Use of Moraxella catarrhalis lipooligosaccharide mutants to identify specific oligosaccharide epitopes recognized by human serum antibodies

Abstract

Moraxella catarrhalis is a causative agent of otitis media in children and lower respiratory tract infections in adults suffering from chronic obstructive pulmonary disease (COPD). This strict human pathogen continues to be a significant cause of disease in this broad spectrum of patients because there is no available vaccine. Although numerous putative vaccine antigens have been described, little is known about the human immune response to M. catarrhalis infection in vivo. Human serum antibodies are directed at a number of surface proteins, and lipooligosaccharides (LOS) and detoxified LOS may be an effective immunogen in mice. In this study, we used a specific LOS-based enzyme-linked immunosorbent assay (ELISA), containing the three major M. catarrhalis serotypes together with a complete series of truncated LOS mutants, to detect the development of new antibodies to specific regions of the oligosaccharide molecule. We compared serum samples from COPD patients who had recently cleared an M. catarrhalis infection to serum samples collected prior to their infection. Variability in the antibody response to LOS was observed, as some patients developed serotype-specific antibodies, others developed antibodies to the LOS of each serotype, others developed broadly cross-reactive antibodies, and some did not develop new antibodies. These newly developed human antibodies are directed at both side chains and core structures in the LOS molecule. This LOS-based ELISA can be used to dissect the human antibody response to both internal and external carbohydrate epitopes, thus providing a better understanding of the humoral immune response to M. catarrhalis LOS epitopes developed during natural infection.

FIG. 1.

Identification of newly developed serum IgG antibodies to the LOS of the homologous M. catarrhalis isolate. ELISAs were used to test the level of serum IgG antibody (abs) in response to LOS obtained from the exacerbating strain of bacterium. COPD patients are indicated in addition to the LOS serotype of the exacerbating strain of M. catarrhalis isolated from that patient. Serum samples obtained preacquisition and postclearance of the infecting M. catarrhalis strain were incubated in a 96-well plate coated with 20 to 30 μg of the homologous LOS per well. The bars represent the mean differences in absorbance for data from at least three independent ELISAs of serum samples taken after clearance of the exacerbating strain to preacquisition of that strain, with the error bars representing the standard errors of the means. Asterisks indicate a P value of ≤0.05 as determined by a one-sided paired t test.

FIG. 2.

COPD patients who developed serotype-specific and externally directed antibody responses to different LOS epitopes. Shown are data from an evaluation of COPD patient IgG serum levels against various truncated LOS molecules. The OS used as antigens are listed along the x axis. Patient 3 (A) was infected with 3P34B1 (serotype A), patient 6 (B) was infected with 6P29B1 (serotype A), patient 19 (C) was infected with 19P54B1 (serotype A), and patient 87 (D) was infected with 87P15B1 (serotype A). ELISAs were used to test the level of COPD serum IgG antibody (abs) in response to truncated LOS obtained from a panel of glycosyltransferase mutants and full-length wild-type structures representing serotype A (25238), serotype B (7169), and serotype C (26391). Serum samples obtained preacquisition and postclearance of the infecting M. catarrhalis strain were incubated in a 96-well plate coated with 20 to 30 μg of LOS per well. The bars represent the mean differences in absorbance for at least three independent ELISAs of serum samples taken postclearance of the exacerbating strain to preacquisition of that strain, with the error bars representing the standard errors of the means. Asterisks indicate a P value of ≤0.05 as determined by a one-sided paired t test.

FIG. 3.

COPD patients who developed more broadly cross-reactive and internally directed antibody responses to different LOS epitopes. Shown are data from an evaluation of levels of IgG against various truncated LOS molecules in sera from COPD patients. The OS used as antigens are listed along the x axis. Patient 7 (A) was infected with 7P33B1 (serotype A), patient 7 (B) was infected with 7P94B1 (serotype C), patient 18 (C) was infected with 18P7B1 (serotype B), and patient 51 (D) was infected with 51P9B1 (serotype A). ELISAs were used to test the level of IgG antibody in sera of COPD patients in response to truncated LOS obtained from a panel of glycosyltransferase mutants and full-length wild-type structures representing serotype A (25238), serotype B (7169), and serotype C (26391). Serum samples obtained preacquisition and postclearance of the infecting M. catarrhalis strain were incubated in a 96-well plate coated with 20 to 30 μg of LOS per well. The bars represent the mean differences in absorbance for at least three independent ELISAs of serum samples taken postclearance of the exacerbating strain to preacquisition of that strain, with the error bars representing the standard errors of the means. Asterisks indicate a P value of ≤0.05 as determined by a one-sided paired t test.