Colonic Immune Stimulation by Targeted Oral Vaccine

Citation: Kathania M, Zadeh M, Lightfoot YL, Roman RM, Sahay B, et al. ( Colonic Immune Stimulation by Targeted Oral Vaccine Mahesh Kathania 0 Mojgan Zadeh 0 Yama L. Lightfoot 0 Robert M. Roman 0 Bikash Sahay 0 Jeffrey R. Abbott 0 Mansour Mohamadzadeh 0 Prosper N. Boyaka, The Ohio State University, United States of America 0 1 Department of Infectious Diseases and Pathology, University of Florida, Gainesville, Florida, United States of America, 2 Division of Hepatology/Gastroenterology and Nutrition, University of Florida, Gainesville, Florida, United States of America, 3 Department of Medicine, Emerging Pathogens Institute, University of Florida , Gainesville, Florida , United States of America Background: Currently, sufficient data exist to support the use of lactobacilli as candidates for the development of new oral targeted vaccines. To this end, we have previously shown that Lactobacillus gasseri expressing the protective antigen (PA) component of anthrax toxin genetically fused to a dendritic cell (DC)-binding peptide (DCpep) induced efficacious humoral and T cell-mediated immune responses against Bacillus anthracis Sterne challenge. Methodology/Principal Finding: In the present study, we investigated the effects of a dose dependent treatment of mice with L. gasseri expressing the PA-DCpep fusion protein on intestinal and systemic immune responses and confirmed its safety. Treatment of mice with different doses of L. gasseri expressing PA-DCpep stimulated colonic immune responses, resulting in the activation of innate immune cells, including dendritic cells, which induced robust Th1, Th17, CD4+Foxp3+ and CD8+Foxp3+ T cell immune responses. Notably, high doses of L. gasseri expressing PA-DCpep (1012 CFU) were not toxic to the mice. Treatment of mice with L. gasseri expressing PA-DCpep triggered phenotypic maturation and the release of proinflammatory cytokines by dendritic cells and macrophages. Moreover, treatment of mice with L. gasseri expressing PADCpep enhanced antibody immune responses, including IgA, IgG1, IgG2b, IgG2c and IgG3. L. gasseri expressing PA-DCpep also increased the gene expression of numerous pattern recognition receptors, including Toll-like receptors, C-type lectin receptors and NOD-like receptors. Conclusion/Significance: These findings suggest that L. gasseri expressing PA-DCpep has substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration and may be used as a safe oral vaccine against anthrax challenge. - Funding: This work was supported in part by National Institutes of Health Grant 1R01AI098833-01. No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Mucosal surfaces are the principal sites of interaction between a microorganism and its host and, as such, represent the major route of entry for microbial pathogens [1]. In recent years, numerous reports of successful vaccination with mucosal vector vaccines have been published. The mucosal immune system functions to protect mucous membranes from invading infectious agents by regulating immune responses through selective, immune effector cascades, all of which are meant to protect the body from pathogen challenge [2]. Live bacteria and viruses are known to be more immunogenic than inactive vectors and thus, represent superior candidates to induce both mucosal and systemic immune responses against pathogens. The development of bacteria as live vaccine vehicles has focused primarily on the use of attenuated strains of pathogenic bacteria, including Salmonella, Bordetella, and Listeria spp. [35]. The pathogenic properties related to these bacteria render them attractive candidates to enhance immunogenicity; however, the potential toxicity and possibility of reversion of these attenuated strains to virulence is a significant safety concern. The strong immunogenicity of these vaccines also makes them less suitable for use in immunocompromised or susceptible individuals. Consumption of fermented products, especially yogurt, has been recognized for centuries to have a positive effect on gastrointestinal health. These effects are now largely attributed to Lactobacillus species that are generally regarded as safe (GRAS) for human consumption. Lactic acid bacteria (LAB) comprise a group of Gram-positive bacteria that include species of Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus [6]. The ability of LAB to survive gastric transit and to assume a close physical association with the intestinal epithelium, in addition to their immunomodulatory properties and safe consumption in large quantities, make lactobacilli attractive candidates for the development of live vaccine vectors targeting immunogens to the intestinal mucosa [7]. Therefore, recent advances in biotechnology and in the understanding of intestinal immunity and microbial-host cell interactions have made it possible to design new mucosal delivery systems. Vaccinations aimed at the mucosal immune system are intended to promote a robust systemic memory immune response that Figure 1. L. gasseri expressing PA-DCpep is safe for mice at high doses. C57BL/6 mice were orally gavaged with 109 CFU of L. gasseri expressing PA-DCpep once and the CFU per gram of feces was determined (A) in MRS plates with erythromycin (5 mg/mL). C57BL/6 mice were orally gavaged with increasing doses of L. gasseri expressing PA-DCpep (107, 109 and 1012 CFU) or PBS, serum was collected after days 1, 3 and 7, and the enzyme activities of ALT/SGPT (B) and AST/SGOT (C) were analyzed by ELISA. CCl4 was used as a positive control for toxicity (B & C). Tissues from C57BL/6 mice orally gavaged with increasing doses of L. gasseri expressing PA-DCpep (107, 109 and 1012 CFU) or PBS were collected and sections stained with H&E at days 1, 3 and 7 of treatment. (D) Photomicrographs of H&E sections of liver AD, kidney EH, spleen IL, colon MP, and cecum QT. Insets of a representative glomerulus and surrounding proximal tubules are included in each of the kidney photomicrographs. Insets of high magnification of the lymphoid tissue are included in each of the spleen photomicrographs. For each of the organs, the PBS control is pictured in the left column A, E, I, M and Q, respectively. The three photomicrographs on the right are from increasing concentrations of bacteria from 107, 109 and 1012 from left to right. doi:10.1371/journal.pone.0055143.g001 provides protection against repeat exposure to the targeted pathogen without causing tissue damage or excessive inflammation. Initiation and propagation of proinflammatory immune responses to infectious agents occurs through the activation of antigen-presenting cells (APCs) via innate receptors that bind conserved molecular patt (...truncated)