Genetic fusion of three tandem copies of murine C3d sequences to diphtheria toxin fragment B elicits a decreased fragment B-specific antibody response.

Previous studies have demonstrated that the covalent modification of target protein and polysaccharide antigens with the activated complement product C3d results in dramatically enhanced immunogenicity of the target antigens. In this paper, we describe our attempts to enhance the immunogenicity of the non-toxic B fragment of diphtheria toxin (DT-B) by genetic fusion to polypeptides derived from the C3d coding sequence. Contrary to expectations, we found that the antibody responses elicited by immunizing mice with DT-B genetically linked to three tandem copies of C3d-derived sequences were markedly reduced relative to the antibody responses elicited by immunizing mice with DT-B alone. These results demonstrate levels of complexity in the immunomodulatory effects of the complement system that were not apparent in earlier reports on the adjuvant effects of C3d administered to mice as genetic fusions to target antigens, such as hen egg lysozyme, human immunodeficiency virus (HIV) gp120 or influenza virus hemaglutinnin. The data presented herein suggest that C3d may act as a negative regulator, in some immunological contexts, for antibody production in the mammalian host.

Enhanced immunogenicity of pneumococcal surface adhesin A (PsaA) in mice via fusion to recombinant human B lymphocyte stimulator (BLyS).

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. CONCLUSION: 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. REVIEWERS: This article was reviewed by Jonathan Yewdell, Rachel Gerstein, and Michael Cancro (nominated by Andy Caton).

Evaluation of antibody responses elicited by immunization of mice with a pneumococcal antigen genetically fused to murine HSP70 and murine interleukin-4.

The heat shock (stress) protein HSP70 has been shown to be a potent stimulator of cellular immune responses. In order to determine whether HSP70 has the ability to stimulate antibody responses, we constructed and expressed fusion proteins consisting of murine HSP70 or murine interleukin (IL)-4 covalently linked to a pneumococcal cell wall-associated protein antigen designated PpmA. Immunization of mice with the PpmA-HSP70 fusion protein (PpmA-70) failed to elicit an increased PpmA-specific serum antibody response. In contrast, mice immunized with PpmA fused to IL-4 (PpmA-IL4), or PpmA fused to both IL-4 and HSP70 (PpmA-IL4-70) fusion proteins elicited high levels of PpmA-specific antibody responses. These data suggest that HSP70 has a limited capacity to stimulate immune responses to heterologous antigens in vivo.

Relationship between surface accessibility for PpmA, PsaA, and PspA and antibody-mediated immunity to systemic infection by Streptococcus pneumoniae.

Antibodies to capsular polysaccharide (PS) are protective against systemic infection by Streptococcus pneumoniae, but the large number of pneumococcal serogroups and the age-related immunogenicity of pure PS limit the utility of PS-based vaccines. In contrast, cell wall-associated proteins from different capsular serotypes can be cross-reactive and immunogenic in all age groups. Therefore, we evaluated three pneumococcal proteins with respect to relative accessibility to antibody, in the context of intact pneumococci, and their ability to elicit protection against systemic infection by encapsulated S. pneumoniae. Sequences encoding pneumococcal surface adhesin A (PsaA), putative protease maturation protein A (PpmA), and the N-terminal region of pneumococcal surface protein A (PspA) from S. pneumoniae strain A66.1 were cloned and expressed in Escherichia coli. The presence of genes encoding PsaA, PpmA, and PspA in 11 clinical isolates was examined by PCR, and the expression of these proteins by each strain was examined by Western blotting with antisera raised to the respective recombinant proteins. We used flow cytometry to demonstrate that PspA was readily detectable on the surface of the pneumococcal strains analyzed, whereas PsaA and PpmA were not. Consistent with these observations, mice with passively or actively acquired antibodies to PspA or type 3 PS were equivalently protected from homologous systemic challenge with type 3 pneumococci, whereas mice with passively or actively acquired antibodies to PsaA or PpmA were not effectively protected. These experiments support the hypothesis that the extent of protection against systemic pneumococcal infection is influenced by target antigen accessibility to circulating host antibodies.

Complement component C3d-antigen complexes can either augment or inhibit B lymphocyte activation and humoral immunity in mice depending on the degree of CD21/CD19 complex engagement.

C3d can function as a molecular adjuvant by binding CD21 and thereby enhancing B cell activation and humoral immune responses. However, recent studies suggest both positive and negative roles for C3d and the CD19/CD21 signaling complex in regulating humoral immunity. To address whether signaling through the CD19/CD21 complex can negatively regulate B cell function when engaged by physiological ligands, diphtheria toxin (DT)-C3d fusion protein and C3dg-streptavidin (SA) complexes were used to assess the role of CD21 during BCR-induced activation and in vivo immune responses. Immunization of mice with DT-C3d3 significantly reduced DT-specific Ab responses independently of CD21 expression or signaling. By contrast, SA-C3dg tetramers dramatically enhanced anti-SA responses when used at low doses, whereas 10-fold higher doses did not augment immune responses, except in CD21/35-deficient mice. Likewise, SA-C3dg (1 microg/ml) dramatically enhanced BCR-induced intracellular calcium concentration ([Ca2+]i) responses in vitro, but had no effect or inhibited [Ca2+]i responses when used at 10- to 50-fold higher concentrations. SA-C3dg enhancement of BCR-induced [Ca2+]i responses required CD21 and CD19 expression and resulted in significantly enhanced CD19 and Lyn phosphorylation, with enhanced Lyn/CD19 associations. BCR-induced CD22 phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1/CD22 associations were also reduced, suggesting abrogation of negative regulatory signaling. By contrast, CD19/CD21 ligation using higher concentrations of SA-C3dg significantly inhibited BCR-induced [Ca2+]i responses and inhibited CD19, Lyn, CD22, and Syk phosphorylation. Therefore, C3d may enhance or inhibit Ag-specific humoral immune responses through both CD21-dependent and -independent mechanisms depending on the concentration and nature of the Ag-C3d complexes.

Enhanced immunogenicity of pneumococcal surface adhesin A by genetic fusion to cytokines and evaluation of protective immunity in mice.

Immunization of mice with pneumococcal surface adhesin A (PsaA) emulsified in complete Freund's adjuvant (CFA) provides protection against systemic infection with Streptococcus pneumoniae. Because the use of CFA is not acceptable in humans, we sought to develop alternative means of enhancing the immunogenicity of protein antigens of potential use in pneumococcal vaccines. We designed a series of genetic constructs in which coding sequences for PsaA were linked to sequences encoding either murine interleukin-2 (mIL-2), mIL-4, or two copies of an immunostimulatory nonapeptide derived from mIL-1beta. The PsaA-cytokine constructs were cloned and expressed in Escherichia coli. Mice immunized twice with PsaA-IL-2, or PsaA-IL-4 responded with PsaA-specific antibody production comparable in magnitude to that of mice primed with PsaA in CFA and boosted with PsaA in incomplete Freund's adjuvant (PsaA-Adj). Antibodies elicited by PsaA-Adj were predominantly of the immunoglobulin G1 (IgG1) subclass, while PsaA-IL-2 and PsaA-IL-4 elicited substantial amounts of IgG2a in addition to IgG1. Mice immunized with PsaA-Adj or PsaA-IL-4 were partially protected against intraperitoneal challenge with virulent S. pneumoniae (30% overall survival beyond 15 days postchallenge). Mice immunized with PsaA and no adjuvant or PsaA-IL-2 exhibited 0 or 5% survival rates, respectively, following challenge. In contrast, mice immunized twice with capsular polysaccharide were 100% protected. The modest levels of protection seen in mice immunized with PsaA and its more immunogenic derivatives may be explained in part by the relative inaccessibility of antibody to PsaA on the surface of encapsulated S. pneumoniae.