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Isolation of highly active monoclonal antibodies against multiresistant gram-positive bacteria
Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extend...
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Published in: | PloS one 2015-02, Vol.10 (2), p.e0118405-e0118405 |
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description | Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials. |
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Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0118405</identifier><identifier>PMID: 25706415</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Acids ; Animal models ; Animals ; Antibiotics ; Antibodies, Monoclonal - genetics ; Antibodies, Monoclonal - immunology ; Antibodies, Monoclonal - isolation & purification ; Bacteria ; Cell culture ; Cell division ; CHO Cells ; Clinical trials ; Cricetinae ; Cricetulus ; DNA Primers ; Drug resistance ; Drug Resistance, Multiple, Bacterial - immunology ; Endocarditis ; Enterococcus faecalis - drug effects ; Enterococcus faecium ; Female ; Gene expression ; Genetic Vectors ; Gram-positive bacteria ; Hospitals ; Humans ; Immunoglobulin G ; Immunoglobulins ; Infections ; Infectious diseases ; Lymphocytes B ; Medical research ; Medicine ; Molecular chains ; Monoclonal antibodies ; Nosocomial infection ; Nosocomial infections ; Pathogens ; Pediatrics ; Peritonitis ; Phagocytosis ; Plasmids ; Polymerase Chain Reaction ; Rats ; Rats, Wistar ; Sepsis ; Staphylococcus aureus ; Staphylococcus aureus - drug effects ; Staphylococcus infections ; Strains (organisms)</subject><ispartof>PloS one, 2015-02, Vol.10 (2), p.e0118405-e0118405</ispartof><rights>2015 Rossmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015 Rossmann et al 2015 Rossmann et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c526t-7efd6f8ed6b6d26848bef88370413817b7e8ba04b12fe79abe89686c9820c1e43</citedby><cites>FETCH-LOGICAL-c526t-7efd6f8ed6b6d26848bef88370413817b7e8ba04b12fe79abe89686c9820c1e43</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.proquest.com/docview/1658095300/fulltextPDF?pq-origsite=primo$$EPDF$$P50$$Gproquest$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/1658095300?pq-origsite=primo$$EHTML$$P50$$Gproquest$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,25753,27924,27925,37012,37013,44590,53791,53793,75126</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25706415$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Kluytmans, Jan</contributor><creatorcontrib>Rossmann, Friederike S</creatorcontrib><creatorcontrib>Laverde, Diana</creatorcontrib><creatorcontrib>Kropec, Andrea</creatorcontrib><creatorcontrib>Romero-Saavedra, Felipe</creatorcontrib><creatorcontrib>Meyer-Buehn, Melanie</creatorcontrib><creatorcontrib>Huebner, Johannes</creatorcontrib><title>Isolation of highly active monoclonal antibodies against multiresistant gram-positive bacteria</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.</description><subject>Acids</subject><subject>Animal models</subject><subject>Animals</subject><subject>Antibiotics</subject><subject>Antibodies, Monoclonal - genetics</subject><subject>Antibodies, Monoclonal - immunology</subject><subject>Antibodies, Monoclonal - isolation & purification</subject><subject>Bacteria</subject><subject>Cell culture</subject><subject>Cell division</subject><subject>CHO Cells</subject><subject>Clinical trials</subject><subject>Cricetinae</subject><subject>Cricetulus</subject><subject>DNA Primers</subject><subject>Drug resistance</subject><subject>Drug Resistance, Multiple, Bacterial - immunology</subject><subject>Endocarditis</subject><subject>Enterococcus faecalis - drug effects</subject><subject>Enterococcus faecium</subject><subject>Female</subject><subject>Gene expression</subject><subject>Genetic Vectors</subject><subject>Gram-positive bacteria</subject><subject>Hospitals</subject><subject>Humans</subject><subject>Immunoglobulin G</subject><subject>Immunoglobulins</subject><subject>Infections</subject><subject>Infectious diseases</subject><subject>Lymphocytes B</subject><subject>Medical research</subject><subject>Medicine</subject><subject>Molecular chains</subject><subject>Monoclonal antibodies</subject><subject>Nosocomial infection</subject><subject>Nosocomial infections</subject><subject>Pathogens</subject><subject>Pediatrics</subject><subject>Peritonitis</subject><subject>Phagocytosis</subject><subject>Plasmids</subject><subject>Polymerase Chain Reaction</subject><subject>Rats</subject><subject>Rats, Wistar</subject><subject>Sepsis</subject><subject>Staphylococcus aureus</subject><subject>Staphylococcus aureus - 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genetics</topic><topic>Antibodies, Monoclonal - immunology</topic><topic>Antibodies, Monoclonal - isolation & purification</topic><topic>Bacteria</topic><topic>Cell culture</topic><topic>Cell division</topic><topic>CHO Cells</topic><topic>Clinical trials</topic><topic>Cricetinae</topic><topic>Cricetulus</topic><topic>DNA Primers</topic><topic>Drug resistance</topic><topic>Drug Resistance, Multiple, Bacterial - immunology</topic><topic>Endocarditis</topic><topic>Enterococcus faecalis - drug effects</topic><topic>Enterococcus faecium</topic><topic>Female</topic><topic>Gene expression</topic><topic>Genetic Vectors</topic><topic>Gram-positive bacteria</topic><topic>Hospitals</topic><topic>Humans</topic><topic>Immunoglobulin G</topic><topic>Immunoglobulins</topic><topic>Infections</topic><topic>Infectious diseases</topic><topic>Lymphocytes B</topic><topic>Medical research</topic><topic>Medicine</topic><topic>Molecular chains</topic><topic>Monoclonal antibodies</topic><topic>Nosocomial infection</topic><topic>Nosocomial infections</topic><topic>Pathogens</topic><topic>Pediatrics</topic><topic>Peritonitis</topic><topic>Phagocytosis</topic><topic>Plasmids</topic><topic>Polymerase Chain Reaction</topic><topic>Rats</topic><topic>Rats, Wistar</topic><topic>Sepsis</topic><topic>Staphylococcus aureus</topic><topic>Staphylococcus aureus - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Rossmann, Friederike S</au><au>Laverde, Diana</au><au>Kropec, Andrea</au><au>Romero-Saavedra, Felipe</au><au>Meyer-Buehn, Melanie</au><au>Huebner, Johannes</au><au>Kluytmans, Jan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Isolation of highly active monoclonal antibodies against multiresistant gram-positive bacteria</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2015-02-23</date><risdate>2015</risdate><volume>10</volume><issue>2</issue><spage>e0118405</spage><epage>e0118405</epage><pages>e0118405-e0118405</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>25706415</pmid><doi>10.1371/journal.pone.0118405</doi><oa>free_for_read</oa></addata></record> |
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subjects | Acids Animal models Animals Antibiotics Antibodies, Monoclonal - genetics Antibodies, Monoclonal - immunology Antibodies, Monoclonal - isolation & purification Bacteria Cell culture Cell division CHO Cells Clinical trials Cricetinae Cricetulus DNA Primers Drug resistance Drug Resistance, Multiple, Bacterial - immunology Endocarditis Enterococcus faecalis - drug effects Enterococcus faecium Female Gene expression Genetic Vectors Gram-positive bacteria Hospitals Humans Immunoglobulin G Immunoglobulins Infections Infectious diseases Lymphocytes B Medical research Medicine Molecular chains Monoclonal antibodies Nosocomial infection Nosocomial infections Pathogens Pediatrics Peritonitis Phagocytosis Plasmids Polymerase Chain Reaction Rats Rats, Wistar Sepsis Staphylococcus aureus Staphylococcus aureus - drug effects Staphylococcus infections Strains (organisms) |
title | Isolation of highly active monoclonal antibodies against multiresistant gram-positive bacteria |
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