Development of influenza vaccine production by means of chromatographic methods

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1 Development of influenza vaccine production by means of chromatographic methods WHO meeting on prospects for influenza vaccine technology transfer to vaccine manufacturers of developing countries March 2012 Belgrade, Serbia Dr. Igor Krasilnikov

2 Chromatography with controlled-size porous silica has been used in production. Types: Rabies vaccine Influenza vaccine Tick-borne encephalitis vaccine Hepatitis B vaccine 2

3 1 2 Elution of influenza viruses from macro porous silica. 1. Influenza virus B 2. Influenza virus A1

4 Сalibration dependence obtained with gel permeation chromatography of viruses on porous silica with different pore sizes Tick-borne encephalitis virus Reo 1 virus Influenza virus Sendai virus RS virus 4

5 Gel-permeated chromatography of virus suspensions Purification of allantois influenza virus on modified macroporous glass CPG 1000 (100 nm) Purification of Rabies virus suspension on macroporous silica glass MPG 1200 (120 nm)

6 Adsorption and gel-permeated chromatography of viruses 6

7 VACCINE PRODUCTION SOLVENT INACTIVATE D VIRUS- CONTAINING SUBSTRATE A.C.U.F INACTIVATED G.P.C INACTIVATED S.F. VIRUS VIRUS-PURIFIED CONCENTRATE CONCENTRATE INACTIVATED VIRUS ADSORBED on AL+++ FINAL VACCINE Virus suspension CONCENTRATION PURIFICATION ADSORPTION 0.5 ML/DOSE Contains allantois fluid by TIMES of 99,5-99,7% ON ALUMINIUM PURIFICATION HYDROXIDE A.C. ADSORPTION CHROMATOGRAPHY ON MACROPOROUS SILICA U.F. ULTRAFILTRATION ON TRACK MEMBRANES G.P.C. GEL-PERMEATED CHROMATOGRAPHY S.F. STERILIZING FILTRATION (0.22 MK)

8 Polypeptide composition of AviFlu (split virosomal) and OrniFlu (subunit) vaccines semi-products. Gel electrophoresis under nonreducing conditions. 1. Sample of purified virions, serotype N5N1, strain Indo/05/ AviFlu (split virosomal) vaccine semi-product, serotype N5N1, strain Indo/05/ OrniFlu (subunit) vaccine semi-product, serotype N5N1, strain A/Vietnam/1194/04 (NIBRG- 14)

9 Development of Split Virosomal Pre-pandemic Influenza Vaccine adjuvanted with Aluminum Hydroxide Inactivated Avian Influenza Split Virosomal Vaccine adjuvanted with aluminum hydroxide (AviFlu) is a pre-pandemic candidate vaccine based on: - A/Indonesia/5/2005 (H5N1), strain provided by СDC - a high-growth reassortant strain A/17/Duck/Potsdam/86/92 (H5N2) [Len17/H5] provided by the Institute of Experimental Medicine (IEM), St.Petersburg. In 2008 phase I double-blind studies in healthy adults were conducted to assess safety, reactogenicity and immunogenicity of the vaccine candidates. Clinical base: Mechnikov Research Institute of Vaccines and Serum, Moscow 9

10 Study s Conduct Procedures: 2 IM doses of the vaccine Given 28 days apart Reactogenicity: - Vaccinated participants screened for good health by Hx, PE and laboratory tests (Hgb, WBC, Plts, Cr, Alt; IgE) - Randomized and vaccinated at the first visit - Observed for 30 minutes after inoculation Memory aid for 7 days (captures of local and systemic symptoms) Call on day 2 to review memory aid Visit on day 7 for repeat laboratory tests and review of memory aid Call on day 14 to solicit AEs 10

11 Frequency (%) of post-vaccination local and systemic reactions V 1 AviFlu (Indo H 5 N 1 15 µg HA\dose) AviFlu (Indo H 5 N 1 30 µg HA\dose) AviFlu ( Duck H 5 N2 15 µg HA\dose) V 2 AviFlu ( Indo H 5 N 1 15 µg HA\dose) AviFlu (Indo H 5 N 1 30 µg HA\dose) AviFlu ( Duck H 5 N2 15 µg HA\dose) Low (t 37,0-37,5 º C) Post-vaccination reactions Systemic reaction (temperature) Local reactions (gyperemia, swelling, infiltrate) Middle (t 37,6-38,5 С) High (t > 38,6 С) Total Pain in the injection site No pain in the injection site % 1,7 % % 1,7 % % 0,8 % % 0,8 % - 5 % % 1,7 % % - 11

12 Frequency (%) of post-vaccination general reactions Symptoms AviFlu ( Indo H 5 N 1 15 µg HA\dose) AviFlu (Indo H 5 N 1 30 µg HA\dose) AviFlu ( Duck H 5 N2 15 µg HA\dose) V1 (n=24 ) V2 (n=24 ) V1 (n=20) V2 (n=20) V 1 (n=20) V 2 (n=20) Undue fatigability 4% - 5% - 5% - Headache 4% - 5% Vertigo - - 5% Rhinitis % - 5% Cough % - - Pharyngitis % - - Myalgia - - 5% Arthralgia 4% Nausea Diarrhea Sleepiness - 5% Total number of subjects 2 (8%) - 3 (15%) 1 (5%) 1 (5%) 1 (5%) 12

13 Immunogenicity and potential cross-reactivity of AviFlu in the Haemaglutination-Inhibition (HAI) Assay SC, % SP,% GMT SCF AviFlu Indo H5N1 15 µg HA\dose Indo H5N1 30 µg HA\dose Duck H5N2 15 µg HA\dose A/Ind. (H 5 N 1 ) NIBRG- 14 A/duck NIBRG- 14 NIBRG- 14 NIBRG- 14 (H 5 N 2 ) (H 5 N 1 ) (H 5 N 2 ) (H 5 N 1 ) (H 5 N 2 ) (H 5 N 1 ) (H 5 N 1 ) (H 5 N 1 ) (H 5 N 1 ) (H 5 N 1 ) 79,2 41,7 37, ,5 8,3 V1 26,6 11,5 10,3 V ,5 70, ,2 8, ,3 V ,6 10,7 11 V ,5 13,2 13, V1 56,6 49,3 56,5 V A/Ind. A/duck A/Ind. A/duck A/Ind. 4,8 2,2 2, ,7 52, ,9 10,5 20,4 9,5 3,7 5,1 3,4 2,6 A/duck (H 5 N 2 ) 3,3 2,1 2,2 2,6 11,3 9,9 11,3 CHMP criteria >40% >70% >2.5 13

14 Conclusions After vaccination with all the series of AviFlu vaccine candidate there were no detections of undue reactions in post-vaccination period. Slightly shaped local reactions were for a short period (1-3 days) and did not significantly influence a good state of health of the vaccine recipients. Two-fold immunization with the inactivated avian influenza split adjuvanted vaccine candidates has induced a strong antibody response to the homologous vaccine strains with the antibody titres above the seroprotection level (HAI and MN titre 40). In addition, the mock-up vaccines have promoted broad and persistent cross-clade immunity, which is a pre-requisite for a pre-pandemic vaccines. 14

15 Immunogenicity and protection of some novel inactivated influenza vaccine candidates. Comparative study. Aim of the Research: To compare immunogenic and protective activity of the several prototype vaccines against HPAIV in mice. FCLP (fish caviar-like particles): the adjuvant represents negatively charged particles with the size of about 100 nm

16 Immunogenicity and protection of some novel live and inactivated influenza vaccine candidates. Comparative study. Vaccine formulations: Inactivated: Split - H5N1 split vaccine bulk, strain A/Vietnam/1194/2004 (VNH5N1) \ 2,5 µg of HA 0,1 FCLP 2,5 µg of HA VNH5N1 adjuvanted with 0,083 mg of fish caviar-like particles FCLP 0,01 FCLP 2,5 µg of HA VNH5N1 adjuvanted with 0,0083 mg of fish caviar-like particles FCLP 0,1 Al 2,5 µg of HA VNH5N1 adjuvanted with 0,083 mg of AL(OH)3 0,01 Al 2,5 µg of HA VNH5N1 adjuvanted with 0,0083 mg of AL(OH)3 Vir H5N1 whole virus reassortant strain VNH5N1-PR8/CDC-RG \ 8 µg of HA PR8 H1N1 whole virus strain A/Puerto Rico/8/34/ \ 8 µg of HA Live: V-L H5N2 cold-adapted reassortant strain A/Vietnam/1203/2004(H5)-Leningrad/134/17/57-R; 5.0 lg TCID/mouse NC H1N1 reassortant strain-a/newcaledonia/20/99(h1n1) and strain A/Leningrad/134/17/57(H2N2);7.0 lg TCID/mouse Control phosphate buffered saline. Route: Inactivated formulations IM; LAIV IN. Challenge: H5N1 Chicken/Kurgan/3/2005; 4.0 lg TCID/mouse (more than 100 LD50%). Clinical base: Chumakov Institute of Poliomyelitis and Viral Encephalitis of RAMS, Moscow.

17 Study s results Groups of vaccine candidates Virus strain Antibody titer to H5N1 Days after control challenge Protectivity (%) Inactivated virus 1 Split H5N1 <20 2 0,1 FCLP H5N ,01 FCLP H5N ,1 Al H5N ,01Al H5N Vir H5N PR8 H1N1 <20 Live attenuated 8 V-L H5N NC` H1N1 <20 10 Control - < , , , , ,

18 Acknowledgements to Virology Center at the Microbiology Research Institute, the RF Ministry of Defense State Scientific Centre «Vector», the RF Ministry of Health Research Institute of experimental medicine of RAMS, St. Petersburg* Mechnikov Research Institute of Vaccines and Serum, Moscow Research Influenza Institute of RAMS, St. Petersburg Chumakov Institute of Poliomyelitis and Viral Encephalitis of RAMS, Moscow.**

19 THANK YOU FOR ATTENTION 19