Slide 1

Innate Imprinting by the Modified Heat-Labile Toxin of Escherichia coli (LTK63) Provides Generic Protection Against Lung Infectious Disease Prepared by: Stewart Schaefer and Kevin Strozyk Nov. 19, 2008

Slide 2

Authors Andrew Evan Williams: Imperial College London National Heart and Lung Institute research associate Lorna Edwards: Respiratory immunopathology Ian Robert Humphreys: Respiratory immunopathology Robert Snelgrove: Respiratory immunopathology National Heart and Lung Institute research associate Aaron Rae: Respiratory immunopathology Rino Rappuoli: Globally renown for his work in vaccines and immunology Co-founded field of cell microbiology Tracy Hussell: British Society for Immunology

Slide 3

Innate Imprinting A subjects history of past infections individualizes their memory T cell pool (heterologous immunity) If we can mimic this heterologous immunity without actual infection via vaccination, we can provide a means to enhance innate immunity

Slide 4

Barriers to Respiratory Tract Vaccination Poor immunogenicity when delivered mucosally Antigen dilution or denaturation Regulation of mucosal surfaces Often requires codelivery of an adjuvant A safe, immunogenic adjuvant is yet to be created Too toxic Induces insufficient immune stimulation

Slide 5

Proposed Adjuvants Cholera toxin (CT) and heat-labile enterotoxin (LT) from E. Coli are among most potent mucosal adjuvants. Codelivery of cytokines? Modified LT with single-point mutations can inhibit enzyme function of A subunit Non-toxic Several useful applications This is our focus today

Slide 6

Heat-Labile Enterotoxin from E. Coli LTK63 has a serine to arginine substitution at a.a. 63 of the A subunit. This mutation detoxifies the protein. A subunit *Van Den Akker, F.; Feil, I.K.; Roach, C.; Platas, A.A.; Merritt, E.A.; and Hol, WGJ. Protein Science 1997 6: 2644-2649 AB5 heterohexamer: A ADP-ribosylating unit B GM1 receptor binding Causes severe diarrhea Potent mucosal adjuvant

Slide 7

Modified Lung Microenvironment Due to LTK63 Administration A)Number of cells increased in lung due to increase in B and T cell expansion. It is recognized as foreign. B) T H and T C cells also increased in number C) The proportion of B cells increased, as did their expression of markers of activation D) Number macrophages remained constant, but population expressing CD80 and MHC class II (markers of activation) increased. E) Increased level of Th1 cytokine profile (TNF-, IFN-) F&G) Changes in T cell clonality analyzed as proportion of cells expressing different V subunits. Increased proportion of CD4+ and CD8+ cells specific for LTK63. *X-axis represents subunit variety Figure 1

Slide 8

Weight Loss Prevention during Viral Infection i.n. ; Intra Nasal (injected via nasal cavity) i.p. ; Intra Peritoneal (inside peritoneal membrane of chest cavity) *Respiratory viral lung infection is usually accompanied with profound weight loss due to excessive cellular infiltration and production of inflammatory cytokines. A)Weight loss prevented in mice infected with G/RSV (attachment protein G of RSV) when given LTK i.n., not i.p. PBS = Phosphate Buffer Solution = control B) Weight Loss prevented in mice given LTK63 due to decrease in leukocyte infiltration in lungs RSV = Respiratory Syncytial Virus C) Reduction in leukocyte infiltration is conc. dependent D) Significant decrease in total number of leukocytes in lung (RSV) (Influenza) (RSV) (Influenza) Figure 2

Slide 9

Reduced Eosinophilic Inflammation A)LTK63 reduces the number of eosinophils in the lung when administered i.n. B) There is a corresponding increase in the proportion of neutrophils entering the lung with the decrease in eosinophils (out of total granulocytes) C) Mice infected w/ RSV 12 weeks after LTK63 administration. LTK63 still promotes reduced weight loss. It is long-lasting. D) LTK63 induces a decrease in the percentage of eosinophils with RSV (12 weeks post-LTK63 admin.). E) LTK63 induces a decrease in the percentage of eosinophils with fungal infection (12 weeks post-LTK63 admin.). *Reduction is not pathogen specific To assess the Long-Term effect of LTK63 Figure 3

Slide 10

Enhanced Proportion of Activated T cells A) CD4+ population not affected by administration of LTK63. B) CD8+ population increase in activation is concentration dependent. C) Following exposure to LTK63, the number of CD4+ cells show a 2-fold increase in activation (influenza) D) Following exposure to LTK63, the number of CD8+ cells show an 8-fold increase in activation (influenza) E) Proliferation is LTK63 conc. dependent Figure 4 F) LTK63 increases specific T-cell proliferation (E&F) - Employed [ 3 H]thymidine incorporation assay to measure proliferation. - Proliferation Index was calculated: (Experimental T-cell count)/(Control T-cell count) RSVInfluenza

Slide 11

Altered Cytokine Secretion in G-RSV Mice A) The increase in LTK63 concentration results in a shift to the Th1 subset via a greater proportion of IFN-(Th1-driven) relative to IL-5 (Th2-driven) B) Intranasal introduction is critical in shift to Th1 cytokine subset C&D) LTK63 administration results in a drop in total cytokine secretion. E) Prior administration of LTK63 results in a greater number of secreted IgA recovered from nasal washes (RSV-G) F) Prior administration of LTK63 results in a greater number of secreted IgA recovered from nasal washes (influenza) Figure 5 (RSV) (Influenza) *IgA is the only immunoglobulin found in mucosal membrane. Therefore, it is critical in lung disease in which the primary site of pathogen infection is across mucosal membrane.*

Slide 12

Improved Lung Pathology Following LTK63 Administration A, B, C) Administration of LTK63 2 weeks prior to infection reduced the prevalence of inflammation. Pfu = Plaque Forming Units Cfu = Colony Forming Units D, E, F) LTK63 does not facilitate the clearance of RSV pathogen, but causes a noticeable decrease in the titer for influenza and C. neoformans Dark Stain higher degree of inflammation Y-axis represents pathogen population CT = something to do with PCR Figure 6

Slide 13

Modified Ag Processing OVA = ovalbumin How does LTK63 facilitate a more efficient response To Ag while simultaneously decreasing total T cell pop? B) The effects of LTK63 are partially T & B cell independent. -LTK63 was administered to RAG knockout mice -Alveolar cells were removed and transferred to immunocompetent mice. -Weight loss is still reduced upon exposure to influenza. A) Whole Ova protein is not processed as efficiently with prior LTK63 administration. -Ova peptide processing was not affected. Figure 7

Slide 14

Summary A LOT of information to process but LTK63 stimulates a mild Th1 environment in the lung and matures APCs by increasing MHC class II, B7 costimulatory molecules, and CD40. The effectiveness of this adjuvant lies in its ability to mature antigen processing cells, thereby decreasing the T cell repertoire required to challenge a pathogen and thus marking a reduced inflammatory response. Overall reduction in total number of lymphocytes. Increased number of activated T and B cells.

Slide 15

Healthy Lung infect Infected Lung Normal Lymphocyte Population No inflammation Amplified Response (many lymphocytes) Lung inflammation, Tissue Damage Healthy Lung infect LTK63 (vaccine adjuvant) Infected Lung Fewer Lymphocytes Reduced Inflammation Inc. Resolution of Infection How? Enhanced T cell activation w/o Increased cell number Inc. Efficiency of APCs Modified Ag Processing Shift to Th1 cytokine subset Reduced eosinophilia Increased IgA secretion Normal Infection Pathology Innate Imprinting Kevin Strozyk and Stewart Schaefer, 2008