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HIV-1 Specific IgA Detected in Vaginal Secretions of HIV Uninfected Women Participating in a Microbicide Trial in Southern Africa Are Primarily Directed Toward gp120 and gp140 Specificities

Identifieur interne : 002B20 ( Main/Exploration ); précédent : 002B19; suivant : 002B21

HIV-1 Specific IgA Detected in Vaginal Secretions of HIV Uninfected Women Participating in a Microbicide Trial in Southern Africa Are Primarily Directed Toward gp120 and gp140 Specificities

Auteurs : Kelly E. Seaton [États-Unis] ; Lamar Ballweber [États-Unis] ; Audrey Lan [États-Unis] ; Michele Donathan [États-Unis] ; Sean Hughes [États-Unis] ; Lucia Vojtech [États-Unis] ; M. Anthony Moody [États-Unis] ; Hua-Xin Liao [États-Unis] ; Barton F. Haynes [États-Unis] ; Christine G. Galloway [États-Unis] ; Barbra A. Richardson [États-Unis] ; Salim Abdool Karim [Afrique du Sud, États-Unis] ; Charlene S. Dezzutti [États-Unis] ; M. Juliana Mcelrath [États-Unis] ; Georgia D. Tomaras [États-Unis] ; Florian Hladik [États-Unis]

Source :

RBID : PMC:4108330

Descripteurs français

English descriptors

Abstract

Background

Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines.

Methods and Findings

We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges) for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env) antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035). We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women.

Conclusion

Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.


Url:
DOI: 10.1371/journal.pone.0101863
PubMed: 25054205
PubMed Central: 4108330


Affiliations:


Links toward previous steps (curation, corpus...)


Le document en format XML

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<name sortKey="Mcelrath, M Juliana" sort="Mcelrath, M Juliana" uniqKey="Mcelrath M" first="M. Juliana" last="Mcelrath">M. Juliana Mcelrath</name>
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<name sortKey="Tomaras, Georgia D" sort="Tomaras, Georgia D" uniqKey="Tomaras G" first="Georgia D." last="Tomaras">Georgia D. Tomaras</name>
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<wicri:regionArea>Department of Medicine, University of Washington, Seattle, Washington</wicri:regionArea>
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</author>
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<addr-line>Duke Human Vaccine Institute, Durham, North Carolina, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Duke Human Vaccine Institute, Durham, North Carolina</wicri:regionArea>
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<region type="state">Caroline du Nord</region>
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</affiliation>
</author>
<author>
<name sortKey="Galloway, Christine G" sort="Galloway, Christine G" uniqKey="Galloway C" first="Christine G." last="Galloway">Christine G. Galloway</name>
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<nlm:aff id="aff3">
<addr-line>Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington</wicri:regionArea>
<placeName>
<region type="state">Washington (État)</region>
</placeName>
</affiliation>
</author>
<author>
<name sortKey="Richardson, Barbra A" sort="Richardson, Barbra A" uniqKey="Richardson B" first="Barbra A." last="Richardson">Barbra A. Richardson</name>
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<addr-line>Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington</wicri:regionArea>
<placeName>
<region type="state">Washington (État)</region>
</placeName>
</affiliation>
<affiliation wicri:level="4">
<nlm:aff id="aff4">
<addr-line>Department of Biostatistics, University of Washington, Seattle, Washington, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Department of Biostatistics, University of Washington, Seattle, Washington</wicri:regionArea>
<placeName>
<region type="state">Washington (État)</region>
<settlement type="city">Seattle</settlement>
</placeName>
<orgName type="university">Université de Washington</orgName>
</affiliation>
</author>
<author>
<name sortKey="Karim, Salim Abdool" sort="Karim, Salim Abdool" uniqKey="Karim S" first="Salim Abdool" last="Karim">Salim Abdool Karim</name>
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<nlm:aff id="aff5">
<addr-line>CAPRISA - Centre for the AIDS Programme of Research in South Africa, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa</addr-line>
</nlm:aff>
<country xml:lang="fr">Afrique du Sud</country>
<wicri:regionArea>CAPRISA - Centre for the AIDS Programme of Research in South Africa, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban</wicri:regionArea>
<wicri:noRegion>Durban</wicri:noRegion>
</affiliation>
<affiliation wicri:level="4">
<nlm:aff id="aff6">
<addr-line>Department of Epidemiology, Columbia University, New York, New York, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Department of Epidemiology, Columbia University, New York, New York</wicri:regionArea>
<placeName>
<region type="state">État de New York</region>
<settlement type="city">New York</settlement>
</placeName>
<orgName type="university">Université Columbia</orgName>
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</author>
<author>
<name sortKey="Dezzutti, Charlene S" sort="Dezzutti, Charlene S" uniqKey="Dezzutti C" first="Charlene S." last="Dezzutti">Charlene S. Dezzutti</name>
<affiliation wicri:level="2">
<nlm:aff id="aff7">
<addr-line>Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania</wicri:regionArea>
<placeName>
<region type="state">Pennsylvanie</region>
</placeName>
</affiliation>
</author>
<author>
<name sortKey="Mcelrath, M Juliana" sort="Mcelrath, M Juliana" uniqKey="Mcelrath M" first="M. Juliana" last="Mcelrath">M. Juliana Mcelrath</name>
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<nlm:aff id="aff3">
<addr-line>Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington</wicri:regionArea>
<placeName>
<region type="state">Washington (État)</region>
</placeName>
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<affiliation wicri:level="4">
<nlm:aff id="aff8">
<addr-line>Department of Medicine, University of Washington, Seattle, Washington, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Department of Medicine, University of Washington, Seattle, Washington</wicri:regionArea>
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<region type="state">Washington (État)</region>
<settlement type="city">Seattle</settlement>
</placeName>
<orgName type="university">Université de Washington</orgName>
</affiliation>
<affiliation wicri:level="4">
<nlm:aff id="aff9">
<addr-line>Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Department of Laboratory Medicine, University of Washington, Seattle, Washington</wicri:regionArea>
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<region type="state">Washington (État)</region>
<settlement type="city">Seattle</settlement>
</placeName>
<orgName type="university">Université de Washington</orgName>
</affiliation>
<affiliation wicri:level="4">
<nlm:aff id="aff10">
<addr-line>Department of Global Health, University of Washington, Seattle, Washington, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Department of Global Health, University of Washington, Seattle, Washington</wicri:regionArea>
<placeName>
<region type="state">Washington (État)</region>
<settlement type="city">Seattle</settlement>
</placeName>
<orgName type="university">Université de Washington</orgName>
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<name sortKey="Tomaras, Georgia D" sort="Tomaras, Georgia D" uniqKey="Tomaras G" first="Georgia D." last="Tomaras">Georgia D. Tomaras</name>
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<nlm:aff id="aff1">
<addr-line>Duke Human Vaccine Institute, Durham, North Carolina, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Duke Human Vaccine Institute, Durham, North Carolina</wicri:regionArea>
<placeName>
<region type="state">Caroline du Nord</region>
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</affiliation>
</author>
<author>
<name sortKey="Hladik, Florian" sort="Hladik, Florian" uniqKey="Hladik F" first="Florian" last="Hladik">Florian Hladik</name>
<affiliation wicri:level="4">
<nlm:aff id="aff2">
<addr-line>Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington</wicri:regionArea>
<placeName>
<region type="state">Washington (État)</region>
<settlement type="city">Seattle</settlement>
</placeName>
<orgName type="university">Université de Washington</orgName>
</affiliation>
<affiliation wicri:level="2">
<nlm:aff id="aff3">
<addr-line>Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington</wicri:regionArea>
<placeName>
<region type="state">Washington (État)</region>
</placeName>
</affiliation>
<affiliation wicri:level="4">
<nlm:aff id="aff8">
<addr-line>Department of Medicine, University of Washington, Seattle, Washington, United States of America</addr-line>
</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Department of Medicine, University of Washington, Seattle, Washington</wicri:regionArea>
<placeName>
<region type="state">Washington (État)</region>
<settlement type="city">Seattle</settlement>
</placeName>
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</analytic>
<series>
<title level="j">PLoS ONE</title>
<idno type="eISSN">1932-6203</idno>
<imprint>
<date when="2014">2014</date>
</imprint>
</series>
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<keywords scheme="KwdEn" xml:lang="en">
<term>Analysis of Variance</term>
<term>Anti-Infective Agents (immunology)</term>
<term>Anti-Infective Agents (therapeutic use)</term>
<term>Bodily Secretions (immunology)</term>
<term>Bodily Secretions (virology)</term>
<term>Cohort Studies</term>
<term>Female</term>
<term>HIV Antibodies (immunology)</term>
<term>HIV Envelope Protein gp120 (immunology)</term>
<term>HIV Infections (diagnosis)</term>
<term>HIV Infections (immunology)</term>
<term>HIV Infections (prevention & control)</term>
<term>HIV Seronegativity (immunology)</term>
<term>HIV Seropositivity (immunology)</term>
<term>HIV-1 (drug effects)</term>
<term>HIV-1 (immunology)</term>
<term>Humans</term>
<term>Immunity, Mucosal (drug effects)</term>
<term>Immunity, Mucosal (immunology)</term>
<term>Immunoglobulin A (immunology)</term>
<term>Immunoglobulin G (immunology)</term>
<term>Pilot Projects</term>
<term>South Africa</term>
<term>Specimen Handling (instrumentation)</term>
<term>Specimen Handling (methods)</term>
<term>Vagina (immunology)</term>
<term>Vagina (virology)</term>
<term>env Gene Products, Human Immunodeficiency Virus (immunology)</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr">
<term>Analyse de variance</term>
<term>Anti-infectieux (immunologie)</term>
<term>Anti-infectieux (usage thérapeutique)</term>
<term>Anticorps anti-VIH (immunologie)</term>
<term>Femelle</term>
<term>Humains</term>
<term>Immunité muqueuse ()</term>
<term>Immunité muqueuse (immunologie)</term>
<term>Immunoglobuline A (immunologie)</term>
<term>Immunoglobuline G (immunologie)</term>
<term>Infections à VIH ()</term>
<term>Infections à VIH (diagnostic)</term>
<term>Infections à VIH (immunologie)</term>
<term>Produits du gène env du virus de l'immunodéficience humaine (immunologie)</term>
<term>Projets pilotes</term>
<term>Protéine d'enveloppe gp120 du VIH (immunologie)</term>
<term>Prélèvement biologique ()</term>
<term>Prélèvement biologique (instrumentation)</term>
<term>République d'Afrique du Sud</term>
<term>Sécrétions corporelles (immunologie)</term>
<term>Sécrétions corporelles (virologie)</term>
<term>Séronégativité VIH (immunologie)</term>
<term>Séropositivité VIH (immunologie)</term>
<term>VIH-1 (Virus de l'Immunodéficience Humaine de type 1) ()</term>
<term>VIH-1 (Virus de l'Immunodéficience Humaine de type 1) (immunologie)</term>
<term>Vagin (immunologie)</term>
<term>Vagin (virologie)</term>
<term>Études de cohortes</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en">
<term>Anti-Infective Agents</term>
<term>HIV Antibodies</term>
<term>HIV Envelope Protein gp120</term>
<term>Immunoglobulin A</term>
<term>Immunoglobulin G</term>
<term>env Gene Products, Human Immunodeficiency Virus</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="therapeutic use" xml:lang="en">
<term>Anti-Infective Agents</term>
</keywords>
<keywords scheme="MESH" type="geographic" xml:lang="en">
<term>South Africa</term>
</keywords>
<keywords scheme="MESH" qualifier="diagnosis" xml:lang="en">
<term>HIV Infections</term>
</keywords>
<keywords scheme="MESH" qualifier="diagnostic" xml:lang="fr">
<term>Infections à VIH</term>
</keywords>
<keywords scheme="MESH" qualifier="drug effects" xml:lang="en">
<term>HIV-1</term>
<term>Immunity, Mucosal</term>
</keywords>
<keywords scheme="MESH" qualifier="immunologie" xml:lang="fr">
<term>Anti-infectieux</term>
<term>Anticorps anti-VIH</term>
<term>Immunité muqueuse</term>
<term>Immunoglobuline A</term>
<term>Immunoglobuline G</term>
<term>Infections à VIH</term>
<term>Produits du gène env du virus de l'immunodéficience humaine</term>
<term>Protéine d'enveloppe gp120 du VIH</term>
<term>Sécrétions corporelles</term>
<term>Séronégativité VIH</term>
<term>Séropositivité VIH</term>
<term>VIH-1 (Virus de l'Immunodéficience Humaine de type 1)</term>
<term>Vagin</term>
</keywords>
<keywords scheme="MESH" qualifier="immunology" xml:lang="en">
<term>Bodily Secretions</term>
<term>HIV Infections</term>
<term>HIV Seronegativity</term>
<term>HIV Seropositivity</term>
<term>HIV-1</term>
<term>Immunity, Mucosal</term>
<term>Vagina</term>
</keywords>
<keywords scheme="MESH" qualifier="instrumentation" xml:lang="en">
<term>Specimen Handling</term>
</keywords>
<keywords scheme="MESH" qualifier="methods" xml:lang="en">
<term>Specimen Handling</term>
</keywords>
<keywords scheme="MESH" qualifier="prevention & control" xml:lang="en">
<term>HIV Infections</term>
</keywords>
<keywords scheme="MESH" qualifier="usage thérapeutique" xml:lang="fr">
<term>Anti-infectieux</term>
<term>Prélèvement biologique</term>
</keywords>
<keywords scheme="MESH" qualifier="virologie" xml:lang="fr">
<term>Sécrétions corporelles</term>
<term>Vagin</term>
</keywords>
<keywords scheme="MESH" qualifier="virology" xml:lang="en">
<term>Bodily Secretions</term>
<term>Vagina</term>
</keywords>
<keywords scheme="MESH" xml:lang="en">
<term>Analysis of Variance</term>
<term>Cohort Studies</term>
<term>Female</term>
<term>Humans</term>
<term>Pilot Projects</term>
</keywords>
<keywords scheme="MESH" xml:lang="fr">
<term>Analyse de variance</term>
<term>Femelle</term>
<term>Humains</term>
<term>Immunité muqueuse</term>
<term>Infections à VIH</term>
<term>Projets pilotes</term>
<term>Prélèvement biologique</term>
<term>République d'Afrique du Sud</term>
<term>VIH-1 (Virus de l'Immunodéficience Humaine de type 1)</term>
<term>Études de cohortes</term>
</keywords>
<keywords scheme="Wicri" type="geographic" xml:lang="fr">
<term>Afrique du Sud</term>
</keywords>
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</teiHeader>
<front>
<div type="abstract" xml:lang="en">
<sec>
<title>Background</title>
<p>Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines.</p>
</sec>
<sec>
<title>Methods and Findings</title>
<p>We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges) for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env) antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035). We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women.</p>
</sec>
<sec>
<title>Conclusion</title>
<p>Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.</p>
</sec>
</div>
</front>
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<author>
<name sortKey="Moodie, Z" uniqKey="Moodie Z">Z Moodie</name>
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<author>
<name sortKey="Dubey, S" uniqKey="Dubey S">S Dubey</name>
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<author>
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<country>
<li>Afrique du Sud</li>
<li>États-Unis</li>
</country>
<region>
<li>Caroline du Nord</li>
<li>Pennsylvanie</li>
<li>Washington (État)</li>
<li>État de New York</li>
</region>
<settlement>
<li>New York</li>
<li>Seattle</li>
</settlement>
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<li>Université Columbia</li>
<li>Université de Washington</li>
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<name sortKey="Seaton, Kelly E" sort="Seaton, Kelly E" uniqKey="Seaton K" first="Kelly E." last="Seaton">Kelly E. Seaton</name>
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<name sortKey="Galloway, Christine G" sort="Galloway, Christine G" uniqKey="Galloway C" first="Christine G." last="Galloway">Christine G. Galloway</name>
<name sortKey="Haynes, Barton F" sort="Haynes, Barton F" uniqKey="Haynes B" first="Barton F." last="Haynes">Barton F. Haynes</name>
<name sortKey="Hladik, Florian" sort="Hladik, Florian" uniqKey="Hladik F" first="Florian" last="Hladik">Florian Hladik</name>
<name sortKey="Hladik, Florian" sort="Hladik, Florian" uniqKey="Hladik F" first="Florian" last="Hladik">Florian Hladik</name>
<name sortKey="Hladik, Florian" sort="Hladik, Florian" uniqKey="Hladik F" first="Florian" last="Hladik">Florian Hladik</name>
<name sortKey="Hughes, Sean" sort="Hughes, Sean" uniqKey="Hughes S" first="Sean" last="Hughes">Sean Hughes</name>
<name sortKey="Karim, Salim Abdool" sort="Karim, Salim Abdool" uniqKey="Karim S" first="Salim Abdool" last="Karim">Salim Abdool Karim</name>
<name sortKey="Lan, Audrey" sort="Lan, Audrey" uniqKey="Lan A" first="Audrey" last="Lan">Audrey Lan</name>
<name sortKey="Liao, Hua Xin" sort="Liao, Hua Xin" uniqKey="Liao H" first="Hua-Xin" last="Liao">Hua-Xin Liao</name>
<name sortKey="Mcelrath, M Juliana" sort="Mcelrath, M Juliana" uniqKey="Mcelrath M" first="M. Juliana" last="Mcelrath">M. Juliana Mcelrath</name>
<name sortKey="Mcelrath, M Juliana" sort="Mcelrath, M Juliana" uniqKey="Mcelrath M" first="M. Juliana" last="Mcelrath">M. Juliana Mcelrath</name>
<name sortKey="Mcelrath, M Juliana" sort="Mcelrath, M Juliana" uniqKey="Mcelrath M" first="M. Juliana" last="Mcelrath">M. Juliana Mcelrath</name>
<name sortKey="Mcelrath, M Juliana" sort="Mcelrath, M Juliana" uniqKey="Mcelrath M" first="M. Juliana" last="Mcelrath">M. Juliana Mcelrath</name>
<name sortKey="Moody, M Anthony" sort="Moody, M Anthony" uniqKey="Moody M" first="M. Anthony" last="Moody">M. Anthony Moody</name>
<name sortKey="Richardson, Barbra A" sort="Richardson, Barbra A" uniqKey="Richardson B" first="Barbra A." last="Richardson">Barbra A. Richardson</name>
<name sortKey="Richardson, Barbra A" sort="Richardson, Barbra A" uniqKey="Richardson B" first="Barbra A." last="Richardson">Barbra A. Richardson</name>
<name sortKey="Tomaras, Georgia D" sort="Tomaras, Georgia D" uniqKey="Tomaras G" first="Georgia D." last="Tomaras">Georgia D. Tomaras</name>
<name sortKey="Vojtech, Lucia" sort="Vojtech, Lucia" uniqKey="Vojtech L" first="Lucia" last="Vojtech">Lucia Vojtech</name>
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<name sortKey="Karim, Salim Abdool" sort="Karim, Salim Abdool" uniqKey="Karim S" first="Salim Abdool" last="Karim">Salim Abdool Karim</name>
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</record>

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