The development of H7N9 influenza vaccine in Taiwan and the strategy for universal flu vaccine design against influenza

Transcription

1 The development of H7N9 influenza vaccine in Taiwan and the strategy for universal flu vaccine design against influenza The Head of R&D Juine-Ruey Chen Ph.D 國光生物科技 ( 股 ) 公司 ADIMMUNE CORPORATION

2 Overview of the Adimmune The only cgmp manufacturer and company of human vaccine in Taiwan Founded in 1965 Company location Business Focus Primary Business Technology Partner 45 years of expertise in Vaccinology Taichung Vaccines, Diagnostics, & Biological Products Developer / Manufacturer / Distributor Crucell, Kitasato Institute Major Products (1) JEV vaccine, domestic market share : 100% (2) Tuberculin PPD, domestic market share : 100% (3) Tetanus toxoid, domestic market share : 50% (4) Influenza vaccine, domestic market share : 30%

3 H7N9 raise pandemic concerns and the vaccine preparation By 29 May 2013, approximately 2 months after the initial report, the number of laboratory-confirmed H7N9 infections reached 132, with 37 deaths. The case fatality proportion reached approximately 25%, which is a provisional value because many patients remain hospitalized as of 8 May 2013 and the number of mild cases remains unknown. However, most patients had a history of recent exposure to poultry, generally at live bird markets, however there is no evidence of sustained onwards virus transmission to other people. The concern for a pandemic caused by a newly emerged avian influenza A virus has led to clinical trials with H7N9 candidate vaccines. The Minister of health, Taiwan supported two grants for the Taiwan vaccine industry to develop the H7N9 vaccines. 國光生物科技 ( 股 ) 公司 ADIMMUNE CORPORATION

4 The challenge of development of H7 influenza vaccine (Sanofi Pasteur Inc) Conclusion: This inactivated subunit influenza A (H7N7) vaccine was safe but poorly immunogenic in humans 國光生物科技 ( 股 ) 公司 ADIMMUNE CORPORATION

5 Investigation of the best incubation condition for the H7N9 virus production Seed virus:e4 or E5 10 7~8 EID dilution 10-4 dilution 10-5 dilution 10-6 dilution 0.2mL/embryo egg 0.2mL /embryo egg 0.2mL /embryo egg 0.2mL /embryo egg 5 eggs/group 34±0.5 30hr 48hr 52hr 30hr 48hr 52hr 30hr 48hr 52hr 30hr 48hr 52hr Allantoic fluid collection 初離心移除卵蛋白 8,000g 4 30min 超高速離心收集病毒 (Pellet down) 130,000g 4 60min To estimate the HAU titers

6 Measurement of the H7 virus vaccine manufacturing yield rate with a small scale production 8,000g 60min 4 Centrifugation 130,000g 1hr 4 (269,000 rpm) Ultra-centrifugation Pellet down SW28 Sample 15mL Answer: 15µg HA/ per egg 25,000rpm 1.5hr 4 Ultra-centrifugation 20% 10mL SW28 50% 10mL 0.01M PBS 稀釋 virus band<30% sucrose 25,000rpm 1.5hr 4 Pellet down Ultra-centrifugation SW28 Sample 30% 25mL 10mL Whole virus Split virus 4 3wks 0.05% BPL 4 overnight 0.1% Tween80 Ether treatment 4 7days 0.01% Formalin

7 Improvement of the H7N9 vaccine efficacy in Adimmune Corp. Inactivated split and whole H7N9 virus vaccine (1) Human 15µg/ dose test in mice= 0.5µg (2) Human 45ug/ dose 1.5 µg (3) Human 90µg/ dose 3.0 µg (1) Human 15µg/ dose test in mice= 0.5µg (2) Human 45ug/ dose 1.5 µg (3) Human 90µg/ dose 3.0 µg (1) Human 15µg/ dose test in mice= 0.5µg (2) Human 45ug/ dose 1.5 µg (3) Human 90µg/ dose 3.0 µg Al(OH) 3 Squalene w/o adjuvant

8 The electron micrograph of A/Mallard/Netherlands/12/2000-IBCDC-1(H7N7) A/Shanghai/2/2013 IDCDC-RG32A (H7N9) inactivated split or whole viruses 國光生物科技 ( 股 ) 公司 ADIMMUNE CORPORATION

9 Chen JR, Ma C, Wong CH. Trends Bio technol Sep;29(9): How to prepare an effective influenza vaccine with glycoprotein HA

10 Purification of HA protein of H7N9 with Ni-NTA column IM(mM)

11 Digestion of H7N9 extracellular domain of HA with N-glycosidase PNGase F M min 130 Endo H M min PNGase F hydrolyzes nearly all types of N-glycan chains from glycopeptides/proteins. [x = H or sugar(s)] Endo H cleave only high mannose structures (n = 2-150, x = (Man)1-2, y = H) Adimmune

12 Purification and characterization of the H7N9 extracellular domain of HA ADIM-rHA ADIM-Whole

13 Quantification of the H7N9 vaccine bulk for clinical trial Split H7N9 HA Standard curve y = x R 2 = OD Standard(RD Split H7N9) 線性 (Standard(RD Split H7N9)) HA Conc (ug/ml) Methods ELISA SRID Sample name Sample HA Conc(μg/ml) Sample total protein(μg/ml) FAS13001 Split H7N FAS13002 Split H7N FAS13003 Split H7N ± ±23.9

14 Immunogenicity of H7N9 vaccine in mice A B C 國光生物科技 ( 股 ) 公司 ADIMMUNE CORPORATION

15 Inactivated H7N7 split and whole virus antigens combined with different adjuvants as a vaccine in mice (1) ug 1.5ug 3ug 1000 Serum HI titers (GMT) (Split) 0.5ug w/o (Split) 0.5ug +Al(OH) 3 (Split) 0.5ug +Addavax (whole) 0.5ug +Al(OH) 3 (whole) 0.5ug +Addavax (Split) 1.5ug w/o (Split) 1.5ug +Al(OH)3 (Split) 1.5ug +Addavax (whole) 1.5ug +Al(OH)3 (whole) 1.5ug +Addavax (Split) 3ug w/o (Split) 3ug +Al(OH)3 (Split) 3ug +Addavax (whole) 3ug +Al(OH)3 (whole) 3ug +Addavax 國光生物科技 ( 股 ) 公司 ADIMMUNE CORPORATION

16 H7N9 (Shanghai-2) vaccine not only elicited neutralizing antibodies against same strain of virus but also conferred cross-protection against H7N9 (Anhui-1) and H7N7 viruses Vaccine: H7N9 Shanghai-2 Virus: H7N9 Shanghai-2 Vaccine: H7N9 Shanghai-2 Virus: H7N9 Anhui-1 Vaccine: H7N9 Shanghai-2 Virus: H7N7 Neutralization titers (GMT) HAI 40 0 (Split) 0.5ug w/o (Split) 0.5ug +Al(OH) 3 (Split) 0.5ug +Addavax (whole) 0.5ug w/o (whole) 0.5ug +Al(OH) 3 (whole) 0.5ug +Addavax Non- immunized control 0 (Split) 0.5ug w/o (Split) 0.5ug Al(OH) 3 (Split) 0.5ug AddaVax (Whole) 0.5ug w/o (Whole) 0.5ug Al(OH)3 (Whole) 0.5ug AddaVax Non-immunized control 0 (Split) 0.5ug w/o (Split) 0.5ug +Al(OH) 3 (Split) 0.5ug +Addavax (whole) 0.5ug w/o (whole) 0.5ug +Al(OH) 3 (whole) 0.5ug +Addavax Non- immunized control 國光生物科技 ( 股 ) 公司 ADIMMUNE CORPORATION

17 H7N9 Virus Challenge studies in vaccine immunized mice AddaVax 0.5 g H7N9-S 0.5 g H7N9-S-Al(OH)3 0.5 g H7N9-S-AddaVax 0.1 g H7N9-S-AddaVax Survial Rate (%) Body weight (%) days after infection 國光生物科技 ( 股 ) 公司 ADIMMUNE CORPORATION

18 AddaVax 0.5 g H7N9-S 0.5 g H7N9-S-Al(OH)3 0.5 g H7N9-S-AddaVax 0.1 g H7N9-S-AddaVax Survial Rate (%)

19 Positive Results Presented from H7N9 vaccine with adjuvants in Clinical Trial

20 The Summary of Inactivated H7N9 Split Clinical Results 國光生物科技 ( 股 ) 公司 ADIMMUNE CORPORATION

21 Summary 1. The H7N9 inactivated virus vaccine is sufficient to confer cross-neutralizing immunity against H7N9 and H7N7 viruses 2. The Squalene-based H7N9 vaccine may be the best formulation for the further vaccine preparation 3. Adimmune alum-based H7N9 vaccine results showed that the seroprotection rate were met with the criteria for Phase II clinical and will conduct the phase III study in 國光生物科技 ( 股 ) 公司 ADIMMUNE CORPORATION

22 Glycoprotein vaccine design against influenza Speaker : Juine-Ruey Chen ( 陳俊叡 ) Ph.D 國光生物科技 ( 股 ) 公司 ADIMMUNE CORPORATION

23 Receptor binding and glycosylation sites on HA structure of H1N1 virus (A/Brisbane/59/2007)

24 Schematic overviews and circular dichroism spectra of HAs with different glycosylations Wang C-C and Chen J-R, et al. (2009), PNAS Vol. 106(43):

25 Glycan microarray analysis of HA with different glycosylations Wang C-C and Chen J-R, et al. (2009), PNAS Vol. 106(43):

26 Immunization of mice Consensus H5N1 Al(OH) 3 ; 5µg protein HA fg X days HA mg X 10 Immunization Recovery Surveillance survival 1 st 2 nd Antiserum collection Virus challenge Control PBS X 10

27 Comparison of HA fg and HA mg as vaccine Wang C-C and Chen J-R, et al. (2009), PNAS Vol. 106(43):

28 Glycosylation site analysis of recent HAs H1, H3, and H5 since The glycosylation sites of H5 in this work are shaded in pink. The comparison reveals similarity between H1 and H5 with regard to their N-glycosylation positions, whereas H3 has less-conserved glycosylation positions and differs from H1 and H5.

29 Seasonal flu whole inactivated virus vaccine can not protect the pandemic Swine flu A nurse vaccinates Barack Obama against H1N1

30 Characterization of the HA proteins from A/California/07/2009(H1N1) and A/Brisbane/59/2007(H1N1)

31 Evaluation of the HA protein vaccine efficacy in different animals model Mice Ferrets Guinea pigs

32 Cross-protection against Cal/09, WSN/33 and PR8/34 viruses with Bris/07 HA protein vaccine in mice Vaccine: Bris/07 Virus: Cal/09 Vaccine: Bris/07 Virus: WSN Vaccine: Bris/07 Virus: PR8

33 Cross-protection against Cal/09, WSN/33 and PR8/34 viruses with Bris/07 HA protein vaccine in ferrets Ferrets Antisera: Bris/07 Virus: RG121(Cal/09) Antisera: Bris/07 Virus: WSN/33 Antisera: Bris/07 Virus: PR8/34 Neutralization (%) 80 P< HA mg HA fg PBS Neutralization (%) 80 P< HA mg HA fg PBS Neutralization (%) P< HA mg HA fg PBS Dilution (log 2 ) Dilution (log 2 ) Dilution (log 2 )

34 The guinea pig as a transmission model for human influenza viruses

35 Transmission of Cal/09 virus from intranasally infected guinea pigs to uninfected contacts Days post inoculation

36 Chen JR, Ma C, Wong CH. Trends Bio technol Sep;29(9): The cover story encouraged our research work

37 Vaccine Design of Hemagglutinin Glycoprotein against Influenza

38 Summary The removal of the N-glycan structures on HA increased SA binding affinities while decreasing specificity toward disparate SA ligands. Antibodies raised against HA protein bearing only a single N-linked GlcNAc at each glycosylation site showed better binding affinity and neutralization activity against influenza subtypes than the fully glycosylated HAs elicited. This strategy opens a new direction for vaccine design and facilitates the development of vaccines against viruses such as influenza, hepatitis C virus, and HIV.