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

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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)

Adsorption and gel-permeated chromatography of

viruses

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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)

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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

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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

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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-

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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

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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.**

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