Lars R. Haaheim ( ) PhD, Professor Emeritus, University of Bergen, Norway

Lars R. Haaheim (1945-2011) PhD, Professor Emeritus, University of Bergen, Norway

Licensing requirements a personal perspective John M Wood Summer School on Influenza, Siena August 1-5 2011 National Institute for Biological Standards and Control Assuring the quality of biological medicines

EU licensing of seasonal influenza vaccines Committee for Proprietary Medicinal Products (CHMP) Note for Guidance on harmonisation of requirements for influenza vaccines 12 March, 1997 CPMP/BWP/214/96 A. Yearly choice of influenza virus strains for vaccine B. Labelling C. Potency of influenza vaccine D. Control Authority Batch Release of influenza vaccine E. Clinical trials related to yearly licensing of influenza vaccine

Calendar of influenza vaccine production F M A M J J A S O Public sector WHO EU Vacc. available Clinical trial Licence HGR Vaccine potency reagents Batch release HGR yield Ag Clinical trial vaccine Seed Vaccine production/formulation/qc Vaccine manufacturer

WHO recommendation on influenza vaccine composition

The WHO process WHO Global Influenza Surveillance Network (GISN) Epidemiological data Antigenic and genetic analysis of virus isolates Serological studies WHO Collaborating Centres WHO CCs, Essential Regulatory Laboratories (e.g. NIBSC) WHO recommendation on composition of influenza virus vaccines

Criteria used to make WHO Recommendation Three vaccine components A(H1N1) A(H3N2) B (1 out of the 2 lineages VIC or YAM) Antigenic variants Drift variants based on HI data (low reactors) and antigenic cartography Geographic/temporal distribution of the variants Genetic clusters Phylogeny Clustering of antigenic drift variants Serology Reactivity of post-vaccination sera with recent influenza viruses ( 50% reduction post-gmt)

WHO Recommendation for NH 2011-2012

Success of WHO recommendations From the seasons 1986/7 to 2009/10 24 recommendations were made for three vaccine strains ie 72 recommendations: H1N1 75% success includes mis-match for 2009 pandemic H3N2 58% success B 83% success Overall 72% success

Production of influenza vaccine viruses by reassortment New virus (WHO strain) New vaccine virus (reassortant) double/mixed infection High-yielding strain PR8

Laboratories developing hgrs

High growth reassortant viruses derived from A/Uruguay/716/2007 - yield Method: Bulk virus production in eggs Virus purification (gradient) Total protein determination virus Yield (mg viral protein / 100 eggs) NIB-50 9.6 +/- 1.2 X-175C 12.1 +/- 1.4 IVR-149 5.4 A/Uruguay/716/2007 1.6 X-175C gave 7.6-fold increase in yield

Pandemic vaccine strains 2003 onwards highly pathogenic H5N1 virus spread around the world in wild and domestic birds and human fatalities increased Great urgency in pandemic preparation 2009 - Pandemic H1N1, although mild caused global panic Pandemic virus Removal of genes encoding basic amino acids at HA cleavage region PR8 Safety tests

Influenza vaccine potency

History of influenza vaccine standardisation P Krag & M Weis Bentzon (1971). International Standard for haemagglutination assays O Ouchterlony (1949); G Mancini (1965). Single Radial Diffusion (SRD) used to measure Diphtheria toxin and albumin G Schild et al (1972,1975). First used SRD for estimates of influenza haemagglutinin antigen and established basic principles of assay J Wood et al (1977, 1981) Improved assay Proposal to estimate vaccine potency in µg HA activity/ml Comparison between haemagglutination titres and µg HA activity Haemagglutination assay unsuitable for split vaccines Use of assay for trivalent vaccines International collaborative study 1978 WHO workshop at University of Bergen

History of SRD Barry et al, 1977; Ennis et al, 1977; MRC, 1977. Clinical trials of swine flu H1N1 vaccines demonstrated that SRD and not haemagglutination assays were suitable for standardising split, subunit and whole virus vaccines and the SRD HA content was proportional to vaccine immunogenicity. WHO, 1979. Recommendation for use of SRD to standardise influenza vaccine potency

Influenza vaccine SRD assay Antigen dilution 1.0 0.75 0.5 0.25 V1 d 2 80 70 60 Std V1 V2 50 V2 40 30 20 10 Std 0 0 0,25 0,5 0,75 1 Antigen dilution

Influenza SRD precipitin reaction Before diffusion After diffusion (stained blue)

% HI 40 A/New Jersey/8/76 (H1N1) whole virus vaccine clinical trials (28 trials USA, UK) 100 80 Unprimed Primed 60 40 20 0 0 10 20 30 40 50 60 70 80 90 100 Vaccine dose (mg/ha) 0 10 20 30 40 50 60 70 80 90 100 Vaccine dose (mg/ha) 1 dose 2 doses

SRD collaborative studies 1989 EU study (NIBSC coordinator) 1990 EU study (NIBSC coordinator) 1998 EU proficiency study (EDQM PTS013) 2003 EU proficiency study (EDQM PTS051) 2009 EU proficiency study (EDQM PTS118)

Overall conclusions from SRD collaborative studies Use of same methods between-laboratory reproducibility <10% Use of local methods - between-laboratory reproducibility from 7-38% High due to outliers, assay invalidity Slope ratio and parallel line analysis give similar results (3 studies) Number of outliers can be reduced after proficiency study Between-laboratory reproducibility approx. 10%

NIBSC reagents for influenza vaccine SRD potency assay

WHO Essential Regulatory Laboratories

Pandemic vaccine potency assays SRD reagents developed for H5N1 and H1N1pdm vaccines H5N1 SRD assay H1N1pdm SRD assay In order to save time, alternative potency assays accepted HPLC; SDS PAGE

Influenza vaccine potency assays the future? Workshop on lessons learned from potency testing of pandemic (H1N1) 2009 influenza vaccines and considerations for future potency tests July 27-29, 2010 Ottawa, Canada SRD: Difficulties and delays in preparation, standardization, and distribution of reference reagents (antigen and antiserum); and uncertain correlation of HA concentration with induction of protective immunity. Pandemic vaccines: alternative potency assays (HPLC, SDS PAGE) used. SRD improvements needed Improve reagent calibration Standardise SRD methods and aim for a single set of reagents worldwide EDQM SRD proficiency study 2009 PTS118 Increase number of ERLs Evaluate alternative methods HPLC, SDS PAGE, MS, SPR

Flu vaccine potency assays the future? Surface plasmon resonance technology

Influenza serology assays Haemagglutination-Inhibition Virus Neutralisation Single-Radial-Haemolysis HI VN SRH

Collaborative studies to evaluate reproducibility of flu serology assays Independent study 1994 Seasonal H1N1, H3N2, B viruses; HI and SRH (Wood et al, 1994) WHO studies 2007 H3N2 virus; HI and VN (Stephenson et al, 2007) 2009 H5N1 virus; HI and VN (Stephenson et al, 2009) 2009-10 H1N1pdm; HI and VN (Wood et al, manuscript in preparation) EU studies 2007-9 Seasonal H1N1, H3N2, B viruses; HI and SRH (EDQM BSP063) 2009-10 Retesting of sera from pandemic influenza vaccine trials; HI and VN (Wagner et al, manuscript in preparation)

Within laboratory reproducibility was generally good Four studies 1994-2009: Most replicate HI and VN assay titres were within a two-fold range One study 1994: Most replicate SRH titres were within a 1.25-fold range

Between laboratory reproducibility was poor HI assay Median GCV 112-261% (16-128-fold) SRH assay Median GCV 57% (3.8-fold) VN assay Median GCV 175-359% (35-724-fold) EDQM study variability affected compliance with licensing criteria

Use of an antibody standard In all studies, use of a human antibody standard reduced variability by approx. 50% An International Standard for antibody to H5N1 clade 1 was established (07/150) A candidate International Standard for antibody to pandemic H1N1 was established (09/194) Use of 09/194 reduced variability of pandemic vaccine clinical trial data between EU vaccine manufacturers

2009 collaborative study to evaluate candidate IS for antibody to pandemic H1N1 Began in September 2009 Antisera Freeze dried candidate International Standard, 09/194 (pool of convalescent and postvaccination sera) Low, medium, high titre human test sera Low serum A - convalescent subject; medium and high sera B and F - post-vaccination subjects) 2 identical sera reproducibility assessment (sera B and E) Liquid pre-09/194 serum (serum C) Negative serum (serum D) Viruses (seed virus supplied if needed) A/California/07/2009 (H1N1)pdm NYMCX-179A Assays VN, HI three assays requested

A candidate IS for antibody to pandemic H1N1 Test serum C HI assay VN assay 15 Sample C - Absolute HI titres 15 Sample C - Absolute VN titres 14 14 13 13 12 12 Antibody titres to X-179A or A/Cal/7/09 virus 11 10 9 8 7 11 10 9 8 7 6 13 6 5 11 10 5 4 08 06 4 13 11 3 13 05 12 3 08 05 2 08 04 03 2 13 05 06 10 1 11 06 04 05 09 1 11 12 04 04 06 09 0 -ve 10 20 40 80 160 320 640 1280 2560 5120 10240 20480 40960 81920 0 -ve 10 20 40 80 160 320 640 1280 2560 5120 10240 20480 40960 81920 HI Titre VN Titre NYMC-X179A A/California NYMC-X179A A/California 15 Sample C - HI relative to 09/194 15 Sample C - VN relative to 09/194 14 13 14 13 11 13 Antibody titres relative to 09/194 12 11 10 9 8 7 10 09 06 05 04 03 12 11 10 9 8 7 13 11 6 13 6 10 12 5 11 5 09 06 4 08 4 08 05 3 06 3 13 04 2 05 2 11 06 1 08 04 12 1 04 05 0 -ve 10 20 40 80 160 320 640 1280 2560 5120 10240 20480 40960 81920 0 -ve 10 20 40 80 160 320 640 1280 2560 5120 10240 20480 40960 81920 HI Titre relative to 09/194 VN Titre relative to 09/194

Summary of between laboratory variation - use of 09/194 GCV % Assay virus HI VN A B C E F A B C E F X-179A 95 294 89 345 105 211 294 204 383 274 Absolute titres Cal/7/09 81 151 81 146 83 228 382 255 412 175 Titres relative to 09/194 X-179A 34 161 36 231 109 77 133 44 214 111 Cal/7/09 20 96 12 92 36 259 152 37 182 250

Correlation between HI and VN titres laboratory trends 16384 4096 1024 256 64 16 4 1 4096 4096 1024 1024 256 256 64 64 16 16 4 4 5 4 6 1 1 A B C E F 09/194 A B C E F 09/194 A B C E F 09/194 VN HI 1024 256 64 16 4 1 65536 16384 16384 4096 4096 1024 1024 256 256 64 64 8 16 16 4 9 4 10 1 1 A B C E F 09/194 A B C E F 09/194 A B C E F 09/194

Conclusions from H1N1pdm study There was a significant degree of variability between H1N1pdm serology data from different laboratories Similar to previous studies with H3N2 and H5N1 In general, candidate International Standard 09/194 reduced by 50%, inter laboratory variability of HI and VN assays for antibody to H1N1pdm viruses The relationship between HI and VN titres varied between laboratories In general there was a correlation between HI and VN within a laboratory Consensus titre for candidate International Standard 09/194 HI 1:183 VN 1:516 IU 1300 09/194 accepted as an International Standard by WHO, October 2010

EMA retesting study CHMP expert meeting on pandemic influenza vaccine serology 31 July 2009 Recognised the likelihood of a large inter-laboratory variability in serology assay results Urgent need to centrally re-test a sub-set of sera from different vaccine clinical trials Centralised re-testing of sera will include the candidate International Standard for antibody to H1N1pdm Letter to vaccine manufacturers 26 October 2009 Two central serology laboratories were identified HI assay NIBSC VN assay PEI Each vaccine manufacturer requested to submit selected sera to each of the central laboratories 30 subjects from each age group: children (<10y), adolescents (10-17y), adults (18-59y), elderly (>60y) total 120 subjects Sera from pre-, post 1 dose, post 2 doses and to include negative, low and high antibody titres (ie selected) Sera were tested blind at NIBSC and PEI and then un-blinded after data sent to vaccine manufacturer As the sera were selected, the study would not allow assessment of CHMP compliance 42

GMT log 2 scale GMT log 2 scale Adults Comparison of raw and adjusted HI data GMT log 2 scale GMT log 2 scale 10240 5120 2560 1280 640 320 160 80 40 20 10 5 Manufacturers raw data 1pr 1p11p2 2pr 2p12p2 3pr 3p13p2 4pr 4p14p2 5pr 5p15p2 10240 5120 2560 1280 640 320 160 80 40 20 10 5 Manufacturers adjusted data 1pr 1p1 1p2 2pr 2p1 2p2 3pr 3p1 3p2 4pr 4p1 4p2 5pr 5p1 5p2 Manufacturers Manufacturers NIBSC raw data NIBSC adjusted data 10240 5120 2560 1280 640 320 160 80 40 20 10 5 1pr 1p11p2 2pr 2p12p2 3pr 3p13p2 4pr 4p14p2 5pr 5p15p2 10240 5120 2560 1280 640 320 160 80 40 20 10 5 1pr 1p1 1p2 2pr 2p1 2p2 3pr 3p1 3p2 4pr 4p1 4p2 5pr 5p1 5p2 Manufacturers Manufacturers 43

No 0 of individual results No 0 of individual results Comparison of raw and adjusted manufacturers and NIBSC HI data 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 Spread of ratios of Manufacturers data to NIBSC data Post 1 and Post 2 doses GMTs - raw data Ratio of manufacturers data to NIBSC data 8 Spread of ratios of Manufacturers data to NIBSC data Post 1 and Post 2 doses GMTs - adjusted data using IS 09/194 6 4 2 0 Ratio of manufacturers data to NIBSC data 44

Conclusions from retesting study HI Some consistent differences between manufacturers and NIBSC NIBSC HI titre of 09/194 (1:167) was close to WHO consensus; manufacturers HI titres ranged from 1:320-1:720 Much better agreement between manufacturers was obtained using 09/194 to adjust data VN Essentially similar data to HI 45

Conclusions from serology Despite technical deficiencies, HI assay is the gold standard for measurement of serum antibody to influenza HI data should be supplemented by VN data (or SRH) whenever possible There is inherent lab to lab variability of both assays May be possible to reduce variability by use of common protocols Use of antibody International Standards should reduce variability even further Adjust laboratory assay results using consensus titre of antibody International Standard Regional networks or laboratories could develop their own serology working standards based on the International Standards

Closing remarks Influenza vaccines are unique Strains in seasonal vaccines are updated in most years Pandemic vaccines use different technologies/strategies yet are needed urgently Constant strain renewal and updates in vaccine technology bring challenges for licensing and vaccine standardisation Safe and effective vaccines are achieved by a combination of WHO, national/regional regulatory authorities, public health authorities and vaccine manufacturers working closely together

Acknowledgements WHO CC Directors WHO secretariat ERL scientists Jackie Katz, CDC, USA Karl Nicholson, Iain Stephenson, U of Leicester, UK NIBSC staff, past and present Maria Zambon, Katja Höschler, HPA CfI, UK Ralf Wagner, PEI, Germany