on colonization of meningitidis serogroup B and strains in a human bronchial epithelial cell culture model

Capsular polysaccharide-protein conjugate vaccines protect individuals from invasive 27 disease and decrease carriage, which reduces spread of the organism in the population. In 28 contrast, antibodies elicited by plain polysaccharide or protein antigen-based meningococcal 29 (Men) vaccines have little or no effect on decreasing carriage. In this study, we investigated the 30 mechanism by which vaccine-induced human IgG antibodies affect colonization by serogroup B 31 (MenB) or C (MenC) strains using a human bronchial epithelial cell culture model (16HBE14o-). 32 Fluorescence microscopy showed bacteria colonizing the apical side of 16HBE14o- monolayers 33 had decreased capsular polysaccharide on the bacterial surface that resulted from shedding the 34 capsule and not decreased production of polysaccharide. Capsular polysaccharide shedding 35 depended on the presence of 16HBE14o- cells and bacteria, but not direct adherence of the 36 bacteria to the cells. Treatment of bacteria and cells with post immunization MenC-conjugate 37 IgG or murine anti-MenB polysaccharide mAbs inhibited capsule shedding, microcolony 38 dispersal, and invasion of the 16HBE14o- cell monolayer. In contrast, IgG elicited by 39 immunization with MenC PS, MenB outer membrane vesicle (OMV)-, or Factor H binding 40 protein (FHbp)-based vaccines were not different than pre-immune IgG or no treatment. The 41 results provide new insights on the mechanism by which high avidity anticapsular antibodies 42 elicited by polysaccharide-conjugate vaccines affect meningococcal colonization. The data also 43 suggest that any effect on colonization by IgG elicited by OMV- or FHbp-based vaccines may 44 involve a different mechanism. 49 To establish a model for the initial stage of meningococcal colonization with wild-type encapsulated MenB and MenC strains, we tested the ability of the bacteria to form colonies on 101 Calu-3, CFBE41o-, H441, and 16HBE14o- airway epithelial cell lines. Bacteria were added to 102 the apical surface of confluent cell monolayers on Transwell TM inserts and incubated in 103 chemically defined cell culture medium containing human serum albumin under immerse culture 104 conditions. CFBE41o- and 16HBE14o- cell lines form tight epithelial cell monolayers, which 105 were confirmed by electrical resistance measurements and expression of zona occludens protein 106 1 (20). The number of bacteria in both the apical (top) and basolateral (bottom) sides of the 107 chamber as well as on the epithelial cell layer was counted after 4 hours, 8 hours, and 16 hours 108 incubation. Using a ratio of approximately ~250 bacteria per ~1 million epithelial cells as a 109 reasonable approximation of human colonization, with respect to the number of epithelial cells 110 being in large excess over the number of bacteria, 16HBE14o- cells gave the greatest and most 111 consistent number of colonizing bacteria after 4 hours incubation (data not shown), which 112 simulates the initial stage of meningococcal colonization. While invasion of the monolayer 113 occurred as early as 4 hours (MenC strain 4243), no bacteria were detected in the lower chamber 114 up to 8 hours after adding the bacteria. The human epithelial cell line 16HBE14o- was originally obtained from a 1-year-old heart- 300 lung transplant patient and immortalized with SV40 large T antigen by using the replication 301 defective pSVori- plasmid (37). 16HBE14o- cells were kindly provided by Dr. Gruenert at 302 passage 2.45 and cultured as previously described for transepithelial chloride ion transport 303 measurements (38). For this study, cells were seeded on clear Transwell TM inserts (polyester 304 membrane, 12 mm diameter, 0.4um or 3.0 μm pore size; Corning, Corning, NY, USA) that were 305 prior coated with a mixture of fibronectin/vitrogen/bovine serum albumin/LHC medium (39, 40). 306 Cells were plated at a density of 1x10 5 cells/cm 2 and grown to confluency in Eagle’s Minimal 307 Essential Medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum 308 (FBS; Hyclone TM Characterized; GE Healthcare Bio-Sciences), 100 U/ml penicillin, 100 µg/ml 309 streptomycin (UCSF Cell Culture Facility, San Francisco, CA, USA), and 200 mM glutamine 310 (UCSF Cell Culture Facility). 16HBE14o- cell monolayers were grown under immerse culture 311 conditions with an apical volume of 500 µL and a basolateral volume of 1 mL to facilitate the 312 formation of higher transepithelial electrical resistance values as pointed out by Ehrhardt et al. 313 (20). Cell monolayers were incubated in antibiotic-free medium (MEM, Glutamine, and FBS) 314 overnight followed by two 30-minute incubation periods with MEM, 4mM D,L- Lactate (Sigma- 315 Aldrich, St. Louis, MO, USA), and 1mg/mL human serum albumin (Sigma-Aldrich) before the 316 start of each experiment.