Antibody-Mediated Protection against Infection withHelicobacter pyloriin a Suckling Mouse Model of Passive Immunity

Abstract

Studies of active immunization againstHelicobacter pylori indicate that antibodies play a minor role in immunity. There is also evidence, however, that the translocation of antibodies in the stomach may be insufficient to achieve functional antibody levels in the gastric lumen. We have used a suckling mouse model of passive immunity to determine if perorally delivered antibodies can protect against infection with H. pylori. Female C57BL/6 mice were immunized parenterally with formalin-fixed cells of three clinical isolates of H. pylori (3HP) or the mouse-adapted H. pylori strain SS1 before mating. Their pups were challenged with the SS1 strain at 4 days of age and left to suckle before determination of bacterial loads 14 days later. Compared to age-matched controls, pups suckled by 3HP-vaccinated dams were significantly protected against infection (>95% reduction in median bacterial load; P < 0.0001). Pups suckled by SS1-vaccinated dams were also significantly protected in terms of both median bacterial load (>99.5% reduction; P < 0.0001) and the number of culture-negative pups (28% versus 2% for immune and nonimmune cohorts, respectively; P < 0.0001). Similar results were obtained with pups suckled by dams immunized with a urease-deficient mutant of SS1. Fostering experiments demonstrated that protection was entirely attributable to suckling from an immunized dam, and antibody isotype analysis suggested that protection was mediated by the immunoglobulin G fraction of immune milk. Analysis of the bacterial loads in pups sampled before and after weaning confirmed that infection had been prevented in culture-negative animals. These data indicate that antibodies can prevent colonization by H. pylori and suppress the bacterial loads in animals that are colonized.

Contribution of Secretory Antibodies to Intestinal Mucosal Immunity against Helicobacter pylori

“…C57BL/6 wild-type and pIgR knockout mice (31) were bred and housed in the Department of Microbiology and Immunology Animal Facility at the University of Melbourne as described previously (32). All animal experiments were compliant with the Australian National Health and Medical Research Council guidelines and approved by the University of Melbourne Animal Ethics Committee.…”

“…All tissue recovered for culture was washed twice with saline solution and blotted before weighing. It was then homogenized in 2 ml sterile saline solution and used for quantitative culture on lysed horse blood agar plates supplemented with Dent selective supplement (Oxoid) and 20 g/ml bacitracin (Sigma-Aldrich) as described previously (32). H. pylori was identified by colony morphology; randomly selected colonies from intestinal tissues were confirmed by measuring urease activity and by colony PCR for species determination as described previously (33).…”

“…For immunohistochemical staining, all antibodies were diluted in 2% (vol/vol) fetal bovine serum (FBS)-PBS, and slides were washed between steps with PBS. Dewaxed tissue sections (3 m thick) were blocked with serum-free protein block (30 min; Dako) before incubation (1 h) with one of the following primary antibodies: goat anti-mouse pIgR antibody (R&D Systems) (2 g/ml); rabbit anti-H. pylori SS1⌬ureAB outer membrane vesicle (OMV) polyclonal sera (Sigma) (diluted 1 in 500) produced by hyperimmunization of a rabbit with 3 doses (125 g protein per dose in Freund's incomplete adjuvant) of OMVs purified from SS1⌬ureAB spent culture media as described previously (32); or horseradish peroxidase (HRP)-conjugated goat anti-mouse IgA (KPL Inc.) (diluted 1 in 200). Following washes (performed once for 5 min and once for 30 min), endogenous peroxidase was blocked by immersing the slides in 0.05% (vol/vol) peroxide (30 min), and the slides were washed (three times for 5 min each time).…”

“…The animals derived from each breeding were handled as independent cohorts in two experiments staggered approximately 3 weeks apart for inoculation and subsequent sample collection (Table 1). H. pylori strain SS1 was used for all experiments and cultured as described previously (32). Mice were inoculated at 6 to 8 weeks of age with 0.1 ml of a suspension of H. pylori SS1 in brain heart infusion broth (BHI) (10 8 CFU/ml), or BHI alone, by gavage using a stainless-steel mouse-feeding needle (Cole-Parmer) (20 gauge; 1.5 in.…”

“…Serum was assayed for H. pylori SS1-reactive IgG and IgA by endpoint titration enzyme immunoassay (EIA). Intestinal washings were assayed for total IgA using a mouse IgA-specific EIA (Bethyl Laboratories) and for SS1-reactive IgG and IgA using a standardized EIA as described previously (32). For the standardized EIA of intestinal contents, EIA units were assigned from a standard curve using pooled serum from mice hyperimmunized with H. pylori SS1.…”