Binding to and opsonophagocytic activity of O-antigen-specific monoclonal antibodies against encapsulated and nonencapsulated Klebsiella pneumoniae serotype O1 strains

Abstract

The high mortality of nosocomial infections caused by Klebsiella spp. has acted as a stimulus to develop immunotherapeutic approaches targeted against surface molecules of these bacteria. Since O-antigen-specific antibodies may add to the protective effect of K antisera, we tested the functional and binding capacity of O-antigen-specific monoclonal antibodies (MAbs) raised against different Klebsiella O antigens. The MAbs tested were specific for the O-polysaccharide partial antigens D-galactan II (MAb Ru-O1), D-galactan I (MAb IV/4-5), or core oligosaccharide (MAb V/9-5) of the Klebsiella serogroup O1 antigen. In enzyme-linked immunosorbent assay binding experiments, we found that all MAbs recognized their epitopes on intact capsule-free bacteria; however, binding to encapsulated wild-type strains belonging to different K-antigen serotypes was significantly reduced. The K2 antigen acted as the strongest penetration barrier, while the K7 and K21 antigens allowed some, though diminished, antibody binding. In vitro phagocytic killing experiments showed that MAb Ru-O1 possessed significant opsonizing activity for nonencapsulated O1 serogroup strains and also, to a much lesser extent, for encapsulated strains belonging to the O1:K7 and O1:K21 serotypes. MAbs or antisera specific for the D-galactan II antigen may thus be the most promising agents for further efforts to develop a second-generation Klebsiella hyperimmune globulin comprising both K- and O-antigen specificities.

FIG. 1

Expression of serotype O1 partial antigens by an encapsulated parent Klebsiella strain and its decapsulated mutant. LPS was prepared from strain 591 (lanes 1, 3, 5, and 7) and its decapsulated mutant (lanes 2, 4, 6, and 8). (A) Silver stain; (B) Western blot reacted with MAb Ru-O1; (C) Western blot reacted with MAb IV/4-5; (D) Western blot reacted with MAb V/9-5. Both the parent strain and the decapsulated mutant expressed all three known partial antigens of the O1 serogroup. Similar reactions were obtained with the other strains used in this study.

FIG. 2

Opsonophagocytic activity on encapsulated parent strains of K. pneumoniae (A, C, and E) and their decapsulated mutants (B, D, and F) of MAbs directed against different O1 antigen epitopes of K. pneumoniae LPS. The assay conditions for encapsulated bacteria included a ratio of neutrophils to bacteria of 32:1 (A and C) or 1:2 (E); the concentration of complement was 10% in all experiments. For decapsulated bacteria, the assay conditions included a ratio of neutrophils to bacteria of 1:2 (B and D) or 1:8 (F); the concentration of complement was 2.5% in all experiments (B, D, and F). The MAbs were used at a concentration of 5 μg/ml in each experiment. Values are given as mean ± standard deviation and are from three (A, C, and E) and four (B, D, and F) independent experiments, each done in duplicate. Controls in each experiment included bacteria with HBSS alone; bacteria and PMN without antibodies; bacteria, complement, and PMN without antibodies; bacteria, complement, and antibodies without PMN; and bacteria, complement, PMN, and irrelevant MAbs of the relevant subclass (to show that there was no nonspecific serum activity), each of which showed no killing of bacteria. For statistical analysis, we compared the percentage of killing for each bacterial strain separately by the Mann-Whitney U test. Thus, six comparisons were made for each panel, and statistically significant results (P < 0.01) are indicated by asterisks. For panels A, C, and E, comparison of MAbs with control antibodies was statistically significant (P < 0.01) for each strain and each comparison and hence is not noted explicitly.