Antigenic diversity of Haemophilus somnus lipooligosaccharide: phase-variable accessibility of the phosphorylcholine epitope

Abstract

The lipooligosaccharide (LOS) of Haemophilus somnus undergoes antigenic phase variation, which may facilitate evasion from the bovine host immune response and/or colonization and dissemination. However, LOS antigenic diversity in H. somnus has not been adequately investigated. In this study, monoclonal antibodies (MAbs) specific to various LOS epitopes were used to investigate antigenic variation and stability in LOS from H. somnus strains and phase variants. Clinical isolates of H. somnus exhibited intrastrain, as well as interstrain, antigenic heterogeneity in LOS when probed with MAbs to outer core oligosaccharide epitopes in an enzyme-linked immunosorbent assay (ELISA). However, epitopes reactive with MAbs directed predominately to the inner core heptose region were highly conserved. At least one epitope, which was expressed in few strains, was identified. One LOS component affected by phase variation was identified as phosphorylcholine (PCho), which is linked to the primary glucose residue. Inhibition ELISA, immunoblotting, and electrospray-mass spectrometry were used to confirm that MAb 5F5.9 recognized PCho. LOS reactivity with MAb 5F5.9 was associated with loss of most of the outer core oligosaccharide, indicating that reactivity with PCho was affected by phase variation of the glucose residues in this region. Our results indicate that outer core epitopes of H. somnus LOS exhibit a high degree of random, phase-variable antigenic heterogeneity and that such heterogeneity must be considered in the design of vaccines and diagnostic tests.

FIG. 1

Reactivity of MAb 5F5.9 with strains of H. somnus by ELISA. Error bars, ±2 standard deviations from the mean of the results from four replicate experiments. E. coli J5 is the negative control.

FIG. 2

Dendrogram obtained from cluster analysis of all H. somnus strains (four replicate experiments) and the relative percent binding by ELISA with each corresponding MAb. Semiquantitative binding was determined as previously described (29). Symbols: −, +, ++, and +++ represent <15%, ≥15 to 50%, >50 to 90%, and >90% of maximum optical density, respectively.

FIG. 3

Modified Haworth projection of the predominant structure of H. somnus strain 738 LOS (9). All outer core hexoses are beta-D oriented and both inner core l-glycero-d-manno heptoses are l-α-d oriented. Abbreviations: Gal, galactose; GlcNAc, N-acetyl-glucosamine; Glc, glucose; Hep, heptose; Kdo, 3-deoxy-d-manno-2-octulosonic acid.

FIG. 4

Silver-stained polyacrylamide gel (A) and immunoblot with MAb 5D7 (B) of H. somnus LOS extracts. Lanes 1 and 4, strain 649; 2 and 5, strain 6948; 3 and 6, strain 7226. Molecular sizes are indicated on the left (23).

FIG. 5

Inhibition ELISA of MAb 5F5.9 by PCho (hatched bars) or PEtn (open bar). H. somnus strain 93 LOS was used as the antigen. Error bars, 2 standard deviations above and below the mean data point from five replicate experiments.

FIG. 6

Silver-stained polyacrylamide gel (A) and immunoblot (B) analyses of LOS extracts from MAb 5F5.9-positive and -negative clonal isolates of H. somnus strain 738. Lanes: 1 and 3, 738-P LOS; 2 and 4, 738 LOS; 5, immunoblot repeated on membrane cut from lane 4 using anti-738 LOS rabbit serum as the primary antibody. Molecular sizes are indicated on the left (23).