1. Adaptive Immunity: Understanding cross-reactive responses Lorena E. Brown The University of Melbourne Australia

2. Is vaccination of everyone from birth onwards with current inactivated vaccines desirable? Would a population devoid of infection-induced cross-protective immunity be worse off in situations of: Vaccine mismatch/shortage Emergence of a new subtype Elderly experiencing OAS or waning B cell immunity Lack of vaccine efficacy in >30% population Modelling of the evolution of influenza virus suggests short-lived crossreactive immunity is essential to explain the linear nature of antigenic drift. Without this would we start to see more strain diversity and corresponding difficulty in infection control?

3. What adaptive immune mechanisms are cross-subtype reactive? CD8 + cytotoxic T cells (CTL) – kill cells infected with all type A viruses – can recognise peptides from internal proteins – thought to speed recovery – if present in high numbers at site of infection as memory cells may lead to subclinical outcome Non-neutralising antibodies that bind to viral antigens on infected cells –Lyse infected cells by Ab +C’ or ADCC –Best studied is M2 ectodomain

4. What adaptive immune mechanisms are cross subtype reactive? Stem domain: –Mabs from human B cells ( Throsby et al. PLoS ONE 3(12): e3942; Ekiert et al. Science. 2009 324:246; Corti et al. J. Clin Invest 2010 120:1663 ) –Induced by DNA/ split vaccine prime boost ( Wei et al., Science 2010 329, 1060) Site B epitope –Yoshida et al. PLoS Pathog. 2009 5(3): e1000350 10.1126/science.1195116 Antibodies to conserved regions of HA Site B

5. Investigating cross-protective mechanisms against H5N1 in the ferret model Days 3-5 mth old male/female seronegative Challenge 10 6 EID 50 wt H5N1 A/Vietnam/1203/2004 Vaccine rgA/Vietnam/1194/2004 or other split virus formulations i.m. serology (HI, micro virus neut.) weight temperature (rectal, transponder) clinical symptoms activity score post mortem tissues for histology virus isolation - rectal swab - nasal wash - oral swab - organs post mortem Days 3, 5 and 7 and 14 or at humane endpoint (systemic and pulmonary disease) 0 3 wk 7 wk

6. H5N1 inactivated split vaccines are fully protective when formulated with adjuvant Alert and playful Alert, play only when induced Alert but not playful Neither alert nor playful Culled at humane endpoint *IMX = ISCOMATRIX TM Middleton et al. J. Virol 2009 83:7770

7. Activity score Day post challenge 165 166 167 168177 178 179 180 161 162 163 164 1 3 5 6 7 14 4 2 PBS Fluvax TM + AlPO 4 Fluvax TM + IMX* Fluvax TM 157 158 159 160 * IMX = ISCOMATRIX TM adjuvant Seasonal influenza vaccine can protect against H5N1 173 174 175 176169 170 171 H3N2 + IMX H1N1 + IMX The H1N1 component is inducing the cross-protection Despite any crossreactive immunity measured by HI or VN assays VaccinePre-challenge HI GMT fraction respond Pre-challenge VN GMT fraction respond H5N1 + AlPO 4 45 4/4 H5N1 + IMX 45 4/4 128 4/4 Fluvax + AlPO 4 <4 0/4 Fluvax + IMX <4 0/4

8. * IMX = ISCOMATRIX TM adjuvant Crossreactive responses potentially against N1 BUT reassortant H3N1 vaccine shows no protection against H5N1 Day post challenge 1 3 5 6 7 14 4 255 256 257 258 H1N1 + IMX 251 252 253 254 H3N1 + IMX 259 260 261 262 H3N2 + IMX PBS 271 272 273 274 Activity score Ferret number

9. There is an imperfect correlation between neuraminidase inhibition activity of ferret serum and protection PBSH1N1H3N1H3N2rNArNP Vaccines formulated with ISCOMATRIX TM adjuvant 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 = survivors NI activity of pre-challenge ferret sera against H5N1

10. Conclusions from ferret data: Seasonal influenza vaccine can provide some protection against H5N1, particularly when formulated with an adjuvant. The H1N1 component provides the cross protective immunity This is not reflected by HI or VN assays but imperfectly by NI assays, so the N1s share epitopes for Ab and we should not discount their role in crossprotection. An H3N1 virus was non-protective, possibly due to low N1 levels, nor was H3N2 virus so these crossreactive responses are NOT HETEROSUBTYPIC

11. To induce true heterosubtypic responses we can prime CD8 + T cells (CTL) These are not induced efficiently by inactivated split virus vaccines A different type of vaccine that delivers antigen to the cytoplasm of dendritic cells is required Can’t adequately study these in ferrets

12. Testing benefits of adding CTL-inducing component to split virus vaccine for seasonal influenza Suboptimal dose of seasonal split vaccine mimics people responding poorly to vaccine mimics situation of vaccine mismatch CD4+ T cell epitope CD8+ T cell epitope K Pam2Cys Suboptimal dose of lipopeptide will induce influenza- specific CD4 + and CD8 + T cells will not prevent infection but aid clearance

13. TLR2 immature DC mature DC lipopeptide costimulatory molecule lipopeptide binds to TLR2 on DC surface via Pam2Cys lipopeptide enters cells by TLR2-mediated endocytosis so Ag can enter the class II processing pathway Ag can also escape the endosome and so can enter the class I processing pathway DC maturation is induced by TLR signalling; costim. and MHC II upregulated Help for: Antibody production priming of long lived memory CD8+ T cells T MHC molecules CD8+ T cells The lipopeptide used here do not have any B cell epitopes Lipopeptides CD4+ T cells T Kill virus- infected cells

14. Mixtures of suboptimal split vaccine and lipopeptide induce improved viral clearance Mice given a single dose of vaccine 3 weeks later mice are bled then challenged with A/Memphis/1.71 virus 5 days later lung virus titres are determined

15. Mixtures of suboptimal split vaccine and lipopeptide induce improved viral clearance Slight enhancement of Ab levels; sub cut combination vac. is approaching live virus levels

16. With even lower doses of split vaccine the improvement is best seen when vaccine is given by the i.n. route Though slightly enhanced, Ab levels are still low – other mechanisms may need to come into play

17. Large numbers of activated CD8 + T cells are present in the lungs of intranasally-primed mice 5 days after challenge with influenza influenza-specific IFN  - producing CD8 + T cells in the lungs measured by intracellular cytokine staining 5 days after infection CTLs expanded by infected cells as not enough antibody to remove virus these are as numerous as when induced by prior virus infection note that 5 days is before any T cells are present as a result of the challenge virus

18. Conclusions from lipopeptide/split virus data: Suboptimal doses (10µg and 0.3µg) of split virus leads to poor antibody production and viral clearance Viral clearance can be improved by the addition of a suboptimal dose of lipopeptide that induces T cell responses Extra “help” from CD4+ T cells plus increased DC activation during priming may provide a greater quantity or quality of Ab to enhance the 10 µg dose of split virus. When antibody is insufficient to control infection rapidly, as with the 0.3 µg dose of split virus, CTL-memory cells in the lungs can be activated and expanded to aid clearance Triggering of CTL memory cells already present in the lung (i.n. delivery) may be more efficient than relying on trafficking of memory cells from other organs (sub. cut. delivery) Different arms of the adaptive immune response are acting in synergy in these responses to the combination vaccines

19. Conclusion Co-induction of T cell and antibody responses by influenza vaccines may provide better protection against disease when seroconvertion has not been adequate due to: –vaccine mismatch, –waning B cell responsiveness, –original antigenic sin, –individuals not recently vaccinated –for emergence of a new subtype. Only through a greater understanding of the mechanisms of cross-protection and their induction can we hope to create vaccines that provide us with the much needed “safety net” of heterosubtypic immunity.

20. University of Melbourne David Jackson Joanna Cobbin Weiguang Zeng WHO Collaborating Centre for Influenza Reference and Research Ian Barr Main contributors CSL Limited Steve Rockman Martin Pearse CSIRO Australian Animal Health Laboratory Deborah Middleton Urgent Research into a Potential Avian Influenza-Induced Pandemic Grant Scheme