Per Brandtzaeg per.brandtzaeg@medisin.uio.no

1. Mucosal immunization: relevance to protection against tuberculosis Per Brandtzaeg per.brandtzaeg@medisin.uio.no Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Department of Pathology, Oslo University Hospital Rikshospitalet, Norway

2. The mucosae are an enormous battlefield Mucosal effector sites provide secretory IgA (SIgA) antibodies Section through skin IgAIgG Normal human colon Hornified layer Epithelial cells Airways and oral cavity Mucus and cilia Epithelial cells Glands Plasma cells IgA Plasma cells H&E At the border of hell! Gastrointestinal mucosa Surface epithelium 80% of all plasma cells are located in gut mucosa An adult exports 3 g of SIgA to the gut lumen per day Glands (crypts) Plasma cells

3. Local formation and export of mucosal immunoglobulins Lumen IgA-coated bacteria Lamina propria Free SC Dimeric IgA (pIgA) Containment Plasma cells SIgA pIgR (mSC) IgA+J Gland Ag ‘The IgA pump’ IgA+J J chain SIgM IgM+J IgA Control, not clearance, provides mutualism Pentameric IgM IgG IgG(±J) Mucus Brandtzaeg Nature & J. Immunol. 1974; Brandtzaeg & Prydz Nature 1984

4. Coating of bacteria with SIgA is an important immune exclusion mechanism (SIgA) • SIgA-coated bacteria are less invasive • Less shedding of SIgA-coated bacteria • Less horizontal spread of SIgA-coated pathogens (provides herd protection) (PRR) pIgR Modified from: Natalia Shulzhenko … Polly Matzinger, Nature Med 2011; 17: 1585-93 IgA-producing cells

5. Trachea and bronchi: Secretory IgA Defence of lung parenchyma relies largely on serum-derived IgG and cell-mediated mechanisms The lung is not a gut that breathes Bronchioles (< 1 mm): Serum-derived IgG

6. Trachea and bronchi: Secretory IgA Defence of lung parenchyma relies largely on serum-derived IgG and cell-mediated mechanisms The lung is not a gut that breathes MarcusGereke (Braunschweig, Germany):Alveolar type II epithelial cells: pneumocytes with regulatory properties, also anti-inflammatory Type II pneumocyte Bronchioles (< 1 mm): Serum-derived IgG ALVEOLUS Gereke et al., Respir. Res. 2007; 8: 47

7. Homeostatic function ofmucosal vaccines • The goal of mucosal vaccines is to stop the pathogen at the portal of entry • This can best be achieved by induction of secretory IgA (SIgA) antibodies • Protection against invasive mucosal pathogens requires, in addition, systemic immunity (IgG antibodies and cytotoxic T cells)

8. Regionalization in the integrated mucosal immune system Dichotomy in the integrated mucosal immune system Tonsils (NALT) BALT Peyer’s patches (GALT) Also systemic Tonsils & adenoids (NALT) Lacrimal, nasal, & salivary glands Also systemic Bronchial glands BALT Small intestinal mucosa Peyer's patches (GALT) Mammary glands Large Intestinal mucosa Isolated lymphoid follicles (GALT) Uro- genital tract

9. Tonsils and adenoids are well designed for antigen trapping Inductive sites Tonsils & adenoids (NALT) Human NALTanlagen: prenatal (19 wks).Nasal ILFs in 40% < 2 yrs (inducible?).Rodent NALT: postnatal organogenesis. Human BALT in 40%-100% at young age (2-15 yrs); rare in adult normal lungs (>20 yrs), but inducible (also rodent BALT is inducible) BALT Human palatine tonsil Reticular crypt epithelium(cytokeratin) BALT GC Peyer's patches, appendix and solitary intestinal lymphoid follicles (GALT) Crypt

10. Inductive mucosa-associated lymphoid tissue (MALT) in human airways Inductive sites Human NALTanlagen prenatal (19 wks).Nasal ILFs in 40% < 2 yrs (inducible?).Rodent NALT: postnatal organogenesis Tonsils & adenoids (NALT) Human BALT in 40%-100% at young age (2-15 yrs); rare in adult normal lungs (>20 yrs), but inducible (also rodent BALT is inducible) BALT Adult lung with chronic infection Human bronchus-associtaed lymphoid tissue (BALT) Submucosal glands

11. HLA-DR HLA-DR DC DC

12. CD3(T)FoxP3(Treg) CD3(T)CD20(B)

13. Human nasal mucosa HLA-DR RFD-7 Jahnsen FL, Gran E, Haye R, Brandtzaeg P. Am J Respir Cell Mol Biol, 2004; 30:31-37 Nasal anatomy and location of regional lymphoid tissue Waldeyer’s ring Organized lymphoid tissue with M cells Airway mucosa is extremely rich in dendritic cells Olfactory region Olfactory region Adenoids Ciliated mucosa Tubal tonsil Middle turbinate Skin Inferior turbinate Oral cavity Palatine tonsil Tounge Lingual tonsil 150-200 cm2 Epiglottis

14. Regional lymph drainage through mesenteric and cervical lymph nodes Mesenteric lymph nodes Thoracic duct Right lymphatic duct Cervical lymph nodes

15. Sublingual vaccination Regional lymph drainage through mesenteric and cervical lymph nodes Mesenteric lymph nodes Thoracic duct Right lymphatic duct Cervical lymph nodes

16. Immune compart-ments of the lung From: Holt et al. Nature Rev. Immunol. 2008; 8:142-52 CD4+ T cells only become activated once M. tuberculosis spreads from the lungs to the lymph nodes and starts to produce antigen Wolf et al. J. Exp. Med. 2008 205: 105-15

17. Airway luminal T cells: a newcomer on the stage of TB vaccination strategies Jeyanathan M, Heriazon A, Xing Z. Airway luminal T cells: a newcomer on the stage of TB vaccination strategies. Trends Immunol. 2010; 31: 247-52

18. pIgR/SC knockout mice lack epithelial IgA transport +/+ IgAIgG –/– Johansen et al. J. Exp. Med. 1999; 190: 915-21

19. Mice i.n. vaccinated with PstS-1 antigen (plus CT) and then i.n. infected Naïve mice infected i.n. with 106 CFUs of BCG Effect of mucophilic SIgA and the ciliary conveyer band?

20. Proceedings • Passive administration of purified secretory IgA from human colostrum induces protection against Mycobacterium tuberculosis in a murine model of progressive pulmonary infection Nadine Alvarez1, Oscar Otero1, Frank Camacho1, Reinier Borrero1, Yanely Tirado1, Alina Puig1, Alicia Aguilar1, Cesar Rivas2, Axel Cervantes2, Gustavo Falero-Díaz1, Armando Cádiz3, María E Sarmiento1, Mohd Nor Norazmi4,5, Rogelio Hernández-Pando2 and Armando Acosta1* • * Corresponding author: Armando Acosta aracosta2001@yahoo.com Author Affiliations 1 Department of Molecular Biology. Finlay Institute. Center of Research – Producction of Vaccines. Ave. 27 No. 19805, La Lisa. Ciudad de la Habana, Cuba. AP. 16017, CP 11600 2 Experimental Pathology Section, Department of Pathology, National Institute of Medical Sciences and Nutrition “Salvador Zubiràn”, D.F. Mexico. CP 14 000 3 Enterprise of Production of Serum and Hemoderivates “Adalberto Pesant González”. Ave 51 No.33 235 km 19 medio ½. Arroyo Arenas, La Lisa. Ciudad de la Habana, Cuba. CP 13400 4 School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Malaysia 5 Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 16150 Kubang Kerian, Malaysia For all author emails, please log on. BMC Immunology 2013, 14(Suppl 1):S3doi:10.1186/1471-2172-14-S1-S3 Authors from Cuba, Mexico and Malaysia

21. Determination of bacterial load (A) and pneumonic area (B) in lungs of mice which were untreated (NT) and those treated with human secretory IgA (hsIgA), after challenge with M. tuberculosis H37Rv by intratracheal route 2 hrs after inoculation. Another group received M. tuberculosis preincubated with hsIgA (preinc). Well-organized granuloma of preincubated group 2 months after challenge with M. tuberculosis, visualized by H&E staining (25x) (C).

22. Such vaccine administration elicits both regional mucosal and systemic immunity • Future strategy: prime-boost approach, e.g. BCG (prime) followed by mucosal boost, or vice versa

23. Acknowledgements Laboratory for Immunohistochemistry and Immunopathology (LIIPAT) is part of Centre for Vaccinology and Immunotherapy (CEVI, 2001) and Centre of Excellence for Immune Regulation (CIR, 2007), funded by the Research Council of Norway, University of Oslo and Rikshospitalet University Hospital