IgA antibodies in saliva as a noninvasive readout for intestinal immune responses

Enterotoixigenic Escherichia coli (ETEC) are among the major causes of diarrhoea in children in low and middle income countries. ETEC adheres to the surface of the intestinal epithelium and causes diarrhoea by the release of enterotoxins, and express colonization factors which promote attachment. There is a need for a non-invasive readout as a proxy for intestinal immune induction in field trials of enteric vaccines. The purpose of this project is to evaluate salivary IgA readout as a correlate of intestinal immunity. We developed flow-cytometric approaches for such measurements and succeeded in identifying that the volunteers mounted very vivid responses to the challenge strain in serum, blood lymphocytes (i.e. ALS), intestinal lavage, and in salivary secretions, and that these immune responses were not directed against the heterologous ETEC strain H10407. While much of these antibodies are likely directed against the lipopolysaccharides (O antigens), a fraction might target protein surface antigens. Altogether, our studies show that IgA plasma cell responses to ETEC elicited in submandibular/sublingual salivary glands quite consistently reflect intestinal IgA responses following ETEC infection. Despite the fact that it is not possible to achieve completely selective sampling of these oral secretions, i.e. with no admixture of fluid from the parotid glands, the responses in the latter did seem to be less consistent, apparently supporting our original hypothesis that targeted sampling of submandibular/sublingual secretions for IgA responses better reflect dissemination of activated plasma cell precursors (B cells) after immune induction in the gut. Finally, we found a significant elevation of total salivary IgA following the ETEC challenge, which apparently to some extent could be ascribed to bystander stimulation of B cells in the gut. This must be taken into account when considering whether to normalize salivary immune responses against total IgA concentrations. A scientific paper reporting these results has been published in Mucosal Immunity 28 October 2015.

Investigators at academic institutions in Oslo and Bergen propose research work to answer whether salivary specimens can be used to measure relevant IgA immune responses following intestinal immunization. Non-parotid salivary secretions have been proposed to reflect immunity induced in the gut-associated lymphoid tissue. The original idea was that this project should be closely integrated with WP6 of the GLOBVAC-funded project EntVac, in cooperation with the Norwegian Institute of Public Health (NIPH), wh ere the various immunoassays are being developed and performed. Appropriate enzyme-linked immunoassays (ELISAs) have been established for measurements of total secretory IgA (SIgA) and IgA as well as specific SIgA and IgA antibodies to the subunit B of Es cherichia coli heat-labile enterotoxin (LTB) and a recombinantly expressed surface antigen, EtpA. Extensive comparisons of different variables have led to the conclusion that the assays are more robust for both total IgA and IgA antibodies than for the SI gA counterparts. Analyses of salivary specimens obtained from ETEC-infected volunteers suggest that our original hypothesis indeed may be true because the specific IgA titres in sublingual/submandibular (SL) secretions of the infected individuals were rel atively high after day 7 compared to the mean titres in parotid secretions, whereas rechallenged individuals showed only a retarded response in SL and, on average, no response in parotid secretion. We found it puzzling, however, that when the IgA antibody titres of SL fluid were normalized against the total IgA level in individual samples, the response pattern disappeared. This result could be explained if ETEC-specific IgA constitutes a major portion of total IgA in SL fluid after enteric infection, or t here might still be an unexplained problem with nonspecific binding of IgA to the ELISA plates in some of the samples. The wide scatter among the ETEC titres favours the latter possibility, and all the problems reported by other researchers who have attem pted to use saliva IgA antibody as a proxy for gut immunity point to the fact that considerable pitfalls exist in ELISA-based measurements of antibodies in saliva. As an alternative method, we used heat-inactivated E. coli and examined the antibody respon se in serum by flow cytometry; a distinct IgG response was regularly observed and usually also a distinct IgA response. We believe that the antibody titres seen by this method are mainly directed against lipopolysaccharide (LPS, endotoxin) in the cell wal l of E. coli, but we cannot exclude that other surface antigens are involved. Our flow-cytometric method proved sensitive and showed that the orally infected volunteers indeed had responded immunologically after exposure to ETEC, although most of them rem ained clinically quite well. Based on these results, we would like to exploit flow cytometry further to establish a highly sensitive and versatile method to measure immune responses against purified E. coli antigens, both in serum and SL saliva specimens as well as in gut lavage fluid and ALS (antibodies in lymphocyte secretions). The best analytical method we could choose is the multiplex assay where reactivity for several antigens can be tested at the same time. With this particle-based assay nonspecifi c staining should be a negligible problem in contrast to our experience with salivary specimens on ELISA plates. We believe that this will be the most productive approach to establish a flexible and robust method for readouts of gut immune responses such as in serum, gut lavage and ALS, as well as in salivary specimens. We plan to purify LPS from the ETEC challenge strains and, if resources and time permit, also test other relevant E. coli antigens.