SummaryMany microbes are surrounded by phagocytosisinhibiting capsules. We took advantage of the large size of the polysaccharide capsule of the pathogenic yeast Cryptococcus neoformans to examine capsular architecture and the relationship between molecular architecture and the interaction of the capsule with potentially opsonic serum proteins. Our experimental design used complementary approaches in which (i) assessment of permeability to macromolecules of different Stokes radii; (ii) determination of the binding of Fab fragments of anticapsular antibodies as a measure of matrix density; (iii) capsular deconstruction by treatment with dimethyl sulphoxide; and (iv) evaluation of capsule plasticity, were used to probe the molecular structure of the capsule. The results showed that the capsule is a matrix with a variable porosity that increases with distance from the cell wall. A high density of the matrix at the capsule interior prevents penetration of large macromolecules to sites near the cell wall. In contrast, the capsular edge that is the interface with phagocytes presents capsular polysaccharide in a very low density that exhibits considerable plasticity and permeability to macromolecules. Notably, the capsule of yeast cells harvested from infected tissue showed a greater matrix density than yeast cells grown in vitro under capsule induction conditions.

Bacillus anthracis is surrounded by an antiphagocytic polypeptide capsule composed of poly ␥-D-glutamic acid (␥DPGA). ␥DPGA has been identified recently as a potential target for vaccine development. Studies of the role of ␥DPGA in disease have been hampered by the poor Ab response to this antigen and the lack of immunochemical reagents. As a consequence, neither the extent of ␥DPGA production during anthrax nor the protective activity of ␥DPGA Abs in inhalation anthrax are known. Here we report production of IgG Abs to ␥DPGA in mice following an immunization regimen using ␥DPGA in combination with agonist mAbs to CD40. mAbs were produced that are specific for ␥DPGA. Passive immunization with ␥DPGA mAbs protected >90% of mice in a pulmonary model of anthrax that was lethal in control mice (P < 0.0001). Use of ␥DPGA mAb in an antigen detection immunoassay found that the appearance of ␥DPGA in serum coincided with the emergence of bacteremia. These studies identify CD40 stimulation as a means for production of Ab and generation of mAbs against a weakly immunogenic antigen and demonstrate that the capsule is an effective target for immunoprotection and for antigen detection in the diagnosis of anthrax.

Serotype-specific antibodies to pneumococcal capsular polysaccharide (PPS) are a critical component of vaccine-mediated immunity to Streptococcus pneumoniae. In this study, we investigated the in vitro opsonophagocytic activities of three PPS-specific mouse immunoglobulin G1 monoclonal antibodies (MAbs), 1E2, 5F6, and 7A9, and determined their in vivo efficacies against intranasal challenge with WU2, a serotype 3 pneumococcal strain, in normal and immunodeficient mice. The MAbs had different in vitro activities in a pneumococcal killing assay: 7A9 enhanced killing by mouse neutrophils and J774 cells in the presence of a complement source, whereas 5F6 promoted killing in the absence, but not the presence, of complement, and 1E2 did not promote killing under any conditions. Nonetheless, all three MAbs protected normal and complement component 3-deficient mice from a lethal intranasal challenge with WU2 in passive-immunization experiments in which 10 g of the MAbs were administered intraperitoneally before intranasal challenge. In contrast, only 1E2 protected Fc␥ receptor IIB knockout (Fc␥RIIB KO) mice and mice that were depleted of neutrophils with the MAb RB6, whereas 7A9 and 5F6 required neutrophils and Fc␥RIIB to mediate protection. Conversely, 7A9 and 5F6 protected Fc␥R KO mice, but 1E2 did not. Hence, the efficacy of 1E2 required an activating Fc␥R(s), whereas 5F6 and 7A9 required the inhibitory Fc␥R (Fc␥RIIB). Taken together, our data demonstrate that both MAbs that do and do not promote pneumococcal killing in vitro can mediate protection in vivo, although their efficacies depend on different host receptors and/or components.