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Francisella tularensis is the causative agent of tularemia, a zoonotic bacterial infection that is often fatal if not diagnosed and treated promptly. Natural infection in humans is relatively rare, yet persistence in animal reservoirs, arthropod vectors, and water sources combined with a low level of clinical recognition make tularemia a serious potential threat to public health in endemic areas. F. tularensis has also garnered attention as a potential bioterror threat, as widespread dissemination could have devastating consequences on a population. A low infectious dose combined with a wide range of symptoms and a short incubation period makes timely diagnosis of tularemia difficult. Current diagnostic techniques include bacterial culture of patient samples, PCR and serological assays, however, these techniques are time consuming and require technical expertise that may not be available at the point of care. In the event of an outbreak or exposure a more efficient diagnostic platform is needed. The lipopolysaccharide (LPS) component of the bacterial outer leaflet has been identified previously by our group as a potential diagnostic target. For this study, a library of ten monoclonal antibodies specific to F. tularensis LPS were produced and confirmed to be reactive with LPS from type A and type B strains. Antibody pairs were tested in an antigen-capture enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay format to select the most sensitive pairings. The antigen-capture ELISA was then used to detect and quantify LPS in serum samples from tularemia patients for the first time to determine the viability of this molecule as a diagnostic target. In parallel, prototype lateral flow immunoassays were developed, and reactivity was assessed, demonstrating the potential utility of this assay as a rapid point-of-care test for diagnosis of tularemia.

Immunoglobulin G3 (IgG3) is the predominant IgG subclass elicited in response to polysaccharide antigens in mice. This specific subclass has been shown to crosslink its fragment crystallizable (Fc) regions following binding to multivalent polysaccharides. Crosslinking leads to increased affinity through avidity, which theoretically should lead to more effective protection against bacteria and yeast displaying capsular polysaccharides on their surface. To investigate this further we have analyzed the binding characteristics of 2 IgG monoclonal antibody (mAb) subclass families that bind to the capsular polysaccharide (CPS) of Burkholderia pseudomallei. The first subclass family originated from an IgG3 hybridoma cell line (3C5); the second family was generated from an IgG1 cell line (2A5). When the Fc region of the 3C5 IgG3 is removed by proteolytic cleavage, the resulting F(ab')2 fragments exhibit decreased affinity compared to the full-length mAb. Similarly, when the parent IgG3 mAb is subclass-switched to IgG1, IgG2b, and IgG2a, all of these subclasses exhibit decreased affinity. This decrease in affinity is not seen when the 2A5 IgG1 mAb is switched to an IgG2b or IgG2a, strongly suggesting the drop in affinity is related to the IgG3 Fc region.

The CDC Tier 1 select agent Francisella tularensis is a small, Gram-negative bacterium and the causative agent of tularemia, a potentially life-threatening infection endemic in the United States, Europe and Asia. Currently, there is no licensed vaccine or rapid point-of-care diagnostic test for tularemia. The purpose of this research was to develop monoclonal antibodies (mAbs) specific to the F. tularensis surface-expressed lipopolysaccharide (LPS) for a potential use in a rapid diagnostic test. Our initial antigen capture ELISA was developed using murine IgG3 mAb 1A4. Due to the low sensitivity of the initial assay, IgG subclass switching, which is known to have an effect on the functional affinity of a mAb, was exploited for the purpose of enhancing assay sensitivity. The ELISA developed using the IgG1 or IgG2b mAbs from the subclass-switch family of 1A4 IgG3 yielded improved assay sensitivity. However, surface plasmon resonance (SPR) demonstrated that the functional affinity was decreased as a result of subclass switching. Further investigation using direct ELISA revealed the potential self-association of 1A4 IgG3, which could explain the higher functional affinity and higher assay background seen with this mAb. Additionally, the higher assay background was found to negatively affect assay sensitivity. Thus, enhancement of the assay sensitivity by subclass switching is likely due to the decrease in assay background, simply by avoiding the self-association of IgG3.

Mannan-binding lectin (MBL) is a component of the innate immune system. The goal of the present study was to evaluate binding of MBL to Candida albicans in vitro and in vivo and to assess the impact of MBL treatment on host resistance. The results showed a variable and often discontinuous pattern of binding to individual yeast cells. MBL bound to cells grown at 37 degrees C but not to cells grown at 23 degrees C. The putative MBL ligand was constitutively present on yeast cells grown at 23 degrees C, but the ligand was masked on such cells, such that MBL could not bind. C. albicans yeasts and hyphae in infected tissue bound MBL. Finally, parenteral administration of MBL increased resistance of mice to hematogenously disseminated candidiasis. These results suggest that MBL is an important component of innate resistance to candidiasis and that MBL therapy may be a means to prevent disseminated candidiasis in high-risk patients.

Glucuronoxylomannan (GXM), the major component of the capsular polysaccharide ofCryptococcus neoformans, is essential to virulence of the yeast. Previous studies found that the interaction between GXM and phagocytic cells has biological consequences that may contribute to the pathogenesis of cryptococcosis. We found that GXM binds to and is taken up by murine peritoneal macrophages. Uptake is dose and time dependent. Examination of the sites of GXM accumulation by immunofluorescence microscopy showed that the pattern was discontinuous and punctate both on the surfaces of macrophages and at intracellular depots. Although resident macrophages showed appreciable accumulation of GXM, uptake was greatest with thioglycolate-elicited macrophages. A modest stimulation of GXM binding followed treatment of resident macrophages with phorbol 12-myristate 13-acetate, but treatment with lipopolysaccharide or gamma interferon alone or in combination had no effect. Accumulation of GXM was critically dependent on cytoskeleton function; a near complete blockade of accumulation followed treatment with inhibitors of actin. GXM accumulation by elicited macrophages was blocked by treatment with inhibitors of tyrosine kinase, protein kinase C, and phospholipase C, but not by inhibitors of phosphatidylinositol 3-kinase, suggesting a critical role for one or more signaling pathways in the macrophage response to GXM. Taken together, the results demonstrate that it is possible to experimentally enhance or suppress binding of GXM to macrophages, raising the possibility for regulation of the interaction between this essential virulence factor and binding sites on cells that are central to host resistance.

Many microbes are surrounded by phagocytosis-inhibiting 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 a spore-forming, gram-positive bacterium that is the causative agent of anthrax. This bacterium is normally found in environmental samples such as soil. B. anthracis causes 3 ...

Cryptococcus neoformansis surrounded by an antiphagocytic capsule whose primary constituent is glucuronoxylomannan (GXM). An epitope shared by GXM serotypes A, B, C, and D is immunodominant when mice are immunized with serotype A GXM. In contrast, an epitope shared only by serotypes A and D is immunodominant when mice are immunized with serotype D. Hybridomas secreting antibodies reactive with subdominant epitopes were identified through a positive-negative screening procedure in which antibody-secreting colonies were characterized by reactivity with both the immunizing polysaccharide and GXMs from each of the four major serotypes. In this manner, a monoclonal antibody (MAb) that was reactive with an epitope shared only by serotypes A and B was identified and designated F10F5. Such an epitope has not been described previously. Immunization of mice with de-O-acetylated serotype A GXM generated a hybridoma that secreted an antibody, designated F12D2, that was reactive with all four serotypes. Unlike previously described monoclonal and polyclonal panspecific antibodies, the reactivity of MAb F12D2 was not altered by de-O-acetylation of GXM. These results indicate that there are at least two panspecific GXM epitopes; one epitope is dependent on O acetylation for antibody reactivity, and the other is independent of O acetylation. This study identifies strategies for production of MAbs that are reactive with subdominant or cryptic GXM epitopes and provides new information regarding the antigenic makeup and the humoral immune response to GXM, an essential virulence factor that is a target for active and passive immunization.

Cryptococcus neoformansis surrounded by an antiphagocytic polysaccharide capsule whose primary constituent is glucuronoxylomannan (GXM). Three prominent structural features of GXM are single xylosyl and glucuronosyl side chains and O acetylation of the mannose backbone. Isogenic pairs ofO-acetyl-positive andO-acetyl-negative strains (cas1Δ) as well as xylose-positive and xylose-negative strains (uxs1Δ) of serotype D have been reported. Thecas1Δstrains were hypervirulent, and theuxs1Δstrains were avirulent. The goal of this study was to examine the effects of thecas1Δanduxs1Δmutations on the following: (i) binding of anti-GXM monoclonal antibodies (MAbs) in capsular quellung reactions, (ii) activation of the complement system and binding of C3, (iii) phagocytosis by neutrophils, and (iv) clearance of GXM in vivo. The results showed that loss of O acetylation produced dramatic changes in the reactivities of five of seven anti-GXM MAbs. In contrast, loss of xylosylation produced a substantive alteration in the binding behavior of only one MAb.O-acetyl-negative strains showed no alteration in activation and binding of C3 from normal serum. Xylose-negative strains exhibited accelerated kinetics for C3 deposition. Loss of O acetylation or xylosylation had no effect on phagocytosis of serum-opsonized yeast cells by human neutrophils. Finally, loss of O acetylation or xylosylation altered the kinetics for clearance of GXM from serum and accumulation of GXM in the liver and spleen. These results show that O acetylation and/or xylosylation are important for binding of anti-GXM MAbs, for complement activation, and for tissue accumulation of GXM but do not impact phagocytosis by neutrophils.

Our laboratory previously described the binding characteristics of the murine IgG3 monoclonal antibody (MuAb) F26G3. This antibody binds the poly-glutamic acid capsule (PGA) of Bacillus anthracis, an essential virulence factor in the progression of anthrax. F26G3 IgG3 MuAb binds PGA with a relatively high functional affinity (10 nM), produces a distinct "rim" quellung reaction, and is protective in a murine model of pulmonary anthrax. This study engineered an IgG subclass family of F26G3 mouse-human chimeric antibodies (ChAb). The F26G3 ChAbs displayed 9- to 20-fold decreases in functional affinity, as compared with the parent IgG3 MuAb. Additionally, the quellung reactions that were produced by the ChAbs all differed from the parent IgG3 MuAb in that they appeared "puffy" in nature. This study demonstrates that human constant domains may influence multiple facets of antibody binding to microbial capsular antigens despite their spatial separation from the traditional antigen-binding site.

Bacillus anthracis is surrounded by an anti-phagocytic capsule that is entirely composed of γ-linked d -glutamic acid (γDPGA). γDPGA is required for virulence and is produced in large quantities following spore germination. We have previously described the isolation of several γDPGA-reactive mAbs. The reagents are effective in both immunoprotection and diagnostic applications. The current work was done to further investigate the specificity of γDPGA-reactive mAbs. The specificity of each mAb was characterized using surface plasmon resonance. Our results indicate that each mAb is stereoselective for binding to d -glutamic acid oligomers, but to varying degrees. In particular, mAb F26G3 is highly selective for γDPGA; alterations in stereochemistry disrupted recognition. These differences in mAb reactivity suggest that binding of γDPGA by mAb F26G3 is more specific than non-directional ionic interactions between a negatively charged antigen and a positively charged antibody.

Burkholderia pseudomallei is a Gram-negative bacillus that is the causative agent of melioidosis. The bacterium is inherently resistant to many antibiotics and mortality rates remain high in endemic areas. The lipopolysaccharide (LPS) and capsular polysaccharide (CPS) are two surface-associated antigens that contribute to pathogenesis. We previously developed two monoclonal antibodies (mAbs) specific to the CPS and LPS; the CPS mAb was shown to identify antigen in serum and urine from melioidosis patients. The goal of this study was to determine if passive immunization with CPS and LPS mAbs alone and in combination would protect mice from a lethal challenge with B. pseudomallei. Intranasal (i.n.) challenge experiments were performed with B. pseudomallei strains 1026b and K96423. Both mAbs provided significant protection when administered alone. A combination of mAbs was protective when low doses were administered. In addition, combination therapy provided a significant reduction in spleen colony forming units (cfu) compared to results when either the CPS or LPS mAbs were administered alone.

The genus Burkholderia includes many bacteria that cause serious human infections. As is the case with other Gram-negative bacteria, Burkholderia species produce LPS, which is an abundant component of the bacterial cell surface. Burkholderia cepacia complex (Bcc) bacteria (which include at least 17 separate species) produce LPS structures that are quite different. In an attempt to determine the degree of LPS epitope variation among Bcc species, a mAb was produced, designated 5D8, specific for the LPS of B. cepacia. Western blot analysis determined that mAb 5D8 was able to produce the classic ‘ladder pattern’ when used to probe B. cepacia and Burkholderia anthina lysates, although 5D8 did not produce this pattern with the other seven Bcc species tested. mAb 5D8 reacted with varying intensity to most but not all of the additional B. cepacia and B. anthina strains tested. Therefore, there seems to be significant epitope variation among Bcc LPS both between and within species. Additionally, mAb 5D8 reacted with a proteinase-K-sensitive 22 kDa antigen in all Bcc strains and also in a strain of Burkholderia pseudomallei.

Serotype-specific antibodies to pneumococcal capsular polysaccharide (PPS) are a critical component of vaccine-mediated immunity toStreptococcus 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 μg of the MAbs were administered intraperitoneally before intranasal challenge. In contrast, only 1E2 protected Fcγ receptor IIB knockout (FcγRIIB KO) mice and mice that were depleted of neutrophils with the MAb RB6, whereas 7A9 and 5F6 required neutrophils and FcγRIIB to mediate protection. Conversely, 7A9 and 5F6 protected FcγR KO mice, but 1E2 did not. Hence, the efficacy of 1E2 required an activating FcγR(s), whereas 5F6 and 7A9 required the inhibitory FcγR (FcγRIIB). 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.

Bacillus anthracis is surrounded by an antiphagocytic capsule composed of poly-γ- d -glutamic acid (γDPGA). Bacterial and fungal capsular polysaccharides are shed into body fluids in large amounts during infection. The goal of our study was to examine the in vivo fate and distribution of the γDPGA capsular polypeptide. Mice were injected via the intravenous route with various amounts of purified γDPGA. Blood, urine, and various organs were harvested at different times after treatment. Sites of γDPGA accumulation were determined by immunoassay using monoclonal antibodies specific for γDPGA. The results showed that the liver and spleen were the primary sites for the accumulation of γDPGA. As found in previous studies of capsular polysaccharides, the Kupffer cells of the liver and splenic macrophages were sites for the cellular accumulation of γDPGA. Unlike capsular polysaccharides, the hepatic sinusoidal endothelial cells were also sites for γDPGA accumulation. γDPGA was rapidly cleared from serum and was excreted into the urine. γDPGA in the urine showed a reduced molecular size relative to native γDPGA. The results indicate that in vivo clearance of the polypeptide capsular antigen of B. anthracis shares several features with the clearance of capsular polysaccharides. Key differences between the in vivo behaviors of γDPGA and capsular polysaccharides include the accumulation of γDPGA in hepatic sinusoidal endothelial cells and a γDPGA clearance rate that was more rapid than the clearance reported for capsular polysaccharides.

Bacillus anthracisis surrounded by a polypeptide capsule composed of poly-gamma-d-glutamic acid (γDPGA). In a previous study, we reported that a monoclonal antibody (MAb F26G3) reactive with the capsular polypeptide is protective in a murine model of pulmonary anthrax. The present study examined a library of six MAbs generated from mice immunized with γDPGA. Evaluation of MAb binding to the capsule by a capsular “quellung” type reaction showed a striking diversity in capsular effects. Most MAbs produced a rim type reaction that was characterized by a sharp increase followed directly by a decrease in refractive index at the capsular edge. Some MAbs produced a second capsular reaction well beneath the capsular edge, suggesting complexity in capsular architecture. Binding of MAbs to soluble γDPGA was assessed by a fluorescence perturbation assay in which a change in the MAb intrinsic fluorescence produced by ligand binding was used as a reporter for antigen-antibody interaction. The MAbs differed considerably in the complexity of the binding curves. MAbs producing rim type capsule reactions typically produced the more complex binding isotherms. Finally, the protective activity of the MAbs was compared in a murine model of pulmonary anthrax. One MAb was markedly less protective than the remaining five MAbs. Characteristics of the more protective MAbs included a relatively high affinity, an immunoglobulin G3 isotype, and a complex binding isotherm in the fluorescence perturbation assay. Given the relatively monotonous structure of γDPGA, the results demonstrate a striking diversity in the antigen binding behavior of γDPGA antibodies.

Many microbes are surrounded by phagocytosis-inhibiting 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 γ- d -glutamic acid (γDPGA). γDPGA has been identified recently as a potential target for vaccine development. Studies of the role of γDPGA 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 γDPGA production during anthrax nor the protective activity of γDPGA Abs in inhalation anthrax are known. Here we report production of IgG Abs to γDPGA in mice following an immunization regimen using γDPGA in combination with agonist mAbs to CD40. mAbs were produced that are specific for γDPGA. Passive immunization with γDPGA mAbs protected >90% of mice in a pulmonary model of anthrax that was lethal in control mice ( P < 0.0001). Use of γDPGA mAb in an antigen detection immunoassay found that the appearance of γDPGA 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.

Incubation of encapsulated cryptococci with monoclonal antibodies (MAbs) specific for glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans , produces two distinct capsular quellung-type reactions termed rim and puffy. The type of capsular reaction that occurs is determined by the epitope specificity of the MAb and the serotype of the yeast cell. Several biological activities, including opsonic activity, complement activation, and protective efficacy, are associated with the type of capsular reaction produced by a MAb. The goal of this study was to examine the reactivities of two families of anti-GXM MAbs with serotype A and D capsular polysaccharides in several immunochemical assays, including agglutination, immunofluorescence, quantitative precipitation, and enzyme-linked immunosorbent assay, in an effort to identify serological assays that are predictive of the capsular quellung reaction. The results showed that the type of capsular reaction (rim versus puffy) is a qualitative assessment of antibody-capsule interaction that cannot be predicted on the basis of a serological assay. The results further showed that antibody reactivity demonstrated in one serological assay is not necessarily predictive of results in another assay, particularly in cases where one assay examines antibody-capsule interactions, e.g., agglutination, and another assay examines interaction of antibody with soluble GXM. Taken together, the results suggest caution in interpretation of immunochemical assays for anti-GXM antibodies and recommend the use of multiple assays formats when studying anticryptococcal antibodies.

Bacterial capsules are common targets for antibody-mediated immunity. The capsule of Bacillus anthracis is unusual among capsules because it is composed of a polymer of poly-γ-d-glutamic acid (γdPGA). We previously generated murine IgG3 monoclonal antibodies (mAbs) to γdPGA that were protective in a murine model of pulmonary anthrax. IgG3 antibodies are characteristic of the murine response to polysaccharide antigens. The goal of the present study was to produce subclass switch variants of the γdPGA mAbs (IgG3→IgG1→IgG2b→IgG2a) and assess the contribution of subclass to antibody affinity and protection. Subclass switch antibodies had identical variable regions but differed in their heavy chains. The results showed that a switch from the protective IgG3 to IgG1, IgG2b or IgG2a was accompanied by i) a loss of protective activity ii) a change in mAb binding to the capsular matrix, and iii) a loss of affinity. These results identify a role for the heavy chain constant region in mAb binding. Hybrid mAbs were constructed in which the CH1, CH2 or CH3 heavy chain constant domains from a non-protective, low binding IgG2b mAb were swapped into the protective IgG3 mAb. The IgG3 mAb that contained the CH1 domain from IgG2b showed no loss of affinity or protection. In contrast, swapping the CH2 or CH3 domains from IgG2b into IgG3 produced a reduction in affinity and a loss of protection. These studies identify a role for the constant region of IgG heavy chains in affinity and protection against an encapsulated bacterial pathogen., Author Summary The ability of an antibody to recognize and bind to its target is classically viewed as a function of the variable region of the molecule; this region distinguishes an antibody with one specificity from an antibody with a different specificity. We examined binding of antibodies to an outer coat of the biothreat Bacillus anthracis that is essential for bacterial virulence. We identified regions of the antibody constant region which contribute to antibody binding and the ability of the antibody to protect the host. These constant regions are distinct from the variable regions that directly mediate antibody binding. The results of the study have implications for i) understanding how antibodies function in protection against anthrax and possibly other diseases, ii) understanding how the host responds to a key bacterial virulence factor, iii) selection of antibodies that might be used to treat anthrax, and iv) design of vaccines to protect against anthrax.