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1.
JCI Insight ; 9(10)2024 May 22.
Article in English | MEDLINE | ID: mdl-38775156

ABSTRACT

Since its emergence, SARS-CoV-2 has been continuously evolving, hampering the effectiveness of current vaccines against COVID-19. mAbs can be used to treat patients at risk of severe COVID-19. Thus, the development of broadly protective mAbs and an understanding of the underlying protective mechanisms are of great importance. Here, we isolated mAbs from donors with breakthrough infection with Omicron subvariants using a single-B cell screening platform. We identified a mAb, O5C2, which possesses broad-spectrum neutralization and antibody-dependent cell-mediated cytotoxic activities against SARS-CoV-2 variants, including EG.5.1. Single-particle analysis by cryo-electron microscopy revealed that O5C2 targeted an unusually large epitope within the receptor-binding domain of spike protein that overlapped with the angiotensin-converting enzyme 2 binding interface. Furthermore, O5C2 effectively protected against BA.5 Omicron infection in vivo by mediating changes in transcriptomes enriched in genes involved in apoptosis and interferon responses. Our findings provide insights into the development of pan-protective mAbs against SARS-CoV-2.


Subject(s)
Antibodies, Viral , COVID-19 , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , SARS-CoV-2/immunology , Humans , COVID-19/immunology , COVID-19/virology , Antibodies, Viral/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Animals , Mice , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Cryoelectron Microscopy , Epitopes/immunology , Broadly Neutralizing Antibodies/immunology , Antibody-Dependent Cell Cytotoxicity/immunology , Female
2.
J Travel Med ; 30(5)2023 09 05.
Article in English | MEDLINE | ID: mdl-37133444

ABSTRACT

BACKGROUND: Exposure to pathogens in public transport systems is a common means of spreading infection, mainly by inhaling aerosol or droplets from infected individuals. Such particles also contaminate surfaces, creating a potential surface-transmission pathway. METHODS: A fast acoustic biosensor with an antifouling nano-coating was introduced to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on exposed surfaces in the Prague Public Transport System. Samples were measured directly without pre-treatment. Results with the sensor gave excellent agreement with parallel quantitative reverse-transcription polymerase chain reaction (qRT-PCR) measurements on 482 surface samples taken from actively used trams, buses, metro trains and platforms between 7 and 9 April 2021, in the middle of the lineage Alpha SARS-CoV-2 epidemic wave when 1 in 240 people were COVID-19 positive in Prague. RESULTS: Only ten of the 482 surface swabs produced positive results and none of them contained virus particles capable of replication, indicating that positive samples contained inactive virus particles and/or fragments. Measurements of the rate of decay of SARS-CoV-2 on frequently touched surface materials showed that the virus did not remain viable longer than 1-4 h. The rate of inactivation was the fastest on rubber handrails in metro escalators and the slowest on hard-plastic seats, window glasses and stainless-steel grab rails. As a result of this study, Prague Public Transport Systems revised their cleaning protocols and the lengths of parking times during the pandemic. CONCLUSIONS: Our findings suggest that surface transmission played no or negligible role in spreading SARS-CoV-2 in Prague. The results also demonstrate the potential of the new biosensor to serve as a complementary screening tool in epidemic monitoring and prognosis.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Respiratory Aerosols and Droplets , Transportation , Pandemics/prevention & control
3.
Sci Rep ; 12(1): 12555, 2022 07 22.
Article in English | MEDLINE | ID: mdl-35869245

ABSTRACT

Antibodies recognize protein antigens with exquisite specificity in a complex aqueous environment, where interfacial waters are an integral part of the antibody-protein complex interfaces. In this work, we elucidate, with computational analyses, the principles governing the antibodies' specificity and affinity towards their cognate protein antigens in the presence of explicit interfacial waters. Experimentally, in four model antibody-protein complexes, we compared the contributions of the interaction types in antibody-protein antigen complex interfaces with the antibody variants selected from phage-displayed synthetic antibody libraries. Evidently, the specific interactions involving a subset of aromatic CDR (complementarity determining region) residues largely form the predominant determinant underlying the specificity of the antibody-protein complexes in nature. The interfacial direct/water-mediated hydrogen bonds accompanying the CDR aromatic interactions are optimized locally but contribute little in determining the epitope location. The results provide insights into the phenomenon that natural antibodies with limited sequence and structural variations in an antibody repertoire can recognize seemingly unlimited protein antigens. Our work suggests guidelines in designing functional artificial antibody repertoires with practical applications in developing novel antibody-based therapeutics and diagnostics for treating and preventing human diseases.


Subject(s)
Amino Acids , Complementarity Determining Regions , Antibody Affinity , Antibody Specificity , Antigen-Antibody Complex , Antigens , Complementarity Determining Regions/chemistry , Humans , Proteins
4.
ACS Appl Mater Interfaces ; 13(50): 60612-60624, 2021 Dec 22.
Article in English | MEDLINE | ID: mdl-34902239

ABSTRACT

New analytical techniques that overcome major drawbacks of current routinely used viral infection diagnosis methods, i.e., the long analysis time and laboriousness of real-time reverse-transcription polymerase chain reaction (qRT-PCR) and the insufficient sensitivity of "antigen tests", are urgently needed in the context of SARS-CoV-2 and other highly contagious viruses. Here, we report on an antifouling terpolymer-brush biointerface that enables the rapid and sensitive detection of SARS-CoV-2 in untreated clinical samples. The developed biointerface carries a tailored composition of zwitterionic and non-ionic moieties and allows for the significant improvement of antifouling capabilities when postmodified with biorecognition elements and exposed to complex media. When deployed on a surface of piezoelectric sensor and postmodified with human-cell-expressed antibodies specific to the nucleocapsid (N) protein of SARS-CoV-2, it made possible the quantitative analysis of untreated samples by a direct detection assay format without the need of additional amplification steps. Natively occurring N-protein-vRNA complexes, usually disrupted during the sample pre-treatment steps, were detected in the untreated clinical samples. This biosensor design improved the bioassay sensitivity to a clinically relevant limit of detection of 1.3 × 104 PFU/mL within a detection time of only 20 min. The high specificity toward N-protein-vRNA complexes was validated both by mass spectrometry and qRT-PCR. The performance characteristics were confirmed by qRT-PCR through a comparative study using a set of clinical nasopharyngeal swab samples. We further demonstrate the extraordinary fouling resistance of this biointerface through exposure to other commonly used crude biological samples (including blood plasma, oropharyngeal, stool, and nasopharyngeal swabs), measured via both the surface plasmon resonance and piezoelectric measurements, which highlights the potential to serve as a generic platform for a wide range of biosensing applications.


Subject(s)
COVID-19 Testing , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , Nasal Mucosa/virology , Polymers/chemistry , RNA, Viral/metabolism , SARS-CoV-2 , Biofouling , Biological Assay , Biosensing Techniques , Humans , Ions , Limit of Detection , Mass Spectrometry , Nasopharynx/virology , Phosphoproteins/chemistry , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Specimen Handling
5.
Sci Rep ; 11(1): 15430, 2021 07 29.
Article in English | MEDLINE | ID: mdl-34326410

ABSTRACT

Mesothelin (MSLN) is an attractive candidate of targeted therapy for several cancers, and hence there are increasing needs to develop MSLN-targeting strategies for cancer therapeutics. Antibody-drug conjugates (ADCs) targeting MSLN have been demonstrated to be a viable strategy in treating MSLN-positive cancers. However, developing antibodies as targeting modules in ADCs for toxic payload delivery to the tumor site but not to normal tissues is not a straightforward task with many potential hurdles. In this work, we established a high throughput engineering platform to develop and optimize anti-MSLN ADCs by characterizing more than 300 scFv CDR-variants and more than 50 IgG CDR-variants of a parent anti-MSLN antibody as candidates for ADCs. The results indicate that only a small portion of the complementarity determining region (CDR) residues are indispensable in the MSLN-specific targeting. Also, the enhancement of the hydrophilicity of the rest of the CDR residues could drastically increase the overall solubility of the optimized anti-MSLN antibodies, and thus substantially improve the efficacies of the ADCs in treating human gastric and pancreatic tumor xenograft models in mice. We demonstrated that the in vivo treatments with the optimized ADCs resulted in almost complete eradication of the xenograft tumors at the treatment endpoints, without detectable off-target toxicity because of the ADCs' high specificity targeting the cell surface tumor-associated MSLN. The technological platform can be applied to optimize the antibody sequences for more effective targeting modules of ADCs, even when the candidate antibodies are not necessarily feasible for the ADC development due to the antibodies' inferior solubility or affinity/specificity to the target antigen.


Subject(s)
GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/metabolism , Immunoconjugates/administration & dosage , Molecular Targeted Therapy/methods , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Stomach Neoplasms/drug therapy , Stomach Neoplasms/metabolism , Xenograft Model Antitumor Assays/methods , Animals , Cell Line, Tumor , Complementarity Determining Regions/immunology , Disease Models, Animal , GPI-Linked Proteins/immunology , Heterografts , Humans , Immunoconjugates/immunology , Immunoglobulin G/immunology , Injections, Intravenous , Male , Mesothelin , Mice , Mice, Inbred NOD , Mice, SCID , Pancreatic Neoplasms/pathology , Protein Engineering/methods , Stomach Neoplasms/pathology , Treatment Outcome , Tumor Burden/drug effects
6.
Sci Rep ; 10(1): 13318, 2020 08 07.
Article in English | MEDLINE | ID: mdl-32770098

ABSTRACT

Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10-15 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. The key components underlying the sandwich immunoassays are antibody-based affinity reagents, for which the conventional sources are mono- or poly-clonal antibodies from immunized animals. The downsides of the animal-based antibodies as affinity reagents arise from the requirement of months of development timespan and limited choices of antibody candidates due to immunodominance of humoral immune responses in animals. Hence, developing animal antibodies capable of distinguishing highly related antigens could be challenging. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phage-displayed synthetic antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90-94% in terms of pairwise amino acid sequence identity. We applied the methodology to attain, within four weeks, a panel of IgGs with distinguishable specificities against a group of representative NPs with pairwise amino acid sequence identities up to more than 90%, and the antibodies derived from the antibody libraries without further affinity refinement had comparable affinity of mouse antibodies to the NPs with the detection limit less than 1 nM of viral NP from lysed virus with sandwich ELISA. The panel of IgGs were capable of rapidly distinguishing infections due to virulent avian influenza virus from infections of seasonal flu, in responding to a probable emergency scenario where avian influenza virus would be transmissible among humans overlapping with the seasonal influenza infections. The results indicate that the in vitro antibody development methodology enables developing diagnostic antibodies that would not otherwise be available from animal-based antibody technologies.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Influenza A virus/immunology , Peptide Library , Viral Core Proteins/immunology , Animals , Dogs , Enzyme-Linked Immunosorbent Assay , Humans , Influenza, Human/diagnosis , Influenza, Human/immunology , Madin Darby Canine Kidney Cells , Mice
7.
MAbs ; 11(2): 373-387, 2019.
Article in English | MEDLINE | ID: mdl-30526270

ABSTRACT

Antibodies provide immune protection by recognizing antigens of diverse chemical properties, but elucidating the amino acid sequence-function relationships underlying the specificity and affinity of antibody-antigen interactions remains challenging. We designed and constructed phage-displayed synthetic antibody libraries with enriched protein antigen-recognition propensities calculated with machine learning predictors, which indicated that the designed single-chain variable fragment variants were encoded with enhanced distributions of complementarity-determining region (CDR) hot spot residues with high protein antigen recognition propensities in comparison with those in the human antibody germline sequences. Antibodies derived directly from the synthetic antibody libraries, without affinity maturation cycles comparable to those in in vivo immune systems, bound to the corresponding protein antigen through diverse conformational or linear epitopes with specificity and affinity comparable to those of the affinity-matured antibodies from in vivo immune systems. The results indicated that more densely populated CDR hot spot residues were sustainable by the antibody structural frameworks and could be accompanied by enhanced functionalities in recognizing protein antigens. Our study results suggest that synthetic antibody libraries, which are not limited by the sequences found in antibodies in nature, could be designed with the guidance of the computational machine learning algorithms that are programmed to predict interaction propensities to molecules of diverse chemical properties, leading to antibodies with optimal characteristics pertinent to their medical applications.


Subject(s)
Machine Learning , Protein Engineering/methods , Single-Chain Antibodies/chemistry , Antibody Affinity , Antibody Specificity , Humans , Peptide Library , Structure-Activity Relationship
8.
MAbs ; 11(1): 153-165, 2019 01.
Article in English | MEDLINE | ID: mdl-30365359

ABSTRACT

HER2-ECD (human epidermal growth factor receptor 2 - extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer, but HER2-specific therapeutic options for treating advanced gastric cancer remain limited. We have developed antibody-drug conjugates (ADCs), comprising IgG1 linked via valine-citrulline to monomethyl auristatin E, with potential to treat HER2-positive gastric cancer in humans. The antibodies optimally selected from the ADC discovery platform, which was developed to discover antibody candidates suitable for immunoconjugates from synthetic antibody libraries designed using antibody-antigen interaction principles, were demonstrated to be superior immunoconjugate targeting modules in terms of efficacy and off-target toxicity. In comparison with the two control humanized antibodies (trastuzumab and H32) derived from murine antibody repertoires, the antibodies derived from the synthetic antibody libraries had enhanced receptor-mediated internalization rate, which could result in ADCs with optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models in vivo and to cultured cells in vitro. The in vivo experiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies' propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our in vivo tests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics.


Subject(s)
Aminobenzoates/pharmacology , Immunoconjugates/pharmacology , Molecular Targeted Therapy/methods , Oligopeptides/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Stomach Neoplasms/pathology , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/pharmacology , Humans , Mice , Xenograft Model Antitumor Assays
9.
Sci Rep ; 7(1): 14455, 2017 10 31.
Article in English | MEDLINE | ID: mdl-29089574

ABSTRACT

Pandemic and epidemic outbreaks of influenza A virus (IAV) infection pose severe challenges to human society. Passive immunotherapy with recombinant neutralizing antibodies can potentially mitigate the threats of IAV infection. With a high throughput neutralizing antibody discovery platform, we produced artificial anti-hemagglutinin (HA) IAV-neutralizing IgGs from phage-displayed synthetic scFv libraries without necessitating prior memory of antibody-antigen interactions or relying on affinity maturation essential for in vivo immune systems to generate highly specific neutralizing antibodies. At least two thirds of the epitope groups of the artificial anti-HA antibodies resemble those of natural protective anti-HA antibodies, providing alternatives to neutralizing antibodies from natural antibody repertoires. With continuing advancement in designing and constructing synthetic scFv libraries, this technological platform is useful in mitigating not only the threats of IAV pandemics but also those from other newly emerging viral infections.


Subject(s)
Antibodies, Neutralizing/immunology , Orthomyxoviridae/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Viral/immunology , Bacteriophages/immunology , Disease Outbreaks , Epitopes/immunology , Hemagglutinin Glycoproteins, Influenza Virus/immunology , High-Throughput Screening Assays/methods , Humans , Influenza A Virus, H1N1 Subtype/immunology , Influenza A virus/immunology , Influenza, Human/virology , Pandemics , Single-Chain Antibodies/immunology
10.
Sci Rep ; 5: 15053, 2015 Oct 12.
Article in English | MEDLINE | ID: mdl-26456860

ABSTRACT

Broadly neutralizing antibodies developed from the IGHV1-69 germline gene are known to bind to the stem region of hemagglutinin in diverse influenza viruses but the sequence determinants for the antigen recognition, including neutralization potency and binding affinity, are not clearly understood. Such understanding could inform designs of synthetic antibody libraries targeting the stem epitope on hemagglutinin, leading to artificially designed antibodies that are functionally advantageous over antibodies from natural antibody repertoires. In this work, the sequence space of the complementarity determining regions of a broadly neutralizing antibody (F10) targeting the stem epitope on the hemagglutinin of a strain of H1N1 influenza virus was systematically explored; the elucidated antibody-hemagglutinin recognition principles were used to design a phage-displayed antibody library, which was then used to discover neutralizing antibodies against another strain of H1N1 virus. More than 1000 functional antibody candidates were selected from the antibody library and were shown to neutralize the corresponding strain of influenza virus with up to 7 folds higher potency comparing with the parent F10 antibody. The antibody library could be used to discover functionally effective antibodies against other H1N1 influenza viruses, supporting the notion that target-specific antibody libraries can be designed and constructed with systematic sequence-function information.


Subject(s)
Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Epitopes/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Peptide Library , Single-Chain Antibodies/chemistry , Amino Acid Sequence , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/immunology , Antibodies, Viral/biosynthesis , Antibodies, Viral/immunology , Cross Reactions , Dogs , Epitope Mapping , Epitopes/immunology , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/immunology , Madin Darby Canine Kidney Cells , Molecular Sequence Data , Neutralization Tests , Protein Binding , Single-Chain Antibodies/biosynthesis , Single-Chain Antibodies/immunology
11.
Sci Rep ; 5: 12411, 2015 Jul 23.
Article in English | MEDLINE | ID: mdl-26202883

ABSTRACT

Humoral immunity against diverse pathogens is rapidly elicited from natural antibody repertoires of limited complexity. But the organizing principles underlying the antibody repertoires that facilitate this immunity are not well-understood. We used HER2 as a model immunogen and reverse-engineered murine antibody response through constructing an artificial antibody library encoded with rudimentary sequence and structural characteristics learned from high throughput sequencing of antibody variable domains. Antibodies selected in vitro from the phage-displayed synthetic antibody library bound to the model immunogen with high affinity and specificities, which reproduced the specificities of natural antibody responses. We conclude that natural antibody structural repertoires are shaped to allow functional antibodies to be encoded efficiently, within the complexity limit of an individual antibody repertoire, to bind to diverse protein antigens with high specificity and affinity. Phage-displayed synthetic antibody libraries, in conjunction with high-throughput sequencing, can thus be designed to replicate natural antibody responses and to generate novel antibodies against diverse antigens.


Subject(s)
Antigen-Antibody Reactions/immunology , Immunity, Innate/immunology , Receptor, ErbB-2/chemistry , Receptor, ErbB-2/immunology , Amino Acid Sequence , Animals , Binding Sites , Humans , Mice , Molecular Sequence Data , Protein Binding , Structure-Activity Relationship
12.
J Gen Virol ; 92(Pt 8): 1760-1768, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21490247

ABSTRACT

Epstein-Barr virus (EBV) expresses two transcription factors, Rta and Zta, during the immediate-early stage of the lytic cycle to activate the transcription of early and late genes. This study finds that 0.31 mM protoapigenone from Thelypteris torresiana (Gaud.) inhibits the expression of EBV lytic proteins, including Rta, Zta, EA-D and VCA, in P3HR1 cells after lytic induction with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate. The lack of expression of EBV lytic proteins after protoapigenone treatment is attributed to the inhibition of the transactivation function of Zta because protoapigenone reduces the transactivation activity of Zta and Gal4-Zta, which contains the transactivation domain of Zta fused with Gal4. In contrast, protoapigenone does not affect the ability of Rta to activate a promoter that contains an Rta-response element, showing that the inhibition is unrelated to Rta. Furthermore, in a lactate dehydrogenase assay, protoapigenone is not toxic to P3HR1 cells at the concentrations that inhibit the function of Zta, showing that protoapigenone is valuable for studying the function of Zta and preventing EBV lytic proliferation.


Subject(s)
Cyclohexanones/pharmacology , Down-Regulation/drug effects , Ferns/chemistry , Flavones/pharmacology , Herpesvirus 4, Human/physiology , Plant Extracts/pharmacology , Cell Line , Epstein-Barr Virus Infections/drug therapy , Epstein-Barr Virus Infections/virology , Gene Expression Regulation, Viral/drug effects , Herpesvirus 4, Human/genetics , Humans , Viral Proteins/genetics , Viral Proteins/metabolism
13.
J Gen Virol ; 88(Pt 9): 2463-2472, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17698655

ABSTRACT

A mutant library of 249 mutants with mutations that span the entire Epstein-Barr virus (EBV) genome was generated by transposition with EZ : : TN and insertion with an apramycin resistance gene by a PCR-targeting method. This study also demonstrates the feasibility of generating deletions and site-specific mutations in the BRLF1 promoter on the EBV genome to determine the regions in the promoter that are crucial to transcription. Analysing BZLF1 and BRLF1 mutants by microarray analysis revealed that these two genes regulate the transcription of EBV lytic genes differently. A BZLF1 mutation affects global expression of EBV lytic genes; almost no lytic gene is expressed by the mutant after lytic induction. However, although a BRLF1 mutant still transcribes most lytic genes, the expression of these lytic genes is inefficient. Furthermore, this study shows that the proximal Zta-response element in the BRLF1 promoter is crucial to BRLF1 transcription from the EBV genome, despite the fact that another work demonstrated that this site was unimportant in transient transfection analysis. Furthermore, mutants with a mutation in BDLF1 and BORF1 cannot assemble viral capsids. Results of this study demonstrate the usefulness of a comprehensive mutant library in genetic analyses of EBV.


Subject(s)
Gene Library , Herpesvirus 4, Human/genetics , Mutation , DNA, Viral/genetics , Drug Resistance, Viral/genetics , Genome, Viral , Genomic Library , Membrane Glycoproteins/genetics , Polymerase Chain Reaction , Transcription, Genetic , Viral Proteins/genetics
14.
J Virol Methods ; 133(2): 158-66, 2006 May.
Article in English | MEDLINE | ID: mdl-16384612

ABSTRACT

Epstein-Barr virus (EBV) genome-chips are employed to determine the EBV infection rate and to reveal the gene expression patterns of EBV in tumor biopsies. These chips are produced with 71 consecutive PCR-amplified EBV DNA fragments of 1-3 kbp covering the entire EBV genome. The specificity of the EBV-chips is determined by hybridizing the DNA on the chips with biotin-labeled cDNA probes reverse transcribed from the mRNA of P3HR1 cells, which were B-cell infected latently by EBV. Hybridization results revealed only the expression of EBNA1, EBNA2, EBER1 and EBER2 in these cells. On the other hand, EBV lytic genes are expressed after the cells are treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate to induce the EBV lytic cycle. Fourty-four tumor biopsies from different organs are assayed with these chips, which showed many defined and interesting EBV gene expression patterns. This study demonstrates that the EBV-chip is useful for screening infection with EBV in tumors, which may lead to insights into tumorigenesis associated with this virus.


Subject(s)
Epstein-Barr Virus Infections/virology , Gene Expression Regulation, Neoplastic , Gene Expression Regulation, Viral , Herpesvirus 4, Human/genetics , Microarray Analysis/methods , Neoplasms/virology , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , B-Lymphocytes/virology , Burkitt Lymphoma/pathology , Butyrates/pharmacology , Cell Line, Tumor , DNA, Viral/genetics , Evaluation Studies as Topic , Gene Expression Profiling , Genes, Viral , Genome, Viral , Humans , RNA, Messenger/metabolism , Tetradecanoylphorbol Acetate/pharmacology
15.
Biochem Biophys Res Commun ; 301(4): 1062-8, 2003 Feb 21.
Article in English | MEDLINE | ID: mdl-12589821

ABSTRACT

(-)-Epigallocatechin gallate (EGCG), abundant in green tea, is a potent anti-microbial and anti-tumor compound. This investigation used immunoblot, flow cytometry, microarray, and indirect immunofluorescence analyses to show that at concentrations exceeding 50 microM, EGCG inhibits the expression of Epstein-Barr virus (EBV) lytic proteins, including Rta, Zta, and EA-D, but does not affect the expression of EBNA-1. Moreover, DNA microarray and transient transfection analyses demonstrated that EGCG blocks EBV lytic cycle by inhibiting the transcription of immediate-early genes, thus inhibiting the initiation of EBV lytic cascade.


Subject(s)
Antiviral Agents/pharmacology , Catechin/analogs & derivatives , Catechin/pharmacology , Herpesvirus 4, Human/drug effects , Herpesvirus 4, Human/physiology , Epstein-Barr Virus Nuclear Antigens/biosynthesis , Epstein-Barr Virus Nuclear Antigens/genetics , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Genes, Immediate-Early/drug effects , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/pathogenicity , Humans , Oligonucleotide Array Sequence Analysis , Transcription, Genetic/drug effects , Transfection , Tumor Cells, Cultured , Viral Proteins/biosynthesis , Viral Proteins/genetics
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