Chimeras between the human immunodeficiency virus (HIV-1) Env and vaccinia virus immunogenic proteins p14 and p39 generate in mice broadly reactive antibodies and specific activation of CD8+ T cell responses to Env
Identifieur interne : 002110 ( Istex/Corpus ); précédent : 002109; suivant : 002111Chimeras between the human immunodeficiency virus (HIV-1) Env and vaccinia virus immunogenic proteins p14 and p39 generate in mice broadly reactive antibodies and specific activation of CD8+ T cell responses to Env
Auteurs : Manuel Collado ; Dolores Rodr Guez ; Juan Ram N Rodr Guez ; Isabel Vázquez ; Rosa M. Gonzalo ; Mariano EstebanSource :
- Vaccine [ 0264-410X ] ; 2000.
English descriptors
- Teeft :
- Aids research, Amino acids, Animals immunised, Assay, Cell cultures, Cell extracts, Cell response, Cell responses, Chimera, Chimeric, Chimeric proteins, Collado, Elisa, Elispot assay, Envelope glycoprotein, Epitope, Esteban, Fusion protein, Fusion proteins, Glycosylation, Human virus, Human virus type, Humoral, Immune, Immune response, Immune system, Immunisation, Immunised, Immunised animals, Immunodominant, Immunogenic, Immunogenic proteins, Infectious diseases, Mice immunised, Monoclonal antibodies, Mung bean, National academy, Neutralisation, Neutralising, Neutralizing, Peptide, Plasmid, Protective immunity, Protein, Reactive, Reactive antibodies, Recombinant, Recombinant virus, Recombinant viruses, Rodriguez, Secreting cells, Strain iiib, Vaccine, Vaccinia, Vaccinia virus, Virology, Western blot.
Abstract
Abstract: A vaccine based on the envelope protein (Env) of the human immunodeficiency virus type 1 (HIV-1) that triggers widely reactive antibodies might be a desirable approach to control virus infection. To expose epitopes which could induce broadly reactive antibodies against HIV-1 Env, we have generated vaccinia virus (VV) recombinants that express Env fused at its N- or C-terminus with two major antigenic proteins of VV, p14 (A27L gene) and p39 (A4L gene). Biochemical analysis of the chimeric proteins in cell cultures revealed that, in all cases, recombinant viruses expressed the correct fusion proteins. When p14 or p39 are fused at the N-terminus of Env the chimeric proteins are poorly glycosylated but when p14 or p39 are fused at the C-terminus of Env, the chimeric proteins are fully glycosylated. In Balb/c mice, immunisation with the referred VV recombinants induced similar levels of CD8+ T cell specific responses to Env as immunisation with the entire Env protein. The humoral immune response triggered by the fusion proteins was broader than in animals immunised with VV expressing the entire Env (VVEnv1), and was directed to epitopes outside of the V3 loop (V1/V2, C1, C2, C4). One of the chimeric constructs induced a better neutralising antibody response than VVEnv1. We conclude that fusing VV proteins p14 or p39 to Env provides an effective means to induce broadly reactive antibodies and CD8+ T cell responses to Env. This approach might have utility against HIV and other pathogens.
Url:
DOI: 10.1016/S0264-410X(00)00112-2
Links to Exploration step
ISTEX:9D75B48FDB06CE7C33BE95519665F4CFA133A0E3Le document en format XML
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Gonzalo</name><affiliation><mods:affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</mods:affiliation></affiliation></author><author><name sortKey="Esteban, Mariano" sort="Esteban, Mariano" uniqKey="Esteban M" first="Mariano" last="Esteban">Mariano Esteban</name><affiliation><mods:affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: mesteban@cnb.uam.es</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:9D75B48FDB06CE7C33BE95519665F4CFA133A0E3</idno><date when="2000" year="2000">2000</date><idno type="doi">10.1016/S0264-410X(00)00112-2</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-RL3VC9T1-F/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002110</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002110</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a">Chimeras between the human immunodeficiency virus (HIV-1) Env and vaccinia virus immunogenic proteins p14 and p39 generate in mice broadly reactive antibodies and specific activation of CD8+ T cell responses to Env</title><author><name sortKey="Collado, Manuel" sort="Collado, Manuel" uniqKey="Collado M" first="Manuel" last="Collado">Manuel Collado</name><affiliation><mods:affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</mods:affiliation></affiliation></author><author><name sortKey="Rodr Guez, Dolores" sort="Rodr Guez, Dolores" uniqKey="Rodr Guez D" first="Dolores" last="Rodr Guez">Dolores Rodr Guez</name><affiliation><mods:affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</mods:affiliation></affiliation></author><author><name sortKey="Rodr Guez, Juan Ram N" sort="Rodr Guez, Juan Ram N" uniqKey="Rodr Guez J" first="Juan Ram N" last="Rodr Guez">Juan Ram N Rodr Guez</name><affiliation><mods:affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</mods:affiliation></affiliation></author><author><name sortKey="Vazquez, Isabel" sort="Vazquez, Isabel" uniqKey="Vazquez I" first="Isabel" last="Vázquez">Isabel Vázquez</name><affiliation><mods:affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</mods:affiliation></affiliation></author><author><name sortKey="Gonzalo, Rosa M" sort="Gonzalo, Rosa M" uniqKey="Gonzalo R" first="Rosa M." last="Gonzalo">Rosa M. Gonzalo</name><affiliation><mods:affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</mods:affiliation></affiliation></author><author><name sortKey="Esteban, Mariano" sort="Esteban, Mariano" uniqKey="Esteban M" first="Mariano" last="Esteban">Mariano Esteban</name><affiliation><mods:affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: mesteban@cnb.uam.es</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Vaccine</title><title level="j" type="abbrev">JVAC</title><idno type="ISSN">0264-410X</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000">2000</date><biblScope unit="volume">18</biblScope><biblScope unit="issue">27</biblScope><biblScope unit="page" from="3123">3123</biblScope><biblScope unit="page" to="3133">3133</biblScope></imprint><idno type="ISSN">0264-410X</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0264-410X</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Aids research</term><term>Amino acids</term><term>Animals immunised</term><term>Assay</term><term>Cell cultures</term><term>Cell extracts</term><term>Cell response</term><term>Cell responses</term><term>Chimera</term><term>Chimeric</term><term>Chimeric proteins</term><term>Collado</term><term>Elisa</term><term>Elispot assay</term><term>Envelope glycoprotein</term><term>Epitope</term><term>Esteban</term><term>Fusion protein</term><term>Fusion proteins</term><term>Glycosylation</term><term>Human virus</term><term>Human virus type</term><term>Humoral</term><term>Immune</term><term>Immune response</term><term>Immune system</term><term>Immunisation</term><term>Immunised</term><term>Immunised animals</term><term>Immunodominant</term><term>Immunogenic</term><term>Immunogenic proteins</term><term>Infectious diseases</term><term>Mice immunised</term><term>Monoclonal antibodies</term><term>Mung bean</term><term>National academy</term><term>Neutralisation</term><term>Neutralising</term><term>Neutralizing</term><term>Peptide</term><term>Plasmid</term><term>Protective immunity</term><term>Protein</term><term>Reactive</term><term>Reactive antibodies</term><term>Recombinant</term><term>Recombinant virus</term><term>Recombinant viruses</term><term>Rodriguez</term><term>Secreting cells</term><term>Strain iiib</term><term>Vaccine</term><term>Vaccinia</term><term>Vaccinia virus</term><term>Virology</term><term>Western blot</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: A vaccine based on the envelope protein (Env) of the human immunodeficiency virus type 1 (HIV-1) that triggers widely reactive antibodies might be a desirable approach to control virus infection. To expose epitopes which could induce broadly reactive antibodies against HIV-1 Env, we have generated vaccinia virus (VV) recombinants that express Env fused at its N- or C-terminus with two major antigenic proteins of VV, p14 (A27L gene) and p39 (A4L gene). Biochemical analysis of the chimeric proteins in cell cultures revealed that, in all cases, recombinant viruses expressed the correct fusion proteins. When p14 or p39 are fused at the N-terminus of Env the chimeric proteins are poorly glycosylated but when p14 or p39 are fused at the C-terminus of Env, the chimeric proteins are fully glycosylated. In Balb/c mice, immunisation with the referred VV recombinants induced similar levels of CD8+ T cell specific responses to Env as immunisation with the entire Env protein. The humoral immune response triggered by the fusion proteins was broader than in animals immunised with VV expressing the entire Env (VVEnv1), and was directed to epitopes outside of the V3 loop (V1/V2, C1, C2, C4). One of the chimeric constructs induced a better neutralising antibody response than VVEnv1. We conclude that fusing VV proteins p14 or p39 to Env provides an effective means to induce broadly reactive antibodies and CD8+ T cell responses to Env. This approach might have utility against HIV and other pathogens.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>recombinant</json:string><json:string>chimeric</json:string><json:string>chimeric proteins</json:string><json:string>epitope</json:string><json:string>rodriguez</json:string><json:string>immunised</json:string><json:string>vaccinia</json:string><json:string>reactive</json:string><json:string>immune</json:string><json:string>esteban</json:string><json:string>vaccine</json:string><json:string>collado</json:string><json:string>neutralising</json:string><json:string>human virus type</json:string><json:string>glycosylation</json:string><json:string>immunisation</json:string><json:string>peptide</json:string><json:string>national academy</json:string><json:string>humoral</json:string><json:string>fusion proteins</json:string><json:string>mice immunised</json:string><json:string>virology</json:string><json:string>chimera</json:string><json:string>reactive antibodies</json:string><json:string>neutralizing</json:string><json:string>immunodominant</json:string><json:string>neutralisation</json:string><json:string>elisa</json:string><json:string>vaccinia virus</json:string><json:string>animals immunised</json:string><json:string>immunogenic</json:string><json:string>plasmid</json:string><json:string>assay</json:string><json:string>immune response</json:string><json:string>elispot assay</json:string><json:string>immune system</json:string><json:string>cell cultures</json:string><json:string>fusion protein</json:string><json:string>recombinant viruses</json:string><json:string>secreting cells</json:string><json:string>cell responses</json:string><json:string>cell response</json:string><json:string>infectious diseases</json:string><json:string>monoclonal antibodies</json:string><json:string>recombinant virus</json:string><json:string>mung bean</json:string><json:string>western blot</json:string><json:string>amino acids</json:string><json:string>strain iiib</json:string><json:string>immunised animals</json:string><json:string>human virus</json:string><json:string>protective immunity</json:string><json:string>envelope glycoprotein</json:string><json:string>cell extracts</json:string><json:string>aids research</json:string><json:string>immunogenic proteins</json:string><json:string>protein</json:string></teeft></keywords><author><json:item><name>Manuel Collado</name><affiliations><json:string>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</json:string></affiliations></json:item><json:item><name>Dolores Rodrı́guez</name><affiliations><json:string>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</json:string></affiliations></json:item><json:item><name>Juan Ramón Rodrı́guez</name><affiliations><json:string>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</json:string></affiliations></json:item><json:item><name>Isabel Vázquez</name><affiliations><json:string>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</json:string></affiliations></json:item><json:item><name>Rosa M. Gonzalo</name><affiliations><json:string>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</json:string></affiliations></json:item><json:item><name>Mariano Esteban</name><affiliations><json:string>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</json:string><json:string>E-mail: mesteban@cnb.uam.es</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Fusion proteins</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Vaccinia virus</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>HIV-1 env</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Humoral and cellular responses</value></json:item></subject><arkIstex>ark:/67375/6H6-RL3VC9T1-F</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: A vaccine based on the envelope protein (Env) of the human immunodeficiency virus type 1 (HIV-1) that triggers widely reactive antibodies might be a desirable approach to control virus infection. To expose epitopes which could induce broadly reactive antibodies against HIV-1 Env, we have generated vaccinia virus (VV) recombinants that express Env fused at its N- or C-terminus with two major antigenic proteins of VV, p14 (A27L gene) and p39 (A4L gene). Biochemical analysis of the chimeric proteins in cell cultures revealed that, in all cases, recombinant viruses expressed the correct fusion proteins. When p14 or p39 are fused at the N-terminus of Env the chimeric proteins are poorly glycosylated but when p14 or p39 are fused at the C-terminus of Env, the chimeric proteins are fully glycosylated. In Balb/c mice, immunisation with the referred VV recombinants induced similar levels of CD8+ T cell specific responses to Env as immunisation with the entire Env protein. The humoral immune response triggered by the fusion proteins was broader than in animals immunised with VV expressing the entire Env (VVEnv1), and was directed to epitopes outside of the V3 loop (V1/V2, C1, C2, C4). One of the chimeric constructs induced a better neutralising antibody response than VVEnv1. We conclude that fusing VV proteins p14 or p39 to Env provides an effective means to induce broadly reactive antibodies and CD8+ T cell responses to Env. This approach might have utility against HIV and other pathogens.</abstract><qualityIndicators><score>9.904</score><pdfWordCount>6123</pdfWordCount><pdfCharCount>37670</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>11</pdfPageCount><pdfPageSize>595 x 792 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>242</abstractWordCount><abstractCharCount>1515</abstractCharCount><keywordCount>4</keywordCount></qualityIndicators><title>Chimeras between the human immunodeficiency virus (HIV-1) Env and vaccinia virus immunogenic proteins p14 and p39 generate in mice broadly reactive antibodies and specific activation of CD8+ T cell responses to Env</title><pmid><json:string>10856792</json:string></pmid><pii><json:string>S0264-410X(00)00112-2</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Vaccine</title><language><json:string>unknown</json:string></language><publicationDate>2000</publicationDate><issn><json:string>0264-410X</json:string></issn><pii><json:string>S0264-410X(00)X0110-7</json:string></pii><volume>18</volume><issue>27</issue><pages><first>3123</first><last>3133</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>2000</json:string></date><geogName></geogName><orgName><json:string>Columbia University</json:string><json:string>Spain Received</json:string><json:string>FIS</json:string><json:string>Elsevier Science Ltd.</json:string><json:string>Ludwig Institute for Cancer Research, ICSM</json:string><json:string>Ministerio de Sanidad</json:string><json:string>Medical Research Council, UK</json:string><json:string>Â Comision Interministerial</json:string></orgName><orgName_funder><json:string>Â Comision Interministerial</json:string></orgName_funder><orgName_provider></orgName_provider><persName><json:string>M. Esteban</json:string><json:string>Dolores RodrõÂ</json:string><json:string>Isabel Vazquez</json:string><json:string>EVA Programme</json:string><json:string>Carmen Rivas</json:string><json:string>R. Axel</json:string><json:string>Victoria Jimenez</json:string><json:string>F. Lam</json:string><json:string>To</json:string><json:string>Juan Ramon</json:string><json:string>Mariano Esteban</json:string><json:string>Rosa M. Gonzalo</json:string><json:string>Margarita</json:string><json:string>R.M. Gonzalo</json:string><json:string>D. Rodriguez</json:string><json:string>J. Alcami</json:string></persName><placeName><json:string>UK</json:string><json:string>London</json:string><json:string>Madrid</json:string><json:string>Norfolk</json:string><json:string>Puri</json:string><json:string>Spain</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>[4]</json:string><json:string>[23]</json:string><json:string>[18]</json:string><json:string>[2,3]</json:string><json:string>[33]</json:string><json:string>[31,32]</json:string><json:string>[14,15]</json:string><json:string>[1]</json:string><json:string>[21]</json:string><json:string>M. Collado et al.</json:string><json:string>[20]</json:string><json:string>[5]</json:string><json:string>[37]</json:string><json:string>[34,35]</json:string><json:string>[36]</json:string><json:string>[2]</json:string><json:string>[13]</json:string><json:string>[27,28]</json:string><json:string>[29,30]</json:string><json:string>[4,5]</json:string><json:string>[19]</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-RL3VC9T1-F</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - medicine, research & experimental</json:string><json:string>2 - immunology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - virology</json:string></scienceMetrix><scopus><json:string>1 - Health Sciences</json:string><json:string>2 - Medicine</json:string><json:string>3 - Infectious Diseases</json:string><json:string>1 - Health Sciences</json:string><json:string>2 - Medicine</json:string><json:string>3 - Public Health, Environmental and Occupational Health</json:string><json:string>1 - Health Sciences</json:string><json:string>2 - Veterinary</json:string><json:string>3 - General Veterinary</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Immunology and Microbiology</json:string><json:string>3 - General Immunology and Microbiology</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Molecular Medicine</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string><json:string>4 - microbiologie</json:string></inist></categories><publicationDate>2000</publicationDate><copyrightDate>2000</copyrightDate><doi><json:string>10.1016/S0264-410X(00)00112-2</json:string></doi><id>9D75B48FDB06CE7C33BE95519665F4CFA133A0E3</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-RL3VC9T1-F/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-RL3VC9T1-F/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-RL3VC9T1-F/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a">Chimeras between the human immunodeficiency virus (HIV-1) Env and vaccinia virus immunogenic proteins p14 and p39 generate in mice broadly reactive antibodies and specific activation of CD8+ T cell responses to Env</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©2000 Elsevier Science Ltd</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>2000</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note type="content">Fig. 1: Schematic diagram of the chimeric proteins. The position of VV p39 and p14 within Env of HIV-1, the variable V regions and the name of the VV recombinants are presented.</note><note type="content">Fig. 2: Expression of the chimeric proteins in cells infected with VV recombinants. BSC-40 cells were infected with 5 pfu/cell of each VV recombinant, at 24 hpi cell extracts were untreated or treated for 30 min with increasing concentrations of the reducing agent DTT (0, 1, 10 and 50 mM), proteins analysed by SDS–PAGE, blotted and reacted with a mixture of antibodies to the V3 loop and to gp41. The Western blot shows oligomers formed in the absence of DTT while different sise proteins are produced by each VV recombinant. The names of VV recombinants and molecular weight markers are indicated.</note><note type="content">Fig. 3: Extent of glycosylation of chimeric proteins produced by VV recombinants. BSC-40 cells were infected (5 pfu/cell) with different VV recombinants in the absence or presence of the glycosylation inhibitor tunicamycin (10 μg/ml) and at 24 hpi cell extracts were fractionated by SDS–PAGE, proteins blotted and reacted with a mixture of antibodies to V3 loop and gp41. A parallel blot was reacted with antibodies against VV proteins p39 and p14. The Western blot shows the sise of the immune reactive bands, with (+) and without (−) tunicamycin treatment. Upper panel, HIV-1 Env immune reactive chimeric proteins; lower panel, VV immune reactive chimeric proteins. Lower panel of part B does not show free p14.</note><note type="content">Fig. 4: Antigen specific CD8+ T cell response after priming with VV recombinants expressing chimeric proteins. Balb/c mice were inoculated i.p. with purified VV recombinants and 21 days later spleen cells were collected and used immediately as effector cells in the ELISPOT assay. The figure shows the number of IFN-γ secreting cells after coculture of the different spleen cell preparations with APCs coated with the peptide (GPGRAFVTI) corresponding to the V3 loop of strain IIIB. Background values from non-pulsed P815 cells were substracted from cells pulsed with the peptide. A negative control is provided by the lack of response observed in splenocytes obtained from mice immunised with a VV recombinant expressing an unrelated antigen, CS of Plasmodium (VVPYCS). The different VV recombinants are indicated.</note><note type="content">Fig. 5: Evaluation of the humoral immune response induced by VV recombinants. Balb/c mice were primed and boosted with purified VV recombinants and pooled sera from animals immunised with the same construct was reacted in an ELISA with either V3 loop peptide or with purified gp160, as described in Section 2. The results show absorbance obtained from three dilutions of sera. Background levels correspond to sera from animals immunised with VVPYCS.</note><note type="content">Fig. 6: Localisation of the regions of gp120 recognised by antibodies derived from mice immunised with VV recombinants expressing chimeric proteins. Pooled sera from Balb/c mice immunised as in Fig. 5, but derived from a different experiment, were tested by ELISA for the ability to bind a collection of peptides (20 amino acids, overlapping by 10 amino acids) spanning the entire gp120. Panel (a), shows that while sera from mice immunised with VV-Env1 recognise largely the V3 loop, antibodies from mice immunised with VV recombinants expressing chimeric Env proteins are directed against other regions. The reactivity of sera to purified gp120 is indicated (+). Panel (b), shows a scheme of gp120 with the extent of peptide recognition by the different sera. Two independent experiments were performed and the results shown are representative.</note><note type="content">Fig. 7: Neutralisation activity of sera from mice immunised with VV recombinants. Pooled sera from mice immunised as in Fig. 6 were mixed with HIV, NL4.3, incubated with MT-2 cells and cell viability determined 7 days later by the MTT assay. A positive control represent pooled sera from several human patients infected with HIV. The virus origin is denoted in symbols.</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">Chimeras between the human immunodeficiency virus (HIV-1) Env and vaccinia virus immunogenic proteins p14 and p39 generate in mice broadly reactive antibodies and specific activation of CD8+ T cell responses to Env</title><author xml:id="author-0000"><persName><forename type="first">Manuel</forename><surname>Collado</surname></persName><note type="biography">Present address: Ludwig Institute for Cancer Research, ICSM at St Mary’s, Norfolk Place, W2 1PG London, UK.</note><affiliation>Present address: Ludwig Institute for Cancer Research, ICSM at St Mary’s, Norfolk Place, W2 1PG London, UK.</affiliation><affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Dolores</forename><surname>Rodrı́guez</surname></persName><affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Juan Ramón</forename><surname>Rodrı́guez</surname></persName><affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</affiliation></author><author xml:id="author-0003"><persName><forename type="first">Isabel</forename><surname>Vázquez</surname></persName><affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Rosa M.</forename><surname>Gonzalo</surname></persName><affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</affiliation></author><author xml:id="author-0005"><persName><forename type="first">Mariano</forename><surname>Esteban</surname></persName><email>mesteban@cnb.uam.es</email><note type="correspondence"><p>Corresponding author. Tel.: +34-91-585-4503; fax: +34-91-585-4506</p></note><affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</affiliation></author><idno type="istex">9D75B48FDB06CE7C33BE95519665F4CFA133A0E3</idno><idno type="ark">ark:/67375/6H6-RL3VC9T1-F</idno><idno type="DOI">10.1016/S0264-410X(00)00112-2</idno><idno type="PII">S0264-410X(00)00112-2</idno></analytic><monogr><title level="j">Vaccine</title><title level="j" type="abbrev">JVAC</title><idno type="pISSN">0264-410X</idno><idno type="PII">S0264-410X(00)X0110-7</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000"></date><biblScope unit="volume">18</biblScope><biblScope unit="issue">27</biblScope><biblScope unit="page" from="3123">3123</biblScope><biblScope unit="page" to="3133">3133</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2000</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: A vaccine based on the envelope protein (Env) of the human immunodeficiency virus type 1 (HIV-1) that triggers widely reactive antibodies might be a desirable approach to control virus infection. To expose epitopes which could induce broadly reactive antibodies against HIV-1 Env, we have generated vaccinia virus (VV) recombinants that express Env fused at its N- or C-terminus with two major antigenic proteins of VV, p14 (A27L gene) and p39 (A4L gene). Biochemical analysis of the chimeric proteins in cell cultures revealed that, in all cases, recombinant viruses expressed the correct fusion proteins. When p14 or p39 are fused at the N-terminus of Env the chimeric proteins are poorly glycosylated but when p14 or p39 are fused at the C-terminus of Env, the chimeric proteins are fully glycosylated. In Balb/c mice, immunisation with the referred VV recombinants induced similar levels of CD8+ T cell specific responses to Env as immunisation with the entire Env protein. The humoral immune response triggered by the fusion proteins was broader than in animals immunised with VV expressing the entire Env (VVEnv1), and was directed to epitopes outside of the V3 loop (V1/V2, C1, C2, C4). One of the chimeric constructs induced a better neutralising antibody response than VVEnv1. We conclude that fusing VV proteins p14 or p39 to Env provides an effective means to induce broadly reactive antibodies and CD8+ T cell responses to Env. This approach might have utility against HIV and other pathogens.</p></abstract><textClass><keywords scheme="keyword"><list><head>Keywords</head><item><term>Fusion proteins</term></item><item><term>Vaccinia virus</term></item><item><term>HIV-1 env</term></item><item><term>Humoral and cellular responses</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2000-02-18">Modified</change><change when="2000">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-RL3VC9T1-F/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="gr6"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="gr7"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla"><item-info><jid>JVAC</jid><aid>2117</aid><ce:pii>S0264-410X(00)00112-2</ce:pii><ce:doi>10.1016/S0264-410X(00)00112-2</ce:doi><ce:copyright type="full-transfer" year="2000">Elsevier Science Ltd</ce:copyright></item-info><head><ce:title>Chimeras between the human immunodeficiency virus (HIV-1) Env and vaccinia virus immunogenic proteins p14 and p39 generate in mice broadly reactive antibodies and specific activation of CD8+ T cell responses to Env</ce:title><ce:author-group><ce:author><ce:given-name>Manuel</ce:given-name><ce:surname>Collado</ce:surname><ce:cross-ref refid="FN1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Dolores</ce:given-name><ce:surname>Rodrı́guez</ce:surname></ce:author><ce:author><ce:given-name>Juan Ramón</ce:given-name><ce:surname>Rodrı́guez</ce:surname></ce:author><ce:author><ce:given-name>Isabel</ce:given-name><ce:surname>Vázquez</ce:surname></ce:author><ce:author><ce:given-name>Rosa M.</ce:given-name><ce:surname>Gonzalo</ce:surname></ce:author><ce:author><ce:given-name>Mariano</ce:given-name><ce:surname>Esteban</ce:surname><ce:cross-ref refid="CORR1">*</ce:cross-ref><ce:e-address>mesteban@cnb.uam.es</ce:e-address></ce:author><ce:affiliation><ce:textfn>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author. Tel.: +34-91-585-4503; fax: +34-91-585-4506</ce:text></ce:correspondence><ce:footnote id="FN1"><ce:label>1</ce:label><ce:note-para>Present address: Ludwig Institute for Cancer Research, ICSM at St Mary’s, Norfolk Place, W2 1PG London, UK.</ce:note-para></ce:footnote></ce:author-group><ce:date-received day="24" month="11" year="1999"></ce:date-received><ce:date-revised day="18" month="2" year="2000"></ce:date-revised><ce:date-accepted day="13" month="3" year="2000"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>A vaccine based on the envelope protein (Env) of the human immunodeficiency virus type 1 (HIV-1) that triggers widely reactive antibodies might be a desirable approach to control virus infection. To expose epitopes which could induce broadly reactive antibodies against HIV-1 Env, we have generated vaccinia virus (VV) recombinants that express Env fused at its N- or C-terminus with two major antigenic proteins of VV, p14 (A27L gene) and p39 (A4L gene). Biochemical analysis of the chimeric proteins in cell cultures revealed that, in all cases, recombinant viruses expressed the correct fusion proteins. When p14 or p39 are fused at the N-terminus of Env the chimeric proteins are poorly glycosylated but when p14 or p39 are fused at the C-terminus of Env, the chimeric proteins are fully glycosylated. In Balb/c mice, immunisation with the referred VV recombinants induced similar levels of CD8+ T cell specific responses to Env as immunisation with the entire Env protein. The humoral immune response triggered by the fusion proteins was broader than in animals immunised with VV expressing the entire Env (VVEnv1), and was directed to epitopes outside of the V3 loop (V1/V2, C1, C2, C4). One of the chimeric constructs induced a better neutralising antibody response than VVEnv1. We conclude that fusing VV proteins p14 or p39 to Env provides an effective means to induce broadly reactive antibodies and CD8+ T cell responses to Env. This approach might have utility against HIV and other pathogens.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Fusion proteins</ce:text></ce:keyword><ce:keyword><ce:text>Vaccinia virus</ce:text></ce:keyword><ce:keyword><ce:text>HIV-1 env</ce:text></ce:keyword><ce:keyword><ce:text>Humoral and cellular responses</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Chimeras between the human immunodeficiency virus (HIV-1) Env and vaccinia virus immunogenic proteins p14 and p39 generate in mice broadly reactive antibodies and specific activation of CD8+ T cell responses to Env</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Chimeras between the human immunodeficiency virus (HIV-1) Env and vaccinia virus immunogenic proteins p14 and p39 generate in mice broadly reactive antibodies and specific activation of CD8+ T cell responses to Env</title></titleInfo><name type="personal"><namePart type="given">Manuel</namePart><namePart type="family">Collado</namePart><affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</affiliation><description>Present address: Ludwig Institute for Cancer Research, ICSM at St Mary’s, Norfolk Place, W2 1PG London, UK.</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Dolores</namePart><namePart type="family">Rodrı́guez</namePart><affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Juan Ramón</namePart><namePart type="family">Rodrı́guez</namePart><affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Isabel</namePart><namePart type="family">Vázquez</namePart><affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Rosa M.</namePart><namePart type="family">Gonzalo</namePart><affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Mariano</namePart><namePart type="family">Esteban</namePart><affiliation>Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologı́a, CSIC, Campus Universidad Autonoma, E-28049 Madrid, Spain</affiliation><affiliation>E-mail: mesteban@cnb.uam.es</affiliation><description>Corresponding author. Tel.: +34-91-585-4503; fax: +34-91-585-4506</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued><dateModified encoding="w3cdtf">2000-02-18</dateModified><copyrightDate encoding="w3cdtf">2000</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: A vaccine based on the envelope protein (Env) of the human immunodeficiency virus type 1 (HIV-1) that triggers widely reactive antibodies might be a desirable approach to control virus infection. To expose epitopes which could induce broadly reactive antibodies against HIV-1 Env, we have generated vaccinia virus (VV) recombinants that express Env fused at its N- or C-terminus with two major antigenic proteins of VV, p14 (A27L gene) and p39 (A4L gene). Biochemical analysis of the chimeric proteins in cell cultures revealed that, in all cases, recombinant viruses expressed the correct fusion proteins. When p14 or p39 are fused at the N-terminus of Env the chimeric proteins are poorly glycosylated but when p14 or p39 are fused at the C-terminus of Env, the chimeric proteins are fully glycosylated. In Balb/c mice, immunisation with the referred VV recombinants induced similar levels of CD8+ T cell specific responses to Env as immunisation with the entire Env protein. The humoral immune response triggered by the fusion proteins was broader than in animals immunised with VV expressing the entire Env (VVEnv1), and was directed to epitopes outside of the V3 loop (V1/V2, C1, C2, C4). One of the chimeric constructs induced a better neutralising antibody response than VVEnv1. We conclude that fusing VV proteins p14 or p39 to Env provides an effective means to induce broadly reactive antibodies and CD8+ T cell responses to Env. This approach might have utility against HIV and other pathogens.</abstract><note type="content">Fig. 1: Schematic diagram of the chimeric proteins. The position of VV p39 and p14 within Env of HIV-1, the variable V regions and the name of the VV recombinants are presented.</note><note type="content">Fig. 2: Expression of the chimeric proteins in cells infected with VV recombinants. BSC-40 cells were infected with 5 pfu/cell of each VV recombinant, at 24 hpi cell extracts were untreated or treated for 30 min with increasing concentrations of the reducing agent DTT (0, 1, 10 and 50 mM), proteins analysed by SDS–PAGE, blotted and reacted with a mixture of antibodies to the V3 loop and to gp41. The Western blot shows oligomers formed in the absence of DTT while different sise proteins are produced by each VV recombinant. The names of VV recombinants and molecular weight markers are indicated.</note><note type="content">Fig. 3: Extent of glycosylation of chimeric proteins produced by VV recombinants. BSC-40 cells were infected (5 pfu/cell) with different VV recombinants in the absence or presence of the glycosylation inhibitor tunicamycin (10 μg/ml) and at 24 hpi cell extracts were fractionated by SDS–PAGE, proteins blotted and reacted with a mixture of antibodies to V3 loop and gp41. A parallel blot was reacted with antibodies against VV proteins p39 and p14. The Western blot shows the sise of the immune reactive bands, with (+) and without (−) tunicamycin treatment. Upper panel, HIV-1 Env immune reactive chimeric proteins; lower panel, VV immune reactive chimeric proteins. Lower panel of part B does not show free p14.</note><note type="content">Fig. 4: Antigen specific CD8+ T cell response after priming with VV recombinants expressing chimeric proteins. Balb/c mice were inoculated i.p. with purified VV recombinants and 21 days later spleen cells were collected and used immediately as effector cells in the ELISPOT assay. The figure shows the number of IFN-γ secreting cells after coculture of the different spleen cell preparations with APCs coated with the peptide (GPGRAFVTI) corresponding to the V3 loop of strain IIIB. Background values from non-pulsed P815 cells were substracted from cells pulsed with the peptide. A negative control is provided by the lack of response observed in splenocytes obtained from mice immunised with a VV recombinant expressing an unrelated antigen, CS of Plasmodium (VVPYCS). The different VV recombinants are indicated.</note><note type="content">Fig. 5: Evaluation of the humoral immune response induced by VV recombinants. Balb/c mice were primed and boosted with purified VV recombinants and pooled sera from animals immunised with the same construct was reacted in an ELISA with either V3 loop peptide or with purified gp160, as described in Section 2. The results show absorbance obtained from three dilutions of sera. Background levels correspond to sera from animals immunised with VVPYCS.</note><note type="content">Fig. 6: Localisation of the regions of gp120 recognised by antibodies derived from mice immunised with VV recombinants expressing chimeric proteins. Pooled sera from Balb/c mice immunised as in Fig. 5, but derived from a different experiment, were tested by ELISA for the ability to bind a collection of peptides (20 amino acids, overlapping by 10 amino acids) spanning the entire gp120. Panel (a), shows that while sera from mice immunised with VV-Env1 recognise largely the V3 loop, antibodies from mice immunised with VV recombinants expressing chimeric Env proteins are directed against other regions. The reactivity of sera to purified gp120 is indicated (+). Panel (b), shows a scheme of gp120 with the extent of peptide recognition by the different sera. Two independent experiments were performed and the results shown are representative.</note><note type="content">Fig. 7: Neutralisation activity of sera from mice immunised with VV recombinants. Pooled sera from mice immunised as in Fig. 6 were mixed with HIV, NL4.3, incubated with MT-2 cells and cell viability determined 7 days later by the MTT assay. A positive control represent pooled sera from several human patients infected with HIV. The virus origin is denoted in symbols.</note><subject><genre>Keywords</genre><topic>Fusion proteins</topic><topic>Vaccinia virus</topic><topic>HIV-1 env</topic><topic>Humoral and cellular responses</topic></subject><relatedItem type="host"><titleInfo><title>Vaccine</title></titleInfo><titleInfo type="abbreviated"><title>JVAC</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued></originInfo><identifier type="ISSN">0264-410X</identifier><identifier type="PII">S0264-410X(00)X0110-7</identifier><part><date>2000</date><detail type="volume"><number>18</number><caption>vol.</caption></detail><detail type="issue"><number>27</number><caption>no.</caption></detail><extent unit="issue-pages"><start>3077</start><end>3206</end></extent><extent unit="pages"><start>3123</start><end>3133</end></extent></part></relatedItem><identifier type="istex">9D75B48FDB06CE7C33BE95519665F4CFA133A0E3</identifier><identifier type="ark">ark:/67375/6H6-RL3VC9T1-F</identifier><identifier type="DOI">10.1016/S0264-410X(00)00112-2</identifier><identifier type="PII">S0264-410X(00)00112-2</identifier><accessCondition type="use and reproduction" contentType="copyright">©2000 Elsevier Science Ltd</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Elsevier Science Ltd, ©2000</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-RL3VC9T1-F/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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