Antibodies to both terminal and internal B-cell epitopes of Francisella tularensis O-polysaccharide produced by patients with tularemia

Abstract

Francisella tularensis, the Gram-negative bacterium that causes tularemia, is considered a potential bioterrorism threat due to its low infectivity dose and the high morbidity and mortality from respiratory disease. We previously characterized two mouse monoclonal antibodies (MAbs) specific for the O-polysaccharide (O antigen [OAg]) of F. tularensis lipopolysaccharide (LPS): Ab63, which targets a terminal epitope at the nonreducing end of OAg, and Ab52, which targets a repeating internal OAg epitope. These two MAbs were protective in a mouse model of respiratory tularemia. To determine whether these epitope types are also targeted by humans, we tested the ability of each of 18 blood serum samples from 11 tularemia patients to inhibit the binding of Ab63 or Ab52 to F. tularensis LPS in a competition enzyme-linked immunosorbent assay (ELISA). Although all serum samples had Ab63- and Ab52-inhibitory activities, the ratios of Ab63 to Ab52 inhibitory potencies varied 75-fold. However, the variation was only 2.3-fold for sequential serum samples from the same patient, indicating different distributions of terminal- versus internal-binding antibodies in different individuals. Western blot analysis using class-specific anti-human Ig secondary 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.

FIG 1

Inhibition of binding of Ab63 and Ab52 MAbs to LPS by blood serum samples of tularemia patients. Threefold serial dilutions of patient serum or a normal human serum pool (NHS) were used in competition ELISA with fixed concentrations of Ab63 or Ab52. The data are averages of 3 independent determinations, and the bars indicate the standard errors of the means.

FIG 2

Western blot analysis of serum samples from tularemia patients. Mouse MAbs Ab63 (IgG3) specific for the nonreducing end of F. tularensis OAg and Ab52 (IgG2a) specific for internal OAg epitopes were used as positive controls, and the reactions were detected with AP-anti-mouse IgG secondary antibody. The reactions on all other strips were detected with AP anti-human Ig class-specific secondary antibodies for IgG, IgM, and IgA, as indicated. The positions of molecular mass standards, in kDa, are indicated. P, patient; S, serum; NHS, normal human serum pool.

FIG 3

Specificity of antibodies to F. tularensis LPS in Western blot analysis. Mouse MAbs Ab63 (IgG3) specific for the nonreducing end of F. tularensis OAg, Ab52 (IgG2a) specific for F. tularensis internal OAg epitopes, and 73/28 (IgG2a) specific for Klebsiella spp. were used as specificity controls, and the reactions were detected with AP-anti-mouse IgG secondary antibody. The reactions on all other strips were detected with AP anti-human Ig class-specific secondary antibodies for IgG, IgM, and IgA, as indicated. The positions of molecular mass standards, in kDa, are indicated. S, serum; NHS, normal human serum pool; H2, H6, H7, individual normal human serum samples.