Characterization of a humanized monoclonal antibody recognizing clumping factor A expressed by Staphylococcus aureus

We report the humanization and characterization of monoclonal antibody (MAb) T1-2 or tefibazumab, a monoclonal antibody that recognizes clumping factor A expressed on the surface of Staphylococcus aureus. We demonstrate that the binding kinetics of MAb T1-2 is indistinguishable compared to that of its murine parent. Furthermore, MAb T1-2 is shown to enhance the opsonophagocytic uptake of ClfA-coated latex beads, protect against an intravenous challenge in a prophylactic model of rabbit infective endocarditis, and enhance the efficacy of vancomycin therapy in a therapeutic model of established infective endocarditis.     

Fibrinogen-binding protein/clumping factor from Staphylococcus aureus.

The binding of staphylococcal components to fibrinogen was studied. Fibrinogen-binding material from lysed staphylococcal cells or culture supernatants was affinity purified on fibrinogen-Sepharose and analyzed on Western (immuno-) blots by the use of fibrinogen and antifibrinogen antibodies. Two main bands of 87 and 19 kilodaltons (kDa) and a weaker band of 35 kDa bound specifically to fibrinogen. A monoclonal antibody bound to all three bands, indicating that these were of the same origin. The yield of these components was much higher in the culture supernatant than on washed cells, suggesting that these molecules are essentially extracellular products. In a plasma coagulase test, the 87-kDa band, but not the 19-kDa band, clotted rabbit plasma, demonstrating that the 87-kDa molecule is coagulase. This was further confirmed by the fact that the 87-kDa band binds specifically to prothrombin. It was shown that the 87- and the 19-kDa molecules were present on the cell surface by surface labeling the cells with 125I. In addition, the fact that killed and washed cells could induce plasma clotting demonstrates that staphylococci have coagulase exposed on the surface. It was concluded that cell-bound coagulase has affinity for fibrinogen also in the absence of prothrombin and thus is responsible for the clumping of staphylococci in fibrinogen.     

Isolation of Staphylococcus aureus clumping factor.

Immunochemically identical components were isolated from water-soluble phases of five Staphylococcus aureus strains by affinity chromatography on fibrinogen-linked Sepharose 4B. The elution was performed with 1 M MgCl2. The component could be isolated from sonicated preparations of whole cells, cell walls, and extracellular products of S. aureus but not from sonicated preparations of staphylococcal L-forms or from Staphylococcus epidermidis. Investigations of the eluted component by immunoelectrophoresis and Western blot analysis by use of different polyspecific antibodies to S. aureus raised in rabbits revealed only one immunoprecipitate or one band. By means of gel filtration on Sepharose CL 6B and sodium dodecyl sulfate-polyacrylamide gel electrophoresis a molecular mass of 420,000 and 360,000 was found, respectively. Chemical analysis showed a carbohydrate content of about 20% by weight. By crossed immunoelectrophoresis the isolated component was demonstrated to bind to human fibrinogen. The finding that this purified component inhibited the fibrinogen-induced clumping of staphylococci strongly suggests that the component is the S. aureus clumping factor.     

Reassessing the role of Staphylococcus aureus clumping factor and fibronectin-binding protein by expression in Lactococcus lactis.

Since Staphylococcus aureus expresses multiple pathogenic factors, studying their individual roles in single-gene-knockout mutants is difficult. To circumvent this problem, S. aureus clumping factor A (clfA) and fibronectin-binding protein A (fnbA) genes were constitutively expressed in poorly pathogenic Lactococcus lactis using the recently described pOri23 vector. The recombinant organisms were tested in vitro for their adherence to immobilized fibrinogen and fibronectin and in vivo for their ability to infect rats with catheter-induced aortic vegetations. In vitro, both clfA and fnbA increased the adherence of lactococci to their specific ligands to a similar extent as the S. aureus gene donor. In vivo, the minimum inoculum size producing endocarditis in > or =80% of the rats (80% infective dose [ID80]) with the parent lactococcus was > or =10(7) CFU. In contrast, clfA-expressing and fnbA-expressing lactococci required only 10(5) CFU to infect the majority of the animals (P < 0.00005). This was comparable to the infectivities of classical endocarditis pathogens such as S. aureus and streptococci (ID80 = 10(4) to 10(5) CFU) in this model. The results confirmed the role of clfA in endovascular infection, but with a much higher degree of confidence than with single-gene-inactivated staphylococci. Moreover, they identified fnbA as a critical virulence factor of equivalent importance. This was in contrast to previous studies that produced controversial results regarding this very determinant. Taken together, the present observations suggest that if antiadhesin therapy were to be developed, at least both of the clfA and fnbA products should be blocked for the therapy to be effective.     

Genetic evidence that bound coagulase of Staphylococcus aureus is not clumping factor.

Staphylococcus aureus Newman cells carry a surface receptor for fibrinogen called clumping factor. The bacteria also express coagulase, an extracellular protein that binds to prothrombin to form a complex with thrombinlike activity which coverts fibrinogen to fibrin. We have confirmed a recent report (M. K. Bodén and J.-I. Flock, Infect. Immun. 57:2358-2363, 1989) that coagulase can bind to fibrinogen as well as to prothrombin and also that a fraction of coagulase is firmly attached to the cell. A mutant with a deletion in the chromosomal coa gene was isolated by allelic replacement. Allelic replacement either was directly selected by electrotransformation of S. aureus R3N4220 with a nonreplicating suicide plasmid, pCOA18, carrying the delta coa::Tcr mutation or occurred after transduction of the integrated pCOA18 plasmid. The coa mutant was completely devoid of coagulase activity but interacted both with soluble fibrinogen and with solid-phase fibrinogen with the same avidity as the parental strain. This strongly suggests that the bound form of coagulase is not clumping factor and is not responsible for the adherence of S. aureus Newman to solid-phase fibrinogen. The fibrinogen binding determinant of coagulase was located in the C terminus of the protein, by analyzing truncated fusion proteins, in contrast to the prothrombin-binding region which was located in the N terminus.     

A monoclonal antibody recognizing selectively rat granulocytes

In the present study, a murine monoclonal antibody, RK-4 (IgG1) specific for rat granulocytes and their precursors in blood and bone marrow is described. Results obtained by immunocytological and flowcytometry analysis showed this mAb to recognize selectively a surface antigen on neutrophils, eosinophils and basophils. Analysis of cryosections of various lymphatic and non-lymphatic organs demonstrates that this mouse antibody does not cross-react with any other structures. By flow cytometric analysis we found peripheral blood leukocytes to consist of about 15-20% granulocytes. RK-4 is therefore a valuable tool for studying differentiation and function of granulocytes under various conditions.     

Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis.

The pathogenic role of staphylococcal coagulase and clumping factor was investigated in the rat model of endocarditis. The coagulase-producing and clumping factor-producing parent strain Staphylococcus aureus Newman and a series of mutants defective in either coagulase, clumping factor, or both were tested for their ability (i) to attach in vitro to either rat fibrinogen or platelet-fibrin clots and (ii) to produce endocarditis in rats with catheter-induced aortic vegetations. In vitro, the clumping factor-defective mutants were up to 100 times less able than the wild type strain to attach to fibrinogen and also significantly less adherent than the parents to platelet-fibrin clots. Coagulase-defective mutants, in contrast, were not altered in their in vitro adherence phenotype. The rate of in vivo infection was inoculum dependent. Clumping factor-defective mutants produced ca. 50% less endocarditis than the parent organisms when injected at inoculum sizes infecting, respectively, 40 and 80% (ID40 and ID80, respectively) of rats with the wild-type strain. This was a trend at the ID40 but was statistically significant at the ID80 (P < 0.05). Coagulase-defective bacteria were not affected in their infectivity. Complementation of a clumping factor-defective mutant with a copy of the wild-type clumping factor gene restored both its in vitro adherence and its in vivo infectivity. These results show that clumping factor plays a specific role in the pathogenesis of S. aureus endocarditis. Nevertheless, the rate of endocarditis with clumping factor-defective mutants increased with larger inocula, indicating the contribution of additional pathogenic determinants in the infective process.     

Contribution of clumping factor B to pathogenesis of experimental endocarditis due to Staphylococcus aureus

Staphylococcus aureus Newman with an insertion mutation in clfB, the gene encoding clumping factor B, only marginally decreased infection rate (P>0.05) in rats with experimental endocarditis. In contrast, clfB complementation on a multicopy plasmid significantly increased infectivity (P<0.05) over the deleted mutants. Although clfB could affect endovascular infection, its importance in experimental endocarditis was limited.     

Both complement- and fibrinogen-dependent mechanisms contribute to platelet aggregation mediated by Staphylococcus aureus clumping factor B

Staphylococcus aureus can stimulate activation and aggregation of platelets, which are thought to be factors in the development of infective endocarditis. Previous studies have identified clumping factor A (ClfA) and fibronectin binding proteins A and B (FnBPA and FnBPB) as potent platelet aggregators. These proteins are able to stimulate rapid platelet aggregation by either a fibrinogen- or a fibronectin-dependent process which also requires antibodies specific to each protein. Slower aggregation has been seen in other systems where specific fibrinogen binding ligands are absent and platelet aggregation is mediated by complement and specific antibodies. Bacteria expressing ClfB aggregate platelets with a longer lag time than ClfA or FnBPA and FnBPB. In order to investigate whether ClfB causes platelet aggregation in a complement- or fibrinogen-dependent manner, a non-fibrinogen-binding mutant of ClfB (ClfB Q235A) was constructed. Lactococcus lactis expressing ClfB Q235A was able to stimulate platelet aggregation in platelet-rich plasma without a significant increase in lag time. The requirements for platelet aggregation were investigated using gel-filtered platelets. Fibrinogen and specific anti-ClfB antibodies were found to be sufficient to allow platelet aggregation mediated by the wild-type ClfB protein. It seems that ClfB causes platelet aggregation by a fibrinogen-dependent mechanism. The non-fibrinogen-binding ClfB mutant was unable to stimulate platelet aggregation under these conditions. However, bacteria expressing ClfB Q235A caused platelet aggregation in a complement-dependent manner which required specific anti-ClfB antibodies.     

Characterization of a monoclonal antibody recognizing a polymorphic epitope mainly on HLA-DPw2 and DPw4 molecules

A monoclonal antibody (MAb), 13.3.B4, was obtained from a murine hybridoma after fusing Sp2/0 myeloma cells with spleen cells from C3H mice immunized with mouse L cells transfected with the A1 and B1 genes of HLA-DPw4. In radiobinding-assays, MAb 13.3.B4 bound to HLA transfectants expressing DPw2 or DPw4 as well as DPw2 or DPw4 homozygous B-cell lines, while most cell lines expressing other DP determinants were negative. MAbs with known DP or other HLA class II-specificities were used to inhibit binding of MAb 13.3.B4 in a radioimmunoassay. Three MAbs demonstrated inhibition, but their pattern of reactivity with HLA homozygous B cell lines differed from that of MAb 13.3.B4. An evaluation of DNA sequence data showed that MAb 13.3.B4 reacts with all cell lines expressing DP beta-chains of type 2.1, 2.2, 4.1 or 4.2. No correlation between 13.3.B4-reactivity and expression of DP alpha-chain variants was found. The results indicate that MAb 13.3.B4 defines a polymorphic epitope which may be determined by the sequence gly-gly-pro-met at residues 84-87 of the DP beta-chain.     

A monoclonal antibody recognizing a conserved epitope in a group of phospholipases A2.

Notexin and nigexine are monomeric phospholipases A2(PLA2s) from the venoms of Notechis scutatus scutatus and Naja nigricollis, respectively. Polyclonal antibodies raised in mice against these antigenic proteins displayed non-reciprocal cross-reactivity; anti-notexin antibodies recognized notexin but not nigexine, whereas anti-nigexine antibodies recognized both antigens. Polyclonal antibodies raised by successive immunization with nigexine and notexin contained cross-reacting antibodies with affinities for both antigens that differed from those of antibodies present in anti-nigexine antiserum. A monoclonal antibody has been obtained from a mouse immunized with both PLA2s. This monoclonal antibody, called MN1, recognized notexin and nigexine with comparable high affinity (Kd = 10(-9) M). It also recognized most purified PLA2s from elapid snake venoms and all PLA2-containing venoms from cobras and sea-snakes. This offers the first demonstration that most PLA2s from cobras and sea-snakes share a fine structure which is not restricted to the common catalytic site.     

Characterization of a monoclonal antibody and its single-chain antibody fragment recognizing the nucleoside Triphosphatase/Helicase domain of the hepatitis C virus nonstructural 3 protein

We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors. Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371-1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.     

A monoclonal antibody recognizing a determinant common to HLA—A3 and All

Spleen cells from a mouse immunized with an AML cell expressing HLA-A3 produced a hybridoma secreting an anti-HLA-A3, A11 monoclonal antibody, 26D3. By complement-dependent cytotoxicity at dilutions to 1:10(4) the ascites antibody lysed 13/13 A3, 15/15 A11, and 8 other lymphocytes from a panel of 98 donors. The 26 D3 immunoprecipitated a molecule consisting of subunits of 44,000 and 12,000 daltons. Monoclonality of the antibody was demonstrated by isoelectric focusing and protein A affinity chromatography.     

The Sbi protein is a multifunctional immune evasion factor of Staphylococcus aureus

The second immunoglobulin-binding protein (Sbi) of Staphylococcus aureus has two N-terminal domains that bind the Fc region of IgG in a fashion similar to that of protein A and two domains that can bind to the complement protein C3 and promote its futile consumption in the fluid phase. It has been proposed that Sbi helps bacteria to avoid innate immune defenses. By comparing a mutant defective in Sbi with mutants defective in protein A, clumping factor A, iron-regulated surface determinant H, and capsular polysaccharide, it was shown that Sbi is indeed an immune evasion factor that promotes bacterial survival in whole human blood and the avoidance of neutrophil-mediated opsonophagocytosis. Sbi is present in the culture supernatant and is also associated with the cell envelope. S. aureus strains that expressed truncates of Sbi lacking N-terminal domains D1 and D2 (D1D2) or D3 and D4 (D3D4) or a C-terminal truncate that was no longer retained in the cell envelope were analyzed. Both the secreted and envelope-associated forms of Sbi contributed to immune evasion. The IgG-binding domains contributed only when Sbi was attached to the cell, while only the secreted C3-binding domains were biologically active.     

Characterization of an unusual cold shock protein from Staphylococcus aureus

Of the three cold shock proteins expressed by Staphylococcus aureus, CspC is induced poorly by cold but strongly by various antibiotics and toxic chemicals. Using a purified CspC, here we demonstrate that it exists as a monomer in solution, possesses primarily β‐sheets, and bears substantial structural similarity with other bacterial Csps. Aggregation of CspC was initiated rapidly at temperatures above 40 °C, whereas, the Gibbs free energy of stabilization of CspC at 0 M GdmCl was estimated to be +1.6 kcal mol^–1, indicating a less stable protein. Surprisingly, CspC showed stable binding with ssDNA carrying a stretch of more than three thymine bases and binding with such ssDNA had not only stabilized CspC against proteolytic degradation but also quenched the fluorescence intensity from its exposed Trp residue. Analysis of quenching data indicates that each CspC molecule binds with ～5 contiguous thymine bases of the above ssDNA and binding is cooperative in nature. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A novel blocking monoclonal antibody recognizing a distinct epitope of human CD40 molecule

CD40, a member of the tumor necrosis factor receptor superfamily, is an important costimulatory molecule during the immune response. Here, we report a blocking mouse antihuman CD40 monoclonal antibody, mAb 3G3, of which the specificity was verified by flow cytometry and Western blot. It was shown by competition test that 3G3 bound to a different site (epitope) of CD40 from the reported CD40 mAbs, including clone mAb89, 3B2, and 5C11. It was also found that mAb 3G3 could inhibit homotypic aggregation of Daudi cells induced by the agonistic anti-CD40 mAb 5C11. Furthermore, mAb 3G3 effectively inhibited the proliferation of peripheral blood mononuclear cells in mixed lymphocyte reaction assay. Finally, a sensitive and specific soluble CD40 (sCD40) ELISA kit was established by matching mAb 3G3 with 5C11, and it was found that the levels of sCD40 in sera from patients with immune disorders such as hyperthyroidism, chronic nephritis, and rheumatoid arthritis were obviously higher than those from normal individuals. Thus, this blocking anti-CD40 mAb provides a novel tool for the study of CD40.     

Radioimmunoassays for protein A of Staphylococcus aureus

Radioimmunoassays have been developed that can detect nanogram amounts of protein A (SpA), a product generated by Staphylococcus aureus that binds selectively to the Fc region of IgG from most mammalian species. Competition assays for fluid phase SpA utilize antibodies produced in chickens, 125I-labeled SpA as the tracer molecule, and either F(ab')2 fragments of rabbit IgG anti-chicken IgG or 40% ammonium sulfate as the precipitating agent to separate antigen-antibody complexes from free antigen. The double antibody assay could be carried out in serum from species that form only soluble complexes with SpA (e.g., rabbit), that react poorly with SpA (e.g., rat), or under appropriate conditions in serum from species (e.g., dog) that show high reactivity with SpA and form precipitating complexes. Chicken antibodies prepared by affinity chromatography on SpA-Sepharose and labeled with 125I were used in a direct binding assay for SpA present either on the cell wall of Cowan strain I or Wood 46 bacteria, in insoluble complexes prepared from SpA and whole serum or purified IgG, or in Clq binding complexes that were formed by passage of serum from normal or tumor bearing humans or dogs over SpA-collodion charcoal. Since both types of assays could detect SpA even in the presence of serum or IgG, they offer advantages over other techniques in which the SpA-Fc interaction may interfere.