development of influenza vaccine production by means of ... · cpg 1000 (100 nm) purification of...
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Development of influenza vaccine production by means of chromatographic methods
Dr. Igor Krasilnikov
WHO meeting on prospects for influenza vaccine technologytransfer to vaccine manufacturers of developing countries.
27-28 March 2012Belgrade, Serbia
Types:
•Rabies vaccine
•Influenza vaccine
•Tick-borne encephalitis vaccine
•Hepatitis B vaccine
2
Chromatography with controlled-size porous silica
has been used in production.
Elution of influenza viruses from
macro porous silica.
1. Influenza virus B2. Influenza virus A1
1 2
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
Gel-permeated chromatography of virus
suspensions
Purification of allantoisinfluenza virus on modified
macroporous glassCPG 1000 (100 nm)
Purification of Rabies virus suspension on macroporous silica
glass MPG 1200 (120 nm)
INACTIVATED VIRUS-
CONTAINING SUBSTRATE
INACTIVATED VIRUS
CONCENTRATE
INACTIVATED VIRUS-PURIFIED CONCENTRATE
INACTIVATED VIRUS
ADSORBED on AL+++
A.C.U.F G.P.C S.F.
FINAL VACCINE
VACCINE PRODUCTION
SOLVENT
Virus suspension CONCENTRATION PURIFICATION ADSORPTION 0.5 ML/DOSE
Contains allantois fluid by 10-20 TIMES of 99,5-99,7% ON ALUMINIUM
PURIFICATION HYDROXIDE
A.C. ADSORPTION CHROMATOGRAPHY ON MACROPOROUS SILICAU.F. ULTRAFILTRATION ON TRACK MEMBRANESG.P.C. GEL-PERMEATED CHROMATOGRAPHYS.F. STERILIZING FILTRATION (0.22 MK)
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/05/20052. AviFlu (split virosomal) vaccine semi-product,serotype N5N1, strain Indo/05/20053. 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
10
Study’s Conduct
Procedures:– 2 IM doses of the vaccine– Given 28 days apartReactogenicity:- 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
11
Frequency (%) of post-vaccination local and systemic reactions
Post-vaccination reactions
Systemic reaction (temperature) Local reactions (gyperemia ,
swelling, infiltrate)Low (t 37,0-37,5 º C) Middle(t 37,6-38,5 С) High(t > 38,6 С) Total Pain in
the injection site
No pain in
the injection
site
V 1
AviFlu (Indo H5N1
15 µg HA\dose)- - - - 52 % 1,7 %
AviFlu (Indo H5N1
30 µg HA\dose)- - - - 20 % 1,7 %
AviFlu ( Duck H5N2
15 µg HA\dose)- - - - 30 % 0,8 %
V 2
AviFlu ( Indo H5N1
15 µg HA\dose)- - - - 24 % 0,8 %
AviFlu (Indo H5N1
30 µg HA\dose)- 5 % - - 15 % 1,7 %
AviFlu ( Duck H5N2
15 µg HA\dose)
- - - - 30 % -
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Frequency (%) of post-vaccination general reactions
Symptoms AviFlu ( Indo H5N1
15 µg HA\dose)
AviFlu (Indo H5N1
30 µg HA\dose)
AviFlu ( Duck H5N2
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% - 5%
Cough - - - 5% - -
Pharyngitis - - - 5% - -
Myalgia - - 5% - - -
Arthralgia 4% - - - - -
Nausea - - - - - -
Diarrhea - - - - - -
Sleepiness - 5% - - -
Total number of
subjects
2 (8%) - 3 (15%) 1 (5%) 1 (5%) 1 (5%)
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Immunogenicity and potential cross-reactivity of AviFlu in the
Haemaglutination-Inhibition (HAI) Assay
AviFluNIBRG-
14
NIBRG-
14
NIBRG-
14
NIBRG-
14
(H5N1) (H5N1) (H5N1) (H5N1)
80 75 90 10,9 10,5 20,4
>2.5>40% >70%CHMP criteria
2,62,65,1
11,3 9,9 11,3
3,7
49,3 56,5
79,2 41,7 37,5 50 12,5 8,3
8,329,2
54,7 52,7 102
Indo H5N1
15 µg
HA\dose
Indo H5N1
30 µg
HA\dose
Duck H5N2
15 µg
HA\dose
11,5 10,326,6
18,6
V2 85 90 100
V1
4,8 2,2 2,1
3,33,49,510070,862,5
V2 25,5 13,2 13,2251550455570
V1 10,7 11
100
70 45 45 35 5 15
V2 52 18 16,3
A/duck
(H5N2)
V1
85 80 85 75 75 75
2,1 2,2
SP,% GMT SCF
A/Ind.
(H5N1)
A/duck
(H5N2)
A/Ind.
(H5N1)
A/duck
(H5N2)
A/Ind.
(H5N1)
A/duck
(H5N2)
A/Ind.
(H5N1)
SC, %
56,6
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.
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
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” FCLP0,01 FCLP – 2,5 µg of HA VNH5N1 adjuvanted with 0,0083 mg of “fish caviar-like particles” FCLP0,1 Al – 2,5 µg of HA VNH5N1 adjuvanted with 0,083 mg of AL(OH)30,01 Al – 2,5 µg of HA VNH5N1 adjuvanted with 0,0083 mg of AL(OH)3Vir – 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/mouseNC–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.
Study’s results
№Groups of vaccine candidates
Virus strain
Antibody titer to H5N1 Days after control challenge Protectivity (%)
1-7 8 9 10 11 12 13 14-17Inactivated virus
1 Split H5N1 <20
19 19 14 12 12 12 12 11 57,92 0,1 FCLP H5N1 800
17 17 16 15 14 14 14 14 82,43 0,01 FCLP H5N1 400
19 19 15 13 13 13 13 13 68,44 0,1 Al H5N1 400
15 11 11 10 10 9 9 9 60,05 0,01Al H5N1 400
19 17 16 14 13 12 12 12 63,26 Vir H5N1 3000
13 13 13 13 13 13 13 13 1007 PR8 H1N1 <20
10 1 1 1 0 0 0 0 0Live attenuated
8 V-L H5N2 800
11 11 11 11 11 11 11 11 1009 NC` H1N1 <20
6 6 6 6 6 6 6 6 10010 Control - <20
20 3 0 0 0 0 0 0 0
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.**