title: Purification and functional characterization of mucosal IgA from vaccinated and SIV-infected rhesus macaques.
abstract: Vaccine-induced mucosal antibodies are often evaluated using small volumes of secretory fluids. However, fecal matter containing mucosal IgA is abundant. We purified fecal IgA from five SIV-vaccinated and five SIV-infected rhesus macaques by sequential affinity chromatography. The purified IgA was dimeric by native PAGE, contained secretory component, and was analogous to IgA in colostrum and vaginal fluid by western blot. IgA from one infected and four vaccinated animals neutralized H9-derived SIV(mac)251 with IC(50)s as low as 1 μg/mL. Purified IgAs inhibited transcytosis and exhibited phagocytic activity, the latter significantly correlated with SIV(mac)251 Env-specific IgA in the purified samples. Among different affinity resins, peptide M was optimal compared to jacalin, anti-monkey IgA and SSL7 for IgA purification, as confirmed using tandem peptide M/anti-monkey IgA columns. Fecal IgA provided material sufficient for several assays relevant to protective efficacy, and was shown to be multifunctional. Our approach is potentially applicable to human clinical studies.
authors.affiliation: Vaccine Branch, NCI, NIH, Bethesda, MD, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Musich
authors.affiliation: Vaccine Branch, NCI, NIH, Bethesda, MD, USA.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Demberg
authors.affiliation: Vaccine Branch, NCI, NIH, Bethesda, MD, USA.
authors.forename: Ian L
authors.initials: IL
authors.lastname: Morgan
authors.affiliation: Frederick National Laboratory for Cancer Research, Frederick, MD, USA.
authors.forename: Jacob D
authors.initials: JD
authors.lastname: Estes
authors.affiliation: Vaccine Branch, NCI, NIH, Bethesda, MD, USA.
authors.forename: Genoveffa
authors.initials: G
authors.lastname: Franchini
authors.affiliation: Vaccine Branch, NCI, NIH, Bethesda, MD, USA. Electronic address: guroffm@mail.nih.gov.
authors.forename: Marjorie
authors.initials: M
authors.lastname: Robert-Guroff
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing; D002460:Cell Line; D004731:Endotoxins; D005243:Feces / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D008253:Macaca mulatta; D009000:Monocytes; D010587:Phagocytosis; D018449:SAIDS Vaccines / Q000276:immunology*; D016097:Simian Acquired Immunodeficiency Syndrome / Q000276:immunology*; D015302:Simian Immunodeficiency Virus / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D004731:Endotoxins; D007070:Immunoglobulin A; D018449:SAIDS Vaccines
keywords: Feces; Neutralization; Non-human primate mucosal IgA; Peptide M; Phagocytosis; Transcytosis inhibition
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