title: Enhanced immunogenicity of pneumococcal surface adhesin A (PsaA) in mice via fusion to recombinant human B lymphocyte stimulator (BLyS).
abstract: BACKGROUND
  B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of ligands that mediates its action through three known receptors. BLyS has been shown to enhance the production of antibodies against heterologous antigens when present at elevated concentrations, supporting an immunostimulatory role for BLyS in vivo.
  
  
  METHODS
  We constructed a fusion protein consisting of human BLyS and Pneumococcal Surface Adhesin A (PsaA) and used this molecule to immunize mice. The immunostimulatory attributes mediated by BLyS in vivo were evaluated by characterizing immune responses directed against PsaA.
  
  
  RESULTS
  The PsaA-BLyS fusion protein was able to act as a co-stimulant for murine spleen cell proliferation induced with F(ab')₂ fragments of anti-IgM in vitro in a fashion similar to recombinant BLyS, and immunization of mice with the PsaA-BLyS fusion protein resulted in dramatically elevated serum antibodies specific for PsaA. Mice immunized with PsaA admixed with recombinant BLyS exhibited only modest elevations in PsaA-specific responses following two immunizations, while mice immunized twice with PsaA alone exhibited undetectable PsaA-specific serum antibody responses. Sera obtained from PsaA-BLyS immunized mice exhibited high titers of IgG1, IgG2a, IgG2b, and IgG3, but no IgA, while mice immunized with PsaA admixed with BLyS exhibited only elevated titers of IgG1 following two immunizations. Splenocytes from PsaA-BLyS immunized mice exhibited elevated levels of secretion of IL-2, IL-4 and IL-5, and a very modest but consistent elevation of IFN-γ following in vitro stimulation with PsaA. In contrast, mice immunized with either PsaA admixed with BLyS or PsaA alone exhibited modestly elevated to absent PsaA-specific recall responses for the same cytokines. Mice deficient for one of the three receptors for BLyS designated Transmembrane activator, calcium modulator, and cyclophilin ligand [CAML] interactor (TACI) exhibited attenuated PsaA-specific serum antibody responses following immunization with PsaA-BLyS relative to wild-type littermates. TACI-deficient mice also exhibited decreased responsiveness to a standard pneumococcal conjugate vaccine.
  
  
  CONCLUSIONS
  This study identifies covalent attachment of BLyS as a highly effective adjuvant strategy that may yield improved vaccines. In addition, this is the first report demonstrating an unexpected role for TACI in the elicitation of antibodies by the PsaA-BLyS fusion protein.
  
  
  METHODS
  This article was reviewed by Jonathan Yewdell, Rachel Gerstein, and Michael Cancro (nominated by Andy Caton).
authors.affiliation: Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4943, USA.
authors.forename: Dennis O
authors.initials: DO
authors.lastname: Gor
authors.forename: Xuedong
authors.initials: X
authors.lastname: Ding
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.forename: Dilara
authors.initials: D
authors.lastname: Sultana
authors.forename: Salamatu S
authors.initials: SS
authors.lastname: Mambula
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Bram
authors.forename: Neil S
authors.initials: NS
authors.lastname: Greenspan
mesh_terms: D018829:Adhesins, Bacterial / Q000276:immunology*; D000818:Animals; D000917:Antibody Formation / Q000276:immunology; D053264:B-Cell Activating Factor / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology*; D016207:Cytokines / Q000096:biosynthesis; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007114:Immunization; D007136:Immunoglobulins / Q000276:immunology; D007156:Immunologic Memory; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000302:isolation & purification; D053303:Transmembrane Activator and CAML Interactor Protein / Q000172:deficiency / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D053264:B-Cell Activating Factor; D001426:Bacterial Proteins; D016207:Cytokines; D000939:Epitopes; D007136:Immunoglobulins; D011993:Recombinant Fusion Proteins; C486361:Tnfrsf13b protein, mouse; D053303:Transmembrane Activator and CAML Interactor Protein; C051592:pneumococcal surface protein A