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2/29/16 Adaptive Immune Responses to Ranaviruses and Immune Evasion - PDF document

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2/29/16 Adaptive Immune Responses to Ranaviruses and Immune Evasion Strategies of Ranaviruses http://www.urmc.rochester.edu/smd/mbi/xenopus What is adaptive immunity anyway? An adaptive Immune System is present in all jawed vertebrates

  1. 2/29/16 Adaptive Immune Responses to Ranaviruses and Immune Evasion Strategies of Ranaviruses http://www.urmc.rochester.edu/smd/mbi/xenopus What is adaptive immunity anyway? An adaptive Immune System is present in all jawed vertebrates Characterized by: Ø a wide somatic diversification of immune receptor repertoires Ø high specificity of immune receptors for antigens, Ø long term immunological memory Ø and a complex cytokine- and chemokine-mediated regulatory network — Immunoglobulin (IgM, IgG or IgG-equivalent IgY, IgD - Fish IgZ, IgT) — T Cell Receptor ( α , β , γ , δ ) — MHC class II, classical class Ia (selection), nonclassical MHC class Ib — RAG-1, 2 mediated gene rearrangement, TdT — Somatic hypermutation (AID-mediated) — Primary and secondary lymphoid tissues (e.g. thymus, spleen, bone marrow, lymph nodes) 1

  2. 2/29/16 B cell receptors and T cell receptors Abs recognize recognize only (bind) peptides bound epitopes on whole to MHC proteins in solution molecules Somatic lymphocyte gene rearrangement Peptide Nature Rev. Immunoloy Antigen presenting cell (APC) MHC haplotypes Allelic polymorphism Some amphibian species have only 1 MHC class I gene per genome ( Xenopus ). Other have 2 or 3 genes per genomes ( Ranidae ) 2

  3. 2/29/16 Organization of the immune system Spleen Lymph node Liver Kidney spleen Evolutionary trajectory of lymphoid tissues in vertebrates General hematopoietic tissues Boehm et al., (2012) Special focus: Structure and function of lymphoid tissues. Trends Immunol. 33:315 Features of an Adaptive Immune System • Ig, TCR, MHC • RAG 1, 2 expression • Lymphoid Compartments Flajnik, Nature Rev. Immunology 2, 688-698 (2002) 3

  4. 2/29/16 Immunoglobulin Evolution Flajnik, Nature Reviews Immunology 2, 688-698 (2002) Chamel, llama Ab IgH only (no IgL chain) IgD IgZ, IgT IgMgi (no TM) No IgL chain Antiviral immune responses Memory Activation Expansion Effector Maturation phase Ags + Memory “danger” CD8 Kill class I + signals targets TLRs Apoptosis Class I CTL CD8 Co-stim Cytokine Cytokine (B7, CD40) Memory release release APC CD4 Inflam- CD4 masome Th Apoptosis Class II M φ Plasma B cells Robert and Ohta, 2009. Dev. Dyn. 238:1249 Metamorphosis ¡ Ø External development , absence of maternal influences on embryos Ø Tadpoles are immunocompetent but immature Ø Immune system develop early (10 days of age) Ø Only about 20,000 T cells, mainly innate T cells, in tadpoles Ø No classical MHC class I protein expression until metamorphosis Ø No NK cells, weaker T cell responses than adults Ø Drastic remodeling of the immune system during metamorphosis Ø Thymocytes degenerate, new thymic education from new progenitors 4

  5. 2/29/16 Urodelean adaptive immunity • Relatively poor adaptive immunity compared to anurans • Low IgM antibody heterogeneity (no specific IgY is produced • Expanded MHC class I repertoire (~100 genes) that may include classical and nonclassical MHC class I as well as a non-polymorphic MHC class II • Based on chronic rejection of allografts and xenografts, weak immune responses appear to characterize most species of salamanders • High susceptibility to ranavirus infection • But still able to survive in pathogen-rich environments Importance of B cells and antibodies in host response to ranavirus Humoral (antibody) response IgY IgY 5

  6. 2/29/16 Humoral (antibody) response Enzyme-Linked Immunosorbant Assay (ELISA) FV3 FV3 Ag + Antiserum Denatured (boiled) FV3 + Antiserum Coated Ag + Pre- immune serum Humoral response — Xenopus and mammals have similar organization and usage of their Ig genes ( RAG-dependent VDJ rearrangements ) — Thymus-dependent switch IgM to IgY (IgG functional equivalent), T-B collaboration — But Xenopus antibodies are limited in heterogeneity, mature poorly in affinity (less than 10 fold) and their serum titer increase only slightly during a secondary response — How important is the humoral response in the resistance against natural pathogens such as FV3 infection? 6

  7. 2/29/16 Anti-FV3 IgY antibody response Priming Boost 1 Boost 2 Immunization by infection without adjuvant d42 d28 d0 2 wks 4 wks d0 d3 d7 d10 d14 d17 d21 d24 d28 Naive d-28 .23 Adjusted absorbance Native .2 Denatured * * .17 * .15 * .13 .1 .08 .05 .03 0 Naïve d-28 0 3 7 10 14 17 21 24 28 Days Xenopus anti-FV3 IgY ( 1:200 dilution, O.D. = 0.4) Rabbit anti-FV3 IgG ( 1:20,000 dil, Oution.D. = 1.1) Maniero et al., Dev Comp Immunol 2006, 30:649 Long lasting B cell memory (Re-infection 15 months after primary infection) 0.6 Anti-FV3 IgY (1:100 dil.) O. D. 0.4 Rabbit serum 0.2 anti-FV3 1:20,000 dilution 0.0 O.D. = 1.1 0 1 2 3 4 5 6 Weeks Xenopus adult produce neutralizing anti-FV3 antibodies Neg. Control (no FV3) Immune serum 10:1 Naïve serum 10:1 ≈ 100 µ Positive Control Immune serum 1:1 Naïve serum 1:1 (FV3, no serum) 7

  8. 2/29/16 Neutralization capacity of Xenopus anti-FV3 serum by TCID50 1.E+06 Initial titer: 3x10 5 pfu/ml Nb. of surviving larvae 1.E+05 Pfu/ml 1.E+04 1.E+03 0 1/10 1/10 1/100 1/1000 1/10000 NXS FV3 antiserum Maniero et al., Dev Comp Immunol 2006, 30:649 Tadpole exhibit poor anti-ranavirus antibody responses Passive protection of anti-FV3 Immunization FV3 antiserum in susceptible larvae Heat inactivated + alum 10 100 p < 0.012 % survival Percent survival 8 80 6 60 4 40 anti-FV3 serum APBS Unimmun. 20 2 pre-immune serum Immunized 1,000 pfu anti-FV3 serum +1d 0 0 0 2 4 6 8 10 0 5 10 15 20 25 30 Days Days post-infection 1,000 pfu/animal ~ 10 ug of protein Summary I • Anuran amphibians like Xenopus are capable to generate effective antibodies (IgM and IgY) against ranaviruses More efficient, IgY , antibody response is elicited during a • secondary infection (No anti-FV3 Ab detected in adult sera during a primary infection in absence of adjuvant in Xenopus ) FV3-specific IgY antibodies (thymus-dependent IgG equivalent) • detected from 10 up to 24 days after re-infection (no adjuvent) • B cell memory lasting at least 15 months after a first infection Serum of immunized frogs contain antibodies that can neutralize • ranavirus ( Xenopus adults can generate potent neutralizing anti- FV3 antibodies, that are able to provide passive protection to susceptible tadpoles • Compared to adult frogs, tadpoles exhibit poor anti-ranavirus antibody response 8

  9. 2/29/16 Importance of T cells in host response to ranavirus Assessing T function by sublethal γ -irradiation ² T cell differentiation in the thymus is dependent on cell division, which is very sensitive to γ -irradiation ² Whole body γ -irradiation 5 to 10 Gray depletes mostly thymocytes and T cells ² This impairs adaptive immunity for 1 to 2 week (e.g., Skin graft rejection) ² Resistant adult Xenopus become susceptible and die from FV3 infection following sublethal γ -irradiation ² Infected γ -irradiated frogs also release more virus into the environment More specific assessment of CD8 T cells by Ab treatment v In vivo CD8 depletion by anti-CD8 mAb-treatment increases susceptibility to FV3 in adults 9

  10. 2/29/16 T cell memory Flow cytometry Detecting in vivo cell proliferation upon FV3 infection, primary response Bromo deoxyUridine (BrdU) Surface labeling Synthetic nucleoside analog of thymidine followed by intracellular BrdU α CD8 α CD5 Cell α Class II proliferation FV3 infection and BrdU incubation Flow cytometry (added in water in obscurity) FV3 (10 6 pfu) 2 days CD8 6 days + BrdU + BrdU 10

  11. 2/29/16 Primary Secondary FV3 + BrdU 2d before harvest FV3 + BrdU 2d before harvest 0 3 6 9 - 1 month - 0 3 6 9 Spleen Kidney 2-color flow cytometry Immuno-histo ( α CD8 or class II ) α CD8 or class II (surface) PCR, RT-PCR, α BrdU (intracellul) TCID50 Flow Cytometry Output C D3 D6 NCD8.013 D3CD8.014 D6CD8.015 1.1% 2.4% 0.51% CD8 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 BrdU FITC BrdU FITC BrdU FITC Brd U CD8 T cell proliferative response * P < 0.05 Total splenic CD8 T cells Proliferating splenic CD8 T cells ** P < 0.01 100 100 Log cell nb *** P < 0.001 10 10 *** * 1 Primary 1 0.1 0.1 0.01 0.01 0.001 0.001 | | | | | | | | C 3 6 9 C 3 6 9 100 100 10 10 ** Log cell nb ** ** 1 1 Secondary 0.1 0.1 0.01 0.01 0.001 0.001 | | | | | | | | C 3 6 9 C 3 6 9 Morales & Robert, J. Virol. 2007 Detection of FV3 and CD8 T cells in the kidney of infected adult frogs Anti-FV3 antibody Anti-CD8 antibody d0 d3 d0 50 µ m d10 d35 d6 Robert ¡et ¡al. ¡(2005). ¡Virology; ¡332: ¡667 ¡ ¡ 11

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