Gut Microbiota Elicits a Protective Immune Response against Malaria Transmission
Identifieur interne : 000350 ( Pmc/Corpus ); précédent : 000349; suivant : 000351Gut Microbiota Elicits a Protective Immune Response against Malaria Transmission
Auteurs : Bahtiyar Yilmaz ; Silvia Portugal ; Tuan M. Tran ; Raffaella Gozzelino ; Susana Ramos ; Joana Gomes ; Ana Regalado ; Peter J. Cowan ; Anthony J. F. D Pice ; Anita S. Chong ; Ogobara K. Doumbo ; Boubacar Traore ; Peter D. Crompton ; Henrique Silveira ; Miguel P. SoaresSource :
- Cell [ 0092-8674 ] ; 2014.
Abstract
Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:β-galactoside-α1-3-galactosyltransferase (
Url:
DOI: 10.1016/j.cell.2014.10.053
PubMed: 25480293
PubMed Central: 4261137
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PMC:4261137Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Gut Microbiota Elicits a Protective Immune Response against Malaria Transmission</title><author><name sortKey="Yilmaz, Bahtiyar" sort="Yilmaz, Bahtiyar" uniqKey="Yilmaz B" first="Bahtiyar" last="Yilmaz">Bahtiyar Yilmaz</name><affiliation><nlm:aff id="aff1">Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</nlm:aff></affiliation></author><author><name sortKey="Portugal, Silvia" sort="Portugal, Silvia" uniqKey="Portugal S" first="Silvia" last="Portugal">Silvia Portugal</name><affiliation><nlm:aff id="aff2">Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, Room 125, 12441 Parklawn Drive, Rockville, MD 20852-8180, USA</nlm:aff></affiliation></author><author><name sortKey="Tran, Tuan M" sort="Tran, Tuan M" uniqKey="Tran T" first="Tuan M." last="Tran">Tuan M. Tran</name><affiliation><nlm:aff id="aff2">Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, Room 125, 12441 Parklawn Drive, Rockville, MD 20852-8180, USA</nlm:aff></affiliation></author><author><name sortKey="Gozzelino, Raffaella" sort="Gozzelino, Raffaella" uniqKey="Gozzelino R" first="Raffaella" last="Gozzelino">Raffaella Gozzelino</name><affiliation><nlm:aff id="aff1">Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</nlm:aff></affiliation></author><author><name sortKey="Ramos, Susana" sort="Ramos, Susana" uniqKey="Ramos S" first="Susana" last="Ramos">Susana Ramos</name><affiliation><nlm:aff id="aff1">Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</nlm:aff></affiliation></author><author><name sortKey="Gomes, Joana" sort="Gomes, Joana" uniqKey="Gomes J" first="Joana" last="Gomes">Joana Gomes</name><affiliation><nlm:aff id="aff1">Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Centro de Malaria e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira, 100, 1349-008 Lisboa, Portugal</nlm:aff></affiliation></author><author><name sortKey="Regalado, Ana" sort="Regalado, Ana" uniqKey="Regalado A" first="Ana" last="Regalado">Ana Regalado</name><affiliation><nlm:aff id="aff1">Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</nlm:aff></affiliation></author><author><name sortKey="Cowan, Peter J" sort="Cowan, Peter J" uniqKey="Cowan P" first="Peter J." last="Cowan">Peter J. Cowan</name><affiliation><nlm:aff id="aff4">Immunology Research Centre, St. Vincent’s Hospital, Fitzroy, Melbourne, VIC 3065, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="aff5">Department of Medicine, University of Melbourne, Parkville, VIC 2900, Australia</nlm:aff></affiliation></author><author><name sortKey="D Pice, Anthony J F" sort="D Pice, Anthony J F" uniqKey="D Pice A" first="Anthony J. F." last="D Pice">Anthony J. F. D Pice</name><affiliation><nlm:aff id="aff4">Immunology Research Centre, St. Vincent’s Hospital, Fitzroy, Melbourne, VIC 3065, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="aff5">Department of Medicine, University of Melbourne, Parkville, VIC 2900, Australia</nlm:aff></affiliation></author><author><name sortKey="Chong, Anita S" sort="Chong, Anita S" uniqKey="Chong A" first="Anita S." last="Chong">Anita S. Chong</name><affiliation><nlm:aff id="aff6">Section of Transplantation, Department of Surgery, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA</nlm:aff></affiliation></author><author><name sortKey="Doumbo, Ogobara K" sort="Doumbo, Ogobara K" uniqKey="Doumbo O" first="Ogobara K." last="Doumbo">Ogobara K. Doumbo</name><affiliation><nlm:aff id="aff7">Mali International Center of Excellence in Research, University of Sciences, Techniques and Technologies of Bamako, 1805 Bamako, Mali</nlm:aff></affiliation></author><author><name sortKey="Traore, Boubacar" sort="Traore, Boubacar" uniqKey="Traore B" first="Boubacar" last="Traore">Boubacar Traore</name><affiliation><nlm:aff id="aff7">Mali International Center of Excellence in Research, University of Sciences, Techniques and Technologies of Bamako, 1805 Bamako, Mali</nlm:aff></affiliation></author><author><name sortKey="Crompton, Peter D" sort="Crompton, Peter D" uniqKey="Crompton P" first="Peter D." last="Crompton">Peter D. Crompton</name><affiliation><nlm:aff id="aff2">Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, Room 125, 12441 Parklawn Drive, Rockville, MD 20852-8180, USA</nlm:aff></affiliation></author><author><name sortKey="Silveira, Henrique" sort="Silveira, Henrique" uniqKey="Silveira H" first="Henrique" last="Silveira">Henrique Silveira</name><affiliation><nlm:aff id="aff3">Centro de Malaria e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira, 100, 1349-008 Lisboa, Portugal</nlm:aff></affiliation></author><author><name sortKey="Soares, Miguel P" sort="Soares, Miguel P" uniqKey="Soares M" first="Miguel P." last="Soares">Miguel P. Soares</name><affiliation><nlm:aff id="aff1">Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25480293</idno><idno type="pmc">4261137</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4261137</idno><idno type="RBID">PMC:4261137</idno><idno type="doi">10.1016/j.cell.2014.10.053</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000350</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Gut Microbiota Elicits a Protective Immune Response against Malaria Transmission</title><author><name sortKey="Yilmaz, Bahtiyar" sort="Yilmaz, Bahtiyar" uniqKey="Yilmaz B" first="Bahtiyar" last="Yilmaz">Bahtiyar Yilmaz</name><affiliation><nlm:aff id="aff1">Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</nlm:aff></affiliation></author><author><name sortKey="Portugal, Silvia" sort="Portugal, Silvia" uniqKey="Portugal S" first="Silvia" last="Portugal">Silvia Portugal</name><affiliation><nlm:aff id="aff2">Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, Room 125, 12441 Parklawn Drive, Rockville, MD 20852-8180, USA</nlm:aff></affiliation></author><author><name sortKey="Tran, Tuan M" sort="Tran, Tuan M" uniqKey="Tran T" first="Tuan M." last="Tran">Tuan M. Tran</name><affiliation><nlm:aff id="aff2">Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, Room 125, 12441 Parklawn Drive, Rockville, MD 20852-8180, USA</nlm:aff></affiliation></author><author><name sortKey="Gozzelino, Raffaella" sort="Gozzelino, Raffaella" uniqKey="Gozzelino R" first="Raffaella" last="Gozzelino">Raffaella Gozzelino</name><affiliation><nlm:aff id="aff1">Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</nlm:aff></affiliation></author><author><name sortKey="Ramos, Susana" sort="Ramos, Susana" uniqKey="Ramos S" first="Susana" last="Ramos">Susana Ramos</name><affiliation><nlm:aff id="aff1">Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</nlm:aff></affiliation></author><author><name sortKey="Gomes, Joana" sort="Gomes, Joana" uniqKey="Gomes J" first="Joana" last="Gomes">Joana Gomes</name><affiliation><nlm:aff id="aff1">Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Centro de Malaria e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira, 100, 1349-008 Lisboa, Portugal</nlm:aff></affiliation></author><author><name sortKey="Regalado, Ana" sort="Regalado, Ana" uniqKey="Regalado A" first="Ana" last="Regalado">Ana Regalado</name><affiliation><nlm:aff id="aff1">Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</nlm:aff></affiliation></author><author><name sortKey="Cowan, Peter J" sort="Cowan, Peter J" uniqKey="Cowan P" first="Peter J." last="Cowan">Peter J. Cowan</name><affiliation><nlm:aff id="aff4">Immunology Research Centre, St. Vincent’s Hospital, Fitzroy, Melbourne, VIC 3065, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="aff5">Department of Medicine, University of Melbourne, Parkville, VIC 2900, Australia</nlm:aff></affiliation></author><author><name sortKey="D Pice, Anthony J F" sort="D Pice, Anthony J F" uniqKey="D Pice A" first="Anthony J. F." last="D Pice">Anthony J. F. D Pice</name><affiliation><nlm:aff id="aff4">Immunology Research Centre, St. Vincent’s Hospital, Fitzroy, Melbourne, VIC 3065, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="aff5">Department of Medicine, University of Melbourne, Parkville, VIC 2900, Australia</nlm:aff></affiliation></author><author><name sortKey="Chong, Anita S" sort="Chong, Anita S" uniqKey="Chong A" first="Anita S." last="Chong">Anita S. Chong</name><affiliation><nlm:aff id="aff6">Section of Transplantation, Department of Surgery, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA</nlm:aff></affiliation></author><author><name sortKey="Doumbo, Ogobara K" sort="Doumbo, Ogobara K" uniqKey="Doumbo O" first="Ogobara K." last="Doumbo">Ogobara K. Doumbo</name><affiliation><nlm:aff id="aff7">Mali International Center of Excellence in Research, University of Sciences, Techniques and Technologies of Bamako, 1805 Bamako, Mali</nlm:aff></affiliation></author><author><name sortKey="Traore, Boubacar" sort="Traore, Boubacar" uniqKey="Traore B" first="Boubacar" last="Traore">Boubacar Traore</name><affiliation><nlm:aff id="aff7">Mali International Center of Excellence in Research, University of Sciences, Techniques and Technologies of Bamako, 1805 Bamako, Mali</nlm:aff></affiliation></author><author><name sortKey="Crompton, Peter D" sort="Crompton, Peter D" uniqKey="Crompton P" first="Peter D." last="Crompton">Peter D. Crompton</name><affiliation><nlm:aff id="aff2">Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, Room 125, 12441 Parklawn Drive, Rockville, MD 20852-8180, USA</nlm:aff></affiliation></author><author><name sortKey="Silveira, Henrique" sort="Silveira, Henrique" uniqKey="Silveira H" first="Henrique" last="Silveira">Henrique Silveira</name><affiliation><nlm:aff id="aff3">Centro de Malaria e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira, 100, 1349-008 Lisboa, Portugal</nlm:aff></affiliation></author><author><name sortKey="Soares, Miguel P" sort="Soares, Miguel P" uniqKey="Soares M" first="Miguel P." last="Soares">Miguel P. Soares</name><affiliation><nlm:aff id="aff1">Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</nlm:aff></affiliation></author></analytic><series><title level="j">Cell</title><idno type="ISSN">0092-8674</idno><idno type="eISSN">1097-4172</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Summary</title><sec><p>Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:β-galactoside-α1-3-galactosyltransferase (<italic>α1,3GT</italic>) gene, which ablated the expression of the Galα1-3Galβ1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against <italic>Plasmodium spp.</italic> infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both <italic>Plasmodium spp.</italic> and the human gut pathobiont <italic>E. coli</italic> O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in <italic>α1,3GT</italic>-deficient mice, which produce protective anti-α-gal Abs when colonized by <italic>E. coli</italic> O86:B7. Anti-α-gal Abs target <italic>Plasmodium</italic> sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by <italic>Anopheles</italic> mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.</p></sec><sec><title>PaperFlick</title><p><supplementary-material content-type="local-data" id="mmc2"><media xlink:href="mmc2.jpg"></media></supplementary-material></p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Avila, J L" uniqKey="Avila J">J.L. Avila</name></author><author><name sortKey="Rojas, M" uniqKey="Rojas M">M. Rojas</name></author><author><name sortKey="Galili, U" uniqKey="Galili U">U. Galili</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Avila, J L" uniqKey="Avila J">J.L. Avila</name></author><author><name sortKey="Rojas, M" uniqKey="Rojas M">M. Rojas</name></author><author><name sortKey="Velazquez Avila, G" uniqKey="Velazquez Avila G">G. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Cell</journal-id><journal-id journal-id-type="iso-abbrev">Cell</journal-id><journal-title-group><journal-title>Cell</journal-title></journal-title-group><issn pub-type="ppub">0092-8674</issn><issn pub-type="epub">1097-4172</issn><publisher><publisher-name>Cell Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25480293</article-id><article-id pub-id-type="pmc">4261137</article-id><article-id pub-id-type="publisher-id">S0092-8674(14)01425-1</article-id><article-id pub-id-type="doi">10.1016/j.cell.2014.10.053</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Gut Microbiota Elicits a Protective Immune Response against Malaria Transmission</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Yilmaz</surname><given-names>Bahtiyar</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Portugal</surname><given-names>Silvia</given-names></name><xref rid="aff2" ref-type="aff">2</xref></contrib><contrib contrib-type="author"><name><surname>Tran</surname><given-names>Tuan M.</given-names></name><xref rid="aff2" ref-type="aff">2</xref></contrib><contrib contrib-type="author"><name><surname>Gozzelino</surname><given-names>Raffaella</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Ramos</surname><given-names>Susana</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Gomes</surname><given-names>Joana</given-names></name><xref rid="aff1" ref-type="aff">1</xref><xref rid="aff3" ref-type="aff">3</xref></contrib><contrib contrib-type="author"><name><surname>Regalado</surname><given-names>Ana</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Cowan</surname><given-names>Peter J.</given-names></name><xref rid="aff4" ref-type="aff">4</xref><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author"><name><surname>d’Apice</surname><given-names>Anthony J.F.</given-names></name><xref rid="aff4" ref-type="aff">4</xref><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author"><name><surname>Chong</surname><given-names>Anita S.</given-names></name><xref rid="aff6" ref-type="aff">6</xref></contrib><contrib contrib-type="author"><name><surname>Doumbo</surname><given-names>Ogobara K.</given-names></name><xref rid="aff7" ref-type="aff">7</xref></contrib><contrib contrib-type="author"><name><surname>Traore</surname><given-names>Boubacar</given-names></name><xref rid="aff7" ref-type="aff">7</xref></contrib><contrib contrib-type="author"><name><surname>Crompton</surname><given-names>Peter D.</given-names></name><xref rid="aff2" ref-type="aff">2</xref></contrib><contrib contrib-type="author"><name><surname>Silveira</surname><given-names>Henrique</given-names></name><xref rid="aff3" ref-type="aff">3</xref></contrib><contrib contrib-type="author"><name><surname>Soares</surname><given-names>Miguel P.</given-names></name><email>mpsoares@igc.gulbenkian.pt</email><xref rid="aff1" ref-type="aff">1</xref><xref rid="cor1" ref-type="corresp">∗</xref></contrib></contrib-group><aff id="aff1"><label>1</label>Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal</aff><aff id="aff2"><label>2</label>Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, Room 125, 12441 Parklawn Drive, Rockville, MD 20852-8180, USA</aff><aff id="aff3"><label>3</label>Centro de Malaria e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira, 100, 1349-008 Lisboa, Portugal</aff><aff id="aff4"><label>4</label>Immunology Research Centre, St. Vincent’s Hospital, Fitzroy, Melbourne, VIC 3065, Australia</aff><aff id="aff5"><label>5</label>Department of Medicine, University of Melbourne, Parkville, VIC 2900, Australia</aff><aff id="aff6"><label>6</label>Section of Transplantation, Department of Surgery, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA</aff><aff id="aff7"><label>7</label>Mali International Center of Excellence in Research, University of Sciences, Techniques and Technologies of Bamako, 1805 Bamako, Mali</aff><author-notes><corresp id="cor1"><label>∗</label>Corresponding author <email>mpsoares@igc.gulbenkian.pt</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>04</day><month>12</month><year>2014</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><day>04</day><month>12</month><year>2014</year></pub-date><volume>159</volume><issue>6</issue><fpage>1277</fpage><lpage>1289</lpage><history><date date-type="received"><day>8</day><month>8</month><year>2014</year></date><date date-type="rev-recd"><day>26</day><month>9</month><year>2014</year></date><date date-type="accepted"><day>30</day><month>9</month><year>2014</year></date></history><permissions><copyright-statement>© 2014 The Authors</copyright-statement><copyright-year>2014</copyright-year><license license-type="CC BY-NC-ND" xlink:href="http://creativecommons.org/licenses/by-nc-nd/3.0/"><license-p> This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).</license-p></license></permissions><abstract><title>Summary</title><sec><p>Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:β-galactoside-α1-3-galactosyltransferase (<italic>α1,3GT</italic>) gene, which ablated the expression of the Galα1-3Galβ1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against <italic>Plasmodium spp.</italic> infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both <italic>Plasmodium spp.</italic> and the human gut pathobiont <italic>E. coli</italic> O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in <italic>α1,3GT</italic>-deficient mice, which produce protective anti-α-gal Abs when colonized by <italic>E. coli</italic> O86:B7. Anti-α-gal Abs target <italic>Plasmodium</italic> sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by <italic>Anopheles</italic> mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.</p></sec><sec><title>PaperFlick</title><p><supplementary-material content-type="local-data" id="mmc2"><media xlink:href="mmc2.jpg"></media></supplementary-material></p></sec></abstract><abstract abstract-type="graphical"><title>Graphical Abstract</title><fig id="undfig1" position="anchor"><graphic xlink:href="fx1"></graphic></fig></abstract><abstract abstract-type="author-highlights"><title>Highlights</title><p><list list-type="simple"><list-item id="u0010"><label>•</label><p>α-gal is expressed at the surface of <italic>Plasmodium</italic> sporozoites</p></list-item><list-item id="u0015"><label>•</label><p>Anti-α-gal Abs recognizing <italic>E. coli</italic> O86:B7 are protective against malaria</p></list-item><list-item id="u0020"><label>•</label><p>Anti-α-gal Abs are cytotoxic to <italic>Plasmodium</italic> sporozoites</p></list-item><list-item id="u0025"><label>•</label><p>Vaccination against α-gal confers sterile protection against malaria</p></list-item></list></p></abstract><abstract abstract-type="teaser"><p>Specific members of the gut microbiota induce antibodies that prevent malaria transmission through recognition of a glycan residue that is shared by the microbiota and the causative agent of malaria, the <italic>Plasmodium</italic>.</p></abstract></article-meta></front><body><sec sec-type="intro" id="sec1"><title>Introduction</title><p>Humans have relatively high levels of circulating antibodies (Abs) recognizing xeno-glycans expressed by pathogens (<xref rid="bib44" ref-type="bibr">Oyelaran et al., 2009</xref>). As for other antigens, xeno-glycans cannot be targeted by the immune system when also expressed as self-glycans. This limitation can be bypassed by natural selection of mutations that inactivate the expression of self-glycans (<xref rid="bib6" ref-type="bibr">Bishop and Gagneux, 2007</xref>). Presumably, natural selection of such loss-of-function mutations tailored the human anti-glycan immune repertoire through evolution (<xref rid="bib6" ref-type="bibr">Bishop and Gagneux, 2007</xref>). This notion is supported by the inactivation of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase-like (<italic>CMAH</italic>) gene in humans, which suppressed the expression of N-glycolylneuraminic acid (Neu5Gc) (<xref rid="bib23" ref-type="bibr">Hayakawa et al., 2001</xref>) and allowed for immune reactivity against Neu5Gc (<xref rid="bib57" ref-type="bibr">Tangvoranuntakul et al., 2003</xref>). In a similar manner, inactivation of the <italic>α1,3GT</italic> gene, which suppressed the expression of the Galα1-3Galβ1-4GlcNAc-R (α-gal) carbohydrate in ancestral anthropoid primates that gave rise to humans (<xref rid="bib17" ref-type="bibr">Galili and Swanson, 1991</xref>), also allowed for immune reactivity against α-gal (<xref rid="bib19" ref-type="bibr">Galili et al., 1984</xref>). While it has been argued that this evolutionary process is driven to a large extent by the acquisition of immune-resistance against pathogens expressing such glycans (<xref rid="bib6 bib10" ref-type="bibr">Bishop and Gagneux, 2007; Cywes-Bentley et al., 2013</xref>), this was never tested experimentally.</p><p>Humans do not express α-gal and up to 1%–5% of the repertoire of circulating immunoglobulin M (IgM) and immunoglobulin G (IgG) in healthy adults is directed against this glycan (<xref rid="bib32" ref-type="bibr">Macher and Galili, 2008</xref>). Production of α-gal-specific Abs is thought to be driven by exposure to bacterial components of the microbiota expressing α-gal (<xref rid="bib32" ref-type="bibr">Macher and Galili, 2008</xref>), including specific members of the <italic>Klebsiella spp.</italic>, <italic>Serratia spp.</italic>, and <italic>Escherichia coli spp.</italic> (<xref rid="bib21" ref-type="bibr">Galili et al., 1988</xref>). Expression of α-gal by these <italic>Enterobacteriaceae</italic> is associated with the bacterial capsule and cell wall glycoproteins, as well as with lipopolysaccharide (LPS) (<xref rid="bib21" ref-type="bibr">Galili et al., 1988</xref>). Gut colonization by the human pathobiont <italic>E. coli</italic> O86:B7 (<xref rid="bib45" ref-type="bibr">Pal et al., 1969</xref>) recapitulates the etiology of anti-α-gal Ab production in mice (<xref rid="bib48" ref-type="bibr">Posekany et al., 2002</xref>) and in primates (<xref rid="bib33" ref-type="bibr">Mañez et al., 2001</xref>), as well as the production of Abs directed against the α-gal-related anti-B blood group glycan in chickens (<xref rid="bib54" ref-type="bibr">Springer et al., 1959</xref>) and humans (<xref rid="bib53" ref-type="bibr">Springer and Horton, 1969</xref>). This argues that gut colonization by <italic>E. coli</italic> O86:B7 may be particularly relevant in triggering the production of α-gal-specific Abs, presumably contributing to the high titers of these circulating Abs in healthy adult humans (<xref rid="bib21" ref-type="bibr">Galili et al., 1988</xref>). Moreover, anti-α-gal Abs may also be produced in response to infection by pathogens expressing α-gal, such illustrated for gram-negative bacteria from <italic>Salmonella spp.</italic> or for protozoan parasites from <italic>Trypanosoma spp.</italic> (<xref rid="bib1" ref-type="bibr">Avila et al., 1989</xref>).</p><p>Anti-α-gal Abs are cytotoxic toward α-gal-expressing pathogens, as demonstrated in vitro for bacteria (<xref rid="bib21" ref-type="bibr">Galili et al., 1988</xref>), protozoan parasites (<xref rid="bib1" ref-type="bibr">Avila et al., 1989</xref>), and viruses enveloped by xenogeneic α-gal-expressing cell membranes (<xref rid="bib56" ref-type="bibr">Takeuchi et al., 1996</xref>). Whether anti-α-gal Abs confer resistance to these and/or other pathogens in vivo has, to the best of our knowledge, not been established. Here, we tested this hypothesis specifically for <italic>Plasmodium spp.</italic> infection, the causative agent of malaria and a major driving force that shaped the evolution of anthropoid primates, including humans.</p><p>Malaria is transmitted to humans by the inoculation of <italic>Plasmodium</italic> sporozoites via the bite of female <italic>Anopheles</italic> (<italic>A.</italic>) mosquitoes (<xref rid="bib34" ref-type="bibr">Ménard et al., 2013</xref>). While transmission may be rather efficient, only a fraction of the inoculated parasites manage to progress toward the establishment of infection (<xref rid="bib49 bib51 bib60" ref-type="bibr">Rickman et al., 1990; Sauerwein et al., 2011; Verhage et al., 2005</xref>), hinting at a natural mechanism of protection that presumably targets the initial phases of the <italic>Plasmodium</italic> life cycle. Here, we demonstrate that production of anti-α-gal Abs in response to the gut <italic>E. coli</italic> O86:B7 pathobiont contributes critically to this natural defense mechanism, reducing malaria transmission by <italic>A.</italic> mosquitoes.</p></sec><sec sec-type="results" id="sec2"><title>Results</title><sec id="sec2.1"><title><italic>Plasmodium spp.</italic> Express the α-Gal Glycan</title><p>The α-gal glycan was detected on the surface of <italic>Plasmodium</italic> sporozoites, as assessed by immunofluorescence for the human pathogen <italic>Plasmodium falciparum</italic> 3D7, as well as for the transgenic GFP-expressing strains of the rodent pathogens <italic>Plasmodium berghei</italic> ANKA (<italic>Pb</italic>A) or <italic>Plasmodium yoelii</italic> 17XNL, using the lectin <italic>Bandeiraea</italic> (<italic>Griffonia</italic>) <italic>simplicifolia</italic>-I isolectin IB<sub>4</sub> (BSI-B<sub>4</sub>) (<xref rid="bib20" ref-type="bibr">Galili et al., 1985</xref>) or an anti-α-gal monoclonal antibody (M86 mAb) (<xref rid="bib18" ref-type="bibr">Galili et al., 1998</xref>) (<xref rid="fig1" ref-type="fig">Figure 1</xref>A; <xref rid="figs1" ref-type="fig">Figures S1</xref>A and S1B available online). Specificity of α-gal detection was confirmed by its enzymatic removal using α-galactosidase (<xref rid="fig1" ref-type="fig">Figures 1</xref>B and 1C). Expression of α-gal was associated with proteins, as assessed by western blot in whole-cell extracts from <italic>P. falciparum</italic> 3D7, <italic>Pb</italic>A, or <italic>P. yoelii</italic> 17XNL sporozoites (<xref rid="fig1" ref-type="fig">Figure 1</xref>D) and confirmed by enzymatic removal of α-gal (<xref rid="fig1" ref-type="fig">Figure 1</xref>D). Residual levels of α-gal were detected in the salivary glands of noninfected mosquitoes, suggesting that this glycan may be generated, at least partially, by <italic>A.</italic> mosquitoes (<xref rid="fig1" ref-type="fig">Figure 1</xref>D).</p><p>Expression of α-gal by <italic>Pb</italic>A sporozoites was reduced by ∼4-fold when the glycosylphosphatidylinositol (GPI) anchor was cleaved by phospholipase C (PLC), as assessed by flow cytometry (<xref rid="fig1" ref-type="fig">Figure 1</xref>E). In contrast, GPI cleavage failed to reduce the expression of circumsporozoite protein (CSP), the main protein expressed at the surface of <italic>Plasmodium</italic> sporozoites (<xref rid="fig1" ref-type="fig">Figure 1</xref>F). This suggests that α-gal is bound to GPI-anchored surface proteins, including or not CSP, which despite being GPI-anchored (<xref rid="bib38" ref-type="bibr">Moran and Caras, 1994</xref>) is resistant to PLC cleavage (<xref rid="bib29" ref-type="bibr">Kimmel et al., 2003</xref>) (<xref rid="fig1" ref-type="fig">Figure 1</xref>F).</p></sec><sec id="sec2.2"><title>α-Gal-Specific IgM Abs Are Associated with Protection from <italic>P. falciparum</italic> Infection in Humans</title><p>We investigated whether a correlation exists between the levels of anti-α-gal Abs in healthy uninfected children and adults before the malaria season (n = 330 for IgG; n = 229 for IgM) and subsequent risk of <italic>P. falciparum</italic> infection (determined by biweekly PCR analysis of fingerprick blood samples) and febrile malaria (determined by weekly physical examination), during the ensuing 6 month malaria season in a cohort study in Mali, where this season is predictable and intense (<xref rid="bib59" ref-type="bibr">Tran et al., 2014</xref>). In children <2 years, the average level of anti-α-gal IgM Abs was 33.4 μg/ml (95% confidence interval [CI]: 18.4–48.3 μg/ml) (<xref rid="fig2" ref-type="fig">Figure 2</xref>A), similar to that reported in children with no history of malaria exposure (<xref rid="bib2 bib12 bib19 bib46" ref-type="bibr">Avila et al., 1992; Doenz et al., 2000; Galili et al., 1984; Parker et al., 1999</xref>). However, anti-α-gal IgM Abs increased with age, reaching an average of 123.03 μg/ml (95% CI: 79.3–166.7 μg/ml) in adults— more than twice the level reported in adults with no malaria exposure, i.e., 51.6 μg/ml (95% CI: 14.9–88.3 μg/ml) (<xref rid="fig2" ref-type="fig">Figure 2</xref>A) (<xref rid="bib2 bib12 bib19 bib46" ref-type="bibr">Avila et al., 1992; Doenz et al., 2000; Galili et al., 1984; Parker et al., 1999</xref>). The average level of anti-α-gal IgM Abs in children >4 years of age who had no <italic>P. falciparum</italic> infections detected during the 6-month malaria season (n = 13) was higher than those who became infected (n = 141) (<xref rid="fig2" ref-type="fig">Figure 2</xref>B). This suggests that there is a positive correlation between the levels of anti-α-gal IgM Abs and incidence of <italic>P. falciparum</italic> infection.</p><p>The average level of anti-α-gal IgG Abs in children <2 years was 1.46 μg/ml (95% CI: 0.22–0.69 μg/ml) and increased in adults to 3.66 μg/ml (95% CI: 3.04–4.28 μg/ml) (<xref rid="fig2" ref-type="fig">Figure 2</xref>C). In contrast to IgM, the levels of circulating α-gal-specific IgG were similar between malaria-exposed and nonexposed adults, suggesting that <italic>P. falciparum</italic> infection fails to drive an IgG response directed against α-gal (<xref rid="fig2" ref-type="fig">Figure 2</xref>D). This also suggests that there is no correlation between anti-α-gal IgG Abs and incidence of <italic>P. falciparum</italic> infection. Time-to-event analysis did not show a correlation between α-gal-specific IgM and IgG levels before the malaria season and subsequent risk of <italic>P. falciparum</italic> infection (p = 0.76 and p = 0.08, respectively) or febrile malaria (p = 0.35 and p = 0.18, respectively).</p></sec><sec id="sec2.3"><title>Gut Colonization by <italic>E. coli</italic> O86:B7 Elicits a Protective α-Gal-Specific IgM Ab Response against Malaria Transmission</title><p>To test whether anti-α-gal IgM Abs are protective against malaria transmission, we took advantage of “human-like” <italic>α1,3Gt</italic>-deficient mice. Unlike humans, wild-type mice have a functional <italic>α1,3Gt</italic> gene and express α-gal on secreted and cell-surface glycoconjugates, suppressing the development of anti-α-gal immunity (<xref rid="bib63" ref-type="bibr">Yang et al., 1998</xref>). Deletion of <italic>α1,3Gt</italic> gene eliminates α-gal (<xref rid="bib58" ref-type="bibr">Tearle et al., 1996</xref>), allowing for anti-α-gal Ab production in <italic>α1,3Gt</italic><sup>−/−</sup> mice (<xref rid="bib8 bib58 bib63" ref-type="bibr">Chiang et al., 2000; Tearle et al., 1996; Yang et al., 1998</xref>). However, <italic>α1,3Gt</italic><sup>−/−</sup> mice are known to produce only residual levels of circulating anti-α-gal Abs when maintained under specific pathogen-free (SPF) conditions (<xref rid="bib8" ref-type="bibr">Chiang et al., 2000</xref>). Production of anti-α-gal Abs can be enhanced upon enteric exposure to <italic>E. coli</italic> O86:B7 (<xref rid="bib48" ref-type="bibr">Posekany et al., 2002</xref>). We confirmed that <italic>E. coli</italic> O86:B7 expresses high levels of α-gal (<xref rid="bib64" ref-type="bibr">Yi et al., 2006</xref>), which is not the case for the <italic>E. coli</italic> K12 strain (<xref rid="fig3" ref-type="fig">Figures 3</xref>A and 3B). Colonization of <italic>α1,3Gt</italic><sup>−/−</sup> mice by <italic>E. coli</italic> O86:B7 after antibiotic treatment (streptomycin sulfate; 5 g/l in drinking water for 7 days prior to colonization) increased the levels of circulating anti-α-gal IgM Abs from 1.4 μg/ml (95% CI: 1.1–1.8 μg/ml) to 162.9 μg/ml (95% CI: 95.89–230.1 μg/ml) before and after colonization, respectively (<xref rid="fig3" ref-type="fig">Figure 3</xref>C). Levels of anti-α-gal IgM Abs in colonized <italic>α1,3Gt</italic><sup>−/−</sup> mice were in the range of adult individuals from a malaria endemic region (<xref rid="fig2" ref-type="fig">Figure 2</xref>A). In contrast, the levels of circulating anti-α-gal IgG Abs remained at residual levels, i.e., <1 μg/ml (<xref rid="figs2" ref-type="fig">Figure S2</xref>A), again in the range of adult individuals from a malaria endemic region (<xref rid="fig2" ref-type="fig">Figure 2</xref>C). Colonization by <italic>E. coli</italic> K12 did not induce the production of circulating anti-α-gal Abs (<xref rid="fig3" ref-type="fig">Figure 3</xref>C). Gut colonization by <italic>E. coli</italic> O86.B7 was associated with protection of <italic>α1,3Gt</italic><sup>−/−</sup> mice from <italic>Pb</italic>A transmission by infected <italic>Anopheles stephensi</italic> mosquitoes (<xref rid="fig3" ref-type="fig">Figure 3</xref>D). This was not the case when <italic>α1,3Gt</italic><sup>−/−</sup> mice were or were not colonized by <italic>E. coli</italic> K12 (<xref rid="fig3" ref-type="fig">Figure 3</xref>D).</p><p>To determine whether the protective effect associated with gut colonization by <italic>E. coli</italic> O86.B7 is mediated by anti-α-gal Abs, we performed similar colonization experiments in <italic>α1,3Gt</italic><sup>−<italic>/</italic>−</sup><italic>J</italic><sub><italic>H</italic></sub><italic>T</italic><sup>−<italic>/</italic>−</sup> lacking B cells (<xref rid="bib22" ref-type="bibr">Gu et al., 1993</xref>), <italic>α1,3Gt</italic><sup>−<italic>/</italic>−</sup><sc><italic>μ</italic></sc><italic>S</italic><sup>−<italic>/</italic>−</sup> mice lacking circulating IgM (<xref rid="bib14" ref-type="bibr">Ehrenstein et al., 1998</xref>) or <italic>α1,3Gt</italic><sup>−<italic>/</italic>−</sup><italic>Aid</italic><sup>−<italic>/</italic>−</sup> mice that fail to undergo Ig class switch recombination or somatic hypermutation (<xref rid="bib40" ref-type="bibr">Muramatsu et al., 2000</xref>). Gut colonization by <italic>E. coli</italic> O86.B7 failed to protect <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup><italic>J</italic><sub><italic>H</italic></sub><italic>T</italic><sup><italic>−/−</italic></sup> and <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup><sc><italic>μ</italic></sc><italic>S</italic><sup><italic>−/−</italic></sup>, but not <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup><italic>Aid</italic><sup><italic>−/−</italic></sup> mice from <italic>Pb</italic>A-infected mosquitoes, as compared to genetic-matched control mice colonized or not by <italic>E. coli</italic> K12 (<xref rid="fig3" ref-type="fig">Figure 3</xref>E). This shows that the protective effect of gut colonization by <italic>E. coli</italic> O86.B7 acts via a mechanism mediated by anti-α-gal IgM Abs that do not undergo somatic hypermutation.</p><p>Germ-free (GF) <italic>α1,3Gt</italic><sup><italic>−</italic>/<italic>−</italic></sup> mice had low but detectable levels of anti-α-gal IgM Abs, i.e., 0.87 μg/ml (95% CI: 0.66–1.1 μg/ml), suggesting that these are natural Abs (<xref rid="fig3" ref-type="fig">Figure 3</xref>F). The production of anti-α-gal IgM Abs in GF <italic>α1,3Gt</italic><sup><italic>−</italic>/<italic>−</italic></sup> mice being driven by expression of these glycans in food components is possible, but this has not been tested. GF <italic>α1,3Gt</italic><sup><italic>−</italic>/<italic>−</italic></sup> mice did not produce anti-α-gal IgG Abs (<xref rid="figs2" ref-type="fig">Figure S2</xref>C). Susceptibility to <italic>Pb</italic>A transmission by infected <italic>A.</italic> mosquitoes was similar in SPF versus GF <italic>α1,3Gt</italic><sup><italic>−</italic>/<italic>−</italic></sup> mice (<xref rid="fig3" ref-type="fig">Figures 3</xref>D and 3G). When GF <italic>α1,3Gt</italic><sup><italic>−</italic>/<italic>−</italic></sup> mice were monocolonized by <italic>E. coli</italic> O86:B7, the levels of circulating anti-α-gal IgM Abs increased to 96.62 μg/ml (95% CI: 59.32–133.9 μg/ml) (<xref rid="fig3" ref-type="fig">Figure 3</xref>F), which is the range in which adult individuals from a malaria endemic region (<xref rid="fig2" ref-type="fig">Figure 2</xref>A), without concomitant induction of anti-α-gal IgG Abs (<xref rid="figs2" ref-type="fig">Figure S2</xref>C). Monocolonization by <italic>E. coli</italic> O86:B7, but not by <italic>E. coli</italic> K12, protected <italic>α1,3Gt</italic><sup><italic>−</italic>/<italic>−</italic></sup> mice from <italic>Pb</italic>A transmission by <italic>A.</italic> mosquitoes (<xref rid="fig3" ref-type="fig">Figure 3</xref>F). This suggests that gut colonization by a specific pathobiont expressing α-gal recapitulates to a large extent the normal etiology of the human anti-α-gal Ab response (<xref rid="fig2" ref-type="fig">Figure 2</xref>) and induces protection against <italic>Plasmodium</italic> infection, such as observed in a malaria endemic region (<xref rid="fig2" ref-type="fig">Figure 2</xref>).</p><p>It should be noted that the percentage of infected red blood cell (RBC), i.e., parasitemia, and incidence of mortality were similar among those <italic>α1,3Gt</italic><sup><italic>−</italic>/<italic>−</italic></sup> mice that were infected by <italic>Pb</italic>A regardless of colonization (<xref rid="figs2" ref-type="fig">Figures S2</xref>B and S2D). This suggests that gut colonization by <italic>E. coli</italic> O86:B7 protects against <italic>Plasmodium</italic> transmission, but not against disease once the erythrocytic stage of infection is established.</p></sec><sec id="sec2.4"><title>Immunization against α-Gal Protects from <italic>Plasmodium</italic> Transmission</title><p>Immunization of <italic>α1,3Gt</italic><sup><italic>−</italic>/<italic>−</italic></sup> mice against α-gal, using rabbit RBC membranes (rRBCM) expressing high levels of α-gal or synthetic α-gal conjugated to BSA (α-gal-BSA) elicited the production of circulating anti-α-gal IgM and IgG Abs (<xref rid="fig4" ref-type="fig">Figure 4</xref>A). Control <italic>α1,3Gt</italic><sup>+/+</sup> mice failed to produce anti-α-gal Abs (<xref rid="bib8 bib58 bib63" ref-type="bibr">Chiang et al., 2000; Tearle et al., 1996; Yang et al., 1998</xref>) (<xref rid="figs3" ref-type="fig">Figure S3</xref>A). Circulating anti-α-gal immunoglobulin A (IgA) and immunoglobulin E (IgE) Abs were undetectable in control or immunized <italic>α1,3Gt</italic><sup><italic>−</italic>/<italic>−</italic></sup> and <italic>α1,3Gt</italic><sup>+/+</sup> mice (data not shown). The concentration of anti-α-gal IgM Abs in the plasma of immunized <italic>α1,3Gt</italic><sup><italic>−</italic>/<italic>−</italic></sup> mice was in the range of adult individuals from malaria endemic regions (<xref rid="fig2" ref-type="fig">Figure 2</xref>A). Circulating anti-α-gal IgG Abs in immunized <italic>α1,3Gt</italic><sup><italic>−</italic>/<italic>−</italic></sup> mice, predominantly from IgG1, IgG2b, and IgG3 subclasses, were present at higher concentrations, as compared to total IgG in adult individuals from malaria endemic regions. Little or no circulating IgG2a (<xref rid="fig4" ref-type="fig">Figure 4</xref>A) or IgG2c (data not shown) were detected in immunized <italic>α1,3Gt</italic><sup><italic>−</italic>/<italic>−</italic></sup> mice.</p><p>Immunization against α-gal protected <italic>α1,3Gt</italic><sup>−/−</sup> mice from <italic>Pb</italic>A (<xref rid="fig4" ref-type="fig">Figure 4</xref>B) and <italic>P. yoelli</italic> 17XNL (<xref rid="fig4" ref-type="fig">Figure 4</xref>C) transmission by infected <italic>A. stephensi</italic> mosquitoes, as well as from <italic>Pb</italic>A transmission by <italic>A. gambiae</italic> mosquitoes (<xref rid="fig4" ref-type="fig">Figure 4</xref>D) versus control nonimmunized <italic>α1,3Gt</italic><sup>−/−</sup> mice. Control immunized <italic>α1,3Gt</italic><sup>+/+</sup> mice were neither protected from <italic>Pb</italic>A (<xref rid="figs3" ref-type="fig">Figure S3</xref>B) nor <italic>P. yoelli</italic> 17XNL (<xref rid="figs3" ref-type="fig">Figure S3</xref>C) transmission by <italic>A. stephensi</italic> mosquitoes nor against <italic>Pb</italic>A transmission by <italic>A. gambiae</italic> mosquitoes (<xref rid="figs3" ref-type="fig">Figure S3</xref>D) versus naive <italic>α1,3Gt</italic><sup>+/+</sup> mice.</p><p>Immunized <italic>α1,3Gt</italic><sup>−/−</sup> mice were protected from artificial transmission of <italic>Pb</italic>A sporozoites via intradermal inoculation versus control nonimmunized <italic>α1,3Gt</italic><sup>−/−</sup> mice (<xref rid="fig4" ref-type="fig">Figure 4</xref>E) or control immunized or nonimmunized <italic>α1,3Gt</italic><sup>+/+</sup> mice (<xref rid="figs3" ref-type="fig">Figure S3</xref>E). Protection was no longer observed when sporozoites were inoculated intravenously (<xref rid="fig4" ref-type="fig">Figures 4</xref>E and <xref rid="figs3" ref-type="fig">S3</xref>E). This suggests that the protective effect of α-gal immunization is exerted in the dermis, presumably via an anti-α-gal Ab driven mechanism that is no longer effective once sporozoites reach the blood.</p><p><italic>Pb</italic>A transmission was associated with accumulation of <italic>Plasmodium</italic> 18S rRNA at the site of inoculation, as quantified in the ear pinna by qRT-PCR (<xref rid="fig4" ref-type="fig">Figure 4</xref>F). The relative amount of <italic>Plasmodium</italic> 18S rRNA was similar in immunized versus control nonimmunized <italic>α1,3Gt</italic><sup>−<italic>/</italic>−</sup> mice (<xref rid="fig4" ref-type="fig">Figure 4</xref>F) or control immunized or nonimmunized <italic>α1,3Gt</italic><sup>+/+</sup> mice (<xref rid="figs3" ref-type="fig">Figure S3</xref>F). Immunized <italic>α1,3Gt</italic><sup>−/−</sup> mice did not accumulate <italic>Plasmodium</italic> 18S rRNA in the liver, when compared to control nonimmunized <italic>α1,3Gt</italic><sup>−/−</sup> mice (<xref rid="fig4" ref-type="fig">Figure 4</xref>F) or control nonimmunized or immunized <italic>α1,3Gt</italic><sup>+/+</sup> mice (<xref rid="figs3" ref-type="fig">Figure S3</xref>F). This suggests that α-gal immunization arrests the transit of inoculated sporozoites from the skin into the liver, without interfering with sporozoite inoculation by <italic>A.</italic> mosquitoes.</p></sec><sec id="sec2.5"><title>TLR9 Agonist Adjuvant Enhances the Protective Effect of α-Gal Immunization</title><p>Immunization of <italic>α1,3Gt</italic><sup>−/−</sup> mice with rRBCM emulsified in complete Freund’s adjuvant (CFA), supplemented with toll-like receptor 9 agonist CpG, enhanced anti-α-gal IgG2b and IgG3 Ab response by 2- to 3-fold (<xref rid="fig4" ref-type="fig">Figure 4</xref>G) versus immunization without adjuvant (<xref rid="fig4" ref-type="fig">Figure 4</xref>A). This was associated with 88% reduction in the relative risk of transmission of <italic>Pb</italic>A infection by <italic>A.</italic> mosquitoes (95% CI: 0.032–0.452) versus 61% reduction upon immunization without adjuvant (95% CI: 0.209–0.726) (<xref rid="fig4" ref-type="fig">Figures 4</xref>B and 4H). This protective effect was not observed in control <italic>α1,3Gt</italic><sup>+/+</sup> mice (<xref rid="figs3" ref-type="fig">Figures S3</xref>G and S3H).</p><p>Parasitemias were similar in immunized <italic>α1,3Gt</italic><sup>−/−</sup> mice not protected from <italic>Pb</italic>A infection versus control nonimmunized <italic>α1,3Gt</italic><sup>−/−</sup> mice as well as control nonimmunized or immunized <italic>α1,3Gt</italic><sup>+/+</sup> mice (data not shown). Moreover, when infected, all mice succumbed to experimental cerebral malaria. This suggests that while protective against malaria transmission, α-gal immunization is not protective against the development of severe disease if <italic>Plasmodium</italic> manages to establish infection. In keeping with this notion, when inoculated with <italic>Pb</italic>A-infected RBC, immunized <italic>α1,3Gt</italic><sup>−/−</sup> mice developed similar levels of parasitemia and disease severity, as compared to control nonimmunized <italic>α1,3Gt</italic><sup>−/−</sup> mice as well as to control nonimmunized or immunized <italic>α1,3Gt</italic><sup>+/+</sup> mice (<xref rid="figs4" ref-type="fig">Figure S4</xref>A).</p><p>We tested further whether the protective effect conferred by α-gal immunization is associated with sterile protection, i.e., inability of <italic>Plasmodium</italic> to establish blood stage of infection. Passive transfer of RBCs harvested from protected immunized <italic>α1,3Gt</italic><sup>−/−</sup> mice at day 8–9 post-<italic>Pb</italic>A transmission by <italic>A.</italic> mosquitoes failed to transmit disease to naive <italic>α1,3Gt</italic><sup>−/−</sup> mice (<xref rid="figs4" ref-type="fig">Figure S4</xref>B). In contrast, passive transfer of RBC harvested from nonprotected immunized <italic>α1,3Gt</italic><sup>−/−</sup> mice, readily transmitted disease to naive <italic>α1,3Gt</italic><sup>−/−</sup> mice (<xref rid="figs4" ref-type="fig">Figure S4</xref>B). This demonstrates that the protective effect of immunization against α-gal is associated with sterile protection against malaria.</p></sec><sec id="sec2.6"><title>Anti-α-Gal IgM and IgG Abs Produced in Response to α-Gal Immunization Confer Protection against Malaria Transmission</title><p>We asked whether the protective effect of α-gal immunization is mediated by anti-α-gal IgM and/or IgG Abs. Immunized <italic>α1,3Gt</italic><sup>−/−</sup><italic>J</italic><sub><italic>H</italic></sub><italic>T</italic><sup>−/−</sup> mice failed to produce anti-α-gal IgM or IgG Abs versus naive <italic>α1,3Gt</italic><sup>−/−</sup><italic>J</italic><sub><italic>H</italic></sub><italic>T</italic><sup>−/−</sup> mice or immunized <italic>α1,3Gt</italic><sup>−/−</sup> mice (<xref rid="fig5" ref-type="fig">Figure 5</xref>A). Moreover, immunized <italic>α1,3Gt</italic><sup>−/−</sup><italic>J</italic><sub><italic>H</italic></sub><italic>T</italic><sup>−/−</sup> mice were not protected against <italic>Pb</italic>A transmission by <italic>A.</italic> mosquitoes versus control nonimmunized <italic>α1,3Gt</italic><sup>−/−</sup><italic>J</italic><sub><italic>H</italic></sub><italic>T</italic><sup>−/−</sup> mice (<xref rid="fig5" ref-type="fig">Figure 5</xref>B). This shows that the protective effect of α-gal immunization is mediated via a B cell-dependent mechanism.</p><p>Immunization of <italic>α1,3Gt</italic><sup>−/−</sup><italic>Aid</italic><sup><italic>−</italic>/−</sup> mice failed to induce the production of anti-α-gal IgG, but not IgM Abs, versus naive <italic>α1,3Gt</italic><sup>−/−</sup><italic>Aid</italic><sup><italic>−</italic>/−</sup> or immunized <italic>α1,3Gt</italic><sup>−/−</sup> mice (<xref rid="fig5" ref-type="fig">Figure 5</xref>A). Immunized <italic>α1,3Gt</italic><sup>−/−</sup><italic>Aid</italic><sup><italic>−</italic>/−</sup> mice were nevertheless protected against <italic>Pb</italic>A transmission by <italic>A.</italic> mosquitoes versus nonimmunized <italic>α1,3Gt</italic><sup>−/−</sup><italic>Aid</italic><sup><italic>−</italic>/−</sup> mice (<xref rid="fig5" ref-type="fig">Figure 5</xref>B). This confirms that anti-α-gal IgM Abs can confer protection against malaria transmission (<xref rid="fig2" ref-type="fig">Figure 2</xref>B) and that the protective effect of α-gal-specific IgM Abs does not require somatic hypermutation.</p><p>Immunization of <italic>α1,3Gt</italic><sup>−/−</sup><sc><italic>μ</italic></sc><italic>S</italic><sup><italic>−</italic>/−</sup> mice failed to induce anti-α-gal IgM Abs, without interfering with anti-α-gal IgG Ab response versus naive <italic>α1,3Gt</italic><sup>−/−</sup><sc><italic>μ</italic></sc><italic>S</italic><sup>−/−</sup> mice or immunized <italic>α1,3Gt</italic><sup>−/−</sup> mice (<xref rid="fig5" ref-type="fig">Figure 5</xref>A). Immunized <italic>α1,3Gt</italic><sup>−/−</sup><sc><italic>μ</italic></sc><italic>S</italic><sup><italic>-</italic>/-</sup> mice were nevertheless protected from <italic>Pb</italic>A transmission by <italic>A.</italic> mosquitoes versus control naive <italic>α1,3Gt</italic><sup>−/−</sup><sc><italic>μ</italic></sc><italic>S</italic><sup><italic>−</italic>/−</sup> mice (<xref rid="fig5" ref-type="fig">Figure 5</xref>B). Immunized <italic>α1,3Gt</italic><sup>−/−</sup> mice did not produce circulating anti-α-gal IgA or IgE Abs (data not shown) and a putative protective effect for these Ig isotypes was excluded. This demonstrates that anti-α-gal IgG Abs produced in response to immunization confer protection against malaria transmission.</p><p>Immunization of <italic>α1,3Gt</italic><sup>−/−</sup><italic>Tcrβ</italic><sup><italic>−</italic>/−</sup> mice lacking mature αβ T cells (<xref rid="bib37" ref-type="bibr">Mombaerts et al., 1992</xref>) compromised anti-α-gal IgM and IgG response versus control immunized <italic>α1,3Gt</italic><sup>−/−</sup> mice (<xref rid="fig5" ref-type="fig">Figure 5</xref>A). Immunized <italic>α1,3Gt</italic><sup>−/−</sup><italic>Tcrβ</italic><sup><italic>−/</italic>−</sup> mice were not protected from <italic>Pb</italic>A transmission by <italic>A.</italic> mosquitoes versus control naive <italic>α1,3Gt</italic><sup>−/−</sup><italic>Tcrβ</italic><sup><italic>−</italic>/−</sup> mice (<xref rid="fig5" ref-type="fig">Figure 5</xref>B). This shows that anti-α-gal Abs produced in response to immunization are T cell-dependent (<xref rid="bib9" ref-type="bibr">Cretin et al., 2002</xref>) and so is their protective effect.</p><p>Naive and immunized <italic>α1,3Gt</italic><sup>−/−</sup><italic>J</italic><sub><italic>H</italic></sub><italic>T</italic><sup>−/−</sup> (<xref rid="figs5" ref-type="fig">Figure S5</xref>A), <italic>α1,3Gt</italic><sup>−/−</sup><italic>Aid</italic><sup><italic>−</italic>/−</sup>, (<xref rid="figs5" ref-type="fig">Figure S5</xref>B) and <italic>α1,3Gt</italic><sup>−/−</sup><sc><italic>μ</italic></sc><italic>S</italic><sup><italic>−</italic>/−</sup> (<xref rid="figs5" ref-type="fig">Figure S5</xref>C) mice, not protected from <italic>Pb</italic>A transmission, developed similar levels of parasitemia and succumbed to experimental cerebral malaria. This was not the case for <italic>α1,3Gt</italic><sup>−/−</sup><italic>Tcrβ</italic><sup><italic>−</italic>/−</sup> mice (<xref rid="figs5" ref-type="fig">Figure S5</xref>D), consistent with the involvement of T cells in the pathogenesis of experimental cerebral malaria (<xref rid="bib4" ref-type="bibr">Belnoue et al., 2002</xref>).</p><p>Passive transfer of anti-α-gal IgM to naive <italic>α1,3Gt</italic><sup>−/−</sup> mice conferred protection against <italic>Pb</italic>A transmission by <italic>A.</italic> mosquitoes (<xref rid="fig5" ref-type="fig">Figure 5</xref>C). This was also the case for passive transfer of anti-α-gal Abs from specific IgG subclasses, namely, IgG2b and IgG3 (<xref rid="fig5" ref-type="fig">Figure 5</xref>C), but not IgG1 or IgG2a (<xref rid="fig5" ref-type="fig">Figure 5</xref>C). Relative binding to α-gal was similar for all mAbs tested, as assessed by ELISA using α-gal-BSA as a solid-phase antigen (<xref rid="figs6" ref-type="fig">Figure S6</xref>A) or by immunofluorescence using <italic>Pb</italic>A sporozoites (<xref rid="figs6" ref-type="fig">Figure S6</xref>B). Specificity of anti-α-gal binding to <italic>Plasmodium</italic> sporozoites was assessed by enzymatic removal of α-gal, confirming that these mAbs recognize specifically and only the α-gal glycan on the surface of <italic>Plasmodium</italic> sporozoites (<xref rid="figs6" ref-type="fig">Figure S6</xref>C). IgG2a, IgG2b, and IgG3 mAbs are class-switched mutants derived from the original anti-α-gal IgG1 clone and as such have similar affinities for α-gal (<xref rid="bib11" ref-type="bibr">Ding et al., 2008</xref>). These data reveal that while IgM anti-α-gal Abs are sufficient per se to confer protection against malaria transmission, this protective effect can be enhanced when specific subclasses anti-α-gal IgG Abs are present at sufficient high levels.</p><p>Once bound to the surface of <italic>Plasmodium</italic> sporozoites, anti-α-gal IgM, IgG2b, and IgG3 mAbs activated the classical pathway of complement, as assessed by C3 deposition (<xref rid="fig5" ref-type="fig">Figure 5</xref>D). Anti-α-gal IgG1 or IgG2a mAbs failed to activate complement (data not shown), and complement activation was also not observed in the absence of anti-α-gal Abs (<xref rid="fig5" ref-type="fig">Figures 5</xref>D and <xref rid="figs7" ref-type="fig">S7</xref>), showing that the alternative and lectin pathways of complement are not activated by <italic>Plasmodium</italic> sporozoites.</p><p>We then asked whether the protective effect exerted by anti-α-gal Abs is mediated via a mechanism involving the activation of the complement cascade (<xref rid="fig5" ref-type="fig">Figure 5</xref>D) (<xref rid="bib11 bib35" ref-type="bibr">Ding et al., 2008; Miyatake et al., 1998</xref>). Passive transfer of anti-α-gal IgM Abs or anti-α-gal IgG2b mAb to <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup><italic>C3</italic><sup><italic>−/−</italic></sup> mice, which lack C3 and cannot activate the complement cascade, failed to confer protection against <italic>Pb</italic>A transmission versus control <italic>α1,3G</italic><sup><italic>−/−</italic></sup><italic>C3</italic><sup><italic>−/−</italic></sup> mice (<xref rid="fig5" ref-type="fig">Figure 5</xref>E). Passive transfer of anti-α-gal IgG3 mAb to <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup><italic>C3</italic><sup><italic>−/−</italic></sup> mice conferred residual but significant protection versus control <italic>α1,3G</italic><sup><italic>−/−</italic></sup><italic>C3</italic><sup><italic>−/−</italic></sup> mice (<xref rid="fig5" ref-type="fig">Figure 5</xref>E). This suggests that the protective effect exerted by anti-α-gal IgM and IgG2b Abs acts via a mechanism that is strictly complement dependent, whereas the protective effect of anti-α-gal IgG3 Abs is partially but probably not strictly dependent on complement activation. Infection incidence was similar in control <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup><italic>C3</italic><sup><italic>−/−</italic></sup> versus <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup><italic>C3</italic><sup><italic>+/+</italic></sup> mice (<xref rid="fig5" ref-type="fig">Figures 5</xref>C and 5E).</p><p>Complement activation generates C3a and C5a chemoattractants that promote IgG-dependent polymorphonuclear (PMN) cell cytotoxicity (<xref rid="bib11 bib42 bib65" ref-type="bibr">Ding et al., 2008; Nimmerjahn and Ravetch, 2008; Yin et al., 2004</xref>). Therefore, we asked whether the protective effect of anti-α-gal Abs involves PMN cells. Passive transfer of anti-α-gal IgG2b Abs to <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup> mice, depleted from PMN cells by the administration of anti-Ly-6G (Gr-1) (<xref rid="bib47" ref-type="bibr">Porcherie et al., 2011</xref>), failed to confer protection against <italic>Pb</italic>A transmission by <italic>A.</italic> mosquitoes, whereas passive transfer of anti-α-gal IgM or IgG3 Abs conferred protection (<xref rid="fig5" ref-type="fig">Figure 5</xref>F). This suggests that the protective effect exerted by anti-α-gal IgM and IgG2b Abs acts via a mechanism strictly dependent on PMN cells, whereas the protective effect of anti-α-gal IgG3 Abs is partially but probably not strictly dependent on PMN cells. Depletion of PMN cells per se did not interfere with <italic>Plasmodium</italic> infection (<xref rid="fig5" ref-type="fig">Figure 5</xref>F) while preventing the onset of cerebral malaria (data not shown), consistent with previous findings (<xref rid="bib7" ref-type="bibr">Chen et al., 2000</xref>).</p></sec><sec id="sec2.7"><title>Anti-α-Gal Abs Are Cytotoxic to <italic>Plasmodium</italic> Sporozoites</title><p>Complement activation by anti-α-gal IgM, IgG2b, or IgG3 mAb was cytotoxic to <italic>Pb</italic>A sporozoites in vitro, as assessed by sporozoite GFP expression (<xref rid="fig6" ref-type="fig">Figure 6</xref>A). Anti-α-gal IgG1 and IgG2a mAbs, which did not activate complement when bound to <italic>Plasmodium</italic> sporozoites (data not shown), were not cytotoxic (<xref rid="fig6" ref-type="fig">Figure 6</xref>A). The cytotoxic effect of anti-α-gal IgM, IgG2b, and IgG3 was similar when using mouse (<xref rid="fig6" ref-type="fig">Figure 6</xref>A) or rabbit (<xref rid="figs7" ref-type="fig">Figure S7</xref>A) complement but was strictly dependent on the presence of complement (<xref rid="figs7" ref-type="fig">Figure S7</xref>B). A similar cytotoxic effect was observed when quantifying viable “crescent-shaped” sporozoites (<xref rid="figs7" ref-type="fig">Figure S7</xref>C), an independent readout for sporozoite viability (<xref rid="bib24" ref-type="bibr">Hegge et al., 2010</xref>). Isotype-matched control anti-dinitrophenyl (DNP) Abs were not cytotoxic to <italic>Pb</italic>A sporozoites in vitro (<xref rid="fig6" ref-type="fig">Figures 6</xref>A and <xref rid="figs7" ref-type="fig">S7</xref>A–S7C).</p></sec><sec id="sec2.8"><title>Anti-α-Gal Abs Inhibit Hepatocyte Invasion by <italic>Plasmodium</italic> Sporozoites</title><p>We asked whether anti-α-gal Abs inhibit hepatocyte transmigration (wounding) and/or hepatocyte invasion by <italic>Plasmodium</italic> sporozoites (<xref rid="bib39" ref-type="bibr">Mota et al., 2001</xref>). Complement activation by anti-α-gal IgM, IgG2b, and IgG3 Abs inhibited hepatocyte transmigration (<xref rid="fig6" ref-type="fig">Figure 6</xref>B) and invasion (<xref rid="fig6" ref-type="fig">Figure 6</xref>C), as assessed in vitro for <italic>Pb</italic>A sporozoites. This inhibitory effect was not observed when using anti-α-gal IgG1 or IgG2a Abs or isotype/subclass-matched control anti-DNP Abs (<xref rid="fig6" ref-type="fig">Figures 6</xref>B and 6C).</p><p>We then assessed whether anti-α-gal Abs inhibit the development of exoerythrocytic forms (EEF) of <italic>Plasmodium</italic>. Complement activation by anti-α-gal IgM, IgG2b, and IgG3 Abs reduced the number of EEF (<xref rid="fig7" ref-type="fig">Figure 7</xref>A), as well as the average EEF size (<xref rid="fig7" ref-type="fig">Figures 7</xref>B and 7C) formed in vitro by <italic>Pb</italic>A sporozoites. Anti-α-gal IgG1 Abs did not show this inhibitory effect, while anti-α-gal IgG2a Abs did not reduce the number of EEF (<xref rid="fig7" ref-type="fig">Figure 7</xref>A) but had a residual inhibitory effect on EEF size (<xref rid="fig7" ref-type="fig">Figures 7</xref>B and 7C). Isotype/subclass-matched control anti-DNP Abs did not modulate EEF numbers (<xref rid="fig7" ref-type="fig">Figure 7</xref>A) or average size (<xref rid="fig7" ref-type="fig">Figures 7</xref>B and 7C).</p></sec></sec><sec sec-type="discussion" id="sec3"><title>Discussion</title><p>When inoculated in humans through the bite of an <italic>A.</italic> mosquito, <italic>Plasmodium</italic> sporozoites are confronted with relatively high levels of cytotoxic anti-α-gal IgM Abs (<xref rid="fig2" ref-type="fig">Figure 2</xref>A). That these Abs are protective against malaria transmission is supported by three independent lines of evidence. First, individuals from a malaria endemic region that show evidence of decreased <italic>P. falciparum</italic> infection risk have higher levels of circulating α-gal-specific IgM Abs, as compared to individuals who are susceptible to <italic>P. falciparum</italic> infection (<xref rid="fig2" ref-type="fig">Figure 2</xref>B). Second, when present at levels similar to those observed in individuals from a malaria endemic region—in <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup> mice colonized by human gut pathobiont <italic>E. coli</italic> O86:B7 expressing α-gal (<xref rid="fig3" ref-type="fig">Figure 3</xref>) or in immunized <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup> mice (<xref rid="fig4" ref-type="fig">Figures 4</xref>A and 4B)—anti-α-gal IgM Abs confer protection against malaria transmission. Third, passive transfer of anti-α-gal IgM Abs is sufficient per se to protect <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup> mice from malaria transmission (<xref rid="fig5" ref-type="fig">Figure 5</xref>C).</p><p>The protective effect exerted by anti-α-gal IgM Abs should be relevant to understand why malaria incidence is higher in children versus adults from malaria endemic regions (<xref rid="bib36" ref-type="bibr">Modiano et al., 1996</xref>). Relative absence of these antibodies in children under the age of 2–3 years should favor malaria transmission, as compared to adults that have higher levels of circulating anti-α-gal IgM Abs (<xref rid="fig2" ref-type="fig">Figure 2</xref>A). This relative absence of anti-α-gal IgM in children may be explained by the (1) kinetics of establishment of an adult-like gut microbiota (<xref rid="bib50" ref-type="bibr">Ringel-Kulka et al., 2013</xref>), (2) requirement of environmental and dietary exposure in the establishment of an adult-like gut microbiota, and/or (3) the kinetics of the establishment of adult-like B cell repertoire, including anti-α-gal B cells.</p><p>The protective effect exerted by anti-α-gal IgM Abs might also contribute to explain why only a small fraction of <italic>Plasmodium</italic> sporozoites inoculated by mosquitoes manage to progress toward the establishment of infection in humans. This is true even when <italic>Plasmodium</italic> sporozoites are inoculated under controlled experimental conditions in adults (<xref rid="bib49 bib51 bib60" ref-type="bibr">Rickman et al., 1990; Sauerwein et al., 2011; Verhage et al., 2005</xref>). Presumably, when present at sufficient high levels in adults, circulating anti-α-gal IgM Abs prevent the large majority of <italic>Plasmodium</italic> sporozoites from establishing a successful infection. However, infection is established if as few as a couple of <italic>Plasmodium</italic> sporozoites manage to escape this natural mechanism of protection.</p><p>Whether α-gal detected at the surface of <italic>Plasmodium</italic> sporozoites (<xref rid="fig1" ref-type="fig">Figure 1</xref>) is produced by <italic>Plasmodium</italic> and/or by the mosquito is not clear. The salivary glands of noninfected mosquitoes express low levels of α-gal, as detected by western blot (<xref rid="fig1" ref-type="fig">Figure 1</xref>) and immunostaining (data not shown). <italic>Plasmodium</italic> sporozoites are masked by mosquito laminin (<xref rid="bib61" ref-type="bibr">Warburg et al., 2007</xref>), an evolutionary conserved glycoprotein that in other species contains α-gal (<xref rid="bib55" ref-type="bibr">Takahashi et al., 2014</xref>). It is possible therefore that anti-α-gal Abs recognize laminin or another mosquito-derived protein expressing α-gal, masking <italic>Plasmodium</italic> sporozoites (<xref rid="bib61" ref-type="bibr">Warburg et al., 2007</xref>).</p><p>It is now well established that specific components of the gut microbiota can modulate immunity and resistance to infection (<xref rid="bib3 bib25" ref-type="bibr">Belkaid and Hand, 2014; Honda and Littman, 2012</xref>). In support of this notion, resistance to viral and bacterial (<xref rid="bib15" ref-type="bibr">Fagundes et al., 2012</xref>) infections is impaired in GF mice (<xref rid="bib13" ref-type="bibr">Dolowy and Muldoon, 1964</xref>) or mice subjected to antibiotic-driven dysbiosis (<xref rid="bib27" ref-type="bibr">Ichinohe et al., 2011</xref>). We reasoned that xeno-glycans expressed by specific components of the gut microbiota might trigger a protective immune response against pathogens expressing the same xeno-glycans. We show that this is the case for α-gal, a xeno-glycan expressed by the human gut pathobiont <italic>E. coli</italic> O86:B7, as well by <italic>Plasmodium spp.</italic> (<xref rid="fig1" ref-type="fig">Figures 1</xref>, <xref rid="fig3" ref-type="fig">3</xref>A, and 3B). When colonized by <italic>E. coli</italic> O86:B7, <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup> mice produce an anti-α-gal IgM Ab response (<xref rid="fig3" ref-type="fig">Figures 3</xref>C and 3F) that confer protection against <italic>Plasmodium</italic> infection (<xref rid="fig3" ref-type="fig">Figures 3</xref>D, 3E, 3G, and <xref rid="fig5" ref-type="fig">5</xref>C) via a lytic mechanism mediated by complement activation (<xref rid="fig5" ref-type="fig">Figures 5</xref>E and <xref rid="fig6" ref-type="fig">6</xref>A). It is worth noticing that in a similar manner to other microbiota-driven resistance mechanisms, the protective effect exerted by <italic>E. coli</italic> O86:B7 acts at the level of a tissue barrier, i.e., the skin, to prevent <italic>Plasmodium</italic> transmission (<xref rid="fig4" ref-type="fig">Figures 4</xref>E and 4F).</p><p>Levels of circulating anti-α-gal IgG Abs in individuals from a malaria endemic region (<xref rid="fig2" ref-type="fig">Figure 2</xref>C), as well as in <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup> mice colonized with <italic>E. coli</italic> O86:B7 (<xref rid="figs2" ref-type="fig">Figures S2</xref>A and S2C), are ∼30-fold and ∼40 to 70-fold, respectively, lower than levels of IgM anti-α-gal Abs. This may explain why basal levels anti-α-gal IgG Abs in individuals from a malaria endemic region are not associated with decreased risk of <italic>P</italic>. <italic>falciparum</italic> infection (<xref rid="fig2" ref-type="fig">Figure 2</xref>D). This also suggests that <italic>P. falciparum</italic> infection fails to induce class switch of the anti-α-gal Ig Ab response in those individuals. It is possible therefore that <italic>P. falciparum</italic> represses Ig class-switch recombination, explaining the residual levels of circulating anti-α-gal IgG Abs (<xref rid="fig2" ref-type="fig">Figure 2</xref>C).</p><p>While anti-α-gal Abs can provide sterile protection against malaria in mice (<xref rid="fig5" ref-type="fig">Figures 5</xref>B, 5C, and <xref rid="figs4" ref-type="fig">S4</xref>B), this is not commonly observed in malaria endemic regions in which adult individuals have circulating anti-α-gal IgM Abs, possibly because the levels of these Abs are below a threshold level required to provide sterile protection (<xref rid="fig2" ref-type="fig">Figure 2</xref>). However, we show that this natural mechanism of protection can be enhanced via immunization using adjuvants that favor the production of T cell-dependent complement fixing anti-α-gal IgG Abs (<xref rid="fig4" ref-type="fig">Figures 4</xref>G and 4H). Moreover, when coupled to <italic>Plasmodium</italic> antigens, this approach should enhance the immunogenicity of such antigens (<xref rid="bib5" ref-type="bibr">Benatuil et al., 2005</xref>) and boost the protective efficacy of candidate malaria vaccines based on such antigens (<xref rid="bib43" ref-type="bibr">Olotu et al., 2013</xref>). This approach should be useful in preventing not only individual infections but also disease transmission given the protective effect of anti-α-gal Abs.</p><p>It is possible that the protective effect of “attenuated” sporozoite vaccine trials against malaria (<xref rid="bib52" ref-type="bibr">Seder et al., 2013</xref>) is driven to some extent by an anti-α-gal Ab response, given the expression of α-gal by <italic>Plasmodium falciparum</italic> sporozoites (<xref rid="fig1" ref-type="fig">Figure 1</xref>). Whether a correlation can be established between the effectiveness of such candidate vaccines and a putative anti-α-gal IgG Ab response has not been established but may be useful to consider as a retrospective analyzes.</p><p>As a final note, we predict that in a similar manner to anti-α-gal Abs other anti-glycan Abs may confer protection against malaria as well as other vector-borne protozoan parasites (<xref rid="bib26 bib30 bib41" ref-type="bibr">Huflejt et al., 2009; Lacroix-Desmazes et al., 1995; Nagele et al., 2013</xref>). Moreover, anti-α-gal Abs may also target other vector-borne protozoan parasites expressing α-gal, such as <italic>Leishmania spp.</italic> and <italic>Trypanosoma spp.</italic>, the causative agents of Leishmaniasis and Trypanosomiasis, respectively (<xref rid="bib1" ref-type="bibr">Avila et al., 1989</xref>). As such, vaccination approaches similar to the one proposed here for malaria may be considered against these diseases as well.</p></sec><sec sec-type="methods" id="sec4"><title>Experimental Procedures</title><sec id="sec4.1"><title>Cohort Study</title><p>For detailed analysis, see the <xref rid="dtbox1" ref-type="boxed-text">Extended Experimental Procedures</xref>.</p></sec><sec id="sec4.2"><title>Immunization against α-Gal</title><p>Eight- to ten-week-old mice received 3 × 10<sup>8</sup> rabbit rRBCM equivalents (100 μl; PBS; intraperitoneal [i.p.]). Adjuvants are described in the <xref rid="dtbox1" ref-type="boxed-text">Extended Experimental Procedures</xref>. Mouse serum was collected 2 weeks after last immunization, and circulating anti-α-gal Abs were quantified by ELISA. See the <xref rid="dtbox1" ref-type="boxed-text">Extended Experimental Procedures</xref> for details on anti-α-gal ELISA.</p></sec><sec id="sec4.3"><title>Passive Transfer of Anti-α-Gal mAbs</title><p><italic>α1,3Gt</italic><sup>−/−</sup> mice received anti-α-gal IgG1, IgG2a, IgG2b, and IgG3 mAbs (<xref rid="bib11 bib65" ref-type="bibr">Ding et al., 2008; Yin et al., 2004</xref>) (150 μg; 100 μl per mouse) or polyclonal IgM (150 μg; 300–400 μl per mouse) via a single intravenous (i.v.) injection 24 hr prior to mosquito exposure.</p></sec><sec id="sec4.4"><title>Plasmodium Strains</title><p>Transgenic <italic>P. berghei</italic> ANKA (<italic>Pb</italic>A) strains expressing GFP under the <italic>eef1α</italic> promoter, i.e., <italic>Pb</italic>A<sup>EEF1a-GFP</sup> (259cl1; MR4; MRA-865) (<xref rid="bib16" ref-type="bibr">Franke-Fayard et al., 2004</xref>), or under the <italic>hsp70</italic> promoter (<xref rid="bib28" ref-type="bibr">Ishino et al., 2006</xref>), i.e., <italic>PbA</italic><sup>Hsp70-GFP</sup> (kindly provided by Robert Menard, Institut Pasteur), transgenic <italic>P. yoelii</italic> 17XNL strain expressing GFP under the <italic>PbA eef1α</italic> promoter (MR4; MRA-817; kindly provided by Robert Menard, Institut Pasteur) (<xref rid="bib62" ref-type="bibr">Weiss et al., 1989</xref>). For sporozoite production, see the <xref rid="dtbox1" ref-type="boxed-text">Extended Experimental Procedures</xref>.</p></sec><sec id="sec4.5"><title><italic>Plasmodium</italic> Transmission</title><p><italic>A. stephensi</italic> or <italic>gambiae</italic> mosquitoes were allowed to feed on anesthetized mice (i.p.; 125 mg/kg ketamine; 12.5 mg/kg xylazine) placed on a warming tray. Two mosquitoes were allowed to probe and feed independently (90–100 s) on restricted to the edge of the mouse ear (10–12/3–4 mm) and dissected thereafter for confirmation of sporozoites in salivary glands. If negative, infection was repeated.</p></sec><sec id="sec4.6"><title>Sporozoites Inoculation</title><p><italic>Pb</italic>A<sup>EEF1a-GFP</sup> sporozoites were inoculated (i.d.) in the ear pinna (750 sporozoites in 20–30 μl; 1% BSA in PBS) or i.v. in the retro-orbital vein (150 sporozoites in 50 μl; 1% BSA in PBS) using a microsyringe (Nanofil 100 μl; 33G beveled needle; World Precision Instruments).</p></sec><sec id="sec4.7"><title>Detection of α-Gal in <italic>Plasmodium</italic> Sporozoites</title><p>Sporozoites were stained with Alexa Fluor 647-conjugated BSI-IB<sub>4</sub> or Alexa Fluor 647-conjugated anti-α-gal mAbs and detected by confocal microscope or flow cytometer. For detection of α-gal in <italic>Pb</italic>A<sup>Hsp70-GFP</sup> by western blotting, see the <xref rid="dtbox1" ref-type="boxed-text">Extended Experimental Procedures</xref>. Green coffee bean α-galactosidase (50–200 μl; 5 U/ml; 60–90 min; 25°C; Sigma Chemical) was used to hydrolyze terminal α-galactosyl moieties from glycolipids and glycoproteins (<xref rid="bib31" ref-type="bibr">Luo et al., 1999</xref>).</p></sec><sec id="sec4.8"><title>Statistical Analysis</title><p>All tests (except human cohort studies) were performed using the GraphPad Prism (v. 6.0) (GraphPad Software). Human analyses were performed in R (v. 3.0.2). Detailed analyses are described in the <xref rid="dtbox1" ref-type="boxed-text">Extended Experimental Procedures</xref>.</p></sec><sec id="sec4.9"><title>Mice</title><p>Experiments in mice were performed in accordance with protocols approved by the Ethics Committee of the Instituto Gulbenkian de Ciência and the Portuguese National Entity (DGAV-Direção Geral de Alimentação e Veterinária). Experiments in mice were performed in accordance with the Portuguese (Decreto-Lei no. 113/2013) and European (directive 2010/63/EU) legislation related to housing, husbandry, and animal welfare. C57BL/6 <italic>J</italic><sub><italic>H</italic></sub><italic>T</italic><sup>−/−</sup> (<xref rid="bib22" ref-type="bibr">Gu et al., 1993</xref>), <italic>Tcrβ</italic><sup><italic>−</italic>/−</sup> (<xref rid="bib37" ref-type="bibr">Mombaerts et al., 1992</xref>), <italic>Aid</italic><sup><italic>−</italic>/−</sup> (<xref rid="bib40" ref-type="bibr">Muramatsu et al., 2000</xref>), <sc><italic>μ</italic></sc><italic>S</italic><sup><italic>−</italic>/−</sup> (<xref rid="bib14" ref-type="bibr">Ehrenstein et al., 1998</xref>), and <italic>C3</italic><sup><italic>−/−</italic></sup> (<xref rid="bib66" ref-type="bibr">Circolo et al., 1999</xref>) mice were crossed with C57BL/6 <italic>α1,3Gt</italic><sup>−/−</sup> mice (<xref rid="bib75 bib58" ref-type="bibr">Shinkel et al., 1997; Tearle et al., 1996</xref>). For the details on genotyping, see the <xref rid="dtbox1" ref-type="boxed-text">Extended Experimental Procedures</xref>.</p><p><boxed-text id="dtbox1"><label>Extended Experimental Procedures</label><sec id="dtbox1sec1"><title>Material</title><p>Synthetic α-gal linked to bovine serum albumin (BSA) or human serum albumin (HSA; Dextra Labs; Reading, UK), α-galactosidase from green coffee beans (Sigma Chemical Co), Fluorescein isothiocyanate (FITC)-conjugated (Sigma-Aldrich Co) and Alexa Fluor 647-conjugated (Invitrogen) <italic>Bandeiraea simplicifolia</italic> lectin (BSI-IB<sub>4;</sub><italic>Griffonia simplicifolia</italic>) (<xref rid="bib69" ref-type="bibr">Kisailus and Kabat, 1978</xref>), rabbit RBC (Patricell Ltd.; Nottingham, UK), incomplete Freund’s Adjuvant (IFA) (BD Difco), inactivated and dried <italic>M. tuberculosis</italic> H37 Ra (BD Difco), Dextran, Tetramethylrhodamine, 10,000 MW, Lysine Fixable (fluoro-Ruby; Life Technologies), mouse anti-α-gal IgM (M86; EnzoLife Sciences or kindly provided by Uri Galili, University of Massachusetts Medical School, Worcester, MA, USA) (<xref rid="bib18" ref-type="bibr">Galili et al., 1998</xref>), IgG1 (GT6-27), IgG2a, IgG2b and IgG3 (GT4-31) mAbs (<xref rid="bib11 bib65" ref-type="bibr">Ding et al., 2008; Yin et al., 2004</xref>), mouse anti-dinitrophenyl IgM (MADNP-5), IgG1 (MADNP-1), IgG2a (MADNP-2), IgG2b (MADNP-3) and IgG3 (MADNP-4) mAbs (kindly provided Hérve Bazin, University of Louvain, Belgium) (<xref rid="bib74" ref-type="bibr">Platteau et al., 1990</xref>). Mouse IgM, IgG1, IgG2a, IgG2b, IgG3, IgA and IgE were detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgA and IgE or a rat mAb anti-mouse IgG3 (Southern Biotechnology Associates). Anti-CSP 3D11 (<xref rid="bib81" ref-type="bibr">Yoshida et al., 1980</xref>) hybridoma (IgG1 mAb directed against the repeat region of <italic>P. berghei</italic> CSP) was kindly provided by Ana Rodriguez (New York Medical School).</p></sec><sec id="dtbox1sec2"><title>Cohort Study</title><p>The Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Sciences, Technique and Technology of Bamako, and the Institutional Review Board of NIAID-NIH approved the cohort study. In May 2011, 695 individuals aged 3 months to 25 years were enrolled in a cohort study in the rural village of Kalifabougou, Mali. Individuals were followed during the malaria season for 7 months. Individuals were invited to participate after random selection from an age-stratified census of the entire village population (n = 4394). Written, informed consent was obtained from adult participants and the parents or guardians of participating children. Enrollment exclusion criteria were hemoglobin level < 7 g/dL, axillary temperature ≥ 37.5°C, acute systemic illness, use of anti-malarial or immunosuppressive medications in the past 30 days and pregnancy. Clinical malaria was detected prospectively by self-referral and weekly active clinical surveillance. All individuals with signs and symptoms of malaria and any level of <italic>Plasmodium</italic> parasitemia detected by light microscopy were treated according to the National guidelines in Mali. The research definition of malaria was parasitemia ≥ 2500 parasites/μL, temperature ≥ 37.5°C and no other cause of fever discernible by physical exam. During scheduled clinic visits, blood was collected by finger prick every two weeks on filter paper. Detection of asymptomatic <italic>Plasmodium</italic> infection by PCR was done retrospectively at the end of the surveillance period. For each participant, PCR was performed on blood samples in chronological order from enrollment onward until the first <italic>P. falciparum</italic> infection was detected. Detailed methods for <italic>P. falciparum</italic> PCR detection have been described (<xref rid="bib78" ref-type="bibr">Tran et al., 2013</xref>). α-gal-specific IgM and IgG Ab levels were determined using ELISA on plasma collected at enrollment from the subset of individuals. Statistical analyses were described in the Statistical analysis’ section.</p></sec><sec id="dtbox1sec3"><title>Genotyping</title><p>Mice were genotyped by PCR using tail genomic DNA and the following primers: i) <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup>: 5′-TCTTGACGAGTTCTTCTGAG-3′, 5′-TCAGCATGATGCGCATGAAGA-3′, 5′-TGGCCGCGTGGTAGTAAAAA-3′; ii) <italic>J</italic><sub><italic>H</italic></sub><italic>T</italic><sup><italic>−/−</italic></sup>: 5′-CAGTGAATGACAGATGGACCTCC-3′, 5′-GCAGAAGCCACAACC ATACATTC-3′, 5′-ACAGTAACTCGTTCTTCTCTGC-3′; iii) <italic>Aid</italic><sup><italic>-</italic>/-</sup>: 5′-GGCCAGCTCATTCCTCCACT-3′, 5′-CACTGAGCGCACCTGTAGCC-3′, 5′-CCTAGTGGCCAAGGTGCAGT-3′, 5′-TCAGGCTGAGGTTAGGGTTCC-3′; iv) <italic>Tcrβ</italic><sup><italic>-</italic>/-</sup>: 5′-TGTCTGAAGGGCAATGACTG-3′, 5′-GCTGATCCGTGGCATCTATT-3′, 5′-CTTGGGTGGAGAGGCTATTC-3′, 5′-AGGTGAGATGACAGGAGATC-3′; v) <italic>C3</italic><sup><italic>−/−</italic></sup>: 5′-ATCTTGAGTGCACCAAGCC-3′, 5′-GGTTGCAGCAGTCTATGAAGG-3′, 5′-GCCAGAGGCCACTTGTGTAG-3′. <sc><italic>μ</italic></sc><italic>S</italic><sup><italic>-</italic>/-</sup> mice were phenotyped by ELISA, as described (<xref rid="bib14" ref-type="bibr">Ehrenstein et al., 1998</xref>). Total genomic DNA was isolated from the tail using a standard protocol. Briefly, tails (0,5 to 1 cm) were obtained and submerged into DirectPCR Lysis Reagent (200-300 μL; Viagen Biotech) containing Proteinase K (100 μg/ml) and incubated (overnight; 55°C) under vigorous agitation. Next day, samples were centrifuged (16,000 g; 5-10 s; RT) and supernatant (1 μl) was amplified by PCR: 95°C (15 min), 30 cycles of 94°C (45 s), 60°C (90 s) (except <italic>Aid</italic><sup><italic>-</italic>/-</sup> genotyping; 63°C), 72°C (90 s) and 72°C (15 min). Visualizations of PCR results were done on 1.5%–2% agarose gel (80-100V; 1-2 hr).</p></sec><sec id="dtbox1sec4"><title>Extraction of Rabbit RBC Membranes</title><p>Rabbit RBC membranes (rRBCM) expressing high levels of α-gal (<xref rid="bib67" ref-type="bibr">Eto et al., 1968</xref>) were prepared from lyzed RBC (50 mM sodium phosphate buffer pH 8), washed (5-7X; sodium phosphate buffer; 10,000 g; 20 min, 4°C) until supernatant was hemoglobin-free, essentially as described (<xref rid="bib71" ref-type="bibr">Matsuzawa and Ikarashi, 1979</xref>). Membranes were collected by centrifugation (20,000 g; 20 min, 4°C), re-suspended in PBS and stored (−80°C) until used.</p></sec><sec id="dtbox1sec5"><title>Immunization</title><p>rRBCM were emulsified in Complete Freund’s Adjuvant (CFA; Incomplete Freund’s Adjuvant (IFA) plus <italic>Mycobacterium tuberculosis</italic> H37 RA; 4 mg/ml; DIFCO) with CpG (50 μg/mouse; ODN M362; Invivogen) and administered subcutaneously (s.c.). Two subsequent immunizations were emulsified in IFA+rRBC+CpG. Emulsions were administered at 200 μl per mouse, 3 times at two weeks intervals. Mice were also immunized (s.c.) with α-gal-BSA (75 μg/mouse) emulsified in CFA with two subsequent immunizations, 2 and 4 weeks thereafter with α-gal-BSA emulsified in IFA.</p></sec><sec id="dtbox1sec6"><title>Anti-α-Gal ELISA</title><p>Mouse serum was collected two weeks after immunization and circulating anti-α-gal antibodies were quantified by ELISA, as described (<xref rid="bib18 bib70" ref-type="bibr">Galili et al., 1998; LaTemple and Galili, 1998</xref>). Briefly, 96-well plates (PolySorp; Nunc) were coated with α-gal (α-gal-HSA or α-gal-BSA; 50 μl; 10 μg/ml in 0.5 M carbonate bicarbonate buffer; pH 9.5; 2 hr at 37°C or overnight at 4°C), blocked with BSA (100 μl; 1% w/v in PBS; 1 hr; RT) and washed (5X; PBS/0.05% Tween-20). Plates were incubated (1 hr; RT) with serum serial dilutions in PBS, 1% BSA and washed (5X; PBS/0.05% Tween-20). Anti-α-gal antibodies were detected using HRP-conjugated anti-mouse IgM, total IgG, IgG1, IgG2a, IgG2b or IgG3 (50 μl, 1:1-2,000 dilution, 1 hr, RT) and washed (5X; PBS/0.05% Tween-20). Purified anti-α-gal IgM (<xref rid="bib18" ref-type="bibr">Galili et al., 1998</xref>), IgG1 (GT6-27), IgG2a and IgG2b and IgG3 (GT4-31) mAb (<xref rid="bib11 bib65" ref-type="bibr">Ding et al., 2008; Yin et al., 2004</xref>) were used as standards. TMB Substrate Reagent Set (BD Biosciences) was used to reveal peroxidase activity (15-30 min; RT) and the reaction was stopped using 2N sulfuric acid. Optical densities (OD) were reported at λ = 450 nm and normalized by subtracting background OD values (λ = 600 nm) (Victor 3; PerkinElmer). Concentration of each anti-α-gal IgG subclass was determined, as described (<xref rid="bib76" ref-type="bibr">Spalter et al., 1999</xref>). Concentration of anti-α-gal antibodies in human plasma was analyzed using a similar assay and a standard curve to transform absorbance into immunoglobulin concentration was obtained by coating 8 duplicate wells in every plate with purified human IgG and IgM (500 to 1.5 ng/ml) and performing the protocol described above in the absence of diluted serum.</p></sec><sec id="dtbox1sec7"><title>Anti-α-Gal mAbs</title><p>Anti-α-gal hybridomas were cultured in RPMI 1640 (Invitrogen Life Technologies) supplemented with 0.1 mM Sodium Pyruvate, 0.01 M HEPES, 0.05 mM 2-mercaptoethanol and 2% FBS (IgG depleted). Anti-α-gal IgG2a, IgG2b and IgG3 hybridomas were derived by sub-cloning of the original IgG1 (GT6-27) hybridoma and as such have similar affinities for α-gal, as described (<xref rid="bib11 bib65" ref-type="bibr">Ding et al., 2008; Yin et al., 2004</xref>). Antibodies were purified by affinity chromatography using HiTrap Protein G columns (GE Healthcare Life Sciences). Anti-α-gal IgG3 purification was carried out by a nonchromatographic method taking advantage of its euglobulin properties, as described (<xref rid="bib68" ref-type="bibr">García-González et al., 1988</xref>). Purified mAbs were extensively dialyzed against PBS. Protein concentration was determined using NanoDrop ND-1000 spectrophotometer (λ = 280 nm; Thermo Scientific) and purity confirmed by SDS-PAGE analysis. When indicated, mAbs were labeled with Alexa Fluor 647 Protein Labeling Kit (A20173), as per manufacturer recommendations (Molecular Probes, Invitrogen). Anti-mouse IgM total-A647 (Clone: R33.24.12) was used to reveal anti-α-gal IgM mAb in immunofluorescence assay.</p></sec><sec id="dtbox1sec8"><title>Passive Anti-α-Gal mAbs Transfer</title><p><italic>α1,3Gt</italic><sup>−/−</sup> mice received anti-α-gal IgG1, IgG2a, IgG2b or IgG3 mAbs via a single i.v. injection (150 μg; 100 μl per mouse), 24 hr prior to mosquito exposure. Passive anti-α-gal IgM transfer was performed using polyclonal IgM. Briefly, eight to ten weeks old <italic>α1,3G</italic><sup>−/−</sup><italic>Aid</italic><sup><italic>−/−</italic></sup> mice received 3x10<sup>8</sup> RBC equivalents of rRBCM in PBS (100 μl; i.p.). Serum was collected two weeks after the last immunization and concentration of anti-α-gal IgM was determined by ELISA (<xref rid="bib18 bib70" ref-type="bibr">Galili et al., 1998; LaTemple and Galili, 1998</xref>). Serum collected from naive <italic>α1,3G</italic><sup>−/−</sup><italic>Aid</italic><sup><italic>−/−</italic></sup> mice was used as control. <italic>α1,3Gt</italic><sup>−/−</sup> mice received the polyclonal IgM via a single i.v. (150 μg; 300-400 μl per mouse), 24 hr prior to mosquito exposure.</p></sec><sec id="dtbox1sec9"><title><italic>Plasmodium</italic> Strains</title><p>For sporozoite production, <italic>P. berghei</italic> ANKA infected RBC (1x10<sup>6</sup>) were administered i.p. to BALB/c mice and the presence of gametocyte-stage parasites capable of exflagellation was monitored in fresh blood preparations. Infected BALB/c mice were used to feed 3 to 4-day-old female mosquitoes (∼1 hr), which were used 18-25 days postinfection for subsequent infections. When infected with <italic>P. berghei</italic> ANKA mosquitoes were maintained at 21°C (IHMT) whereas mosquitoes infected with <italic>P. yoelii</italic> 17XNL-GFP(<xref rid="bib73" ref-type="bibr">Ono et al., 2007</xref>) were maintained (24-25°C; Centre for Production and Infection of <italic>Anopheles</italic>; CEPIA, Pasteur Institute, France). Alternatively, <italic>P. yoelii</italic> 17XNL (MR4; MRA-593) (<xref rid="bib62" ref-type="bibr">Weiss et al., 1989</xref>) infected mosquitoes were purchased from Radboud University Nijmegen Medical Centre (Nijmegen, Netherlands). <italic>P. falciparum</italic> 3D7 strain (kindly provided by Robert Menard, Institut Pasteur, France) (<xref rid="bib80" ref-type="bibr">Walliker et al., 1987</xref>) were used for α-gal detection in infected RBC by immunofluorescence assay.</p></sec><sec id="dtbox1sec10"><title>Sporozoites Isolation and Inoculation</title><p>Mosquitoes were narcotized (−20°C; 3-5 min), washed in 70% ethanol (1X; 10-20 s) and PBS (3X; 10-20 s; Ambion). Salivary glands were obtained by dissection under a zoom stereomicroscope (3X magnification; Nikon SMZ800, Japan) and preserved in RPMI 1640 medium (GIBCO BRL) or PBS. Salivary glands were smashed using Pellet pestles cordless motor (10-15 s; Sigma) to release sporozoites and centrifuged (100 g; 5 min) in LoBind microfuge tubes (Eppendorf). Supernatant was filtered (100 μm cell strainers; BD Falco) to exclude debris. Sporozoites were counted using KOVA Glasstic Slide 10 with quantitative grid (Fisher Scientific GMBH). <italic>Pb</italic>A<sup>EEF1a-GFP</sup> sporozoites were inoculated i.d. in the ear pinna (750 sporozoites in 20-30 μl; 1% BSA in PBS) or i.v. (retro-orbital; 150 sporozoites in 50 μl; 1% BSA in PBS) using a microsyringe (Nanofil 100 μl; 33G beveled needle; World Precision Instruments).</p></sec><sec id="dtbox1sec11"><title>Passive RBCs Transfer</title><p><italic>α1,3Gt</italic><sup><italic>−/−</italic></sup> mice immunized with rRBCM and age matched naive <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup> mice were exposed to <italic>Pb</italic>A<sup>EEF1a-GFP</sup> infected mosquitoes, as described above. Parasitemia was monitored by flow cytometry in FacScan analyzer (BD Biosciences). Nine days post-mosquito feeding, blood was collected and injected i.p. (100-200 μl) to naive <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup> mice. Infection was monitored daily for parasitemia and clinical symptoms, starting from day 3–4 postinfection RBC administration.</p></sec><sec id="dtbox1sec12"><title>Quantification of Sporozoite mRNA</title><p>The mouse ear was excised immediately after and the whole liver was harvested 40 hr after mosquito biting. Samples were frozen in liquid nitrogen, tissues were homogenized in TRIzol (Life Technologies), total RNA isolated according to manufacturer’s instructions (RNeasy Mini kit; QIAGEN). Briefly, RNA (2 μg; 10 μl) was mixed to random primers (1 μl) and dNTPs (10 mM; 1 μl), incubated (65°C; 5 min.), placed on ice and incubated in PCR Buffer (0.1 M DTT; 40 units/μl RNaseOU; 2 min.; 42°C). Superscript RT (SSIIRT) was added (1 μl; 50 min. 42°C) and samples were heated to finalize synthesis (70°C; 15 min). qRT-PCR was performed using <italic>Pb</italic>A 18S rRNA specific primers (5′-AAGCATTAAATAAAGCGAATACATCCTTAC-3′ and 5′-GGAGA TTGGTTTTGACGTTTATGTG-3′). Mouse housekeeping <italic>Arbp0</italic> gene (5′-CTTTGGGCATCACCACGAA-3′ and 5′-GCTGGCTCCCACCTTGTCT-3′) was amplified as control. Applied Biosystems’ Power SYBR Green PCR Master Mix was used as per manufacturer’s instructions (ABI Prism 7000 system; Applied Biosystems). Data are presented as relative expression of <italic>P. berghei</italic> ANKA 18S rRNA normalized to mouse <italic>Arbp0</italic> mRNA.</p></sec><sec id="dtbox1sec13"><title>Detection of α-Gal in <italic>Plasmodium</italic> Sporozoites</title><p><italic>Pb</italic>A<sup>EEF1a-GFP</sup>, <italic>Pb</italic>A<sup>Hsp70-GFP</sup>, <italic>P. falciparum</italic> 3D7 or <italic>P. yoelii</italic> 17XNL-GFP sporozoites, isolated from the salivary glands of <italic>Anopheles stephensi</italic> or <italic>Anopheles gambiae</italic> mosquitoes, 18-25 days postinfection were allowed to attach to Teflon printed (10 wells, 8 mm; no-adherent surface; Immuno-Cell Int.) or to diagnostic glass slides. Sporozoites were fixed (20-50 μl; 4% PFA; 20-30 min; RT or 37°C) and washed gently (1X; PBS). Sporozoites were stained with Alexa Fluor 647 conjugated BSI-IB<sub>4</sub> (100 μl; 200 μg/ml; 2 hr, RT), Alexa Fluor 647 conjugated anti-α-gal IgG1, IgG2a, IgG2b, IgG3 mAb (100 μl; 50 μg/ml; overnight; 4°C) or with nonconjugated anti-α-gal IgM (M86) mAb (100 μl; 50 μg/ml; overnight; 4°C) and washed (1X; PBS). Nonconjugated IgM antibodies were detected using Alexa Fluor 647 conjugated goat anti-mouse IgM (100 μl; 10 μg/ml; overnight, 4°C) (Molecular Probes, Invitrogen). After washing (1X; PBS), slides were incubated with 4’,6-diamidino-2-phenylindole (DAPI) (100 μl; 10 μg/ml; 10 min; RT) (Molecular Probes, washed (1X; PBS) and dried in dark room without covering with coverslip (RT). Images of <italic>Pb</italic>A sporozoites were obtained by DeltaVision Core immunofluorescence microscopy (Applied Precision/Olympus) or Spinning Disk Confocal microscopy Revolution xD (Andor Technology) at 100 × magnification. Actin was detected using Alexa Fluor 488 Phalloidin (Molecular Probes; 100 μl; 3 units/ml; 1 hr; RT) in <italic>P. falciparum</italic> 3D7 and <italic>P. yoelii</italic> 17XNL-GFP sporozoites to which was added ProLong Gold Antifade Reagent (Invitrogen) and wells were covered with coverslips. Slides were visualized with Axiovert II fluorescence microscope (Zeiss). Images were analyzed using bicubic interpolation and rescaling with ImageJ software (NIH).</p><p>For detection of α-gal by flow cytometry, <italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites were isolated from the salivary glands of <italic>A. stephensi</italic> mosquitoes 19-25 days postinfection (10<sup>5</sup>; 30-40ul; PBS; ice cold: 30 min), fixed (30-40 μl; 4% PFA in PBS; 20-30 min; 37°C) and washed (1 ×; PBS; 9,300 g; 2 min). Staining was performed with Alexa Fluor 647 conjugated BSI-IB<sub>4</sub> (100 μg/ml; 1 hr; 37°C). Sporozoites were washed (1 ×; PBS; 9,300 g; 2 min) and Alexa Fluor 647 BSI-IB<sub>4</sub> signal was detected in CyAn™ ADP flow cytometry (Beckman Coulter; USA) with Summit Software (v4.3; Beckman Coulter), gating on FITC (for sporozoite detection) and APC (for α-gal detection).</p><p>For detection of α-gal by western blotting <italic>Pb</italic>A<sup>Hsp70-GFP</sup>, <italic>P. falciparum</italic> 3D7 and <italic>P. yoelii</italic> 17XNL-GFP sporozoites were isolated from the salivary glands of 40-70 mosquitoes, 21-25 days after infection. Briefly, salivary glands were collected into the LoBind microfuge tubes (PBS; on ice), smashed using pellet pestles cordless motor (10-15 s; 2X) and a short spin was applied to pellet debris. Supernatant (30-50 μl; 1-2.5 × 10<sup>5</sup> sporozoites) was transferred into LoBind microfuge tube and aliquots were stored at −80°C until used. Number of <italic>Plasmodium</italic> sporozoites (equivalent to 10<sup>5</sup> per sample) was normalized to corresponding number of salivary glands from noninfected mosquitoes. Samples were lysed (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, Roche complete EDTA-free protease inhibitor cocktail; 1 hr; on ice) and centrifuged (16,000 g, 10 min.; 4°C). Samples (supernatant) were denatured with Laemmli buffer (1% β-mercaptoethanol; 2%SDS; 95°C; 2 min) and separated in SDS-PAGE gradient gel (12% acrylamide/bisacrylamide gel, 29:1; 100V; 2 hr). Proteins were transferred into a PVDF membrane (90 min; 12V), blocked (3% BSA) and incubated overnight with anti-α-gal IgG2b mAb (1 μg/ml; 20 ml). Membrane was washed with 20 mM Tris/HCl pH7.5, 150 mM NaCl and 0.1% Tween-20 buffer (TBST, 3 ×; 5 min; RT) and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG2b-HRP (SouthernBiotech, 20 ng/ml; 1 hr; 50 ml; RT). Membrane was washed (TBST; 1 hr; RT) and developed (SuperSignal Chemiluminescent detection kit; West Pico, Thermo Scientific).</p></sec><sec id="dtbox1sec14"><title>GPI Anchor Cleavage from <italic>Plasmodium</italic> Sporozoites</title><p><italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites were fixed (4% PFA in PBS; 20-30 min; 37°C) and treated with phosphatidylinositol-specific phospholipase C (from <italic>Bacillus cereus</italic>; 5U/ml; 14-16 hr; 37°C). Detection of α-gal is described above and CSP was detected using anti-CSP conjugated to Alexa Fluor 647 in CyA ADP Analyzer (Beckman Coulter).</p></sec><sec id="dtbox1sec15"><title>Bacterial Strains and Growth Conditions</title><p>Frozen <italic>E. coli</italic> O86:B7 (ATCC 12701™; Rockville, Md.) and <italic>E. coli</italic> K12 (ATCC 10798) (∼10<sup>7</sup> CFU/ml; 50% glycerol solution; −80°C) stocks were inoculated into Luria broth (LB; 50 ml; 37°C; overnight) on a shaker rack. Spectrophotometric absorbance was measured at 600 nm (Optical Density-OD<sub>600</sub>) and adjusted to OD<sub>600</sub> ∼0.2 in order to harvest bacteria during exponential growth corresponding to a OD<sub>600</sub> of 2. The approximate cell number was determined according to spectrophotometric measurement and confirmed with most probable numbers (MPN) technique, also known as the method of Poisson zeroes (<xref rid="bib72" ref-type="bibr">Oblinger and Koburger, 1975</xref>).</p></sec><sec id="dtbox1sec16"><title>Detection of α-Gal on Bacterial Cultures</title><p><italic>E. coli</italic> O86:B7 and <italic>E.coli</italic> K12 were cultured (LB; 50 ml; 37°C; overnight), washed (2 ×; PBS; 4,000 g; 5 min; 4°C) and re-suspended in PBS. Bacteria were fixed (200 μl; 4% PFA in PBS; 20-30 min; RT) and washed (1X; PBS; 4000 g; 5 min; RT). 10<sup>8</sup>-10<sup>9</sup> CFU/ml were stained with BSI-IB<sub>4</sub>-FITC (200 μl; 50 μg/ml; 2 hr, RT) for flow cytometry analysis using FacScan analyzer (BD Biosciences). In parallel, immunofluorescence assays were performed using 10 μl stained samples after air-drying (10-20 min; RT). Samples were washed (1x; PBS), incubated with 4’,6-diamidino-2-phenylindole (DAPI) (200 μl; 10 μg/ml; 10 min; RT; Molecular Probes) and washed (1 ×; PBS). Immunofluorescence images were obtained by Spinning Disk Confocal microscopy Revolution xD (Andor Technology; USA) at 100 × magnification and images were processed with ImageJ (NIH) software.</p></sec><sec id="dtbox1sec17"><title>Rederivation of GF <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup> Mice</title><p>Briefly, pregnant female <italic>α1,3Gt</italic><sup>−/−</sup> mice were euthanized (20 days postcoitum), uteri were immersed in 1% VirkonS, rinsed in sterile water and pups were transferred to surrogate mothers kept in GF isolators (Gettinge-La Calhéne, France). GF status was monitored every third week onward.</p></sec><sec id="dtbox1sec18"><title>Gnotobiotic and SPF-Colonized Mice</title><p><italic>α1,3Gt</italic><sup>−/−</sup> mice were re-derived via caesarean section from SPF into GF conditions at the Instituto Gulbenkian de Ciência, as described (<ext-link ext-link-type="uri" xlink:href="http://strains.emmanet.org/protocols/GermFree_0902.pdf" id="intref0010">http://strains.emmanet.org/protocols/GermFree_0902.pdf</ext-link>). For gnotobiotic colonization, 8-12 weeks GF <italic>α1,3Gt</italic><sup>−/−</sup> mice were transferred into sterile micro isolator Ventiracks (Biozone, Margate, UK), fed <italic>ad libitum</italic> with a standard autoclaved chow diet and water and GF status was monitored every third week onward. GF and SPF <italic>α1,3Gt</italic><sup>−/−</sup> mice treated with streptomycin sulfate (5 g/l in drinking water for 7 days; GIBCO), were starved for 12 hr and colonized with <italic>E. coli</italic> O86:B7 or <italic>E. coli</italic> K12 (∼10<sup>7</sup> CFU/100 μl Luria-Bertani - LB - medium) via oral gavage, using a 20-gauge stainless steel animal feeding needle (Cadence Science, Japan). Control mice were inoculated with sterile LB medium. Colonization protocol was administered 3 times at two weeks intervals.</p></sec><sec id="dtbox1sec19"><title>Complement Activation</title><p><italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites were exposed to anti-α-gal mAbs (150 μg/ml; in 50 μl DMEM; 60 min; 4°C) and subsequently to naive C57BL/6 mouse plasma (1:5 in 0.1% gelatin in Veronal Buffer (VB)<sup>2+</sup>; 50 μl; 60 min; 37°C) (Lonza), used as a source of complement. Samples were washed (1 ×; PBS). C3 was detected using APC labeled anti-C3/C3b/iC3b (Clone: 6C9; 1:100; 50 μl; 45 min; 4°C) and analyzed in CyA ADP Analyzer (Beckman Coulter).</p></sec><sec id="dtbox1sec20"><title>Sporozoite Cytotoxicity Assays</title><p><italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites (10-50x10<sup>3</sup>) were incubated with anti-α-gal or isotype matched anti-DNP mAbs (150 μg/ml mAb in 10 μl DMEM GlutaMA; 60 min; 4°C). Naive C57BL/6 mouse plasma (1:5) or baby rabbit complement (1:10; Cedarlane Laboratories) was added (0.1% gelatin in VB<sup>2+</sup>) as a source of complement (60 min; 37°C). Cytotoxicity was quantified according to GFP expression in Andor Spinning Disk Confocal Microscopy (Andor Technology). Alternatively, <italic>Pb</italic>A<sup>EEF1a-GFP</sup> sporozoites (10-30 × 10<sup>3</sup>) were isolated from the salivary glands of <italic>Anopheles stephensi</italic> mosquitoes, 19-25 days postinfection and incubated with anti-α-gal or isotype matched anti-DNP mAbs (150 μg/ml mAb in 10 μl PBS, Life Technologies) (60 min; on ice). Plasma from C57BL/6 mice (1:5) or baby rabbit complement (bRC; 1:10; Cedarlane Laboratories) was added (0.1% gelatin in VB<sup>2+</sup> buffer; Lonza) as a source of complement (60 min; 37°C). Alternatively, sporozoite cytotoxicity was evaluated according to loss of crescent-shaped morphology (%).</p></sec><sec id="dtbox1sec21"><title>Invasion Assay</title><p>Human hepatoma cells (HepG2; kindly provided from Robert Menard, Institut Pasteur, France) were cultured (4x10<sup>4</sup>/well; 200 μl in 96-well plates) in DMEM (GlutaMA, 10% FBS, 100U/ml penicillin/streptomycin; Life Technologies) (37°C; 5%CO<sup>2</sup>; 2 days). <italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites (19-25 days postinfection) were preincubated with anti-α-gal or isotype matched control anti-DNP mAbs (150 μg/ml in 20-50 μl; 60 min; 4°C). Plasma from C57BL/6 mice (1:5 in 20-50 μl 0.1% gelatin in VB<sup>2+</sup> buffer; Lonza) was added (60 min.; 37°C) as a source of complement and sporozoites were immediately transferred onto HepG2 cells at a 1:4 parasite/cell ratio. Cocultures were incubated (120 min; 37°C) with Tetramethylrhodamine-Dextran (10,000 MW, 1:1; 2 mg/ml; Molecular Probes), washed (2X; PBS) and trypsinized (30-50 μl; 0.05% Trypsin-EDTA (1X), phenol red (GIBCO). Percentage of wounded and parasite-invaded cells was determined by flow cytometry analysis (FACScan, BD Biosciences), gating on FL1 (GFP; invasion) and FL2 (Dextran-Red; wounding). Sporozoite maturation was determined by quantifying the number of EEFs per field and EEF area after 40 hr coculture in 15 μ-Slide 8 well (ibiTreat; IBIDI; Germany) using fluorescence microscopy (Screening Microscopy; Nikon Eclipse TE2000-S). Images were obtained at 20x magnification. Pictures were analyzed using Image J software (NIH).</p></sec><sec id="dtbox1sec22"><title>PMN Cell Depletion</title><p>Anti-Gr1 mAb (Clone: RB6-8C5; RB6-8C5 hybridoma) was produced at the IGC under serum-free conditions using a CELLine (Integra, Switzerland) (<xref rid="bib77" ref-type="bibr">Tepper et al., 1992</xref>). Neutrophils were depleted in <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup> mouse by a single intravenous anti-Gr1 mAb injection (250 μg), 48 hr prior to mosquito biting. PMN depletion was confirmed by staining for CD11b-FITC (Integrin α[M] chain, Mac-1 α chain, CR3, BD PharMingen™) and Anti-Gr1-PE (1A8, BD PharMingen™) and analyzed in flow cytometry(<xref rid="bib79" ref-type="bibr">Tsiganov et al., 2014</xref>) (CyA ADP Analyzer, Beckman Coulter).</p></sec><sec id="dtbox1sec23"><title>Statistical Analysis</title><p>All tests (except human cohort studies) were performed using the GraphPad Prism v6.0 (GraphPad Software). Human analyses were performed in R version 3.0.2 (<ext-link ext-link-type="uri" xlink:href="http://www.r-project.org" id="intref0015">http://www.R-project.org</ext-link>). Ab levels between groups were compared with the Kruskal-Wallis test. The relationship between Ab levels and time to <italic>P. falciparum</italic> infection and febrile malaria was determined by Kaplan-Meier survival analysis and the log-rank test. Age dependent differences in anti-α-gal IgM and IgG antibody concentrations in human serum were determined using nonparametric Mann-Whitney test. Differences in anti-α-gal antibody concentration in mouse serum were determined using nonparametric Kruskal-Wallis test with Dunn’s multiple comparison posttest. Incidence of infection was compared using log rank (Mantel-Cox) test. Survival curves were plotted using Kaplan-Meier plot and significant differences among experimental groups were determined using the Log-Rank (Mantel-Cox) test. Data corresponding to relative amount of <italic>Plasmodium</italic> in the skin and liver obtained from qRT-PCR was analyzed using nonparametric Mann–Whitney test. Cytotoxic effect of anti-α-gal antibodies was analyzed using repeated-measures ANOVA followed by Tukey’s multiple comparison posttest. A p value equal to or below 0.05 was considered statistically significant.</p></sec></boxed-text></p></sec></sec><sec sec-type="acknowledgement" id="sec5"><title>Author Contributions</title><p>B.Y. contributed to study design, performed, and/or contributed critically to all experiments, analyzed data, and contributed to writing of the manuscript. In some experiments, B.Y. was assisted by S.R., S.P., T.M.T., and P.D.C. designed, performed, and analyzed the human studies. R.G. performed the western blot experiments. H.S. supervised J.G. in the establishment and maintenance of <italic>Plasmodium</italic>-infected <italic>A.</italic> mosquitoes. A.R. produced and troubleshooted all mAb production. P.J.C. and A.J.F.A. generated <italic>α1,3Gt</italic><sup>−/−</sup> mice. A.S.C. provided anti-α-gal hybridomas. 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Article plus Supplemental Information</title></caption><media xlink:href="mmc1.pdf"></media></supplementary-material></p></sec><ack id="ack0010"><title>Acknowledgments</title><p>The authors thank the Inflammation Group (IGC) for insightful discussions and review of the manuscript, Sofia Rebelo and Silvia Cardoso for mouse breeding and genotyping, Pedro Almada and Nuno Pimpão Martins (IGC Imaging Facility) for technical support, Karen Berman de Ruiz and Joana Bom (IGC Animal Facility) for germ-free breeding, Joana Tavares, Rogerio Amino, and Robert Ménard (Institute Pasteur) for technical support, Alekos Athanasiadis and Jocelyne Demengeot for insightful discussions, Pascal Gagneaux (University of California San Diego), and Daniel Mucida (Rockefeller University) for critical review of the initial version of the manuscript. Financial support from the Bill and Melinda Gates Foundation (OPP1024563), Fundação para a Ciência e Tecnologia (RECI-IMI-IMU-0038-2012), and European Research Council (ERC-2011-AdG 294709-DAMAGECONTROL) (to M.P.S.) and Fundação para a Ciência e a Tecnologia (SFRH/BD/51176/2010) within the PhD Program of Integrative Biomedical Science of the Instituto Gulbenkian de Ciência (to B.Y.) is gratefully acknowledged. The Division of Intramural Research, National Institute of Allergy and Infectious Diseases, and NIH supported the Mali cohort study. Mouse axenization was supported by the EMMA, EU FP7 Capacities Specific Program.</p></ack><fn-group><fn id="d32e830"><p>This is an open access article under the CC BY-NC-ND license (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc-nd/3.0/" id="ccintref0005">http://creativecommons.org/licenses/by-nc-nd/3.0/</ext-link>).</p></fn></fn-group></back><floats-group><fig id="fig1"><label>Figure 1</label><caption><p>Detection of α-Gal in <italic>Plasmodium</italic> Sporozoites</p><p>(A) Composite images of GFP/actin (green), α-gal (red; white arrows), and DNA (blue) in <italic>Plasmodium</italic> sporozoites.</p><p>(B) Same staining as (A), after removal of α-gal by α-galactosidase. Images are representative of 2–3 independent experiments. Scale bar, 5 μm.</p><p>(C) Detection of α-gal in <italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites by flow cytometry, representative of three independent experiments.</p><p>(D) Detection of α-gal in proteins extracted from salivary glands of noninfected (NI), <italic>P. falciparum</italic> 3D7 (<italic>Pf</italic>), <italic>Pb</italic>A<sup>Hsp70-GFP</sup> (<italic>Pb</italic>), or <italic>P. yoelii</italic> 17XNL (<italic>Py</italic>)-infected <italic>A</italic>. mosquitoes. Histone H3 (Hist3) and GFP were detected as loading controls. When indicated, α-gal was digested using α-galactosidase (E).</p><p>(E and F) Detection of α-gal (E) and CSP (F) in <italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites treated or not with phospholipase C (+PLC). Control is not stained. Data representative of 2–4 independent experiments.</p><p>See also <xref rid="figs1" ref-type="fig">Figure S1</xref>.</p></caption><graphic xlink:href="gr1"></graphic></fig><fig id="fig2"><label>Figure 2</label><caption><p>Anti-α-Gal IgM Abs Are Associated with Protection against Malaria Transmission in Individuals from a Malaria Endemic Region</p><p>(A) Anti-α-gal IgM Abs in individuals from a malaria endemic region in Mali (gray dots) or from the United States (black dots). Mean (red bars) ± SD.</p><p>(B) Levels of anti-α-gal IgM Abs in <italic>P. falciparum</italic>-infected (<italic>Pf</italic>+) versus noninfected (<italic>Pf</italic>−) children >4 years of age are shown as box plots in the same population as in (A).</p><p>(C) Anti-α-gal IgG Abs in individuals from a malaria endemic region in Mali (gray dots) or from the United States (black dots). Mean (red bars) ± SD.</p><p>(D) Levels of anti-α-gal IgG Abs in <italic>P. falciparum</italic>-infected (<italic>Pf</italic>+) versus noninfected (<italic>Pf</italic>−) children >4 years of age are shown as box plots in the same population as in (C).</p></caption><graphic xlink:href="gr2"></graphic></fig><fig id="fig3"><label>Figure 3</label><caption><p>Gut Colonization by <italic>E. coli</italic> Expressing α-Gal Protects against <italic>Plasmodium</italic> Infection</p><p>(A and B) Detection of α-gal in <italic>E. coli</italic> strains by (A) flow cytometry and (B) immunofluorescence. Representative of 2–3 independent experiments. Composite images in (B), i.e., α-gal (green) and DNA (blue) at 100× magnification. Scale bar, 10 μm.</p><p>(C and D) <italic>α1,3Gt</italic><sup>−/−</sup> mice maintained under SPF were treated with streptomycin for 7 days. (C) Anti-α-gal IgM Abs levels were measured in <italic>α1,3Gt</italic><sup>−/−</sup> mice not colonized (SPF), colonized with <italic>E. coli</italic> K12, or colonized with O86.B7 strains (2–3 experiments; n = 12). (D) Incidence of blood stage of <italic>Plasmodium</italic> infection (%) in mice colonized as in (C) and exposed to <italic>Pb</italic>A<sup>EEF1a-GFP</sup>-infected <italic>A. stephensi</italic> mosquitoes (four experiments; n = 17–34).</p><p>(E) Incidence of blood stage of <italic>Plasmodium</italic> infection (%) in <italic>α1,3Gt</italic><sup>−/−</sup><italic>J</italic><sub><italic>H</italic></sub><italic>T</italic><sup>−/−</sup>, <italic>α1,3Gt</italic><sup>−/−</sup><italic>Aid</italic><sup>−/−</sup>, and <italic>α1,3Gt</italic><sup>−/−</sup><sc><italic>μ</italic></sc><italic>S</italic><sup>−/−</sup> mice colonized as in (C) and exposed to <italic>Pb</italic>A<sup>Hsp70-GFP</sup>-infected <italic>A. stephensi</italic> mosquitoes (1–2 experiments; n = 4–10).</p><p>(F) Anti-α-gal IgM Abs were measured in GF <italic>α1,3Gt</italic><sup>−/−</sup> mice not colonized (GF), colonized with <italic>E. coli</italic> K12, or colonized with O86.B7 strains (2–3 experiments; n = 12). (G) Incidence of blood stage of <italic>Plasmodium</italic> infection (%) in mice colonized as in (F) and exposed to <italic>Pb</italic>A<sup>EEF1a-GFP</sup>-infected <italic>A. stephensi</italic> mosquitoes (four experiments; n = 9–13).</p><p>Mean (red bars).</p><p>See also <xref rid="figs2" ref-type="fig">Figure S2</xref>.</p></caption><graphic xlink:href="gr3"></graphic></fig><fig id="fig4"><label>Figure 4</label><caption><p>Protective Effect of α-Gal Immunization</p><p>(A) Anti-α-gal Abs in the serum of control (−) versus rRBCM (+) or α-gal-BSA (+) immunized <italic>α1,3Gt</italic><sup>−/−</sup> mice (2–3 experiments; n = 12–29).</p><p>(B–D) Incidence of blood stage of infection (%) in <italic>α1,3Gt</italic><sup>−/−</sup> mice treated as in (A) and exposed to (B) <italic>Pb</italic>A<sup>EEF1a-GFP</sup>-infected <italic>A. stephensi</italic> mosquitoes (seven experiments; n = 27–44), (C) <italic>P. yoelii</italic> 17XNL-infected <italic>A. stephensi</italic> mosquitoes (five experiments; n = 28–39), or (D) <italic>Pb</italic>A<sup>EEF1a-GFP</sup>-infected <italic>A. gambiae</italic> mosquitoes (four experiments; n = 27–34).</p><p>(E) Incidence of blood stage of infection (%) in nonimmunized (control) versus immunized (rRBCM) <italic>α1,3Gt</italic><sup>−/−</sup> mice receiving <italic>Pb</italic>A<sup>EEF1a-GFP</sup> sporozoites (3–4 experiments; n = 17–28).</p><p>(F) <italic>Plasmodium</italic> 18 s rRNA<italic>/Arbp0</italic> mRNA in skin and liver of nonimmunized (control) versus immunized (rRBCM) <italic>α1,3Gt</italic><sup>−/−</sup> mice exposed to <italic>Pb</italic>A<sup>EEF1a-GFP</sup>-infected <italic>A. stephensi</italic> mosquitoes (3–5 experiments). Infected/total mice (gray nbrs).</p><p>(G) Same as (A) in control (−) versus immunized (+; rRBCM emulsified in CFA+CpG) <italic>α1,3Gt</italic><sup>−/−</sup> mice (two experiments; n = 6–23).</p><p>(H) Incidence of blood stage of infection (%) in <italic>α1,3Gt</italic><sup>−/−</sup> mice treated as in (G) and infected as in (B) (three experiments; n = 16–19). In (A), (F), and (G), dots are individual mice and mean (red bars).</p><p>See also <xref rid="figs3" ref-type="fig">Figures S3</xref> and <xref rid="figs4" ref-type="fig">S4</xref>.</p></caption><graphic xlink:href="gr4"></graphic></fig><fig id="fig5"><label>Figure 5</label><caption><p>Protective Effect of Anti-α-Gal Abs</p><p>(A) Relative absorbance of anti-α-gal Abs (Mean ± SD) in serial serum dilutions from nonimmunized (NI) or rRBCM-immunized (I) <italic>α1,3Gt</italic><sup>−/−</sup> mice (two experiments; n = 10).</p><p>(B) Incidence of blood stage infection (%) in specific immune component-deleted <italic>α1,3Gt</italic><sup>−/−</sup> mice immunized (I) or not (NI) as in (A) and exposed to <italic>Pb</italic>A<sup>EEF1a-GFP</sup>-infected mosquitoes (3–7 experiments; n = 13–41).</p><p>(C) Incidence of blood stage of infection (%) in <italic>α1,3Gt</italic><sup>−/−</sup> mice after passive transfer of anti-α-gal Abs versus controls (no passive transfer; ctr.) exposed to <italic>Pb</italic>A<sup>EEF1a-GFP</sup>-infected mosquitoes (4–7 experiments; n = 19–32).</p><p>(D) C3 deposition in <italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites not exposed (ctr.) or exposed to anti-α-gal Abs plus mouse complement (C). Representative of three independent experiments.</p><p>(E) Incidence of blood stage of infection (%) in <italic>α1,3Gt</italic><sup>−/−</sup><italic>C3</italic><sup>−/−</sup> mice after passive transfer of anti-α-gal IgM (μ), IgG2b (γ2b), or IgG3 (γ3) Abs versus controls (ctr.; no passive transfer) not receiving Abs, exposed to <italic>Pb</italic>A<sup>EEF1a-GFP</sup>-infected mosquitoes (four experiments; n = 21–37).</p><p>(F) Same as (E) in PMN-depleted <italic>α1,3Gt</italic><sup><italic>−/−</italic></sup> mice (four experiments; n = 15–25).</p><p>See also <xref rid="figs5" ref-type="fig">Figures S5</xref> and <xref rid="figs6" ref-type="fig">S6</xref>.</p></caption><graphic xlink:href="gr5"></graphic></fig><fig id="fig6"><label>Figure 6</label><caption><p>Protective Effect of Anti-α-Gal Abs against Hepatocyte Infection</p><p>(A) Mean percentage (%) of viable GFP<sup>+</sup><italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites ± STD (3–4 experiments) after exposure in vitro to anti-α-gal or control anti-DNP mAbs in the presence of mouse complement.</p><p>(B and C) Mean percentage (%) of HepG2 cells (B) wounded (Dextran-Red<sup>+</sup>) or (C) invaded (GFP<sup>+</sup>) by <italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites treated as in (A) ± SD (six experiments).</p></caption><graphic xlink:href="gr6"></graphic></fig><fig id="fig7"><label>Figure 7</label><caption><p>Protective Effect of Anti-α-Gal Abs against <italic>Plasmodium</italic> Maturation in Hepatocytes</p><p>(A) Number of EEF per field (dots; 20–23 fields).</p><p>(B) Area of individual EEF (dots) (n = 111–256 EEFs counted in 20–23 fields).</p><p>(C) Total area of EEF (dots) per field (20–23 fields).</p><p>HepG2 cells were incubated with <italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites, previously exposed to anti-α-gal or control anti-DNP mAbs in the presence of complement (A–C).</p><p>See also <xref rid="figs7" ref-type="fig">Figure S7</xref>.</p></caption><graphic xlink:href="gr7"></graphic></fig><fig id="figs1"><label>Figure S1</label><caption><p>Detection of α-Gal in <italic>Plasmodium</italic> Sporozoites, Related to <xref rid="fig1" ref-type="fig">Figure 1</xref></p><p>(A and B) Expression of GFP (<italic>P. berghei</italic> ANKA) and actin (<italic>P. falciparum</italic> 3D7 and <italic>P. yoelli</italic> 17XNL) shown in green, α-gal shown in red and DNA (DAPI) shown in blue. The α-gal epitope was detected with (A) anti-α-gal mAb (M86) or (B) the BSI-B<sub>4</sub> lectin. Representative of 2-3 experiments. Arrows indicate α-gal staining. Scale bar, 5 μm.</p></caption><graphic xlink:href="figs1"></graphic></fig><fig id="figs2"><label>Figure S2</label><caption><p>Anti-α-Gal IgG Abs in Gut-Colonized <italic>α1,3Gt</italic><sup>−/−</sup> Mice and Disease Assessment after Exposure to Infected Mosquitoes, Related to <xref rid="fig3" ref-type="fig">Figure 3</xref></p><p>(A) Anti-α-gal IgG subclass Ab levels in <italic>α1,3Gt</italic><sup>−/−</sup> mice not-colonized (SPF), colonized with <italic>E. coli</italic> K12 or O86.B7 strains after streptomycin treatment (2-3 experiments; n = 12).</p><p>(B) Parasitemia (%) and survival (%) in same mice as (A) after exposure to <italic>Pb</italic>A<sup>EEF1a-GFP</sup> infected <italic>A. stephensi</italic> mosquitoes (4 experiments; n = 17-34).</p><p>(C) Anti-α-gal IgG subclass Ab levels in GF <italic>α1,3Gt</italic><sup>−/−</sup> mice not colonized (GF), colonized with <italic>E. coli</italic> K12 or O86.B7 strains (2-4 experiments; n = 12).</p><p>(D) Parasitemia (%) and survival (%) in same mice as (C) after exposure to <italic>Pb</italic>A<sup>EEF1a-GFP</sup> infected <italic>A. stephensi</italic> mosquitoes (4 experiments; n = 7-10 per group).</p><p>Mean (red bars).</p></caption><graphic xlink:href="figs2"></graphic></fig><fig id="figs3"><label>Figure S3</label><caption><p><italic>α1,3Gt</italic><sup>+/+</sup> Mice Are Not Protected against Malaria Transmission, Related to <xref rid="fig4" ref-type="fig">Figure 4</xref></p><p>(A) Anti-α-gal antibodies in the serum of control (-) versus rRBCM (+) or α-gal-BSA (+) immunized <italic>α1,3Gt</italic><sup>+/+</sup> mice (2-3 experiments; n = 12-16).</p><p>(B–D) Incidence of blood stage of infection (%) in <italic>α1,3Gt</italic><sup>+/+</sup> mice treated as in (A) and exposed to (B) <italic>Pb</italic>A<sup>EEF1a-GFP</sup> infected <italic>A. stephensi</italic> mosquitoes (7 experiments; n = 19-48), (C) <italic>P. yoelii</italic> 17XNL infected <italic>A. stephensi</italic> mosquitoes (5 experiments; n = 26-30) or (D) <italic>Pb</italic>A<sup>EEF1a-GFP</sup> infected <italic>A. gambiae</italic> mosquitoes (4 experiments; n = 27-28).</p><p>(E) Incidence of blood stage of infection (%) in nonimmunized (Control) versus immunized (rRBCM) <italic>α1,3Gt</italic><sup>+/+</sup> mice receiving <italic>Pb</italic>A<sup>EEF1a-GFP</sup> sporozoites (3-4 experiments; n = 15-26).</p><p>(F) <italic>Plasmodium</italic> 18 s rRNA<italic>/Arbp0</italic> mRNA in skin and liver of nonimmunized (Control) versus immunized (rRBCM) <italic>α1,3Gt</italic><sup>+/+</sup> mice exposed to <italic>Pb</italic>A<sup>EEF1a-GFP</sup> infected <italic>Anopheles stephensi</italic> mosquitoes (3-5 experiments). Infected/total mice (gray Nbrs).</p><p>(G) Same as (A) in control (-) versus immunized (+; rRBCM emulsified in CFA plus CpG) <italic>α1,3Gt</italic><sup>+/+</sup> mice (2 experiments; n = 5).</p><p>(H) Same as (B) in mice treated as in (G) (3 experiments; n = 15). In (A, F and G) dots are individual mice and mean (red bars).</p></caption><graphic xlink:href="figs3"></graphic></fig><fig id="figs4"><label>Figure S4</label><caption><p>Immunization against α-Gal Is Ineffective in Protecting against Blood Stage of Infection but Confers Sterile Protection against Malaria Transmission, Related to <xref rid="fig4" ref-type="fig">Figure 4</xref></p><p>(A) Incidence of blood stage of infection (%; left panel), parasitemia (%; middle panel) and survival (%; right panel) upon inoculation of <italic>Pb</italic>A<sup>EEF1a-GFP</sup> infected RBC. Mice were either immunized (I) with rRBCM or not immunized (NI) (One experiment; n = 3-5).</p><p>(B) Schematic representation of infection protocol (left panel) and incidence of blood stage of infection (%; right panel) in <italic>α1,3Gt</italic><sup>−/−</sup> mice transferred with RBC from <italic>α1,3Gt</italic><sup>−/−</sup> mice infected <italic>Pb</italic>A<sup>EEF1a-GFP</sup> or protected from transmission of <italic>Pb</italic>A<sup>EEF1a-GFP</sup> infection by <italic>A. stephensi</italic> mosquitoes (2 experiments; n = 19).</p></caption><graphic xlink:href="figs4"></graphic></fig><fig id="figs5"><label>Figure S5</label><caption><p>Blood Stage Infection and Lethality, Related to <xref rid="fig5" ref-type="fig">Figure 5</xref></p><p>(A–D) Parasitemia (%; left panels) and survival (%; right panels) are shown for infected (A) <italic>α1,3Gt</italic><sup>−/−</sup><italic>Jht</italic><sup>−/−</sup>, (B) <italic>α1,3Gt</italic><sup>−/−</sup><italic>Aid</italic><sup>−/−</sup>, (C) <italic>α1,3Gt</italic><sup>−/−</sup><italic>μS</italic><sup>−/−</sup> and (D) <italic>α1,3Gt</italic><sup>−/−</sup><italic>Tcrβ</italic><sup>−/−</sup> mice immunized with rRBCM (I) <italic>vs</italic>. control nonimmunized (NI) (3-7 experiment; n = 11-22).</p></caption><graphic xlink:href="figs5"></graphic></fig><fig id="figs6"><label>Figure S6</label><caption><p>Specificity of Anti-α-Gal mAbs, Related to <xref rid="fig5" ref-type="fig">Figure 5</xref></p><p>(A) Comparison of relative binding of anti-α-gal mAbs (125 ng/ml of mAb) determined by ELISA using α-gal-BSA as a solid phase antigen.</p><p>(B) Comparison of relative binding capacity of anti-α-gal mAbs (50 μg/ml of mAb) determined by immunofluorescence using <italic>Pb</italic>A<sup>EEF1a-GFP</sup> sporozoites as an antigen. mAb (red), GFP (green) and DNA (blue) staining. Merged images show composite of the three staining. Representative of 2-3 experiments.</p><p>(C) Similar staining as in (<italic>B</italic>) for <italic>Pb</italic>A<sup>EEF1a-GFP</sup> sporozoites treated with α-galactosidase. Composite images are shown with mAb (red), GFP (green) and DNA (blue) staining. White arrows in (B) and (C) indicate binding of different mAb to the α-gal epitope. Scale bar, 5 μm.</p></caption><graphic xlink:href="figs6"></graphic></fig><fig id="figs7"><label>Figure S7</label><caption><p>Cytotoxic Effect of Anti-α-Gal Antibodies, Related to <xref rid="fig7" ref-type="fig">Figure 7</xref></p><p>(A) Mean percentage (%) of viable GFP<sup>+</sup><italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites ± STD (3-4 experiments) after in vitro exposure to anti-α-gal (α-gal) or anti-DNP mAbs in the presence of rabbit complement.</p><p>(B) Mean percentage (%) of viable GFP<sup>+</sup><italic>Pb</italic>A<sup>Hsp70-GFP</sup> sporozoites ± STD (3-4 experiments) after in vitro exposure to anti-α-gal (α-gal) or anti-DNP mAbs in the absence of complement.</p><p>(C) Mean percentage (%) of viable crescent shaped <italic>Pb</italic>A<sup>EEF1a-GFP</sup> sporozoites ± STD (3 experiments) after in vitro exposure to anti-α-gal (α-gal) or anti-DNP (DNP) mAb in the presence of mouse complement.</p></caption><graphic xlink:href="figs7"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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