Pneumolysin fusion proteins as novel vaccine candidates against experimental pneumococcal disease

1. Pneumolysin fusion proteins as novel vaccine candidates against experimental pneumococcal disease Dr Kirsty Ross

2. Introduction • Streptococcus pneumoniae • The burden of pneumococcal disease • Current and novel vaccine development • PATH project • Serology • Protection from colonisation • Protection from invasive disease • Impact on host

3. Pneumococcal disease • Non-invasive disease • Otitis media • Sinusitis • Invasive disease • Pneumonia • Bacteræmia • Meningitis • http://courses.cit.cornell.edu/biomi290/microscopycases/OtitisBiofilm/images/earDiagram.gif • http://img.thebody.com/sfaf/2008/winter08_pneumonia1.jpg

4. Pneumococcal disease • Non-invasive disease • Otitis media • Sinusitis • Invasive disease • Pneumonia • Bacteræmia • Meningitis • O’Brien et al Lancet 2009; 374: 893-902

5. Streptococcus pneumoniae • Gram positive, -haemolytic, aerotolerant anaerobe • Capsule-main virulence factor • Phosphorylcholine • Pneumolysin • Protein virulence factors • http://www.laek-rlp.de/pictures/pneumokokken.png • Mitchell et alNature Reviews Microbiology1, 219-230 (December 2003)

6. Current vaccines Pneumovax® Capsular polysaccharide only 23 capsular types (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F & 33F) Not efficacious in children under 2

7. Current vaccines Pneumovax® Capsular polysaccharide only 23 capsular types (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F & 33F) Not efficacious in children under 2 Prevenar/Prevnar® (PCV7) Capsular polysaccharide chemically conjugated to CRM197 7 capsular types (4, 6B, 9V, 14, 18C, 19F & 23F) Versions with increased valency also available with additional serotypes (1, 3, 5, 6A, 7F, & 19A) Efficacious in children under 2

8. Current vaccines Pneumovax® Capsular polysaccharide only 23 capsular types (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F & 33F) Not efficacious in children under 2 Prevenar/Prevnar® (PCV7) Capsular polysaccharide chemically conjugated to CRM197 7 capsular types (4, 6B, 9V, 14, 18C, 19F & 23F) Increased valency versions also available with additional serotypes (1, 3, 5, 6A, 7F, & 19A) Efficacious in children under 2 Pros Major decrease IPD caused by vaccine serotypes (VT) Protein based conjugation improves efficacy in children under 2 Herd immunity Cons Expensive to produce Replacement disease with non-vaccine serotypes (NVT) Polysaccharide only vaccines require boosters every 5-10 years

9. Next generation vaccines • Subunit based • Many potentially protective antigens have been identified using whole genome analysis and signature tagged mutagenesis screens

10. Next generation vaccines • Subunit based • Many potentially protective antigens have been identified using whole genome analysis and signature tagged mutagenesis screens • Cheap to manufacture • Protein based vaccines would exploit existing manufacturing technologies for high expression levels of protein

11. Next generation vaccines • Subunit based • Many potentially protective antigens have been identified using whole genome analysis and signature tagged mutagenesis screens • Cheap to manufacture • Protein based vaccines would exploit existing manufacturing technologies for high expression levels of protein • Ease of distribution • Ideally would be freeze dried or lack a ‘cold chain supply’, which would simplify delivery in developing countries

12. Next generation vaccines • Subunit based • Many potentially protective antigens have been identified using whole genome analysis and signature tagged mutagenesis screens • Cheap to manufacture • Protein based vaccines would exploit existing manufacturing technologies for high expression levels of protein • Ease of distribution • Ideally would be freeze dried or lack a ‘cold chain supply’, which would simplify delivery in developing countries • Mucosal delivery • Avoid potential for contamination with blood-borne diseases and increase patient acceptability

13. Next generation vaccines • Subunit based • Many potentially protective antigens have been identified using whole genome analysis and signature tagged mutagenesis screens • Cheap to manufacture • Protein based vaccines would exploit existing manufacturing technologies for high expression levels of protein • Ease of distribution • Ideally would be freeze dried or lack a ‘cold chain supply’, which would simplify delivery in developing countries • Mucosal delivery • Avoid potential for contamination with blood-borne diseases and increase patient acceptability • Life-long immunity • Ideally protection would be conferred for life

14. Concept behind PATH project: • Antigens fused to the N terminus of • pneumolysin (PLY) become immunogenic

15. Concept behind PATH project: • eGFP and PsaA were fused to PLY and 6PLY • One 200ng dose sufficient to stimulate systemic antigen specific IgG • Antigens had to be fused to PLY to elicit a response • Antigen specific IgA detected in nasal lavage and BALF • Douce et al Vaccine

16. Objectives Clone and fuse four vaccine candidates (PsaA, PspA, PspC & PhtD) from S. pneumoniae TIGR4 to PLY & 6PLY Purify protein & vaccinate i.n. & s.c. to evaluate immune response to fused antigens Challenge vaccinated mice with homologous and heterologous pneumococcal strains in models of colonisation & pneumonia

17. Cloning & protein purification • Infusion system • Resulting constructs: • For further details, see Jiangtao Ma

18. Purified protein & native protein from cell lysates • Pooled antisera from animals vaccinated with PsaA6PLY • Sera recognises both recombinant & native forms of each protein partner • Jiangtao Ma

19. Single antigens & PLY group • 10xMixture6PLY group • Vaccinated with either a mixture of single antigens or PLY • This was administered three times intranasally at fortnightly intervals • This group received 8µg 10xMix6PLY • This was administered three times intranasally at fortnightly intervals • S. pneumoniae invasion • S. pneumoniae colonisation • All mice were given ~ 107 cfu/50 µl of D39 S. pneumoniae • Mice were monitored for 72hrs using the following: • Tail bleeds for development of bacteræmia • Mice were culled at 72hpi and processed for blood, brain, nasal wash, & lungs. • All mice were given ~ 107 cfu/10 µl of Xen 10 A66.1, D39 or Xen 35 TIGR4 S. pneumoniae • Mice were monitored for 72hrs using the following: • Initial images taken using a Xenogen IVIS 200 using 5 min on large bin, FOV E • Tail bleeds for development of bacteræmia • Mice were culled at 72hpi and processed for blood, brain, nasal wash, & lungs.

20. In conclusion • PLY & 6PLY are capable of significant adjuvant activity at nanogram doses

21. In conclusion • PLY & 6PLY are capable of significant adjuvant activity at nanogram doses • Fusing other antigens to the N terminus appears not to affect haemolytic activity (max. 96kDa tested so far)

22. In conclusion • PLY & 6PLY are capable of significant adjuvant activity at nanogram doses • Fusing other antigens to the N terminus appears not to affect haemolytic activity (max. 96kDa tested so far) • High titres of antigen specific IgG and IgA are generated in response to i.n. delivery

23. In conclusion • PLY & 6PLY are capable of significant adjuvant activity at nanogram doses • Fusing other antigens to the N terminus appears not to affect haemolytic activity (max. 96kDa tested so far) • High titres of antigen specific IgG and IgA are generated in response to i.n. delivery • Potential use as a platform delivery system for other antigens of interest

24. In conclusion • PLY & 6PLY are capable of significant adjuvant activity at nanogram doses • Fusing other antigens to the N terminus appears not to affect haemolytic activity (max. 96kDa tested so far) • High titres of antigen specific IgG and IgA are generated in response to i.n. delivery • Potential use as a platform delivery system for other antigens of interest The response to mixed fusion proteins included:

25. In conclusion • PLY & 6PLY are capable of significant adjuvant activity at nanogram doses • Fusing other antigens to the N terminus appears not to affect haemolytic activity (max. 96kDa tested so far) • High titres of antigen specific IgG and IgA are generated in response to i.n. delivery • Potential use as a platform delivery system for other antigens of interest The response to mixed fusion proteins included: • Increased clearance of three different serotypes of pneumococcus during colonisation

26. In conclusion • PLY & 6PLY are capable of significant adjuvant activity at nanogram doses • Fusing other antigens to the N terminus appears not to affect haemolytic activity (max. 96kDa tested so far) • High titres of antigen specific IgG and IgA are generated in response to i.n. delivery • Potential use as a platform delivery system for other antigens of interest The response to mixed fusion proteins included: • Increased clearance of three different serotypes of pneumococcus during colonisation • Reduction in bacterial burden & protection from invasive disease caused by a heterologous serotype

27. Thank you Prof. Tim Mitchell Dr Gill Douce Jiangtao Ma Dr Andrea Mitchell Ashleigh Holmes Catherine Dalziel Carol McInally Jenny Herbert M. Kashif Mughal Matt Horsham Ryan Ritchie M. Sultan Al-Sharif M. Yahya Noori