognizes the PSA protein. We demonstrate that the synthetic PSA peptide mimics identified in our study are immunologically active and produce neutralizing activity and this has relevance and utility for prostate cancer disease progression.
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Affiliation(s)
- Arulkumaran Shanmugam
- Department of Microbiology and Immunology, New York Medical College, Valhalla, New York 10595, USA
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Pniewski T, Kapusta J, Bociąg P, Wojciechowicz J, Kostrzak A, Gdula M, Fedorowicz-Strońska O, Wójcik P, Otta H, Samardakiewicz S, Wolko B, Płucienniczak A. Low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B surface antigen for prototype plant-derived vaccine tablet formulation. J Appl Genet 2011; 52:125-36. [PMID: 21107787 PMCID: PMC3088802 DOI: 10.1007/s13353-010-0001-5] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2010] [Revised: 10/11/2010] [Accepted: 10/12/2010] [Indexed: 02/07/2023]
Abstract
Efficient immunization against hepatitis B virus (HBV) and other pathogens with plant-based oral vaccines requires appropriate plant expressors and the optimization of vaccine compositions and administration protocols. Previous immunization studies were mainly based on a combination of the injection of a small surface antigen of HBV (S-HBsAg) and the feeding with raw tissue containing the antigen, supplemented with an adjuvant, and coming from plants conferring resistance to kanamycin. The objective of this study was to develop a prototype oral vaccine formula suitable for human immunization. Herbicide-resistant lettuce was engineered, stably expressing through progeny generation micrograms of S-HBsAg per g of fresh weight and formed into virus-like particles (VLPs). Lyophilized tissue containing a relatively low, 100-ng VLP-assembled antigen dose, administered only orally to mice with a long, 60-day interval between prime and boost immunizations and without exogenous adjuvant, elicited mucosal and systemic humoral anti-HBs responses at the nominally protective level. Lyophilized tissue was converted into tablets, which preserved S-HBsAg content for at least one year of room temperature storage. The results of the study provide indications on immunization methodology using a durable, efficacious, and convenient plant-derived prototype oral vaccine against hepatitis B.
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Affiliation(s)
- Tomasz Pniewski
- Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479, Poznań, Poland.
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Shanmugam A, Suriano R, Goswami N, Chaudhuri D, Ashok BT, Rajoria S, George AL, Mittelman A, Tiwari RK. Identification of peptide mimotopes of gp96 using single-chain antibody library. Cell Stress Chaperones 2011; 16:225-34. [PMID: 20953748 PMCID: PMC3059791 DOI: 10.1007/s12192-010-0234-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2010] [Revised: 09/17/2010] [Accepted: 09/21/2010] [Indexed: 10/18/2022] Open
Abstract
Heat shock proteins such as gp96 are immunogenic and are widely used as vaccines in immunotherapy of cancers. The present study focuses on the use of peptide mimotopes as immunotherapeutic vaccines for prostate cancer. To this end, we developed a 15-mer gp96 peptide mimotope specifically reactive to MAT-LyLu gp96-peptide complex using combinatorial single-chain antibody and peptide phage display library. The immunogenicity of the synthesized gp96 mimotope was analyzed initially in normal BALB/c mice in combination with various adjuvants such as complete Freund's adjuvant (CFA), aluminum salts (ALUM), granulocyte-macrophage colony-stimulating factor (GM-CSF), and liposome, of which CFA served as a positive control. The antibody response was determined and found that the gp96 mimotope with ALUM showed a significant increase in antibody titer, followed by GM-CSF and liposomes. Further, the T cell (CD4(+) and CD8(+)) populations from splenocytes, as well as IgG isotypes, interleukin-4, and interleukin-5 of gp96 mimotope with ALUM-immunized animals, were analyzed. The results suggest that the gp96 mimotope may elicit a potent and effective antitumor antibody response. Further, the study identifies ALUM and GM-CSF as adjuvant options to drive an appropriate protective immune response as these adjuvants have prior use in humans.
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Affiliation(s)
- Arulkumaran Shanmugam
- Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595 USA
| | - Robert Suriano
- Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595 USA
| | - Neha Goswami
- Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595 USA
| | - Devyani Chaudhuri
- Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595 USA
| | - Badithe T. Ashok
- Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595 USA
| | - Shilpi Rajoria
- Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595 USA
| | - Andrea L. George
- Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595 USA
| | - Abraham Mittelman
- Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595 USA
| | - Raj K. Tiwari
- Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595 USA
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Expression of Hepatitis B Virus Surface Antigen Containing Y100C Variant Frequently Detected in Occult HBV Infection. HEPATITIS RESEARCH AND TREATMENT 2011; 2011:695859. [PMID: 21331286 PMCID: PMC3038563 DOI: 10.1155/2011/695859] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/22/2010] [Accepted: 01/04/2011] [Indexed: 12/27/2022]
Abstract
Small hepatitis B virus surface protein (S-HBsAg) variant Y100C has been associated with HBsAg-negative phenotype. To determine whether Y100C substitution yields impaired HBsAg or small amounts of HBsAg that may reduce HBsAg detection by commercial anti-HBsAg antibodies, two eukaryotic expression plasmids, one containing a wild-type S and the other an S gene from a Y100C variant, were constructed and their levels of HBsAg compared by ELISA after transfection of HuH7 cells. Unexpectedly, the extracellular HBsAg levels detected with Y100C plasmid were higher than those observed with the wild-type plasmid, but without statistical significance. We concluded that the Y100C substitution alone did not play a role in reducing HBsAg amounts or HBsAg affinity by commercial ELISA assay. Further studies on in vitro replication fitness with the complete genome of HBV isolates displaying or not Y100C substitution may elucidate whether this mutation affects HBV replication and consequently HBsAg production.
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Characterization of occult hepatitis B virus from blood donors carrying genotype A2 or genotype D strains. J Hepatol 2008; 49:537-47. [PMID: 18602718 DOI: 10.1016/j.jhep.2008.04.017] [Citation(s) in RCA: 98] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/06/2008] [Revised: 04/08/2008] [Accepted: 04/19/2008] [Indexed: 02/06/2023]
Abstract
BACKGROUND/AIMS Nucleic acid testing (NAT) for hepatitis B virus (HBV) DNA in blood donations identified occult HBV infection (OBI) as a potential threat to blood safety. METHODS A collaborative study was undertaken to explore the molecular basis of OBIs prevalent in Europe in relation to clinical and serological data. RESULTS Ninety-one percent of 77 donor samples of European origin HBV DNA positive but HBV surface antigen (HBsAg) negative were confirmed. Viral load ranged between unquantifiable and 5640 IU/mL (median 25 IU/mL). Fifty-two strains were genotyped (14 HBV(A2) and 38 HBV(D)). Compared to HBsAg+ samples, genotype D was significantly more frequent than genotype A2 in OBIs from Poland or Italy (P<0.04). Amino acid substitutions were concentrated in the immunologically active parts of the Pre-S/S proteins (P<0.0001) affecting both cellular CD8 T-cell epitopes and B-cell neutralizing Major Hydrophilic Region epitopes. Substitutions were more frequent in OBIs than in HBsAg+ strains of both genotype D (P<0.001) and A2 (P<0.01), in OBIs of genotype D than A2 in the 'a' region (P<0.001) but not cellular epitopes, and in anti-HBs+ than anti-HBs- OBIs (P<0.001). CONCLUSIONS Results support the hypothesis that humoral and cellular immune pressure on the HBV envelope proteins are major mechanisms generating OBI.
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Roohi A, Yazdani Y, Khoshnoodi J, Jazayeri SM, Carman WF, Chamankhah M, Rashedan M, Shokri F. Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens. World J Gastroenterol 2006; 12:5368-74. [PMID: 16981270 PMCID: PMC4088207 DOI: 10.3748/wjg.v12.i33.5368] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the “a” region.
METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).
RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the “a” determinant at positions 120 (P→S), 123(T→N) and 161 (M→T) were found to affect reactivity of these mAbs.
CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.
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Affiliation(s)
- Azam Roohi
- Department of Immunology, School of Public Health, Tehran University of Medical Sciences, PO Box 6446-14133, Tehran, Iran
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Cuestas ML, Mathet VL, Ruiz V, Minassian ML, Rivero C, Sala A, Corach D, Alessio A, Pozzati M, Frider B, Oubiña JR. Unusual naturally occurring humoral and cellular mutated epitopes of hepatitis B virus in a chronically infected argentine patient with anti-HBs antibodies. J Clin Microbiol 2006; 44:2191-8. [PMID: 16757620 PMCID: PMC1489447 DOI: 10.1128/jcm.00057-06] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Serum hepatitis B virus (HBV) DNA was extracted from a chronically infected patient with cocirculation of hepatitis B surface antigen (HBsAg) and anti-HBs antibodies. Direct PCR and clone-derived sequences of the S and overlapped P genes were obtained. DNA sequences and phylogenetic analysis ascribed this isolate to genotype A (serotype adw2). Five of six HBV DNA clones exhibited point mutations inside and outside the major hydrophilic region, while the sixth clone exhibited a genotype A "wild-type" amino acid sequence. Observed replacements included both humoral and/or cellular (major histocompatibility complex class I [MHC-I] and MHC-II) HBV mutated epitopes, such as S45A, P46H, L49H, C107R, T125A, M133K, I152F, P153T, T161S, G185E, A194T, G202R, and I213L. None of these mutants were individually present within a given clone. The I213L replacement was the only one observed in the five clones carrying nonsynonymous mutations in the S gene. Some of the amino acid substitutions are reportedly known to be responsible for the emergence of immune escape mutants. C107R replacement prevents disulfide bonding, thus disrupting the first loop of the HBsAg. Circulation of some of these mutants may represent a potential risk for the community, since neither current hepatitis B vaccines nor hyperimmune hepatitis B immune globulin are effectively prevent the liver disease thereto associated. Moreover, some of the recorded HBsAg variants may influence the accuracy of the results obtained with currently used diagnostic tests.
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Affiliation(s)
- María L Cuestas
- Departamento Microbiología, Facultad de Medicina, UBA, Buenos Aires, Argentina
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Abstract
Hepatitis B viral mutants can emerge in patients as a result of selection pressure from either immune response or treatment options. Mutations that occur within the immunodominant epitopes of hepatitis B surface antigen (HBsAg) allow mutant virus to propagate in the presence of a neutralizing immune response, while wild-type virus is reduced to undetectable levels. HBsAg mutants present as false-negative results in some immunoassays. An understanding of immunoassay reactivity with HBsAg mutants is key to establishing an appropriate testing algorithm for hepatitis B virus detection programs.
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Affiliation(s)
- Paul F Coleman
- Abbott Laboratories, Abbott Park, Illinois 60064-6015, USA.
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Mao L, Koser H. Towards ferrofluidics for μ-TAS and lab on-a-chip applications. NANOTECHNOLOGY 2006; 17:S34-S47. [PMID: 21727352 DOI: 10.1088/0957-4484/17/4/007] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/31/2023]
Abstract
In this paper, we show that ferrofluids can be pumped very effectively in closed-channel geometries both in the macro- and micro-scales using spatially travelling, sinusoidally time-varying magnetic fields. The results from numerical modelling demonstrate that the optimum pumping frequency is the reciprocal of the Brownian relaxation time constant of the magnetic nanoparticles inside the ferrofluid. Since the Brownian time constant depends in part on the overall hydrodynamic volume of the magnetic nanoparticles, this work has been carried with a view to developing functionalized ferrofluids that can be used as sensitive pathogen detectors in the context of ferrohydrodynamic pumping via travelling magnetic fields. A micro-ferrofluidic device has been designed and fabricated in order to demonstrate the potential development of this technology for pathogen detection. A cost-effective fabrication process combining insulated metal substrate etching and soft lithography is used to realize the prototype micro-ferrofluidic device. Results show good agreement between simulation and experiment. We finally propose a ferrofluid-based pathogen detection scheme that is expected to be insensitive to temperature and viscosity differences between the ferrofluid and the sample to be tested.
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Affiliation(s)
- Leidong Mao
- Department of Electrical Engineering, Yale University, 15 Prospect Street, New Haven, CT 06520, USA
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van Roosmalen MH, de Jong JJ, Haenen W, Jacobs T, Couwenberg F, Ahlers-de Boer GJCM, Hellings JA. A new HBsAg screening assay designed for sensitive detection of HBsAg subtypes and variants. Intervirology 2006; 49:127-32. [PMID: 16428888 DOI: 10.1159/000089373] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2004] [Accepted: 03/29/2005] [Indexed: 01/14/2023] Open
Abstract
The design of a new HBsAg screening assay, the Hepanostika HBsAg Ultra is based on the use of monoclonal antibodies raised against native wild-type HBsAg and reactive with HBsAg in which the common 'a'-determinant is modified by site-directed mutagenesis of four of the cysteine moieties. The design was checked using the same cysteine variants and samples from patients known to be infected with HBsAg variants. The results found were compared with other state-of-the-art commercial screening assays. The design of the Hepanostika HBsAg Ultra enabled detection of all variant HBsAg-positive samples in contrast to the other commercial assays. An additional 980 samples were tested to assess the specificity and sensitivity of the Hepanostika HBsAg Ultra. Screening of presumed negative serum and plasma samples resulted in a specificity of 100%. This makes the Hepanostika HBsAg Ultra the first screening assay with a design able to detect HBsAg variants with high sensitivity and specificity.
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Abstract
Hepatitis B viral mutants can emerge in patients as a result of selection pressure from either immune response or treatment options. Mutations that occur within the immunodominant epitopes of hepatitis B surface antigen (HBsAg) allow mutant virus to propagate in the presence of a neutralizing immune response, while wild-type virus is reduced to undetectable levels. HBsAg mutants present as false-negative results in some immunoassays. An understanding of immunoassay reactivity with HBsAg mutants is key to establishing an appropriate testing algorithm for hepatitis B virus detection programs.
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Affiliation(s)
- Paul F Coleman
- Abbott Laboratories, Abbott Park, Illinois 60064-6015, USA.
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Ferrieu-Weisbuch C, Bettsworth F, Becquart L, Paranhos-Baccala G, Michel S, Arnaud M, Jolivet-Reynaud C. Usefulness of the phage display technology for the identification of a hepatitis C virus NS4A epitope recognized early in the course of the disease. J Virol Methods 2006; 131:175-83. [PMID: 16183141 DOI: 10.1016/j.jviromet.2005.08.008] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2005] [Revised: 08/02/2005] [Accepted: 08/03/2005] [Indexed: 11/17/2022]
Abstract
A dodecapeptide phage-displayed library was screened with the mouse monoclonal antibody (mAb) 2E3C2 which competed with human antibodies for the binding to the HCV c100 recombinant protein. Four mimotopes shared a consensus motif with the HCV 1701-1707 sequence corresponding to the carboxyl-terminal domain of the non-structural protein NS4A. However, these mimotopes reacted with 2E3C2 only, whereas the corresponding NS4 epitope defined at the sequence 1698-1709 and displayed on phage was recognized by both 2E3C2 and sera from HCV infected patients. Using the Spot method of multiple peptide synthesis and alanine replacement analysis, the respective reactivities of mAb 2E3C2 and anti-NS4A human antibodies against NS4 were shown to be directed against two slightly different overlapping minimal linear sequences and to involve different critical residues. The phage clone displaying the NS4 epitope was used to study the specific recognition of this epitope by different individual HCV positive sera as well as by two seroconversion panels of sera from HCV infected patients. Compared with the detection by RIBA of the different HCV antigens and c100 particularly, these results indicated that the antibodies directed against the NS4 (1698-1709) epitope were produced early during the course of the disease and decreased later.
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Affiliation(s)
- Catherine Ferrieu-Weisbuch
- Unité Mixte de Recherche UMR 2714 CNRS-bioMérieux, IFR 128 BioSciences Lyon Gerland, 21 avenue Tony Garnier, 69365 Lyon Cedex 07, France
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Roohi A, Khoshnoodi J, Zarnani AH, Shokri F. Epitope mapping of recombinant hepatitis B surface antigen by murine monoclonal antibodies. Hybridoma (Larchmt) 2005; 24:71-7. [PMID: 15857170 DOI: 10.1089/hyb.2005.24.71] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Hepatitis B surface antigen (HBsAg) induces a potent protective antibody response in immunized healthy individuals. The antibody response in humans is largely directed to a restricted conformational immunodominant region of HBsAg, identified as "a" determinant. Our aim was generation and characterization of murine monoclonal antibodies (MAbs) against recombinant HBsAg and their use for epitope mapping of the antigen. Hybridoma cells were established from Balb/c mice immunized with recombinant HBsAg of the "adw" subtype and cloned by limiting dilution. Specificity of MAbs was studied by indirect ELISA and immunoblotting. Topology of the epitopes was analyzed by competitive and inhibition ELISA. Eight hybridoma clones producing MAbs specific for the immunogen were established. Five of the MAbs recognized overlapping conformational epitopes, whereas the remaining three MAbs were found to identify linear epitopes. Cross-inhibition studies suggest recognition of mutually exclusive epitopes by these MAbs. Our data suggest that, similar to the human system, the mouse antibody response is largely directed to restricted conformational overlapping epitopes of HBsAg.
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Affiliation(s)
- A Roohi
- Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
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20
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Ma CL, Fang DX, Yao K, Li FQ, Jin HY, Li SQ, Tan WG. Incidence of HBV variants with a mutation at nt551 among hepatitis B patients in Nanjing and its neighbourhood. World J Gastroenterol 2005; 11:299-302. [PMID: 15633237 PMCID: PMC4205423 DOI: 10.3748/wjg.v11.i2.299] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the epidemiology of hepatitis B virus (HBV) strains with a mutation at nt551 in surface gene among hepatitis B patients in Nanjing and its neighbourhood.
METHODS: By using mutation-specific polymerase chain reaction (msPCR) established by our laboratory for amplifying HBV DNAs with a mutation at nt551, 117 serum samples taken from hepatitis B patients were detected.
RESULTS: The results showed that 112 samples were positive for nt551A, 4 samples were positive for nt551G. One sample was positive for nt551T. No nt551C of HBV DNA was found. The incidence of HBsAg mutants with G, C, T, A at nt551 among 117 samples was 3.42%, 0%, 0.85%, 95.73%, respectively.
CONCLUSION: In Nanjing and its neighbourhood, hepatitis B patients are mainly infected with wild genotype HBV. The incidence of mutants with a mutation at nt551 in HBV genome is significantly lower than that in wild genotype HBV DNA (P<0.01). The necessity of adding components of HBsAg mutants to HBV vaccine needs further investigation.
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Affiliation(s)
- Chun-Ling Ma
- Huadong Research Institute for Medical Biotechnics, Nanjing 210029, Jiangsu Province, China.
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21
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Ferrieu-Weisbuch C, Michel S, Collomb-Clerc E, Pothion C, Deléage G, Jolivet-Reynaud C. Characterization of prostate-specific antigen binding peptides selected by phage display technology. J Mol Recognit 2005; 19:10-20. [PMID: 16312021 DOI: 10.1002/jmr.762] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer. Free PSA has been shown to be more extensively cleaved in sera from benign prostatic hyperplasia patients than in sera from prostate cancer patients. Moreover, the presence of enzymatically activatable PSA was characterized previously in sera from patients with prostate cancer by the use of the specific anti-free PSA monoclonal antibody (mAb) 5D3D11. As an attempt to obtain ligands for the specific recognition of different PSA forms including active PSA, phage-displayed linear and cyclic peptide libraries were screened with PSA coated directly into microplate wells or presented by two different anti-total PSA mAbs. Four different phage clones were selected for their ability to recognize PSA and the inserted peptides were produced as synthetic peptides. These peptides were found to capture and to detect specifically free PSA, even in complex biological media such as sera or tumour cell culture supernatants. Alanine scanning of peptide sequences showed the involvement of aromatic and hydrophobic residues in the interaction of the peptides with PSA whereas Spotscan analysis of overlapping peptides covering the PSA sequence identified a peptide binding to the kallikrein loop at residues 82-87, suggesting that the peptides could recognize a non-clipped form of PSA. Moreover, the PSA-specific peptides enhance the enzymatic activity of PSA immobilized into microplate wells whereas the capture of PSA by the peptides inhibited totally its enzymatic activity while the peptide binding to PSA had no effect in solution. These PSA-specific peptides could be potential tools for the recognition of PSA forms more specifically associated to prostate cancer.
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Affiliation(s)
- Catherine Ferrieu-Weisbuch
- Unité Mixte de Recherche UMR 2714 CNRS-bioMérieux, IFR 128 BioSciences Lyon-Gerland, 21 Avenue Tony Garnier, 69365 LYON Cedex 07, France
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22
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Hu H, Peng XM, Huang YS, Gu L, Xie QF, Gao ZL. Yeast expression and DNA immunization of hepatitis B virus S gene with second-loop deletion of α determinant region. World J Gastroenterol 2004; 10:2989-93. [PMID: 15378779 PMCID: PMC4576258 DOI: 10.3748/wjg.v10.i20.2989] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: Immune escape mutations of HBV often occur in the dominant epitope, the second-loop of the a determinant of hepatitis B surface antigen (HBsAg). To let the hosts respond to the subdominant epitopes in HBsAg may be an effective way to decrease the prevalence of immune escape mutants. For this reason, a man-made clone of HBV S gene with the second-loop deletion was constructed. Its antigenicity was evaluated by yeast expression analysis and DNA immunization in mice.
METHODS: HBV S gene with deleted second-loop, amino acids from 139 to 145, was generated using splicing by overlap extension. HBV deleted S gene was then cloned into the yeast expression vector pPIC9 and the mammalian expression vector pcDNA3 to generate pHB-SDY and pHB-SD, respectively. The complete S gene was cloned into the same vectors as controls. The deleted recombinant HBsAg expressed in yeasts was detected using Abbott IMx HBsAg test kits, enzyme-linked immunoadsorbent assay (ELISA) and immune dot blotting to evaluate its antigenicity in vitro. The anti-HBs responses to DNA immunization in BALB/c mice were detected using Abbott IMx AUSAB test kits to evaluate the antigenicity of that recombinant protein in vivo.
RESULTS: Both deleted and complete HBsAg were successfully expressed in yeasts. They were intracellular expressions. The deleted HBsAg could not be detected by ELISA, in which the monoclonal anti-HBs against the α determinant was used, but could be detected by Abbott IMx and immune dot blotting, in which multiple monoclonal anti-HBs and polyclonal anti-HBs were used, respectively. The activity of the deleted HBsAg detected by Abbott IMx was much lower than that of complete HBsAg (the ratio of sample value/cut off value, 106 ± 26.7 vs 1814.4 ± 776.3, P < 0.01, t = 5.02). The anti-HBs response of pHB-SD to DNA immunization was lower than that of complete HBV S gene vector pHB (the positive rate 2/10 vs 6/10, 4.56 ± 3.52 mIU/mL vs 27.60 ± 17.3 mIU/mL, P = 0.02, t = 2.7).
CONCLUSIONS: HBsAg with deleted second-loop of the α determinant still has antigenicity, and can also raise weak anti-HBs response in mice to DNA immunization, suggesting that it is possible to develop a subdominant vaccine for preventing infections of immune escape mutants of HBV.
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Affiliation(s)
- Hui Hu
- Department of Infectious Diseases, the Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630, Guangdong Province, China
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23
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Jolivet-Reynaud C, Adida A, Michel S, Deléage G, Paranhos-Baccala G, Gonin V, Battail-Poirot N, Lacoux X, Rolland D. Characterization of mimotopes mimicking an immunodominant conformational epitope on the hepatitis C virus NS3 helicase. J Med Virol 2004; 72:385-95. [PMID: 14748062 DOI: 10.1002/jmv.20002] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
The hepatitis C virus (HCV) nonstructural 3 (NS3) protein is composed of an amino terminal protease and a carboxyl terminal RNA helicase. NS3 contains major antigenic epitopes. The antibody response to NS3 appears early in the course of infection and is focused on the helicase region. However, this response cannot be defined by short synthetic peptides indicating the recognition of conformation-dependent epitopes. In this study, we have screened a dodecapeptide library displayed on phage with anti-NS3 mouse monoclonal antibodies (mAbs) that compete with each other and human anti-HCV NS3 positive sera. Two peptides (mimotopes) were selected that appeared to mimic an immunodominant epitope since they were recognized specifically by the different anti-NS3 mAbs of the study and by human sera from HCV infected patients. Homology search between the two mimotopes and the NS3 sequence showed that one of the two peptides shared amino acid similarities with NS3 at residues 1396-1398 on a very accessible loop as visualized on the three-dimensional structure of the helicase domain whereas the other one had two amino acids similar to nearby residues 1376 and 1378. Reproduced as synthetic dodecapeptides, the two mimotopes were recognized specifically by 19 and 22, respectively, out of 49 sera from HCV infected patients. These mimotopes allowed also the detection of anti-NS3 antibodies in sera of HCV patients at the seroconversion stage. These results suggest that the two NS3 mimotopes are potential tools for the diagnosis of HCV infection.
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24
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Ma CL, Fang DX, Chen HB, Li FQ, Jin HY, Li SQ, Tan WG. A mutation specific polymerase chain reaction for detecting hepatitis B virus genome mutations at nt551. World J Gastroenterol 2003; 9:509-12. [PMID: 12632507 PMCID: PMC4621571 DOI: 10.3748/wjg.v9.i3.509] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: The hepatitis B surface antigen (HBsAg) is considered to be one of the best markers for the diagnosis of acute and chronic HBV infection. But in some patients, this antigen cannot be detected by routine serological assays despite the presence of virus. One of the most important explanations for the lack of detectable HBsAg is that mutations which occur within the “a” determinant of HBV S gene can alter expression of HBsAg and lead to changes of antigenicity and immunogenicity of HBsAg accordingly. As a result, these mutants cannot be detected by diagnosis assays. Thus, it is essential to find out specific and sensitive methods to test the new mutants and further investigate their distribution. This study is to establish a method to investigate the distribution of the HBsAg mutant at nt551.
METHODS: A mutation specific polymerase chain reaction (msPCR) was established for amplifying HBV DNA with a mutation at nt551. Four sets of primer pairs, P551A-PPS, P551G-PPS, P551C-PPS and P551T-PPS, with the same sequences except for one base at 3’ terminus were designed and synthesized according to the known HBV genome sequences and the popular HBV subtypes, adr and adw, in China. At the basis of regular PCR method, we explored the specific conditions for amplifying HBV DNAs with a mutation at nt551 by regulating annealing temperature and the concentration of these primers. 126 serum samples from patients of hepatitis B were collected, among which 16 were positive for HBV S DNA in the nested PCR amplification. These 16 HBV S DNAs were detected by using the msPCR method.
RESULTS: When the annealing temperature was raised to 71 °C, nt551A and nt551G were amplified specifically by P551A-PPS and P551G-PPS; At 72 °C and 5 pmole of the primers (each) in reaction of 25 μl volume, nt551C and nt551T were amplified specifically by P551C-PPS and P551T-PPS. 16 of HBV S gene fragments were characterized by using this method. 14 of them were positive for nt551A, one was positive for nt551G, and the other one was positive for nt551T. The results were confirmed by nucleotide sequencing.
CONCLUSION: The mutation specific polymerase chain reaction is a specific and sensitive method for detecting the mutations of HBV genome at nt551.
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Affiliation(s)
- Chun-Ling Ma
- Huadong Research Institute for Medical Biotechnics, Nanjing 210002, China.
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25
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Zheng Y, Rundell A. Biosensor immunosurface engineering inspired by B-cell membrane-bound antibodies: modeling and analysis of multivalent antigen capture by immobilized antibodies. IEEE Trans Nanobioscience 2003; 2:14-25. [PMID: 15382418 DOI: 10.1109/tnb.2003.810158] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Immobilized antibodies are used by many biosensors and diagnostic tests as specific receptors for the presence of targeted substances in clinical, biological, or environmental samples. The antibodies used in these devices are the soluble form of the antibodies presented on the B-cell membrane: they have the same specificity, but they may differ from those presented on the B cell in orientation, flexibility, mobility, and support-membrane properties. These properties influence the formation of noncovalent bonds between the pathogen antigenic determinants (epitopes) and the amino acids of the antibodies. This paper extends the theoretical modeling foundation addressing multivalent antigen binding to cell surface receptors to account for local and far-field antibody surface density effects, immobilized antibodies, and the flexibility and range of motion of immobilized antibodies. An analysis of the derived model provides insight into the design of biosensor immunosurfaces to enhance pathogen capture capability.
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Affiliation(s)
- Yanan Zheng
- Department of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
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26
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Vanlandschoot P, Van Houtte F, Roobrouck A, Farhoudi A, Stelter F, Peterson DL, Gomez-Gutierrez J, Gavilanes F, Leroux-Roels G. LPS-binding protein and CD14-dependent attachment of hepatitis B surface antigen to monocytes is determined by the phospholipid moiety of the particles. J Gen Virol 2002; 83:2279-2289. [PMID: 12185283 DOI: 10.1099/0022-1317-83-9-2279] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
It was observed recently that recombinant yeast-derived hepatitis B surface antigen (rHBsAg) particles, which contain the S protein only, bind almost exclusively to monocytes. It is shown here that binding requires the presence of the LPS receptor CD14. Furthermore, evidence is presented that a domain on CD14 that is identical to or largely overlaps with the LPS-binding pocket is instrumental for the attachment of rHBsAg. Additionally, it is shown that the heat-labile LPS-binding protein (LBP) catalyses the binding of rHBsAg to the cells. Remarkably, natural plasma-derived HBsAg (pHBsAg) does not have this property. pHBsAg devoid of its lipids and reconstituted with phosphatidylserine or phosphatidylglycerol acquires the characteristic of yeast-derived HBsAg. Clearly, the interaction of rHBsAg with the cell membrane is determined by the presence of charged phospholipids that are absent in pHBsAg. Although a lipid-receptor interaction is suggested, antibody-inhibition experiments suggest a possible involvement of the C-terminal region of the S protein in the interaction with monocytes. The possible implications of these observations for hepatitis B virus (HBV) infection and HBV vaccine efficiency are discussed.
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Affiliation(s)
- Peter Vanlandschoot
- Center for Vaccinology, Department of Clinical Biology, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium1
| | - Freya Van Houtte
- Center for Vaccinology, Department of Clinical Biology, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium1
| | - Annelies Roobrouck
- Center for Vaccinology, Department of Clinical Biology, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium1
| | - Ali Farhoudi
- Center for Vaccinology, Department of Clinical Biology, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium1
| | - Felix Stelter
- Institute of Immunology and Transfusion Medicine, Ernst Moritz Arndt University, Greifswald, Germany2
| | - Darell L Peterson
- Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond, VA, USA3
| | - Julian Gomez-Gutierrez
- Departamento de Bioquimica y Biologia Molecular, Universidad Complutense, Madrid, Spain4
| | - Francisco Gavilanes
- Departamento de Bioquimica y Biologia Molecular, Universidad Complutense, Madrid, Spain4
| | - Geert Leroux-Roels
- Center for Vaccinology, Department of Clinical Biology, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium1
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