Biomedical subjects
Publications and source records attributed to D Rowley.
Cholera immunization schedules were investigated in mice, with emphasis placed on obtaining an immune response in the intestine. The most effective schedule for producing a good local response was found to be several orally-given priming doses of the organism followed after 14 days by an intravenous boosting dose. Major differences between the immune responses in the spleen and the intestine were noted.
The immune responses of mice following oral vaccination with two strains of live V. cholerae have been examined. A strain which persisted in the small intestine was a superior local immunogen by comparison with another non-persisting strain. Local persistence and the ability to induce a local immune response appeared to be correlated, since the two vibrio strains elicited identical responses when given parenterally.
A re-evaluation was made of the efficiency with which some of the commonly used assays would detect intestinal antibodies. The data indicate that the most sensitive assays for the detection of intestinal antibodies are the baby mouse protection test and the radioimmunoassay. The reasons for the lack of sensitivity with other assay methods are discussed.
The immune response of the mouse to priming and booster doses of V. cholerae was studied to establish whether serum antibody could be used as a correlate of local immunity. Serum antibody titres following oral boosting of orally-primed animals were shown to reflect the state of local intestinal immunity. This was not the case when the same oral booster dose was given to parenterally-primed animals. These results were discussed in relation to the human endemic situation. The highest titres of intestinal protective antibodies were found following combination of the oral and parenteral routes of immunisation. Various killed or extracted preparations of V. cholerae were used as oral vaccines to test their ability to induce protective antibodies in the gut. Only Boivin antigen was capable of inducing as good an intestinal antibody response as would the living organism.
After oral feeding of mice with avirulent Salmonella, Escherichia coli, or hybrid strains, only certain bacterial strains were able to multiply and persist within the small intestinal Peyer's patches. After oral vaccination alone, or oral priming and subsequent parenteral boosting, antibody class and titers were detected, using a radioimmunoassay on serum and intestinal fluid or a plaque-forming cell assay on spleens. Only those strains that persisted in the Peyer's patches stimulated the production of serum and intestinal immunoglobulin A antibodies against their respective O antigens. Nonpersistent strains were weakly immunogenic, and antibodies, when present, were largely non-immunoglobulin A and confined to the serum.
Oral immunisation studies in germfree, specific pathogen-free (SPF) and conventionalised mice illustrated that the autochthonous gut flora can have a suppressive effect on the induction of a local intestinal immune response to Vibrio cholerae. Temporary colonisation of the small bowel by viable vibrios occurred only in the germfree animal. The lack of colonisation in SPF and conventionalised mice was presumably a cause of their lower coproantibody responses. Prevention of colonisation was probably due to bacterial antagonism rather than to cross-reaction antibodies. This conclusion was reinforced by studies involving oral immunisation of SPF mice maintained on streptomycin, and of conventionalised ex germfree mice. In addition to the increased protective coporantibody response of animals with reduced gut flora, there were increased levels of non-complement-fixing protective antibodies in their serum, which were probably derived from the guy lamina propria.
Purified antibodies to Vibrio cholerae from mouse, rabbit and dog were digested in vitro by homologous intestinal secretions. When assessed with regard to their complement-dependent vibriocidal activity, IgG antibodies were generally more susceptible to degradation than IgM antibodies, High levels of tryptic inhibitors were required to inhibit this digestion. Rabbit IgG was unusual in being quite resistant to digestion. Gel filtration studies demonstrated that secretory IgA, isolated from mouse intestinal secretions, was resistant to proteolysis. Similar studies on dog IgG and mouse IgM demonstrated production of F(ab') 2-like fragments. Digestss of these antibodies, while devoid of Fc-mediated vibriocidal activity, retained significant protective activity for baby mice.
The source of IgM and IgG2a,b in the dog intestine was investigated, using labelled dog immunoglobulins and chronic Thiry-Vella loops of dog small intestine. Clearancess of IgM and IgG2a,b from serum into intestinal secretions were similar, reflecting transfer of a greater mass of IgG2a,b than of IgM. Only 20% of the intestinal IgM derived from serum, irrespective of the marker used. About one third of the total intestinal IgG, of all sub-classes and accrued from all sources, was serum-derived IgG2a,b. This indicates that serum is a major contributor to dog intestinal IgG2a,b.
Increasess in all immunoglobulin classes, except IgM, were observed in the sera of specific pathogen-free (SPF) mice beginning 10 days after their removal from barrier conditions. Concentrations of serum immunoglobulins, comparable with those of conventional mice, were obtained in these animals between 21 and 35 days. Following the removal of germ-free mice from their sterile isolaters, their intestinal IgA levels increased over 35 days to attain levels found in conventional animals. A marked increase in serum immunoglobulin occurred within one day following intravenous administration of live Salmonella typhimurium organisms to SPF animals, and this persisted for longer than 7 weeks (the duration of the study), This rapid elevation in serum immunoglobulin was not elicited by nonbacterial antigens, killed Salmonellae, or viable Vibrio cholerae. Negligible amounts of this immunoglobulin increase could be attributed to specific antibody.
Within 3 h after oral challenge of mice with Salmonella typhimurium, foci of infection developed in the Peyer's patches of the small intestine. The numbers of organisms in the cecum, although in excess of those found in the small intestine, were not firmly associated with the cecal wall but were present largely in the cecum's contents. The Peyer's patches at first were remarkably incapable of eliminating even small numbers of Salmonella, but at about 7 days after infection developed the ability to eliminate a less virulent strain of S. typhimurium. Selected strains of Salmonella of varied virulence, and hybrid Escherichia coli/Salmonella typhimurium with varied O-antigens, revealed that those of low virulence could multiply within the intestinal Peyer's patches at nearly the same rate as a virulent strain, and the ability to multiply within the Peyer's patches was not dependent upon O-antigen type or smooth lipopolysaccharide. The ability of these strains to adhere to intestinal mucosa in vitro did not reflect on their ability to colonize the Peyer's patches, although strains of high in vitro adhesive ability appeared in greater numbers initially after oral challenge. Anti-O serum, ineffective in reducing the in vitro adhesive ability of virulent S. typhimurium, when given with the oral challenge prevented Peyer's patch colonization but was unable to prevent the appearance of a systemic infection. Anti-H serum, although effective in vitro in preventing adherence, had no effect in vivo. These experiments suggest that adhesiveness is neither essential nor sufficient for the virulence of Salmonella and that the usual development of a systemic infection after colonization of the small intestinal Peyer's patches may be subverted by the presence of O-antibody.
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The rapid disposal of antibody-treated vibrios by peristalsis in whole animal models makes it difficult to obtain clear evidence for a simultaneous intra-intestinal bactericidal effect. In this paper we have re-examined the evidence for direct intra-intestinal killing. We have found that the data can be explained adequately by peristaltic effects without postulating a direct bactericidal action in vivo.
The results given below show that, whilst it is not possible to transfer immunity to infections with Nematospiroides dubius passively with serum from immune mice to normal adult mice, young born to immune females are resistant to this infection. The immunity is dependent on an intake of immunoglobulin via the milk for a period longer than 24 h. The passive transfer of immunity from immune mothers to neonatal mice does not appear to be dependent on a specific class of immunoglobulins.
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Enzymic fragments of IgG antibody were tested for their protective abilities in the infant mouse cholera model. F(ab')2 retained the full protective activity of the parent IgG molecule despite losses in complement fixation and opsonic functions. Fab' and Fab fragments also contained significant protective activity but at a level of only 10% of the intact IgG or F(ab')2. Self-recombinant univalent F(ab')2 also contained about 10% of the protective activity of the divalent F(ab')2 parent molecule. These results are interpreted as evidence that cross-linking is an important mechanism by which specific antibody protects mucosal surfaces against experimental infection with Vibrio cholerae.
Several antibody preparations were tested for their ability to reduce adsorption of Vibrio cholerae to isolated intestinal epithelial cells, and this ability was related to agglutination and protective activity in infant mice. The results demonstrate that (1) the reduction in adsorption of V. cholerae to epithelial cells correlates with the degree of agglutination for given antibody preparation; (2) intact tantibodies protect infant mice from cholera only at concentrations that agglutinate the bacteria; and (3) purified antibodies to flagellar antigens protect infant mice from cholera. These results indicate that cross-linking of bacteria by antibody causes a reduction in the number of organisms adsorbed to the intestinal wall. Thus antibody cross-linking plays an important role in immunity to cholera in infant mice.
Preparations of rabbit, dog and sheep IgA, IgA and IgM were examined for their antibacterial effects using a complement-dependent bactericidal assay. IgM and IgG were efficient bactericidal antibodies in the presence of complement; IgA, however, contained negligible activity. Except for sheep IgG no enhancement of bactericidal activity was observed in the presence of added lysozyme.