Proc

Proc. antibodies showed that both terminal- and internal-binding OAg antibodies were of the IgG, IgM, and IgA isotypes. These results support the use of a mouse model to discover protective B-cell epitopes for tularemia vaccines or prophylactic/therapeutic antibodies, and they present a general strategy for interrogating the antibody responses of patients and vaccinees to microbial carbohydrate epitopes that have been characterized in experimental animals. INTRODUCTION but is not currently licensed due to safety concerns (5, 6). The development of alternative vaccines and of immunotherapeutics must take into account both the T- and B-cell components that contribute to immune protection against (7,C12). Antibodies to the lipopolysaccharide (LPS) have been shown to be protective against respiratory tularemia in BALB/c, C3H/HeN, C57BL/6, and C57BL/10 Tap1 mice, and antibodies, most of which are directed to LPS, have been shown to ameliorate tularemia in humans (13,C22). Lipopolysaccharide (LPS), the main component of the outer membrane, is identical in the type A and B strains (23,C27). It is composed of lipid A, a core oligosaccharide (C, mainly Hex4HexNAcKdo), and an capsular polysaccharide also consists of OAg (28, 29). We previously reported that anti-LPS mouse monoclonal antibodies (MAbs) can confer survival to BALB/c mice infected intranasally (i.n.) with an otherwise lethal dose of LVS (30). Subsequently, we found that the anti-LPS MAbs target OAg, and we characterized two types of OAg epitopes: repeating internal epitopes targeted by the vast majority of mouse OAg MAbs and a nonoverlapping less immunogenic unique epitope at the nonreducing end (31). The two types of MAbs are distinguished by their Western blot reactivities with LPS, where terminal binders react equally with short and long chains, all of which have one nonreducing-end epitope, whereas internal binders show increased reactivity with increasing LPS chain length (31, 32). Despite the much higher number of epitopes per OAg chain that can be engaged by the internal binders, all four available terminal-binding MAbs have higher bivalent avidity than the most potent internal-binding MAb, Ab52 (32), and higher agglutination Araloside X titers (31, 32). Using oligosaccharides of defined OAg repeat length as molecular rulers in competition enzyme-linked immunosorbent assay (ELISA), the epitope targeted by the terminal-binding MAb Ab63 was shown to span a single tetrasaccharide repeat (32), whereas the epitope targeted by Ab52 was shown to span two tetrasaccharide repeats (33). The X-ray crystal structures of the Fab fragments (the light chain plus the variable and first constant domains of the heavy chain) of Ab52 and of a closely related clonal variant of Ab63 were determined, and a 2-repeat Araloside X computational model of the OAg chain was docked into the binding sites, guided by the immunochemical constraints (32, 34). These studies revealed which the binding site of Ab63 is normally a little cavity that may accommodate the initial and area of the second terminal glucose residues of OAg with restricted envelopment from the terminal Qui4NFm glucose by aromatic proteins, which may describe the bigger affinity of terminal-binding MAbs (32). The binding site of Ab52 is normally a big groove using a central pocket that accommodates a V-shaped epitope comprising six glucose residues that period two tetrasaccharide do it again systems, BCDABC (34). Ab63 and Ab52 had been proven to prolong the success of and decrease bloodstream bacterial burden in BALB/c mice contaminated i.n. using the extremely virulent type A stress SchuS4 (32, 33). To see whether human beings generate both terminal- and internal-binding antibodies in response to an infection with LVS. Many had been 2- to 3-week convalescent-phase sera. The usage of the serum examples was reviewed with the Boston School INFIRMARY institutional review plank and determined to become exempt. Pooled regular individual serum (NHS) (individual supplement serum; Sigma, St. Araloside X Louis, MO) was utilized being a control. Three person human serum examples were extracted from regular volunteers under an accepted institutional review plank process. Mouse MAbs. The anti-MAbs Ab63 (IgG3) (32) and Ab52 (IgG2a) (31) Araloside X and their purification had been previously defined. Anti-MAb 73/28 (IgG2a) was bought being a purified antibody from Life expectancy Biosciences (Seattle, WA). LPS and.

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