Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes

Summary Human papillomavirus (HPV) causes cervical cancer in women worldwide, which is currently prevented by vaccines based on virus‐like particles (VLPs). However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. Concerning the h...

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Veröffentlicht in:Plant biotechnology journal 2011-08, Vol.9 (6), p.651-660
Hauptverfasser: Waheed, Mohammad T., Thönes, Nadja, Müller, Martin, Hassan, Syed W., Gottschamel, Johanna, Lössl, Elke, Kaul, Hans-Peter, Lössl, Andreas G.
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container_end_page 660
container_issue 6
container_start_page 651
container_title Plant biotechnology journal
container_volume 9
creator Waheed, Mohammad T.
Thönes, Nadja
Müller, Martin
Hassan, Syed W.
Gottschamel, Johanna
Lössl, Elke
Kaul, Hans-Peter
Lössl, Andreas G.
description Summary Human papillomavirus (HPV) causes cervical cancer in women worldwide, which is currently prevented by vaccines based on virus‐like particles (VLPs). However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. Concerning the highest burden of disease in resource‐poor countries, development of an improved mucosal and cost‐effective vaccine is a necessity. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as vaccine candidate. Furthermore, coupling of an adjuvant like Escherichia coli heat‐labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate co‐administration of adjuvants. Our study demonstrates the expression of two pentameric proteins: the modified HPV‐16 L1 (L1_2xCysM) and LTB as a fusion protein in tobacco chloroplasts. Homoplasmy of the transplastomic plants was confirmed by Southern blotting. Western blot analysis showed that the LTB‐L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2% of total soluble protein. Proper folding and display of conformational epitopes for both LTB and L1 in the fusion protein was confirmed by GM1‐ganglioside binding assay and antigen capture ELISA, respectively. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, plants reached maturity and produced seeds by pollination with wild‐type plants. Taken together, these results pave the way for the possible development of a low‐cost adjuvant‐coupled vaccine with potentially improved immunogenicity against cervical cancer.
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However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. Concerning the highest burden of disease in resource‐poor countries, development of an improved mucosal and cost‐effective vaccine is a necessity. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as vaccine candidate. Furthermore, coupling of an adjuvant like Escherichia coli heat‐labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate co‐administration of adjuvants. Our study demonstrates the expression of two pentameric proteins: the modified HPV‐16 L1 (L1_2xCysM) and LTB as a fusion protein in tobacco chloroplasts. Homoplasmy of the transplastomic plants was confirmed by Southern blotting. Western blot analysis showed that the LTB‐L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2% of total soluble protein. Proper folding and display of conformational epitopes for both LTB and L1 in the fusion protein was confirmed by GM1‐ganglioside binding assay and antigen capture ELISA, respectively. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, plants reached maturity and produced seeds by pollination with wild‐type plants. 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Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd</rights><rights>2011 The Authors. 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However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. Concerning the highest burden of disease in resource‐poor countries, development of an improved mucosal and cost‐effective vaccine is a necessity. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as vaccine candidate. Furthermore, coupling of an adjuvant like Escherichia coli heat‐labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate co‐administration of adjuvants. Our study demonstrates the expression of two pentameric proteins: the modified HPV‐16 L1 (L1_2xCysM) and LTB as a fusion protein in tobacco chloroplasts. Homoplasmy of the transplastomic plants was confirmed by Southern blotting. Western blot analysis showed that the LTB‐L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2% of total soluble protein. Proper folding and display of conformational epitopes for both LTB and L1 in the fusion protein was confirmed by GM1‐ganglioside binding assay and antigen capture ELISA, respectively. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, plants reached maturity and produced seeds by pollination with wild‐type plants. 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development</subject><subject>Nicotiana - metabolism</subject><subject>Oncogene Proteins, Viral - chemistry</subject><subject>Oncogene Proteins, Viral - genetics</subject><subject>Oncogene Proteins, Viral - immunology</subject><subject>Papillomavirus Vaccines - genetics</subject><subject>Papillomavirus Vaccines - immunology</subject><subject>Papillomavirus Vaccines - metabolism</subject><subject>Phenotype</subject><subject>Plant Infertility - genetics</subject><subject>Plants, Genetically Modified - genetics</subject><subject>plastid transformation</subject><subject>Plastids - genetics</subject><subject>Pollination</subject><subject>Protein Conformation</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - immunology</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Seeds - physiology</subject><subject>Uterine Cervical Neoplasms - immunology</subject><issn>1467-7644</issn><issn>1467-7652</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNUcuO1DAQjBCIfcAvIN84JdiO4ySIC7uC3ZVGsEiDBnGxnLiz4yGxg-3M48v293CYZc744lJXVXerK0kQwRmJ790mI4yXackLmlFMSIYxJzTbP0vOT8TzE2bsLLnwfoMxJbzgL5MzShgrcUHOk8f7XvqgFYL96MB7bQ2yHZJI2anpIR3BBDmA0y3ayrbVBlArjdJKhohsJLXR5gGtp0EaNMpR970d5Fa7yaeEowVB0gT9AAZ1kweFdjqs0WJ5haRHUm2mbaTfo-Ck8eO8ih3iqIhM8Miv7S5i0DY4O871NRgbDiP4V8mLTvYeXj_9l8n3z5-W17fp4uvN3fXHRdoyXNCUFBWpc1LmUnWKdTWTmBGaU8qANV3Oy7wreAktsKKSTMVb8pw0dUsbqIACzi-Tt8e-o7O_J_BBDNq30McFwU5eVCWr6orjPCqro7J11nsHnRidHqQ7CILFnJrYiDkQMYcj5tTE39TEPlrfPA2ZmgHUyfgvpij4cBTsdA-H_24s7q_uIoj29GjXPsD-ZJful4gXKAux-nIjVnx5W69-_hDf8j9zRrkj</recordid><startdate>201108</startdate><enddate>201108</enddate><creator>Waheed, Mohammad T.</creator><creator>Thönes, Nadja</creator><creator>Müller, Martin</creator><creator>Hassan, Syed W.</creator><creator>Gottschamel, Johanna</creator><creator>Lössl, Elke</creator><creator>Kaul, Hans-Peter</creator><creator>Lössl, Andreas G.</creator><general>Blackwell Publishing Ltd</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>201108</creationdate><title>Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes</title><author>Waheed, Mohammad T. ; Thönes, Nadja ; Müller, Martin ; Hassan, Syed W. ; Gottschamel, Johanna ; Lössl, Elke ; Kaul, Hans-Peter ; Lössl, Andreas G.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4052-158193173adfd4f94a04123224e4bf3673f567ece458a4d201631b9c2be8e2e03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>adjuvant</topic><topic>Adjuvants, Immunologic - genetics</topic><topic>Antigens, Viral - genetics</topic><topic>Bacterial Toxins - chemistry</topic><topic>Bacterial Toxins - genetics</topic><topic>Bacterial Toxins - metabolism</topic><topic>Capsid Proteins - chemistry</topic><topic>Capsid Proteins - genetics</topic><topic>Capsid Proteins - immunology</topic><topic>capsomeres</topic><topic>Chloroplasts - genetics</topic><topic>Chloroplasts - metabolism</topic><topic>Enterotoxins - chemistry</topic><topic>Enterotoxins - genetics</topic><topic>Enterotoxins - metabolism</topic><topic>Epitopes</topic><topic>Escherichia coli Proteins - chemistry</topic><topic>Escherichia coli Proteins - genetics</topic><topic>Escherichia coli Proteins - metabolism</topic><topic>Female</topic><topic>G(M1) Ganglioside - metabolism</topic><topic>HPV-16 L1</topic><topic>Humans</topic><topic>low-cost vaccine</topic><topic>LTB</topic><topic>Nicotiana - genetics</topic><topic>Nicotiana - growth &amp; development</topic><topic>Nicotiana - metabolism</topic><topic>Oncogene Proteins, Viral - chemistry</topic><topic>Oncogene Proteins, Viral - genetics</topic><topic>Oncogene Proteins, Viral - immunology</topic><topic>Papillomavirus Vaccines - genetics</topic><topic>Papillomavirus Vaccines - immunology</topic><topic>Papillomavirus Vaccines - metabolism</topic><topic>Phenotype</topic><topic>Plant Infertility - genetics</topic><topic>Plants, Genetically Modified - genetics</topic><topic>plastid transformation</topic><topic>Plastids - genetics</topic><topic>Pollination</topic><topic>Protein Conformation</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - immunology</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Seeds - physiology</topic><topic>Uterine Cervical Neoplasms - immunology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Waheed, Mohammad T.</creatorcontrib><creatorcontrib>Thönes, Nadja</creatorcontrib><creatorcontrib>Müller, Martin</creatorcontrib><creatorcontrib>Hassan, Syed W.</creatorcontrib><creatorcontrib>Gottschamel, Johanna</creatorcontrib><creatorcontrib>Lössl, Elke</creatorcontrib><creatorcontrib>Kaul, Hans-Peter</creatorcontrib><creatorcontrib>Lössl, Andreas G.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Plant biotechnology journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext_linktorsrc</fulltext></delivery><addata><au>Waheed, Mohammad T.</au><au>Thönes, Nadja</au><au>Müller, Martin</au><au>Hassan, Syed W.</au><au>Gottschamel, Johanna</au><au>Lössl, Elke</au><au>Kaul, Hans-Peter</au><au>Lössl, Andreas G.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes</atitle><jtitle>Plant biotechnology journal</jtitle><addtitle>Plant Biotechnol J</addtitle><date>2011-08</date><risdate>2011</risdate><volume>9</volume><issue>6</issue><spage>651</spage><epage>660</epage><pages>651-660</pages><issn>1467-7644</issn><eissn>1467-7652</eissn><abstract>Summary Human papillomavirus (HPV) causes cervical cancer in women worldwide, which is currently prevented by vaccines based on virus‐like particles (VLPs). However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. Concerning the highest burden of disease in resource‐poor countries, development of an improved mucosal and cost‐effective vaccine is a necessity. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as vaccine candidate. Furthermore, coupling of an adjuvant like Escherichia coli heat‐labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate co‐administration of adjuvants. Our study demonstrates the expression of two pentameric proteins: the modified HPV‐16 L1 (L1_2xCysM) and LTB as a fusion protein in tobacco chloroplasts. Homoplasmy of the transplastomic plants was confirmed by Southern blotting. Western blot analysis showed that the LTB‐L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2% of total soluble protein. Proper folding and display of conformational epitopes for both LTB and L1 in the fusion protein was confirmed by GM1‐ganglioside binding assay and antigen capture ELISA, respectively. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, plants reached maturity and produced seeds by pollination with wild‐type plants. Taken together, these results pave the way for the possible development of a low‐cost adjuvant‐coupled vaccine with potentially improved immunogenicity against cervical cancer.</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>21447051</pmid><doi>10.1111/j.1467-7652.2011.00612.x</doi><tpages>10</tpages></addata></record>
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subjects adjuvant
Adjuvants, Immunologic - genetics
Antigens, Viral - genetics
Bacterial Toxins - chemistry
Bacterial Toxins - genetics
Bacterial Toxins - metabolism
Capsid Proteins - chemistry
Capsid Proteins - genetics
Capsid Proteins - immunology
capsomeres
Chloroplasts - genetics
Chloroplasts - metabolism
Enterotoxins - chemistry
Enterotoxins - genetics
Enterotoxins - metabolism
Epitopes
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - genetics
Escherichia coli Proteins - metabolism
Female
G(M1) Ganglioside - metabolism
HPV-16 L1
Humans
low-cost vaccine
LTB
Nicotiana - genetics
Nicotiana - growth & development
Nicotiana - metabolism
Oncogene Proteins, Viral - chemistry
Oncogene Proteins, Viral - genetics
Oncogene Proteins, Viral - immunology
Papillomavirus Vaccines - genetics
Papillomavirus Vaccines - immunology
Papillomavirus Vaccines - metabolism
Phenotype
Plant Infertility - genetics
Plants, Genetically Modified - genetics
plastid transformation
Plastids - genetics
Pollination
Protein Conformation
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - immunology
Recombinant Fusion Proteins - metabolism
Seeds - physiology
Uterine Cervical Neoplasms - immunology
title Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes
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